Ultrastructure and fate of the nephridial anlagen in the ... - CiteSeerX

Arthropod Structure & Development 34 (2005) 471–480 www.elsevier.com/locate/asd

Ultrastructure and fate of the nephridial anlagen in the antennal segment of Epiperipatus biolleyi (Onychophora, Peripatidae)—evidence for the onychophoran antennae being modified legs Georg Mayer*, Markus Koch Institut fu¨r Biologie/Zoologie, Systematik und Evolution der Tiere, Freie Universita¨t Berlin, Ko¨nigin-Luise-Str. 1-3, D-14195 Berlin, Germany Received 8 February 2005; accepted 31 March 2005

Abstract In this study, new ultrastructural data on the nephridiogenesis in Epiperipatus biolleyi (Onychophora) are provided, and the general distribution of nephridial organs, their vestigia, and derivatives within the onychophoran body is revised. Transient anlagen of nephridial organs proved to be present in the anteriormost segment bearing the antennae. These nephridial anlagen were never found to open to the exterior in any developmental stage studied, but are nevertheless equipped with well-developed cilia. The ciliated nephridial canals are situated at the antennal bases, hence in a more dorsal position than in the remaining body segments. In postantennal segments, the nephridial anlagen constantly arise at the bases of the presumptive legs or their derivatives. These results provide the first evidence that onychophoran antennae are modified legs that retained the original arrangement of the nephridial anlagen at their bases, despite the evolutionary change in position and function of these legs. Current assumptions are accordingly confirmed that the onychophoran antennae and the first antennae in Mandibulata (Euarthropoda) are non-homologous, since they have to be attributed to different head segments. Although the fate of the homologue of the onychophoran antennae in the Euarthropoda remains to be clarified, insights obtained in the present study question previous claims that an ocular segment is absent in extant Mandibulata. q 2005 Elsevier Ltd. All rights reserved. Keywords: Nephridial organ; Nephridiogenesis; Serial homology; Antenna; Body appendage; Arthropoda

1. Introduction The differentiation of mesoderm in the Onychophora (Arthropoda) has always been a matter of controversial discussion (cf. Balfour, 1883; Moseley and Sedgwick, 1883; Kennel, 1883, 1885, 1888; Sedgwick, 1887, 1888; Sheldon, 1888a; Evans, 1901a; Glen, 1918; Manton, 1949; Pflugfelder, 1962; Walker, 1995; Bartolomaeus and Ruhberg, 1999; reviews: Anderson, 1973; Nielsen, 2001). Some contentious issues of mesoderm development were recently clarified by ultrastructural investigations (Mayer et al., 2004, 2005). Regarding the development of nephridia within the onychophoran head, however, the interpretations of earlier authors deviate from each other. This especially * Corresponding author. Tel.: C49 30 838 54 885; fax: C49 30 838 54 869. E-mail address: [email protected] (G. Mayer).

1467-8039/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2005.03.004

concerns the question whether transitory nephridial organs occur in the anteriormost segment bearing the antennae. Embryonic structures of this kind were described by Kennel (1888) as being situated dorsally with respect to the developing eye, at the bases of the presumptive antennae. Other authors, in contrast, described a pair of simple coelomoducts in the ventral part of the antennal segment (Sedgwick, 1887; Sheldon, 1888a; Evans, 1901a). Whether any of these structures really represent vestigia of nephridial organs is still uncertain. In representatives of the Onychophora, the anlagen of nephridial organs and their derivatives can be identified with certainty by the presence of well-developed cilia within their canals (see, e.g. Storch et al., 1978, 1979; Storch and Ruhberg, 1993; Mayer et al., 2004, 2005). In the antennal segment, however, cilia within the nephridial anlagen were not mentioned by the earlier authors (cf. Sedgwick, 1887; Kennel, 1888; Sheldon, 1888a; Evans, 1901a), potentially due to technical limitations of resolution at that time. In order to clarify this subject, mesoderm differentiation in the

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G. Mayer, M. Koch / Arthropod Structure & Development 34 (2005) 471–480

antennal segment was studied ultrastructurally in Epiperipatus biolleyi, a neotropical species of the Peripatidae. In the following, the results of this study are presented and their significance is discussed with respect to the nature of the onychophoran antennae. Insights obtained in this study have implications on our general understanding of head segmentation in arthropods. In addition, available data on the distribution of nephridial organs, their vestigia, and their derivatives within the onychophoran body are summarized.

2. Materials and methods All specimens of Epiperipatus biolleyi (Bouvier, 1902) used for the present study were collected in July 2003 in Costa Rica. Two gravid females were obtained from Cascajal de Coronado, near San Jose´, and one female was found in Coopesilencio, near Quepos. The genital tracts were dissected out of the females and placed into a fixative consisting of 2% paraformaldehyde and 2.5% glutaraldehyde buffered in 0.1 M sodium phosphate (pH 7.2). They were left therein for 2–3 weeks. Ruthenium red was added to the fixative after fixation in order to stain components of the extracellular matrix. The genital tracts were then washed several times in 0.1 M sodium phosphate buffer and kept therein for 2 days at 4 8C. Within the same buffer, the uterine chambers containing the embryos were severed from each other. Only the oldest stages were dissected out. The complete uterine chambers and dissected embryos were postfixed in 1% osmium tetroxide (buffered in 0.1 M sodium phosphate), dehydrated in an acetone series, and embedded in araldite. They were cut with diamond knives into series of semi-thin (1 mm) and silver interferencecolored (55–65 nm) sections on a Reichert Ultracut microtome. Semi-thin sections were placed on glass slides and stained with Toluidine Blue. Ultra-thin sections were mounted on formvar covered, single-slot copper grids and automatically stained with uranyl acetate and lead citrate in a Leica EM ultrostainer. Light microscopy was used for orientation within the embryos. The ultra-thin sections were imaged on a Philips CM 120 transmission electron microscope. Image intensity histograms of the electron micrographs were adjusted using the program AnalySIS. Montages of electron micrographs and final plates were produced using the Adobe Photoshop CS and Adobe Illustrator CS software.

3. Results In E. biolleyi the paired uteri of gravid females have a necklace-like appearance due to regularly arranged swellings caused by embryos in successive developmental stages. The dissected uteri of the three females contained 12, 14, and 15 embryos, respectively. The age of the embryos increases towards the vagina. Accordingly, the

youngest stages are situated near the ovary. A time table on the sequence of embryogenesis cannot be given, since the specimens were not cultured. Instead, specific landmarks are provided to identify the different developmental stages of the antennal segment described in the following. 3.1. Early (coiled) stages comprising coelomogenesis During the early mesoderm development the first pair of embryonic coelomic cavities being formed is that of the anteriormost, i.e. the antennal segment. At this stage no antennae are present yet. They arise first when the embryo elongates and appear as a pair of flattened ectodermal bulbs (Fig. 1A). During the elongation of the embryo, the first pair of coelomic cavities maintains its position at the anteriormost end. Their coelomic linings are represented by thin monolayered epithelia (Fig. 1B). In the posterior region of the antennal segment, the coelomic cavities flank the embryonic pharynx and the anterior protrusion of the presumptive gut (Fig. 1C). The pharynx represents an ectodermal invagination that connects the precursory mouth with the presumptive gut. The walls of the gut (endoderm) are strongly vacuolated and, therefore, easily distinguished from other structures within the investigated embryos. 3.2. Changes during early stage of eye development Like in all other embryonic segments, the linings of coelomic cavities within the antennal segment undergo considerable changes during the further development. These changes are along with the differentiation of other organ systems within the antennal segment, the brain and the eyes in particular. At the stage when the precursory antennae are already innervated, the presumptive brain occupies most of the space within the antennal segment. The presumptive antennal lobes, that represent posterior prolongations of the antennal nerves within the embryonic brain, are already formed (Fig. 2A). The future infracerebral (or ‘hypocerebral’) organs have already been separated from the ectoderm and are embedded into the ventral brain tissue. At this stage, the anlage of the eye represents a simple vesicle that is already separated from the ectoderm (Fig. 2A). The lumen of the eye vesicle is electron-light and appears empty. It is lined by epithelial cells connected by apical junctions. These epithelial cells rest on a basal lamina towards the embryonic hemocoel (Fig. 3A). Muscular cells are already present within the antennal segment, though they do not form any distinctive layers. The muscular cells can only be recognized as constituents of connective tissue (Fig. 2A and C). At this stage of eye development, a small remnant of the embryonic coelomic cavities (cf. Fig. 1C) is found dorsomedially to the eyes and opposite to the antennal lobes (Fig. 2A). The lining cells of these remaining coelomic cavities are still connected by apical junctions and rest on a basal lamina (Fig. 3C). Posteriorly, the coelomic cavities end


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Fig. 1. Ultra-thin sections through the anteriormost, i.e. antennal segment of an embryo of Epiperipatus biolleyi in the early coiled stage (posterior end is still in process of coelomogenesis). A: The anlage of antenna (an) is seen as dorso-lateral thickening of ectoderm in the anteriormost segment. B: Coelomic lining cells (cl) compose a monolayered epithelium. The lining cells are connected by apical junctions (aj) towards the coelomic cavity (co) and rest on a basal lamina (arrowheads). C: Complete cross-section of the antennal segment (montage of several electron micrographs). Dorsal is to the top. The embryo is situated within the lumen of an embryonic sac (es) enclosed by the maternal uterine wall (ut). Internally, paired, crescent-shaped coelomic cavities (co) are found on both sides of the presumptive gut. The arrow points to the embryonic mouth leading to the presumptive pharynx (ph). Aj, apical junction; an, earliest anlage of the antenna; cl, coelomic lining; co, embryonic coelomic cavity; ec, ectoderm; en, endoderm; es, lumen of the embryonic sac; gl, lumen of the presumptive gut; ne, neuroectoderm; nu; nucleus; ph, presumptive pharynx; ut, maternal uterine wall.

blindly. Anteriorly, they bear narrow protrusions that extend into the dorsal part of the presumptive antennae. Another prominent structure within the antennal segment is a paired ciliated canal, which is situated dorsal to the anlagen of the eyes at the bases of the antennae (Fig. 2A and C). The wall of the canal represents a true epithelium, since its cells are connected by apical junctions and rest on a basal lamina, whereas extracellular matrix (lamina densa) is lacking within the lumen of the canal (Figs. 2D and 3A). The canal

does not open to the exterior nor into the embryonic hemocoel, though its distal wall is thin. No podocytes were detected in association with the canal at this or any other investigated stage. The cilia within the lumen of the canal show the typical 9!2C2 distribution of the microtubules (Fig. 2D). The ciliated canals and the described coelomic remnants are the only epithelial structures of mesoderm within the antennal segment. They were found in six embryos of similar developmental stage.

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3.3. Advanced stage of eye development At a slightly later developmental stage, no epithelial structures of mesoderm can be detected within the antennal segment. At this stage, the cuticle that covers the ectoderm is more prominent (Fig. 2B). The lumen of the eye anlage is filled with extracellular, electron-dense material, which represents the precursory lens. The position of the lens is shifted laterally, towards the ectoderm. In this part, the lens is covered by a flattened layer of cells. In contrast, the cells of the remaining eye anlagen are of a columnar shape. Their apical region is filled with electron-dense pigment forming globular vesicles. At this described late developmental stage, the coelomic remnants have disappeared, as have their epithelial protrusions inside the antennae. The same is true for the ciliated canals that have also vanished. Only mesenchymatous muscular cells are found within the antennal segment. Among them, a prominent musculature is formed dorsal to the presumptive eye (Fig. 2B).

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(see Ruhberg and Storch, 1976, 1978; Storch et al., 1978, 1979; Lavallard, 1981; Lavallard and Campiglia, 1983; Storch and Ruhberg, 1990, 1993). The detection of welldeveloped cilia within the transitory canals of the antennal segment in the present study accordingly provides evidence that these canals in E. biolleyi represent nephridial organs as well. Since they disappear later during development, the nephridial organs within the antennal segment are transitory structures. Their position at the antennal bases exactly corresponds to that stated by Kennel (1888) for the coelomic sacs situated near the presumptive antennae in two other neotropical species of the Onychophora. The author’s interpretation of these sacs as the anlagen of ‘segmental organs’ is accordingly supported by our study. Whether the transient nephridial anlagen of the antennal segment are restricted to the neotropical onychophorans, however, remains to be clarified by further ultrastructural investigations. 4.2. Distribution of nephridial organs in Onychophora

3.4. Nephridiogenesis in trunk segments Compared with the antennal segment, ciliated canals also occur within the trunk segments in embryos of E. biolleyi. They arise segmentally at the bases of the presumptive walking legs (Fig. 3B). These ciliated canals represent the anlagen of nephridial organs. At the stage when the ciliated canals occur within the antennal segment (cf. Fig. 2A) the nephridial anlagen of the trunk segments already open to the exterior and bear the presumptive sacculus lined by podocytes (Fig. 3D). However, an excretory end bladder is not yet formed, nor is the nephridial canal differentiated into several regions. The excretory end bladder and the different regions of the canal arise later during embryogenesis.

4. Discussion 4.1. Presence of antennal nephridial anlagen Within the onychophoran body, ciliated canals exclusively occur within the nephridial organs or their derivatives

According to the present study, nephridial organs or their derivatives/rudiments are primarily found in every segment of the onychophoran body (Fig. 4). Within the head, transient nephridial anlagen occur in the antennal segment and the segment of the jaws (present study; Sedgwick, 1887; Kennel, 1888). In the segment of the slime papillae, the nephridial organs are modified into the salivary glands (Kennel, 1885, 1888; Sedgwick, 1885, 1887, 1888, 1895; Evans, 1901a; Storch et al., 1979; Mayer et al., 2005). Within the trunk, the nephridial organs of the first three legbearing segments are small (Balfour, 1883; Sedgwick, 1887, 1888, 1895; Buxton, 1913; Gabe, 1957; Storch et al., 1978), whereas those of the next two segments are large, and open in the distal instead of the proximal part of the legs (Fig. 4; Evans, 1901b; Buxton, 1913; Dakin, 1920; Gabe, 1957; Storch et al., 1978; Lavallard and Campiglia, 1988). In the following trunk segments, the nephridial organs are usually regarded as ‘typical’ nephridia in, e.g. bearing an excretory end bladder (Evans, 1901b; Dakin, 1920; Gabe, 1957; Birket-Smith, 1974; Storch et al., 1978; Lavallard, 1981; Lavallard and Campiglia, 1983; Storch and Ruhberg, 1993).

Fig. 2. Ultra-thin sections through the antennal segment of embryos of Epiperipatus biolleyi in succeeding developmental stages. A: Early stage of eye development, cross-section of eye region. Dorsal is to the top. The eye anlage (ey) consists of an epithelial vesicle that has already been separated from the overlying ectoderm (ec). Asterisk: lumen of the eye vesicle, which appears electron-light, as the lens is not formed yet. Arrowhead: mesenchymatous muscular cells (constituents of the connective tissue). Between the antennal lobe (al) and the eye vesicle, a small remnant of the coelomic cavity (co) is still present. Above the presumptive eye, opposite to the antennal lobe, lies a ciliated canal (ca). B: Advanced stage of eye development, cross-section of eye region. Dorsal is to the top. The cuticle (cu) is more prominent than at the earlier stages of eye development. The precursory lens (le) is already seen as electron-dense content within the eye vesicle. Differentiating optic nerve connecting the presumptive eye with the embryonic brain (br) is present but not shown in this image. No epithelial structures of mesoderm are retained at this stage. Mesoderm is mainly represented by musculature (mu) situated around the eye anlage. C: Early stage of eye development, overview of the ciliated canal (ca) situated above the eye vesicle. The arrow points to the lumen of the canal. The arrowheads indicate connective tissue composed of muscular cells embedded into extracellular matrix (mainly composed of collagen). D: Early stage of eye development. The lumen of the canal (lu) contains well-developed cilia (ci) showing the typical 9!2C2 axonemal pattern. The cells of the canal wall are connected by apical junctions (aj). Aj, apical junction; al, presumptive antennal lobe (Zposterior prolongation of the antennal nerve); br, embryonic brain; ca, ciliated canal; ci, cilia; co, remnant of the embryonic coelomic cavity; cu, embryonic cuticle; ec, ectoderm; ex, exterior; ey, anlage of the eye (Zeye vesicle); he, presumptive hemocoel; le, precursory lens within the eye vesicle; ln, presumptive lateral neuropil of the brain; lu, lumen of the ciliated canal; mu, musculature surrounding the eye vesicle; mv, microvilli.

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Fig. 3. Ultra-thin sections through embryos of Epiperipatus biolleyi at different developmental stages. A: Early stage of eye development, high magnification of the space between the epithelia of eye vesicle (ey) and wall of ciliated canal (cw). Each epithelium rests on its own basal lamina (arrowheads). The space inbetween is continuous with the remaining embryonic hemocoel (he) (Z‘mixocoel’). B: Advanced stage of eye development, partial cross-section of a trunk segment bearing the anlage of the 17th pair of walking legs. Dorsal is to the top. At the basis of the presumptive leg (la) a nephridial anlage is seen, which consists of a presumptive excretory duct opening to the exterior (arrow), a coiled canal (nc) containing well-developed cilia (inset), and a narrow sacculus (sa) composed of podocytes. No excretory end bladder is formed yet. C: Early stage of eye development, high magnification of the epithelial lining of the coelomic remnant (co) near the eye vesicle (cf. Fig. 2A). The lining cells of the coelomic remnant are still connected by apical junctions (aj) and rest on a basal lamina (arrowheads). D: Advanced stage of eye development, high magnification of podocytes (po) from the presumptive nephridial sacculus. The filtrating unit is already formed and consists of pedicels connected by diaphragms and resting on a basal lamina (inset). Asterisks mark the lumen of the sacculus; arrowhead points to the basal lamina. Aj, apical junction; ci, cilia; co, remnant of the coelomic cavity; cw, wall of the ciliated canal (basal part); ec, ectoderm; en, endoderm; ex, exterior; ey, cells of the eye vesicle; he, embryonic hemocoel; la, anlage of the 17th walking leg; nc, anlage of the nephridial canal; nv, presumptive nerve cord (neuropil is already formed); po, podocytes; sa, sacculus of the nephridial anlage.


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1885). Transient nephridial organs are known in this segment in the females, whereas they have been modified into the accessory genital glands in the males (Kennel, 1888; Evans, 1901a; Ruhberg and Storch, 1978, 1988; Storch and Ruhberg, 1993). 4.3. Implications for the nature of onychophoran antennae

Fig. 4. General distribution of nephridial organs, their rudiments, and derivatives within the onychophoran body (segments are numbered). Schematic representation of anterior and posterior ends of adult individuals of both sexes. Transient anlagen of nephridial organs (indicated in red) occur during embryogenesis within the segments of antennae and jaws as well as the female posteriormost segment. Nephridial organs have been modified into salivary glands (indicated in brown) in the segment of slime papillae. Within the first three leg-bearing segments, nephridial organs (in yellow) are small, whereas those of the next two segments are large (in light-blue) and open in the distal instead of the proximal part of legs. Both types lack excretory end bladders. Nephridial organs of the following segments are usually regarded as ‘typical’ nephridia (indicated in green). Within the genital segment, nephridial organs have been modified into the gonoducts of genital tracts (indicated in white). A pair of either welldeveloped or rudimentary nephridial organs (orange-colored) occurs in postgenital, last leg-bearing segment (marked by dotted vertical lines) in representatives of Peripatidae. The corresponding segment and its nephridial organs are completely lacking in representatives of Peripatopsidae. In the ultimate, appendage-less segment, nephridia are postembryonically only maintained in the male, but are modified into a pair of accessory genital glands (indicated in dark-blue).

However, the end bladder may be lacking in the more posterior segments (Evans, 1901b). At the posterior end of the onychophoran body, the nephridial structure deviates from the typical pattern. Within the genital segment, the nephridial organs have been modified into the gonoducts of the genital tract (Sedgwick, 1887; Kennel, 1888; Sheldon, 1888b; Evans, 1901a; Storch and Ruhberg, 1993). The postgenital, last leg-bearing segment is completely lacking in adults of the Peripatopsidae, though Sedgwick (1887) attributed a pair of embryonic coelomic cavities to this segment. In the Peripatidae, a postgenital leg-bearing segment generally exists including a pair of nephridial organs that in some cases might be rudimentary (see, e.g. Buxton, 1913; Daiber, 1913). The following, ultimate segment does not bear any body appendages (e.g. Kennel,

At first sight, the dorsal position of the nephridial anlagen in the antennal segment of E. biolleyi seems to be peculiar, as it differs from that of the remaining nephridial anlagen. Compared to the other body segments, however, there is a striking correspondence in that all nephridial anlagen lie at the bases of the corresponding segmental appendages. In the case of the anteriormost body segment, these appendages are represented by the antennae. The most obvious explanation of the state in the antennal segment accordingly is that the onychophoran antennae represent modified legs that retained nephridial anlagen at their base. During the evolutionary transformation of these legs into sensory structures, the nephridial anlagen of the corresponding segment apparently followed the coincident shift of these legs from a formerly ventral towards an antero-dorsal position on the head. This view is supported by the fact that a conservative retention of the nephridiopore at the leg bases is not only found in the onychophoran trunk segments, but also holds true for the modified nephridia in representatives of the Euarthropoda (see, e.g. Woodring, 1973; Clarke, 1979; Sekiguchi, 1988; Gruner, 1993; Olesen et al., 2003). In this respect, overall independence of the evolution of nephridia and limbs (cf. Scholtz, 2002) seems to be questionable in at least the Arthropoda and deserves to be reconsidered in future developmental studies. Yet, no further evidence was provided for the view that the onychophoran antennae are modified legs. Comparative analyses of the antennal muscular equipment and its nerve supply are still wanting. Previous analyses of gene expression patterns also yielded no definite conclusion. Although the expression pattern of the homeobox gene Distal-less is similar in the onychophoran antennae and legs, insights remain preliminary, as Distal-less is also expressed in non-appendicular body outgrowths (see Panganiban et al., 1997). This still causes much speculation on whether the onychophoran antennae may rather correspond to the prostomial palps as present in some polychaetes (see, e.g. Holmgren, 1916; Hanstro¨m, 1928, 1935; Henry, 1948; Scholtz and Edgecombe, 2005: footnote 2). Recent phylogenetic analyses, however, leave hardly any doubt that neither prostomial, nor peristomial palps belong to the polychaete ground pattern (see Rouse and Fauchald, 1997; Bartolomaeus et al., 2005). Nor does a (nonsegmental) ‘prostomium’ or ‘acron’ seem to be involved in the onychophoran head formation (Eriksson et al., 2003), which is confirmed in our study. We accordingly consider the position of the antennal nephridial anlagen in E. biolleyi to provide the first evidence for the origin of the

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onychophoran antennae from the paired appendages of the anteriormost segment. 4.4. Non-homology of onychophoran and euarthropod antennae The present evidence for the appendicular nature of the onychophoran antennae supports the conclusions drawn by Eriksson et al. (2003) on the existence of an originally appendage-bearing ocular segment in the Arthropoda (Z antennal segment in the Onychophora). The recent distinction of primary and secondary antennae in arthropods by Scholtz and Edgecombe (2005) accordingly seems to be justified due to the implication that the antennae have to be attributed to different head segments (protocerebral in the Onychophora, deutocerebral in at least the Recent Mandibulata). Presence of protocerebral antennae in the arthropod stem species may be supported by some recent experimental studies on gene expression patterns (see, e.g. Panganiban et al., 1997), which indicate that antennae are the default arthropod appendage (for critical discussion see Minelli, 2003). According to this view, the protocerebral antennae represent a plesiomorphic feature of the Onychophora. With respect to the fossil record, however, no unambiguous decision is apparently possible at present on whether protocerebral antennae already belong to the arthropod ground pattern. Irrespective of whether one considers the Articulata or Ecdysozoa concept, protocerebral antennae are absent in the arthropod sister group (i.e. Annelida versus Nematoida/Cycloneuralia; see, e.g. Giribet, 2003) as well as in the Tardigrada, which are traditionally attributed to the still unresolved basal lobopodian assemblage within the Arthropoda (see, e.g. Maas et al., 2004). Absence of protocerebral antennae also seems to be true for several Cambrian lobopodians (see, e.g. Budd, 1997; Ramsko¨ld and Chen, 1998; Dzik, 2003), though their still lobopod-like anteriormost appendages—if homologous to the onychophoran antennae—in part may already be shifted to a more lateral position. We therefore presently tend to consider the antennae of the extant Onychophora to represent an apomorphy of this group, derived from still lobopod-like anteriormost appendages in the arthropod ground pattern. Their fate in the Euarthropoda (and the stem lineage representatives of this group) presently remains unclear (see Budd, 2002, versus Chen et al., 2004, versus Cotton and Braddy, 2004, versus Scholtz and Edgecombe, 2005). Our results support the view that at least in extant forms the protocerebral antennae are either completely lost or transformed into a labrum (Eriksson et al., 2003; Scholtz and Edgecombe, 2005; for a different view on the evolution of the labrum, see, e.g. Walossek, 1999). Insights obtained in the present study encourage comparable analyses of mesoderm differentiation in euarthropods to further contribute to a clarification of this issue.

Acknowledgements We are indebted to Instituto Nacional de Biodiversidad (INBio), Costa Rica, for collecting the animals, dissecting, fixing, and sending the material to us. We are grateful to Ina Mayer, Thomas Bartolomaeus, Gregory Edgecombe and Gerhard Scholtz for critical discussions on the subject and improving the manuscript. Andreas Maas and an anonymous referee also gave some useful comments. Hilke Ruhberg and Ira Richling kindly helped to get contact to the staff of the National Institute of Biodiversity in Costa Rica. We thank Bjo¨rn Quast for writing software for a more comfortable handling of the electron microscopic data. This work was supported by the Studienstiftung des deutschen Volkes to Georg Mayer (D/2002 0033) and the Deutsche Forschungsgemeinschaft (BA 1520/8-1, 8-2, RU 358/4-1, 4-2).

References Anderson, D.T., 1973. Embryology and Phylogeny in Annelids and Arthropods, 50th ed. Pergamon Press, Oxford. Balfour, F.M., 1883. The anatomy and development of Peripatus capensis. Quarterly Journal of Microscopical Science 23, 213–259. Bartolomaeus, T., Ruhberg, H., 1999. Ultrastructure of the body cavity lining in embryos of Epiperipatus biolleyi (Onychophora, Peripatidae)—a comparison with annelid larvae. Invertebrate Biology 118, 165–174. Bartolomaeus, T., Purschke, G., Hausen, H., 2005. Polychaete phylogeny based on morphological data—a comparison of current attempts. Hydrobiologia 535/536, 339–354. Birket-Smith, S.J.R., 1974. The anatomy of the body wall of Onychophora. Zoologische Jahrbu¨cher Abteilung fu¨r Anatomie und Ontogenie der Tiere 93, 123–154. Budd, G.E., 1997. Stem group arthropods from the Lower Cambrian Sirius Passet fauna of North Greenland. In: Fortey, R.A., Thomas, R.H. (Eds.), Arthropod Relationships. Systematics Association, vol. 55. Chapman and Hall, London, pp. 125–138. Budd, G.E., 2002. A palaeontological solution to the arthropod head problem. Nature 417, 271–275. Buxton, B.H., 1913. Coxal glands of arachnids. Zoologische Jahrbu¨cher, Abteilung fu¨r Anatomie und Ontogenie der Tiere suppl. 14, 231–282. Chen, J., Waloszek, D., Maas, A., 2004. A new ‘great appendage’ arthropod from the Lower Cambrian of China and homology of chelicerate chelicerae and raptorial antero-ventral appendages. Lethaia 37, 3–20. Clarke, K.U., 1979. Visceral anatomy and arthropod phylogeny. In: Gupta, A.P. (Ed.), Arthropod Phylogeny. Van Nostrand Reinhold Company, New York, pp. 467–549. Cotton, T.J., Braddy, S.J., 2004. The phylogeny of arachnomorph arthropods and the origin of the Chelicerata. Transactions of the Royal Society of Edinburgh, Earth Sciences 94, 169–193. Daiber, M., 1913. Protracheata (Onychophora). In: Lang, A. (Ed.), Handbuch der Morphologie der wirbellosen Tiere. Gustav Fischer, Jena, pp. 351–371. Dakin, W.J., 1920. Fauna of Western Australia—III. Further contributions to the study of the Onychophora. The anatomy and systematic position of Western Australian Peripatoides, with an account of certain histological details of general importance in the study of Peripatus, Proceedings of the Zoological Society of London 1920. pp. 367–389. Dzik, J., 2003. Early Cambrian lobopodian sclerites and associated fossils from Kazakhstan. Palaeontology 46, 93–112.

G. Mayer, M. Koch / Arthropod Structure & Development 34 (2005) 471–480 Eriksson, B.J., Tait, N.N., Budd, G.E., 2003. Head development in the onychophoran Euperipatoides kanangrensis. With particular reference to the central nervous system. Journal of Morphology 255, 1–23. Evans, R., 1901a. On the Malayan species of Onychophora. Part II—the development of Eoperipatus weldoni. Quarterly Journal of Microscopical Science 45, 41–88. Evans, R., 1901b. On two new species of Onychophora from the Siamese Malay States. Quarterly Journal of Microscopical Science 44, 473–538. Gabe, M., 1957. Donneés histologiques sur les organes segmentaires des Peripatopsidae (Onychophores). Archives d’Anatomie Microscopique et de Morphologie Experimentale 46, 283–305. Giribet, G., 2003. Molecules, development and fossils in the study of metazoan evolution; Articulata versus Ecdysozoa revisited. Zoology 106, 303–326. Glen, E.H., 1918. A revision of certain points in the early development of Peripatus capensis. Quarterly Journal of Microscopical Science 63, 283–292. Gruner, H.-E., 1993. Stamm Arthropoda. In: Gruner, H.-E. (Ed.), Lehrbuch der Speziellen Zoologie, Band I (Wirbellose Tiere) 4. Teil (Arthropoda ohne Insecta). Gustav Fischer, Jena, pp. 11–63. Hanstro¨m, B., 1928. Onychophora. In: Vergbeichende Anatomie des Nervensystems der Wirbellosen Tiere unter Beru¨cksichtigung seiner Funktion. J. Springer, Berlin, pp. 341–351. Hanstro¨m, B., 1935. Bemerkungen u¨ber das Gehirn und die Sinnesorgane der Onychophoren. Kungliga Fysiografiska Sa¨llskapets i Lund Handlingar, Ny Fo¨ljd 46, 1–37. Henry, L.M., 1948. The nervous system and the segmentation of the head in the Annulata. Microentomology 13, 27–48. Holmgren, N.F., 1916. Zur vergleichenden Anatomie des Gehirns von Polychaeten, Onychophoren, Xiphosuren, Arachniden, Crustaceen, Myriapoden, und Insekten. Vorstudien zu einer Phylogenie der Arthropoden, Kungl. Svenska Vetenskapsakademien Handlingar [Series 2], vol. 56, pp. 1–303. Kennel, J. von, 1883. Entwicklungsgeschichte von Peripatus. Zoologischer Anzeiger 6, 531–537. Kennel, J. von, 1885. Entwicklungsgeschichte von Peripatus edwardsii Blanch. und Peripatus torquatus n.sp. I. Theil. Arbeiten aus dem Zoologisch-Zootomischen Institut in Wu¨rzburg, 7, pp. 95–229. Kennel, J. von, 1888. Entwicklungsgeschichte von Peripatus edwardsii Blanch. und Peripatus torquatus n.sp. II. Theil. Arbeiten aus dem Zoologisch-Zootomischen Institut in Wu¨rzburg, 8, pp. 1–93. Lavallard, R., 1981. Donneés ultrastructurales sur les organes segmentaires de Peripatus acacioi Marcus et Marcus (Onychophora: Peripatidae). Annales des Sciences Naturelles, Zoologie et Biologie Animale, 13e Se´rie 3, 23–62. Lavallard, R., Campiglia, S., 1983. Sur la ciliature des nephridies chez Peripatus acacioi Marcus et Marcus (Onychophora: Peripatidae). Archives d’Anatomie Microscopique et de Morphologie Experimentale 72, 183–197. Lavallard, R., Campiglia, S., 1988. Le tubule distal des 4e et 5e paires d’organes reńaux chez Peripatus acacioi Marcus et Marcus (Onychophora: Peripatidae): E´tude infrastructurale. Comptes Rendus de l’Acade´mie des Sciences, Serie III: Sciences de la Vie, 307, pp. 415– 421. Maas, A., Waloszek, D., Chen, J., Braun, A., Wang, X., Huang, D., 2004. Phylogeny and life habits of early arthropods—predation in the early Cambrian sea. Progress in Natural Science 14, 158–166. Manton, S.M., 1949. Studies on the Onychophora VII. The early embryonic stages of Peripatopsis, and some general considerations concerning the morphology and phylogeny of the Arthropoda. Philosophical Transactions of the Royal Society of London, B Biological Sciences 233, 483–580. Mayer, G., Ruhberg, H., Bartolomaeus, T., 2004. When an epithelium ceases to exist—an ultrastructural study on the fate of the embryonic coelom in Epiperipatus biolleyi (Onychophora, Peripatidae). Acta Zoologica 85, 163–170. Mayer, G., Bartolomaeus, T., Ruhberg, H., 2005. Ultrastructure of

479

mesoderm in embryos of Opisthopatus roseus (Onychophora, Peripatopsidae)—revision of the ‘long germ band’ hypothesis for Opisthopatus. Journal of Morphology 263, 60–70. Minelli, A., 2003. The origin and evolution of appendages. International Journal of Developmental Biology 47, 573–581. Moseley, H.N., Sedgwick, A., 1883. Peripatus. Nature 29, 196. Nielsen, C., 2001. Animal evolution, 2nd, Interrelationships of the Living Phyla. University Press, Oxford. Olesen, J., Richter, S., Scholtz, G., 2003. On the ontogeny of Leptodora kindtii (Crustacea, Branchiopoda, Cladocera), with notes on the phylogeny of the Cladocera. Journal of Morphology 256, 235–295. Panganiban, G., Irvine, S.M., Lowe, C., Roehl, H., Corley, L.S., Sherbon, B., Grenier, J.K., Fallon, J.F., Kimble, J., Walker, M., Wray, G.A., Swalla, B.J., Martindale, M.Q., Carroll, S.B., 1997. The origin and evolution of animal appendages. Proceedings of the National Academy of Science USA 94, 5162–5166. Pflugfelder, O., 1962. Onychophora. In: Pflugfelder, O. (Ed.), Lehrbuch der Entwicklungsgeschichte und Entwicklungsphysiologie der Tiere. Gustav Fischer, Jena, pp. 139–144. Ramsko¨ld, L., Chen, J., 1998. Cambrian lobopodians: morphology and phylogeny. In: Edgecombe, G.D. (Ed.), Arthropod Fossils and Phylogeny. Columbia University Press, New York, pp. 107–150. Rouse, G.W., Fauchald, K., 1997. Cladistics and polychaetes. Zoologica Scripta 26, 139–204. Ruhberg, H., Storch, V., 1976. Zur Ultrastruktur von ma¨nnlichem Genitaltrakt, Spermiocytogenese und Spermien von Peripatopsis moseleyi (Onychophora). Zoomorphologie 85, 1–15. Ruhberg, H., Storch, V., 1978. Zur Ultrastruktur der accessorischen Genitaldru¨sen von Opisthopatus cinctipes (Onychophora, Peripatopsidae). Zoologischer Anzeiger 200, 289–299. Ruhberg, H., Storch, V., 1988. Onychophora. In: Adiyodi, K.G., Adiyodi, R.G. (Eds.), Reproductive Biology of Invertebrates Accessory Sex Glands, vol. 3. Oxford and IBH Publishing, New Delhi, pp. 251– 260. Scholtz, G., 2002. The Articulata hypothesis—or what is a segment? Organisms Diversity and Evolution 2, 197–215. Scholtz, G., Edgecombe, G.D., 2005. Heads, hox and the phylogenetic position of trilobites. In: Koenemann, S., Jenner, R., Vonk, R. (Eds.), Crustacea and Arthropod Phylogeny Crustacean Issues, vol. 17, pp. 139–165. Sedgwick, A., 1885. The Development of Peripatus capensis. Part I. Quarterly Journal of Microscopical Science 25, 449–468. Sedgwick, A., 1887. The Development of the Cape species of Peripatus. Part III. On the changes from stage A to stage F. Quarterly Journal of Microscopical Science 27, 467–550. Sedgwick, A., 1888. The Development of the Cape species of Peripatus. Part IV. The changes from stage G to birth. Quarterly Journal of Microscopical Science 28, 373–396. Sedgwick, A., 1895. Peripatus. In: Harmer, S.F., Shipley, A.E. (Eds.), Peripatus, Myriapods and Insects, part I. MacMillan, London, pp. 3–26. Sekiguchi, K., 1988. Arthropoda. II. Arachnida. In: Kume´, M., Dan, K. (Eds.), Invertebrate Embryology. Garland Publishing, New York, pp. 389–404. Sheldon, L., 1888a. On the development of Peripatus novae-zealandiae. Quarterly Journal of Microscopical Science 29, 283–294. Sheldon, L., 1888b. Notes on the anatomy of Peripatus capensis and Peripatus novae-zealandiae. Quarterly Journal of Microscopical Science 28, 495–499. Storch, V., Ruhberg, H., 1990. Electron microscopic observations on the male genital tract and sperm development in Peripatus sedgwicki (Peripatidae, Onychophora). Invertebrate Reproduction and Development 17, 47–56. Storch, V., Ruhberg, H., 1993. Onychophora. In: Harrison, F.W., Rice, M.E. (Eds.), Microscopic Anatomy of Invertebrates Microscopic Anatomy of Invertebrates, vol. 12. Wiley-Liss, New York, pp. 11–56. Storch, V., Ruhberg, H., Alberti, G., 1978. Zur Ultrastruktur der

480


Segmentalorgane der Peripatopsidae (Onychophora). Zoologische Jahrbu¨cher, Abteilung fu¨r Anatomie und Ontogenie der Tiere 100, 47–63. Storch, V., Alberti, G., Ruhberg, H., 1979. Light and electron microscopical investigations on the salivary glands of Opisthopatus cinctipes and Peripatopsis moseleyi (Onychophora: Peripatopsidae). Zoologischer Anzeiger 203, 35–47. Walker, M., 1995. Relatively recent evolution of an unusual pattern of early embryonic development (long germ band?) in a South African

onychophoran, Opisthopatus cinctipes Purcell (Onychophora: Peripatopsidae). Zoological Journal of the Linnean Society 114, 61–75. Walossek, D., 1999. On the Cambrian diversity of Crustacea. In Schram, F.R., von Vaupel Klein, J.C. (Eds.), Crustaceans and the Biodiversity Crisis vol. 1. Koninklijke Brill Publisher, Leiden, pp. 3–27. Woodring, J.P., 1973. Comparative morphology, functions, and homologies of the coxal glands in oribatid mites (Arachnida: Acari). Journal of Morphology 139, 407–429.