Zoomorphology (2006) 125: 1–12 DOI 10.1007/s00435-005-0006-5
O R I GI N A L A R T IC L E
Georg Mayer
Origin and differentiation of nephridia in the Onychophora provide no support for the Articulata
Received: 15 April 2005 / Accepted: 3 August 2005 / Published online: 26 October 2005 Springer-Verlag 2005
Abstract Comparative morphology currently permits no unambiguous decision on the primary homology of the nephridia of Annelida and Arthropoda. In order to obtain additional information on this subject, ultrastructure of morphogenesis and further differentiation of nephridia was studied in the onychophoran Epiperipatus biolleyi (Peripatidae). In this species, the nephridial anlage develops by reorganization of the lateral portion of the embryonic coelomic wall that initially gives rise to a ciliated canal. All other structural components, including the sacculus, merge after the nephridial anlage has been separated from the remaining mesodermal tissue. The nephridial sacculus does not represent a ‘persisting coelomic cavity’, since it arises de novo during embryogenesis. There is no evidence for ‘nephridioblast‘ cells participating in the nephridiogenesis of Onychophora, which is in contrast to the general mode of nephridial formation in Annelida. Available data on nephridiogenesis in euarthropods (Chelicerata, Myriapoda, Crustacea, and Hexapoda) also provide no evidence for nephridia of Annelida and Arthropoda being a synapomorphy of these taxa. These findings accordingly weaken the traditional Articulata hypothesis. Keywords Nephridiogenesis Æ Onychophora Æ Ecdysozoa Æ Articulata Æ Homology
Introduction One of the currently most debated issues in phylogenetics is the Articulata/Ecdysozoa controversy (see, e.g., Giribet 2003; Schmidt-Rhaesa 2003, and references therein). In general, the Articulata hypothesis proposes a G. Mayer (&) Systematik und Evolution der Tiere, Institut fu¨r Biologie/Zoologie, Freie Universita¨t Berlin, Ko¨nigin-Luise-Str. 1-3, 14195 Berlin, Germany E-mail:
[email protected] Tel.: +49-30-83854885 Fax: +49-03-83854869
sister group relationship between the Arthropoda and Annelida. This hypothesis implies that onychophorans maintained a higher number of plesiomorphic features than euarthropods (i.e. Chelicerata, Myriapoda, Crustacea, and Hexapoda) (Storch and Ruhberg 1993; Ax 2000; Nielsen 2001; Brusca and Brusca 2003; Ruppert et al. 2004). In contrast, a close relationship between the Arthropoda and Cycloneuralia (nematodes, priapulids, and allies) has been suggested by the Ecdysozoa hypothesis. This hypothesis was initially based on a phylogenetic analysis of 18S ribosomal DNA sequences (Aguinaldo et al. 1997), but until now has received additional support by several independent approaches (e.g., Boore and Brown 2000; Haase et al. 2001; Mallatt and Winchell 2002; Philippe et al. 2005). Morphological support for this assemblage of molting animals, however, is still limited (see discussion by Jenner and Scholtz 2005). Unlike the Ecdysozoa hypothesis, the Articulata concept is commonly believed to be well-founded on morphology. In fact, however, segmentation represents the only major synapomorphy of Annelida and Arthropoda (see Nielsen 1997, 2001; Wa¨gele et al. 1999; Ax 2000; Wa¨gele and Misof 2001; Scholtz 2002, 2003). The homology of segmentation in the Articulata is mainly supported by segmentally arranged coelomic cavities and nephridia. All other characters that are sometimes interpreted as synapomorphies of the Annelida and Arthropoda (e.g., paired body appendages, ladder-like nervous system, and mushroom bodies) are problematic in the assessment of their homology (see discussions by Schmidt-Rhaesa et al. 1998; Budd 2001; Giribet 2003; Schmidt-Rhaesa 2003, 2004). But even the main characters supporting the homology of segmentation in annelids and arthropods do not withstand a careful consideration. Although segmentally arranged coelomic cavities are present in post-embryonic stages of annelids, they only occur in embryos of arthropods (reviewed by Anderson 1973). During arthropod development, embryonic coelomic cavities fuse with primary body cavity by mix-
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ocoely (see Mayer et al. 2004). Traditionally, only small remnants of coelomic cavities are assumed to persist as nephridial sacculi in adult arthropods (e.g., Storch and Ruhberg 1993; Bartolomaeus and Ruhberg 1999; Ruppert et al. 2004). The sacculi represent terminal compartments of nephridia that are composed of podocytes and are sites of ultrafiltration of the hemolymph. Among arthropods, true segmental arrangement of nephridia is only present in onychophorans whereas nephridial derivatives are restricted to merely one or a few anterior body segments in euarthropods (Fig. 1a, b). Furthermore, cilia occur within nephridial canals of onychophorans, which resembles the state in annelids but contrasts with the state in other arthropods (see Storch et al. 1978; Lavallard and Campiglia 1983; Storch and Ruhberg 1993; Nielsen 1997). Onychophorans accordingly play a pivotal role in the current discussion on the evolution of segmentation in annelids and arthropods (see Budd 2001; Scholtz 2002, 2003; Balavoine and Adoutte 2003; Seaver 2003). Apart from segmental arrangement and ciliation of nephridial canals, however, there seem to be no further correspondences between the nephridia of adult onychophorans and annelids. In general, the inclusion of developmental characters can additionally strengthen the confidence in homology of characters under consideration (see Scholtz 2002). Recent ultrastructural studies on nephridiogenesis in the Onychophora, however, are incomplete (see Mayer et al. 2004, 2005; Mayer and Koch 2005) and the results of earlier authors are contradictory (see Sedgwick 1887, 1888; Kennel 1888; Evans 1901). To close the gap, nephridiogenesis and further differentiation of nephridia has been studied in the onychophoran Epiperipatus biolleyi, a representative of the Neotropical Peripatidae. In the present paper, results of this ultrastructural study are compared to what is known on nephridial formation in other arthropods and in annelids. Insights obtained permit general conclusions on the homology of nephridia in Annelida and Arthropoda.
4C. Within the buffer, the uterine chambers containing the embryos were severed from each other. Only the oldest stages were dissected out of the uteri. The complete uterine chambers and dissected embryos were postfixed in 1% osmium tetroxide (buffered in 0.1 M sodium cacodylate or sodium phosphate), dehydrated in an acetone series, and embedded in araldite. They were cut with diamond knives into series of semi-thin (1 lm) and silver interference-colored (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 self-written software, and the program AnalySIS. Montages of electron micrographs and final plates were produced using the Adobe Photoshop CS and Adobe Illustrator CS software. The present study mainly deals with the nephridiogenesis in the Onychophora. Throughout the onychophoran body, however, the nephridia may differ in their anatomy (cf. Fig. 1a). Because the investigation of each separate segment would require a huge amount of material, the distinct antero-posterior developmental gradient within the investigated embryos was partially used in this study. Such a procedure is justifiable, since the early development of nephridia and their derivatives is similar, irrespective of their position within the onychophoran body (see, e.g., Sedgwick 1887, 1888; Kennel 1888; Evans 1901). Only the late differentiation of nephridia and their derivatives can differ. For the sake of clarity, the different stages and corresponding segments are indicated in the legend of each micrograph.
Results Materials and methods Specimens of Epiperipatus biolleyi (Bouvier 1902) were collected in February 1997 and July 2003 in Costa Rica. Three 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. Two alternative fixatives have been used: (1) 2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate at pH 7.0, and (2) 2% paraformaldehyde and 2.5% glutaraldehyde buffered in 0.1 M sodium phosphate at pH 7.2. Ruthenium red was added to each fixative in order to stain components of the extracellular matrix. After fixation, the genital tracts were washed several times in 0.1 M sodium cacodylate or sodium phosphate buffer, respectively, and kept therein for several days at
The uteri of gravid females of E. biolleyi contain embryos in successive developmental stages. The youngest stages occur near the ovary whereas the age of embryos increases towards the vagina. Because the investigated specimens were not cultured, a developmental time table cannot be given. Differentiation of mesoderm is, therefore, described in parallel with other morphogenetic changes in order to provide additional ‘landmarks’ for characterization of different developmental stages. General Changes during Mesoderm Differentiation During mesoderm proliferation, paired coelomic cavities arise along an antero-posterior gradient within the mesodermal strands in the investigated embryos. These
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Fig. 1 a-b Distribution of nephridia and their derivatives and vestigia in arthropods, schematic representation. a In adult onychophorans, nephridia occur within most trunk segments of the body (numbered from 1 to n, since the number of segments can vary intra-specifically). The nephridia have been modified into salivary glands in the segment of slime papillae (sp), and into posterior accessory genital glands in the male ultimate segment (n). Further modifications are the genital ducts within the segment (n2), and the enlarged nephridia within the segments of the fourth and fifth walking legs. The dotted vertical lines demarcate a segment that is present in the Peripatidae but is lacking in the Peripatopsidae (see Mayer and Koch 2005, for a more detailed description). b Distribution of nephridia and their derivatives in arthropods. Rudimentary anlagen and vestigia are represented by dotted lines. In cases where reports are uncertain a question mark is added. The segments are characterized by their appendages. In Onychophora, precursors of nephridia are initially present in every
segment but disappear later within the anteriormost two segments. During ontogeny, the nephridial anlagen of the third segment become modified into salivary glands. In Euarthropoda, nephridial anlagen usually occur in various segments, though nephridial derivatives only persist as ‘coxal glands‘ (chelicerates), ‘antennary glands’ (crustaceans), ‘maxillary glands’ (crustaceans, myriapods, proturans), and ‘labial glands’ (apterygotan insects) in adult stages. Data compiled from various sources (see Brauer 1895; With 1904; Buxton 1917; Siewing 1953; Yoshikura 1955; Feustel 1958; Moritz 1959; Weygoldt 1964, 1965, 1996; Benesch 1969; Franc¸ois 1969; Anderson 1973; Woodring 1973; Dohle 1980, 1996; Franc¸ois and Dallai 1986; Hessler and Elofsson 1991; Dunger 1993; Klausnitzer 1996; Schminke 1996; Ax 2000; Richter and Scholtz 2001, and references therein). an Antenna, ch chelicera, cl chilarium, ic intercalary segment, jw jaw, la labium, le walking legs, md mandible, mx maxilla, pd pedipalp, sp slime papilla
coelomic cavities are the first serially arranged structures that are recognized. At a more advanced stage, the linings of coelomic cavities consist of regular monolayered epithelia (Fig. 2a). The epithelia are conventionally subdivided into a somatic and splanchnic portion according to their position either beneath the ectoderm or adjacent to the wall of presumptive gut, respectively. In general, endoderm is easily distinguished from other embryonic tissues by electron-light vacuoles and electron-dense vesicles that are common within the endodermal cells (Figs. 2a-c, 4). After segmentation of
mesodermal strands the crescent-shaped coelomic cavities occupy the majority of space between the ectoderm and endoderm (Fig. 2a). At this stage, all mesodermal cells apically face the corresponding coelomic lumen. This situation changes, however, along with neurogenesis. Neurogenesis becomes evident when the ventrolateral ectoderm (neuroectoderm) begins to thicken (Fig. 2b). At the same time the somatic portion of coelomic wall forms a lateral pouch. The coelomic cavity thereby acquires a trilobed shape in being composed of dorsal, lateral, and median portions that are contiguous
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Fig. 2 a-d Differentiation of mesoderm in Epiperipatus biolleyi. Montages of electron micrographs of cross-sectioned embryos at different developmental stages. Dorsal is to the top, median is to the left. The colored insets indicate the topography of different germ layers for orientation: ectoderm in yellow, mesoderm in red, endoderm/proctodaeum in green. a Early stage shortly after the formation of embryonic coelomic cavity (eleventh body segment of an embryo bearing 14 embryonic segments). The mono-layered coelomic lining (cl) encloses a crescent-shaped coelomic cavity (co), which is situated lateral with respect to the presumptive gut. b Advanced ‘trilobed’ condition of the embryonic coelomic cavity from the fifth segment of an embryo bearing 14 embryonic segments. The coelomic cavity consists of a dorsal, median, and lateral portion (arrows). Note the thickened latero-ventral portion of coelomic wall that begins to form folds. The neuroectoderm (ne) has also thickened considerably. c Advanced stage of mesoderm differentiation (seventh body segment of an embryo bearing 32 embryonic segments). Mixocoely (= fusion of coelomic and primary body cavities) occurs by disrupture of the former medioventral coelomic wall (arrow). The nephridial anlage (na) is formed
by a reorganized lateral portion of the former coelomic wall. The nephridial lumen communicates with the newly formed ‘mixocoel’ (the wide transitional area is marked by an asterisk). The cells of precursory nerve cord (nc) have already delaminated from the ectoderm. d Advanced stage of nephridial differentiation. Posterior end [segment (n-1): see Fig. 1a] of an embryo bearing a full number of embryonic segments (posterior growth zone has already disappeared). The nerve cord (nc) already bears a medio-dorsal neuropil (np). The anlage of nephridial canal (ca) begins to coil. A precursory terminal sacculus (sa) is in process of formation, though podocytes are not fully differentiated yet (cf. Figs. 3c, d). ca Presumptive nephridial canal, cl coelomic lining, co embryonic coelomic cavity, ds dorsal sinus (part of the primary body cavity), ec ectoderm, en endoderm, gl lumen of presumptive gut, he presumptive hemocoel, lp latero-ventral portion of coelomic wall forming folds, ‘mx’ ‘mixocoel’, na nephridial anlage, nc presumptive nerve cord, ne neuroectoderm, np presumptive neuropil, pr proctodaeum, sa developing nephridial sacculus, vs ventral sinus (part of the primary body cavity)
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Fig. 3 a-d Formation and differentiation of nephridia in Epiperipatus biolleyi. a-b Electron micrographs of sagittal tangential sections through a nephridial anlage (fifth body segment of an embryo bearing 32 embryonic segments) at different levels. Dorsal is to the top, anterior is to the right. Dotted vertical lines within insets indicate sectional planes (cf. Fig. 2c). a Towards the lateral segmental part, the nephridial anlage bears a large hollow space (asterisk). b Towards the middle, the nephridial lumen communicates with the remaining ‘mixocoel’ by a narrow constriction (asterisk). c Disintegrating septate junction (arrow) between two cells of differentiating nephridial sacculus from the posterior end
[segment (n-1)] of an embryo bearing a full number of embryonic segments. Arrowheads point to the basal lamina. d Disintegrating apical junctions (arrows) between cells of differentiating nephridial sacculus from the posterior end [segment (n-1)] of an embryo bearing a full number of embryonic segments. Diaphragms are not formed yet. Arrowheads point to the basal lamina. ca Ciliated canal of nephridial anlage, cp presumptive pedicels of differentiating podocytes, ec ectoderm, he presumptive hemocoel, me mesenchymatic mesoderm tissue, ‘mx’ ‘mixocoel’, nu nucleus, sl lumen of presumptive sacculus
to each other (Fig. 2b). Simultaneously, the lateral coelomic wall becomes stratified whereas the ventrolateral part is thrown into folds. In this folded area, at least those cells that are situated within the deeper mesodermal layers do not face the lumen of the coelomic cavity, since they have lost their apical junctions and
migrated beneath the coelomic linings. Another strand of immigrated mesodermal cells extends from the median part of coelomic wall into a hollow space located beneath the presumptive gut (Fig. 2b). This hollow space (herein referred to as ‘ventral sinus’) is merely lined by extracellular matrix and represents part of the
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Fig. 4 Embryo of Epiperipatus biolleyi at an advanced stage of eye development (eye lens is already present). Cross-section of a midbody region, segment of the 17th pair of walking legs (montage of several electron micrographs). Dorsal is to the top. The colored inset indicates the topography of different germ layers: ectoderm (yellow), mesoderm (red), and endoderm (green). The presumptive nephridium consists of a coiled ciliated canal (ca) and sacculus (sa).
The nephridial lumen opens by a short ectodermal duct to the exterior. The nephridial opening (arrow) is situated at the basis of the presumptive walking leg (wl). ca Presumptive nephridial canal, ec ectoderm, en endoderm, ex exterior, gl lumen of the presumptive gut, he presumptive hemocoel, nc presumptive nerve cord, np presumptive neuropil, sa presumptive sacculus, wl anlage of the walking leg
primary body cavity that is restricted to narrow spaces between ectoderm, mesoderm, and endoderm (for further details, see Mayer et al. 2004). During further development, fusion between the primary body cavity and embryonic coelomic cavities occurs in the investigated embryos. This fusion, or mixocoely, takes place when the neuroectoderm has al-
ready given rise to segmental precursors of nerve cords (Fig. 2c) that now begin to form dorso-median neuropils. Mixocoely comprises two major events: (1) disintegration of coelomic linings, and (2) conversion of epithelia into mesenchymatic tissue. Disintegration initially occurs in the ventro-median region of each coelomic cavity, where the folds of mesoderm are most
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Fig. 5 a-d Electron micrographs of various parts of almost fully differentiated ‘typical’ nephridium from the mid-body region of Epiperipatus biolleyi (oldest investigated embryo, which was situated near the vagina within the uterus). a The presumptive sacculus is lined by podocytes (po). Their perikarya bulge into the lumen of the sacculus (sl). Within the wall of the nephridial neckpiece, a mitotic cell division (mi) is seen. b The proximal nephridial part bears a large end bladder (eb), which is connected with a short, cuticle-lined ectodermal duct (arrows). c Section through the wall of presumptive nephridial sacculus. The wall consists of podocytes
that are resting on a basal lamina (arrowheads). The pedicels of podocytes are connected by diaphragms (arrows). d Within the nephridial ‘funnel’ (transitional area between the nephridial duct and sacculus), more than one cilium (ci) per cell are sometimes detected. The cilia bear no striated ciliary rootlets yet. aj Apical junction, bb basal bodies, ci cilia, cu embryonic cuticle, eb lumen of the presumptive end bladder, ec ectoderm, ex exterior, he hemocoel, mc muscular cell, mi mitotic cell, nf neck-piece of presumptive nephridium, po podocytes, sl lumen of presumptive sacculus, wl presumptive walking leg
obvious, and proceeds until complete separation (disruption) of lateral and median portions of coelomic wall (Fig. 2c). After this separation the coelomic and primary body cavities become confluent with each other, while cells of the former coelomic linings begin to lose their epithelial character, viz. the apico-basal polarity. The apical junctions connecting them disappear whereas the former apical surface acquires an extracellular matrix (see Mayer et al. 2004 for a more detailed description). Some mesodermal cells remain in clusters, being connected by desmosome-like junctions, others invade the presumptive hemocoel as singular cells or groups of cells, which makes their further fate difficult to trace. Except for the lateral part, all other parts of the remaining coelomic linings disintegrate in the described
manner, i.e. by immigration and/or conversion of the remaining epithelial cells. Nephridiogenesis The fate of the lateral part of coelomic linings is different from that of other parts. In contrast to the dorsal and median coelomic portions, the majority of lateral coelomic cells maintain their epithelial character. During mesoderm differentiation, they contribute to the formation of nephridial anlage which is represented by a ciliated canal (Fig. 2c). The canal originates shortly after the formation of the lateral coelomic pouch. The nephridial anlage is formed by a reorganization (folding)
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and further differentiation of the latero-ventral coelomic wall whereas the lateral coelomic pouch partially becomes incorporated into the nephridial lumen. The cells of the presumptive nephridial canal are initially monociliated. Apart from well-developed cilia, large numbers of microvilli occur within the nephridial lumen. The nephridial canal has not acquired an opening to the exterior yet. Instead, it ends blindly towards the ventral ectoderm. In the opposite (dorsal, or distal) direction, the lumen of nephridial anlage communicates with the newly formed ‘mixocoel’, since no terminal sacculus has been formed yet (Fig. 2c). The lumen of the nephridial anlage becomes closed up against the developing hemocoel (=‘mixocoel’) at a more advanced developmental stage. This closure is better understood from the perspective of sagittal sections through the distal part of the nephridial canal (Fig. 3a, b). In general, further reorganization of mesoderm follows a stereotypic pattern. A small globular space is left out dorsally, i.e., in the distal part of nephridial anlage (Fig. 3a). This space is lined by remaining epithelial cells that are underlain by mesenchymatic tissue. This tissue has been formed during the subsequent enlargement and folding of the ventro-lateral coelomic wall. The nephridial anlage is thus embedded into the mesenchymatic tissue (Fig. 3a). The globular hollow space at the distal end of nephridial anlage is still in connection with the remaining ‘mixocoel’. The connecting region, however, has decreased in size in the anterior segments of the same embryo (cf. Figs. 2c, 3b). The decrease in size thus follows the antero-posterior developmental gradient. The connecting region is only represented by a 5 lm wide, diaphragm-like gap at a more advanced stage (Fig. 3b), as opposed to a 20 lm wide space between the dorsal and ventro-lateral coelomic lining at an earlier stage (Fig. 2c). But even this small gap does not persist, as revealed by the investigation of further anteriorly located segments. In these segments, the connection between the lumen of nephridial anlage and ‘mixocoel’ is abolished completely whereas the presumptive nephridium becomes separated from the remaining mesoderm. During further development, the nephridial canal elongates and its proximal part fuses with the ectoderm. The nephridial lumen, thus, acquires an opening to the exterior. At this stage, muscular cells are abundant within the embryonic hemocoel. They are partially organized into transverse septa, though these septa are only weakly developed. The nerve cords are almost entirely differentiated and bear large dorso-median neuropils that lie opposite to the ventro-lateral walls of the presumptive gut (Fig. 2d). Several commissures per segment are present. After the precursory nephridium has acquired an opening to the exterior, the distal part bearing scarcely distributed microvilli and cilia begins to differentiate into a sacculus (Fig. 2d). The epithelial cells give rise to pseudopodia-like processes whereas their apical contacts and septate-like junctions degenerate (Fig. 3c, d). No diaphragms that are characteristic of
fully differentiated podocytes are formed yet. They occur later during development. Further differentiation of nephridia The further development of nephridia is accompanied by a considerable increase in embryonic body size as well as differentiation of walking limbs. When limbs are formed on the ventro-lateral surface of the body they are invaded by mesodermal cells. During further limb growth, the nephridial sacculus comprising a narrow elongated lumen extends into the limb base (Fig. 4). However, no stereotypical position of the sacculus is apparent, as the location of this structure depends on the developmental status of nephridial anlage. In early stages, precursory cells of the sacculus occur dorsally (Figs. 2c, d, 3a, b) whereas in embryos of a more advanced stage (when podocytes are already present) the sacculus acquires a ventro-lateral position (Fig. 4). At this latter stage, the presumptive nephridium has acquired a typical triple loop due to the extreme elongation of the nephridial canal. Nevertheless, the nephridium is not fully differentiated yet as it lacks, apart from the sacculus, other distinct regions characteristic of nephridia in adult individuals. Almost entirely differentiated nephridia are found in more advanced though still unpigmented embryos, when the presumptive eyes are already located at the antennal bases and the embryonic cuticle has thickened considerably (cf. Fig. 5b). A prominent epicuticle is also present. In the nephridia of these stages the lumen of the sacculus has widened and is clearly lined by podocytes bearing pedicels connected by diaphragms (Fig. 5a, c). Within the nephridial ‘funnel’, which opens into the sacculus, more than one cilium per cell is found (Fig. 5d). The proximal end bladder is already present, the lumen of which opens via a short ectodermal, cuticle-lined duct to the exterior (Fig. 5b). Within different nephridial regions mitotic cells indicate that further growth of the nephridium is still taking place (Fig. 5a, b).
Discussion Comparative morphology of nephridia Apart from the segmental arrangement and ciliation of nephridial canals there seems to be no further correspondence between the nephridia of adult onychophorans and annelids. In contrast to onychophorans (and other arthropods), the metanephridial system of annelids consists of two spatially separated units (see Bartolomaeus and Quast 2005). The podocytes are restricted to particular regions of coelomic linings that are associated with the blood vascular system. The metanephridial duct opens by a ciliated funnel into the coelom of one segment and pierces the septum, thus opening to the exterior within the following segment
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(Ruppert and Smith 1988; Nielsen 1997, 2001; Bartolomaeus and Quast 2005). This composition is correlated with function of metanephridia. Ultrafiltration between podocytes is followed by modification of primary urine within the nephridial duct (Ruppert and Smith 1988; Smith and Ruppert 1988; Bartolomaeus and Quast 2005). Similarly organized functional nephridia also occur in other bilaterian groups, e.g., phoronids, brachiopods, molluscs, and deuterostomes (see Ruppert and Smith 1988; Nielsen 2001; Brusca and Brusca 2003; Ruppert et al. 2004). At present, neither structural features nor the function of metanephridial systems provide support for the nephridia of Annelida and Arthropoda being a synapomorphy of these groups. Common features of nephridial formation in arthropods The present study revealed that in E. biolleyi the terminal sacculus arises from the distal part of the nephridial anlage. During differentiation of the sacculus, all other remnants of the former coelomic linings have already been converted into mesenchymatic tissue. In this respect, the described pattern does not correspond to the simplified scheme, according to which the sacculus of adult onychophorans and other arthropods represents a ‘persisting coelomic cavity’ (see, e.g., Dohle 1979; Pflugfelder 1980; Storch and Ruhberg 1993; Ax 2000; Ruppert et al. 2004). Based on the present findings both the nephridial canal and sacculus are derivatives of the embryonic coelomic wall. This observation disproves the assumptions of Kennel (1888) and Glen (1918) who stated that almost the entire nephridium of Onychophora arises as an ingrowth from ectoderm. Although the early nephridiogenesis varies among different onychophoran species, the nephridia and their derivatives always originate from a portion of the coelomic wall (see Sheldon 1887; Sedgwick 1887, 1888; Evans 1901; Pflugfelder 1948, 1980; Mayer et al. 2004, 2005). Only a short duct leading to the exterior is produced by the ectoderm. Like in the Onychophora, the early formation of nephridial derivatives is highly diverse among euarthropods. The ‘coxal glands’ of chelicerates (Fig. 1b) originate from embryonic coelomic walls in various segments and might acquire a common duct leading to the exterior (Kingsley 1885, 1893; Lebedinsky 1892; Patten and Hazen 1900; Moritz 1959; Scholl 1977; Sekiguchi 1988). In myriapods, the walls of certain coelomic cavities give rise to the ‘maxillary glands’ (Fig. 1b) and paired pre-mandibular ‘lymphoid masses’ that are interpreted as nephridial vestiges (see Heymons 1901; Tiegs 1940, 1947; Anderson 1973; Dohle 1980). In contrast, the sacculi and ducts of the crustacean ‘antennary’ and ‘maxillary glands’ (Fig. 1b) are formed by two initially solid blocks of cells that hollow out and fuse together during further development (Manton 1928, 1934; Weygoldt 1958; Scholl 1963; Benesch 1969; Shiino 1988). The origin of ‘labial glands’ of apterygotan
insects (Fig. 1b) is less-well studied, though it is usually accepted that these organs represent nephridial derivatives (see, e.g., Johannsen and Butt 1941; Feustel 1958; Haupt 1969; Seifert 1979). With respect to this high diversity in nephridial formation within Euarthropoda, it is difficult to infer a common pattern of nephridiogenesis for Arthropoda. One feature shared by all arthropods is the origin of nephridia from segmented mesoderm. In onychophorans, chelicerates, and myriapods the nephridia or their derivatives arise from walls of embryonic coelomic cavities. Irrespective of the present uncertainty on the phylogenetic position of Myriapoda (see, e.g., Friedrich and Tautz 1995; Hwang et al. 2001; Dove and Stollewerk 2003; Harzsch 2004; Kadner and Stollewerk 2004; Mallatt et al. 2004; Giribet et al. 2005; Harzsch et al. 2005), the present insight on the state in Onychophora allows such a formation of nephridia to be considered as the ancestral mode in Arthropoda. Nephridial formation in annelids—no correspondences with arthropods In the past, the ontogenetic origin of annelid metanephridia has been a matter of controversy (see Anderson 1973; Okada 1988; Bunke 2003). According to some authors (Vejdovsky 1892; Goodrich 1895, 1945; Staff 1910; Bahl 1922) these organs arise from ectoderm whereas others (Bergh 1898; Lillie 1905; Penners 1923; Vanderbroek 1935) attributed them to mesoderm. Recent cell lineage studies revealed, however, that metanephridia of clitellates are mesodermal in origin (see, e.g., Kitamura and Shimizu 2000a, 2000b; Shimizu and Nakamoto 2001; Shimizu et al. 2001). Based on the position of nephridial anlage, the same origin has also been assumed for metanephridia of polychaetes (see Bartolomaeus and Quast 2005). Embryologists have long recognized that each metanephridium of annelids develops from a single, large ‘nephridioblast’ cell (Penners 1923; Meyer 1929; Goodrich 1932, 1945; review by Anderson 1973). Recent ultrastructural studies on polychaetes and oligochaetes revealed, however, that the earliest stage of nephridial formation that can be discriminated from the surrounding mesoderm tissue consists of three to four precursory nephridial cells (Bartolomaeus 1989, 1997, 1999; Bunke 2003; Bartolomaeus and Quast 2005). In clitellates, these cells initially occur in the intersegmental septum and give rise to the metanephridial canal and funnel during further development (Bahl 1922; Penners 1923; Meyer 1929; Goodrich 1932, 1945; Bunke 2003). In polychaetes, precursory nephridial cells composing the early nephridial anlage are recognized in a corresponding position but prior to the formation of coelomic cavities (Bartolomaeus 1997, 1999). In accordance with earlier assumptions, Bunke (2003) proposed that the metanephridia of polychaetes and clitellates originate from an identical mesodermal stem cell.
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The special course of nephridial development by precocious determination and arrangement of nephridioblast cells in both, polychaetes and clitellates, suggests that the formation of metanephridia in annelids is independent from embryonic coelomic linings. Correspondingly, there is no evidence that the coelomic linings contribute to the formation of the nephridial funnel in polychaetes (see Lillie 1905; Bartolomaeus 1989, 1997, 1999; Bartolomaeus and Ax 1992). This is in strong contrast to the present findings, since the nephridia of E. biolleyi originate from walls of the embryonic coelomic cavities. Unlike in annelids, a distinction between nephridium and coelom is not justified by embryogenesis of Onychophora, because both structures are causally connected. Such a causal-temporal relationship between embryonic coelomic cavities and nephridial anlagen is a common feature of arthropods (see, e.g., Sedgwick 1887, 1888; Heymons 1901; Tiegs 1940, 1947; Moritz 1959; Scholl 1977). In this respect, there is a fundamental difference in the formation of nephridia between the Annelida and Arthropoda.
Conclusions Although we can never offer proof for non-homology, it must be stressed that among homologous features only synapomorphies can be used to support the monophyly of a given taxon whereas symplesiomorphies are uninformative in this respect (see, e.g., Sudhaus and Rehfeld 1992; Ax 1996; Wiesenmu¨ller et al. 2003; Wa¨gele 2005). The nephridial organization in adults of annelids and arthropods presently provides no characters that might be regarded as clear synapomorphies. Serially arranged mesodermal nephridia with ciliated, tubular ducts occur in other bilaterians as well and are therefore not specific for annelids and arthropods only (see, e.g., Schaefer and Haszprunar 1997; Nielsen 2001; Balavoine and Adoutte 2003; Ruppert et al. 2004). Regarding nephridiogenesis, I was also unable to find any correspondence that might be regarded as a potential synapomorphy of the Arthropoda and Annelida. Based on prevalent differences in morphogenesis and organization of nephridia between annelids and arthropods, it is currently impossible to decide a priori whether or not these organs are the result of convergent evolution. However, they can only be regarded as a synapomorphy, if other characters clearly indicate a sister group relationship between the Annelida and Arthropoda. The structure and development of the nephridial organs themselves, however, do not reveal identities that would substantiate such a hypothesis. The findings of the present study accordingly demonstrate that the presence of ‘segmentally arranged nephridia’ (see, e.g., Dohle 1979; Weygoldt 1986; Rouse and Fauchald 1995, 1997; Nielsen 1997, 2001; Scholtz 1997, 2002, 2003; Wa¨gele et al. 1999; Ax 2000; Jenner and Scholtz 2005) can no longer be considered to provide strong support for the homology hypoth-
esis of segmentation or for the monophyly of the Articulata. Acknowledgements My sincere thanks are expressed to the staff of the Instituto Nacional de Biodiversidad (INBio) in Costa Rica, especially to Alvaro Herrera, for collecting the animals, dissecting, fixing, and sending the material to me. I thank Thomas Bartolomaeus, Gregory Edgecombe, Markus Koch, Hilke Ruhberg and Gerhard Scholtz for giving some critical comments and useful suggestions on the manuscript. Ira Richling kindly helped to get contact to the staff of the INBio. I am thankful to Bjo¨rn Quast for writing software for a more comfortable handling of the electron microscopic data. This study was supported by the Studienstiftung des deutschen Volkes (D/2002 0033) and the Deutsche Forschungsgemeinschaft (BA 1520/8-1, 8-2; RU 358/4-1, 4-2).
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