Old trees, new trees – is there any progress?1

Zoology 106 (2003): 291–301 © by Urban & Fischer Verlag http://www.urbanfischer.de/journals/zoology

REVIEW

Old trees, new trees – is there any progress?** Andreas Schmidt-Rhaesa* Zoomorphology and Systematics, University Bielefeld, Germany

Summary The amount of comparative data for phylogenetic analyses is constantly increasing. Data come from different directions such as morphology, molecular genetics, developmental biology and paleontology. With the increasing diversity of data and of analytical tools, the number of competing hypotheses on phylogenetic relationships rises, too. The choice of the phylogenetic tree as a basis for the interpretation of new data is important, because different trees will support different evolutionary interpretations of the data investigated. I argue here that, although many problematic aspects exist, there are several phylogenetic relationships that are supported by the majority of analyses and may be regarded as something like a robust backbone. This accounts, for example, for the monophyly of Metazoa, Bilateria, Deuterostomia, Protostomia (= Gastroneuralia), Gnathifera, Spiralia, Trochozoa and Arthropoda and probably also for the branching order of diploblastic taxa (“Porifera”, Trichoplax adhaerens, Cnidaria and Ctenophora). Along this “backbone”, there are several problematic regions, where either monophyly is questionable and/or where taxa “rotate” in narrow regions of the tree. This is illustrated exemplified by the probable paraphyly of Porifera and the phylogenetic relationships of basal spiralian taxa. Two problems span wider regions of the tree: the position of Arthropoda either as the sister taxon of Annelida (= Articulata) or of Cycloneuralia (= Ecdysozoa) and the position of tentaculate taxa either as sister taxa of Deuterostomia (= Radialia) or within the taxon Spiralia. The backbone makes it possible to develop a basic understanding of the evolution of genes, molecules and structures in metazoan animals. Key words: phylogeny, phylogenetic systematics, evolution, Metazoa, Bilateria

Introduction The comparative aspect, i.e. the evolutionary perspective, is one of the uniting dimensions of diverse biological disciplines. Any comparative data, sampled from different species, can be used in two ways. First, to supply the data basis for the reconstruction of a hypothesis on phylogenetic relationships (= tree), but second, data can also be mapped on existing trees and thus be interpreted in an evolutionary context. This has been stated as one major goal of evolutionary developmental biology by Arthur (1997, p. 150).

If one looks for a tree to map characters onto, searching different sources will reveal trees differing significantly in topology. A superficial view into systematical journals will support this confusion: problems with finding well-supported hypotheses on relationships seem to be more abundant than satisfying solutions to systematical problems. What does this mean? Is there a recognizable progress in systematics? First of all, it has to be stated that progress in systematics is different from progress in experimental biological disciplines. Systematics deals with the reconstruction of historical processes. This is done by evaluating com-

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Corresponding author: Andreas Schmidt-Rhaesa, Zoomorphology and Systematics, University Bielefeld, PO Box 100131, 33501 Bielefeld, Germany; phone: +49-521-1062720; fax: +49-521-1066426; e-mail: [email protected] ** Presented at the 96th Annual Meeting of the Deutsche Zoologische Gesellschaft, in association with the Deutsche Gesellschaft für Parasitologie, in Berlin, June 9–13, 2003 0944-2006/03/106/04-291 $ 15.00/0

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peting hypotheses, but since each hypothesis is a product of the current data basis, new data always have the potential to falsify existing hypotheses. This makes systematics a highly complex and dynamic field. Progress in systematics should not be expected to manifest in singular analyses or publications, but is more like a picture emerging from various, often contradicting sources. To illustrate some differences in the general tree topology, I will give a few examples. Following Schimkevitch (1891), Libbie H. Hyman adopted a division of Bilateria into three “grades”, acoelomate, pseudocoelomate and coleomate bilaterians, in her five volume series “The Invertebrates”. Despite her earlier statement that this division “stands firmly on a realistic anatomical basis” (Hyman, 1940, p. 35), she later stated that “these groupings do not […] entirely correspond to taxonomic relationships” (Hyman, 1951, p. 23). This tripartition of Bilateria served as a basis for several following authors and is, for example, expressed in Willmer (1990) and Ruppert and Barnes (1994) as well as in general textbooks such as Campbell and Markl (1997). An alternative would be to divide Bilateria according to their cleavage pattern into two taxa, Spiralia (with autapomorphic spiral cleavage) and Radialia (with plesiomorphic radial cleavage) (e.g. Ax, 1989). “Aschelminthes”, which include nematodes and related groups, posed a large phylogenetic problem and were often excluded from analyses, because it was not clear if they are monophyletic and where they belong to. This changed in the mid 1990s, when Ahlrichs (1995) and Ehlers et al. (1996) hypothesized a monophyletic taxon Nemathelminthes as the sister group of Spiralia. Some recent textbooks (e.g. Gilbert, 2000; Carroll et al., 2001; Campbell et al., 2003) have adopted the hypothesis that Arthropoda are the sister taxon of a part of Nemathelminthes (together named Ecdysozoa), but other sources present Arthropoda as the sister group of Annelida in a taxon called Articulata (Ax, 2000; Westheide and Rieger, 2001; Brusca and Brusca, 2003). So, depending on which textbook you follow, you will map your data on different trees and therefore get different interpretations of your data. In the following, I will briefly review current developments in systematics and will show that, apart from numerous problematic areas, we have something like a robust backbone in the tree topology along which problematic areas can be precisely located and thus serve as a target for focussed questions and investigations.

animal organisms occurred, at least successfully, only once (see e.g. Ax, 1996; Nielsen, 2001). This is supported by characters connected to multicellularity such as the existence of an extracellular matrix (ECM), cellcell connections (spot desmosomes and septate junctions), somatic differentiation as well as a development including (radial) cleavage and a blastula stage. Other characters are the existence of haploid gametes with a characteristic gametogenesis, sperm structure and the existence of at least one Hox-gene (Peterson and Davidson, 2000). Among diploblastic animals, morphological characters support a clear hypothesis of a sequential branching of Porifera, the sponges, first, followed by Trichoplax adhaerens, Cnidaria and Ctenophora (Fig. 1). This is supported by characters from tissue organization, the presence or absence of tissues and cell-cell contacts (see Ax, 1996; Nielsen, 2001 for details). Molecular data do not decisively support this branching pattern, but instead imply a monophyly of diploblastic animals (e.g. Lafay et al., 1992; Philippe et al., 1994; Winnepenninckx et al., 1995; Kobayashi et al., 1996), or a sister-group relationship of Porifera and Ctenophora (e.g. Wainwright et al., 1993; Winnepenninckx et al., 1998; Peterson and Eernisse, 2001). Recently, Rokas et al. (2003) reinvestigated the molecular support for basal metazoan relationships and concluded that it is questionable whether DNA sequences can be used to resolve these relationships.

Bilateria There is no doubt that bilaterally symmetrical animals have a common ancestor. This bilaterian ancestor seems to be of enormous significance to us, probably because it is the first representative expressing the symmetrical proportions so familiar to us. However, we still have no clear impression how this bilaterian ancestor looked like, or better, we have a number of differing hypotheses. It may have been microscopically small

Metazoa and their basal relationships The monophyly of Metazoa is hardly questioned today. All data, morphological and molecular, indicate that the transition from unicellular organisms to multicellular 292

Fig. 1. Basal phylogenetic relationships within Metazoa, according to morphological characters. Zoology 106 (2003) 4

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(Ax, 1996) or macroscopic (Dewel, 2000), solitary or colonial (Dewel, 2000), it had a benthic way of life (Ax, 1996) or a biphasic life cycle (Rieger, 1994), to name just a few conflicting thoughts (for an overview on different concepts see Rieger et al., 1991). While most authors imagine the bilaterian ancestor to be relatively simple, findings of homologous genes in “model” organisms such as Mus musculus, Caenorhabditis elegans and Drosophila spp. have led to the postulation of a complex bilaterian ancestor (e.g. Carroll et al., 2001) which was segmented (De Robertis, 1997), had eyes (Arendt and Wittbrod, 2001), appendages (Panganiban et al., 1997) and a heart (De Robertis and Sasai, 1996). Even the “typical” bilaterian characteristics can be discussed controversially, because bilateral symmetry is also present in ctenophores and some cnidarians, and because individualized muscle cells which some interpret as mesoderm are present in ctenophores (Martindale et al., 2002).

Deuterostomia Within Bilateria, two taxa seem to be very well supported: Deuterostomia and Spiralia (Fig. 2). Deuterostomia include the taxa Echinodermata, Pterobranchia, Enteropneusta and Chordata. Their relationships have recently been under debate. This is caused mainly by problems with the position of pterobranchs and enteropneusts, which are often, but not always (see Ax, 2003, Nielsen, 2001) considered as sister taxa in the monophyletic Hemichordata. While Hemichordata were originally thought to be the sister taxon of Chordata, molecular analyses place them as the sister taxon of Echinodermata (Turbeville et al., 1994; Bromham and Degnan, 1999; Cameron et al., 2000). The monophyly of Chordata and Echinodermata seems to be well supported in all analyses.

Fig. 2. Consensus on phylogenetic relationships within Bilateria. Exceptions from this consensus are a different position of the basal taxon of Nemathelminthes, Gastrotricha, and a probable paraphyly of Plathelminthes (see text). Zoology 106 (2003) 4

Spiralia Although Spiralia seem to be monophyletic, their basal relationships (i.e. taxa Plathelminthes, Gnathifera and Nemertini) are not well resolved (Fig. 2). This is especially due to recent discussions about the monophyly of Plathelminthes (see below), because the taxon Acoelomorpha might not belong into Spiralia. Another monophyletic taxon within Spiralia is Trochozoa (Fig. 2), which is characterized by the presence of a planctonic trochophore larva. In this context I would like to remind you that under the term trochophora we summarize larvae of different groups which share characters such as the prototroch and the apical ciliary tuft, but not characters such as a mesotroch and a metatroch which evolved later (see van den Biggelaar et al., 1997; Rouse, 1999). Trochozoa include at least Mollusca, Annelida and some smaller groups such as Kamptozoa (which are probably the sister group of Mollusca, see Ax, 2000), Sipunculida and Echiurida. While Sipunculida are probably the sister group of Annelida, Echiurida may belong within Annelida (for further discussion see Hessling and Westheide, 2002), as certainly do Pogonophora and Vestimentifera (McHugh, 1997; Bartolomaeus, 1995). For another taxon, Myzostomida, contradictory results exist. Their nervous system leads to their interpretation as derived annelids (Müller and Westheide, 2000), but according to molecular data they are more basal within Spiralia (Eekhaut et al., 2000).

Gnathifera “Pseudocoelomate” taxa, the so-called Aschelminthes, were regarded a phylogenetic problem until the mid1990s. Their monophyly was doubted (e.g. Ruppert, 1991; Kristensen, 1995), but there were no alternative concepts. A varying number of taxa with a “core group” including Nematoda, Rotifera, Acanthocephala, Gastrotricha, Nematomorpha, Priapulida, Kinorhyncha and (since 1983) Loricifera were summarized as Aschelminthes. It has long been recognized that rotifers and acanthocephalans share some characters, as do the remaining six taxa. Ahlrichs (1995) and Rieger and Tyler (1995) came to the conclusion that the ultrastructure of cuticular hard elements in the pharynx of rotifers corresponds in ultrastructure to the jaws in gnathostomulids. A jaw is not present in acanthocephalans, but a syncytial epidermis with an intrasyncytial dense layer is unique in rotifers and acanthocephalans, uniting them as Syndermata. Syndermata and Gnathostomulida form the Gnathifera (Fig. 3). In fact, rotifers in the classical sense seem to be paraphyletic, with Seison being closer related to Acanthocephala (Ahlrichs, 1995, 1997; Herlyn et al., 2003; for a summary of competing hypothe293

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Fig. 3. Phylogenetic relationships of Gnathifera. 1. Jaw, 2. internal layer, 3. syncytial epidermis, 4. dense bodies in spermatozoon.

ses see Garey et al., 1998). The phylogenetic relationships within Gnathifera were confirmed by the discovery of Limnognathia maerski (Micrognathozoa) from cold springs in Greenland. Limnognathia possesses a jaw with an ultrastructure similar to the remaining Gnathifera and also an internal layer, but in a cellular epidermis (Kristensen and Funch, 2000). It therefore fit perfectly “between” gnathostomulids and Syndermata (Fig. 3) and confirmed the previously hypothesized relationships (see also Sørensen et al., 2000; Sørensen, 2003).

Nemathelminthes For the remaining taxa, Ahlrichs (1995; see also Ehlers et al., 1996), Nielsen (1995; see also 2001) and Wallace et al. (1995, 1996) synchronously arrived at corresponding hypotheses about their phylogenetic relationships (Fig. 4). Priapulida, Kinorhyncha and Loricifera all share an anterior body part that can be invaginated and withdrawn and that is equipped with cuticular structures called scalids which include receptor cells. They are named Scalidophora (Lemburg, 1995). Among Scalidophora, relationships are discussed controversially; a Kinorhyncha-Loricifera relationship is favoured by Nielsen (2001) while a Priapulida–Loricifera relationship is favoured by Ax (2003; see Lemburg, 1999 for a review). A sister-group relationship of Nematoda and Nematomorpha (together: Nematoida) is well supported (Schmidt-Rhaesa, 1998), and Nematoida and Scalidophora together form the Cycloneuralia, based on the structure of the brain and the cuticule as well as the moulting of the cuticle (but see “Ecdysozoa” below for similar cuticular structure and moulting). According to morphological characters, Gastrotrichs are the sister taxon of Cycloneuralia (together forming the Nemathelminthes), but in molecular analyses (e.g. Winnepenninckx et al., 1995; Giribet et al., 2000) gastrotrich taxa appear in close relationship to Plathelminthes (see Schmidt-Rhaesa, 2002; Zrzavy, 2003). The names used here refer to the ones introduced by Ahlrichs (1995), Lemburg (1995) and 294

Fig. 4. Phylogenetic relationships of Nemathelminthes indicating names introduced by Ahlrichs (1995) and Lemburg (1995) in regular script, and by Nielsen (1995), in italics.

Schmidt-Rhaesa (1996). Nielsen (1995; see also 2001) introduced different names, which I regard to be less well suited (compare Fig. 4). Cephalorhyncha, which is used instead of Scalidophora, refers to a hypothesized relationship of Nematomorpha and Scalidophora (Malakhov, 1980). Introverta, used for Cycloneuralia, implies a homology of the anterior part of the body, which I doubt, based on different ultrastructural and symmetrical patterns (Schmidt-Rhaesa, 1998). Nemathelminthes are called Cycloneuralia by Nielsen. Cycloneuralia (sensu Ahlrichs) have a ring-shaped brain, while in gastrotrichs, there is a dominant dorsal portion and a weak subpharyngeal commissure (Teuchert, 1977; Wiedermann, 1995). Therefore, the gastrotrich brain resembles the brain in Plathelminthes and represents the plesiomorphic position. It seems better to use the name Cycloneuralia in connection with the autapomorphic condition, i.e. the ring-shaped brain of nematodes, kinorhynchs and relatives, but not including Gastrotricha. The evidence that arthropods are the sister taxon of Cycloneuralia (together: Ecdysozoa) is briefly discussed below (problematic regions). Depending on the validity of Ecdysozoa and the position of Gastrotricha as the sister taxon of Cycloneuralia or Ecdysozoa, either Cycloneuralia, Ecdysozoa or Nemathelminthes are probably the sister taxon of Spiralia, together forming Protostomia (= Gastroneuralia). This is supported by most molecular analyses (e.g. Zrzavy et al., 1998; Giribet et al., 2000; Peterson and Eernisse, 2001). From the morphological perspective, the presence of a supraintestinal brain with subintestinal commissures would be an autapomorphy of Protostomia.

Problematic regions The tree as presented up until now seems to be well supported by the majority of analyses (morphological Zoology 106 (2003) 4

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and molecular) and therefore serves as a framework for the more problematic questions. There are several such problematic regions along the tree, but alternating hypotheses usually span relatively narrow regions of the tree. They may, however, have an enormous impact on character interpretation and on our reconstruction of ancestral character states. One example is the possible paraphyly of sponges. Most authors consider Porifera, the sponges, to be monophyletic and list a number of autapomorphies such as the life cycle with a macroscopic, sessile adult and a microscopic, planctonic larva (biphasic life cycle), choanocytes and their organization into water chambers with inhalant and exhalant pores (Böger, 1988; Reitner and Mehl, 1996; Ax, 1996; Nielsen, 2001; see Fig. 5A). Some of these characters have been discussed controversially, such as the biphasic life cycle, which is regarded by some (e.g. Jägersten, 1972; Rieger, 1994) as a character already present in the metazoan ancestor, while others doubt this (e.g. Ax, 1996; Nielsen, 1998). Choanocytes of sponges have lateral extensions, the vanes (Mehl and Reiswig, 1991), which also occur in choanoflagellates (Hibberd, 1975), one candidate for the sister group of Metazoa. Ultrastructural differences have led Ax (1996) to assume a convergent evolution of such vanes. The monophyly of sponges has been challenged by several molecular analyses of the 18S rDNA gene (Cavalier-Smith et al., 1996; Collins, 1998; Borchiellini et al., 2001; Peterson and Eernisse, 2001), protein kinase C (Kruse et al.,

1998) and probably also 28S rDNA (Lafay et al., 1992). According to these analyses, Calcarea (one subtaxon of Porifera) are closer related to Epitheliozoa (Fig. 5B). This is supported by the finding that striated ciliary rootlets have been found in larvae of calcareous sponges (Woollacott and Pinto, 1995) and are present in Epitheliozoa (Rieger, 1976), while they are lacking in the remaining (siliceous) sponges. If paraphyly of sponges is further supported, this would change our reconstruction of the metazoan ancestor dramatically, because the “typical” sponge characters would have to be assumed to have already been present in the metazoan ancestor (see Fig. 5B). Another example are the basal spiralian taxa. The monophyly of Plathelminthes (see Ehlers, 1985; Ax, 1996) has been challenged by morphological (Smith et al., 1986; Haszprunar, 1996) and molecular data (RuizTrillo et al., 1999). The relationships of the three subtaxa Acoelomorpha, Catenulida and Rhabditophora are recently under discussion (for a summary, see Tyler, 2001). In molecular analyses some representatives of the Acoela appear as the sister taxon of all Bilateria (Ruiz-Trillo et al., 1999; Fig. 6), which is in accordance with their lack of protonephridia, an epithelial intestine and ECM (but see Tyler and Rieger, 1999, for probable remnants of ECM). However, the small taxon Nemertodermatida seems to be closely related to Acoela (together: Acoelomorpha) on the basis of a complex network of epidermal ciliary rootlets and probably the presence of a statocyst (Ehlers, 1985). Nemertodermatida have ECM (Smith, 1981; Lundin and Sterrer, 2001) and an epithelial intestine, which makes the primary absence of these two characters in Acoela not probable (if they are sister taxa). In molecular analyses Nemertodermatida also seem to appear as basal bilaterians, but according to the 18S rDNA gene they are not the sister group of Acoela (Jondelius et al., 2002) and

Fig. 5. Conflicting hypotheses concerning monophyly versus paraphyly of sponges and some consequences for the interpretation of character evolution.

Fig. 6. Polyphyly of Plathelminthes, with a basal placement of Acoelomorpha (Acoela + Nemertodermatida). See text for explanation.

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according to the myosin-heavy-chain type II gene Acoelomorpha are monophyletic (Ruiz-Trillo et al., 2002). The probable polyphyly of Plathelminthes makes it difficult to currently establish patterns of relationship between Plathelminthes, Nemertini and Gnathifera. However, Protostomia and Spiralia remain monophyletic taxa, even if Acoela/Acoelomorpha are excluded. There are two other problematic areas where conflicting hypotheses span wider areas of the tree. One is the relationship of arthropods, the other that of the tentaculate taxa. With the exception of gastrotrichs, all remaining Nemathelminthes (= Cycloneuralia) moult their cuticle. This ecdysis seemed to explain the clustering of arthropod and cycloneuralian taxa in molecular analyses, while the traditional Articulata were never supported. The taxon (Arthropoda + Cycloneuralia) was named Ecdysozoa (see also review by Giribet, 2003). Meanwhile, further data have been found in favor of Ecdysozoa: correspondences in the cuticular ultrastructure and in the hormonal regulation of molting (summarized in Schmidt-Rhaesa et al., 1998), gene structure data such as three repeats of an amino acid motif in β-Thymosin (Manuel et al., 2000) or probably a four amino acid insertion in the elongation factor-2 gene (Regier and Schultz, 2001) and the presence of anti-horseradish peroxidase sensitive glycoproteins in the nervous system (Haase et al., 2001). The taxon Ecdysozoa has already been adopted in several current textbooks, but Wägele et al. (1999), Wägele and Misof (2001) and Scholtz (2002), among others, present a critical discussion and point out that, although molecular support is lacking for a relationship between arthropods and annelids, the morphological characters supporting this relationship are so complex that the convergent evolution of, in particular, segments of these two taxa is hardly imaginable. Recently, Nielsen (2003) suggested regarding Ecdysozoa as valid and as the sister taxon of Annelida. I suspect that the discussion has not yet come to an end. New data, such as insights into the hormonal control of molting in cyclonauralian taxa or further comparative data on the development of segmental patterns could help in approaching a solution to the “Ecdysozoa problem”. The uncritical adoption as well as the rigid rejection of a particular hypothesis is currently hindering more than being helpful. The second problem consists of the tentaculate (= lophophorate) taxa Brachiopoda, Bryozoa and Phoronida. There is a current debate whether these taxa are related to Deuterostomia (together forming the taxon Radialia; e.g. Ax, 2003) or whether they belong within the taxon Spiralia (e.g. Halanych, 1995). A number of characters support a relationship of the three tentaculate taxa with Deuterostomia, e.g., two coelomic 296

cavities, a tentacular apparatus and composite metanephridia (Lüter and Bartolomaeus, 1997; Ax, 2003). Recent reinvestigations have shown that neither in Brachiopoda (Lüter, 2000) nor in Phoronida (Bartolomaeus, 2001; Gruhl, unpublished diploma thesis, Bielefeld 2002) and in Bryozoa (Wegener, unpublished diploma thesis, Bielefeld 2002), there are more than two coelomic cavities. This corrects earlier light-microscopical investigations which hypothesized three cavities. The archicoelomate hypothesis, which was based upon the presence of three coelomic cavities (Masterman, 1898; Ulrich, 1973; Siewing, 1980), is therefore without support. The “reduction” from three to two coelomic cavities does, however, not change the quality of arguments supporting a relationship to Deuterostomia, because one coelom supporting the tentacular crown and one body coelom remain putative homologous structures. Molecular analyses, in contrast, never place tentaculates close to deuterostomes, but in close association with spiralian taxa (e.g. Halanych, 1995; Zrzavy et al., 1998; Giribet et al., 2000). There are no morphological characters supporting this hypothesis. Some see the fossil halkieriids as representatives of the stem lineage of and between molluscs, annelids and brachiopods (Conway Morris, 1998). Halkieriids possess two shells, one in the anterior and one in the posterior region (Conway Morris and Peel, 1995), and a sequence has been drawn in which the shell plates approach and finally fold to form a brachiopod-like shell (Conway Morris, 1998). Even if such a folding seems possible regarding the origin of the brachiopod shell (both brachiopod shells seem to represent the dorsal side) (“brachiopod fold hypothesis; Cohen et al., 2003), this scenario appears quite speculative. There are some taxa for which even new methods have not helped much in revealing their phylogenetic position. This is especially true for chaetognaths, which in molecular analyses are prone to long-branch artifacts (e.g. Halanych, 1996). Also controversially discussed is Xenoturbella, which is alternatively hypothesized as the sister taxon of all Bilateria (Ehlers and SopottEhlers, 1997) or as a member of Deuterostomia (Bourlat et al., 2003). The relationship to bivalves (Norén and Jondelius, 1997; Israelsson, 1997, 1998) has turned out to have been an artifact due to contamination (Bourlat et al., 2003). Hopefully, the mysterious Buddenbrockia will turn out to be a more positive example , since morphological and molecular reinvestigations have shown that it likely belongs to the taxon Myxozoa (Monteiro et al., 2002; Okamura et al., 2002), which itself seems to represent highly derived cnidarians (Siddall et al., 1995; Zrzavy, 2001). “Mesozoa” are likely to be paraphyletic and its two taxa Rhombozoa and Orthonectida are not “intermediates” between uniZoology 106 (2003) 4

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cellular and multicellular organisms (e.g. Hyman, 1940). Although their exact position is not yet clear, characters such as the presence of cilia with striated rootlets (Furuya et al., 1997 for Rhombozoa; Slyusarev, 1994 for Orthonectida), belt desmosomes (Slyusarev, 1994 for O), gap junctions (Furuya et al., 1997 for R) and a cuticle (Slyusarev, 2000, 2003 for O) will help in developing phylogenetic hypotheses. According to these characters, Orthonectida are members of Epitheliozoa, and Rhombozoa are members of Eumetazoa. Investigations of Hox-genes in dicyemids (Rhombozoa) seem to indicate that they belong to Spiralia (Kobayashi et al., 1999).

Conclusions Systematics has become an integrative discipline, managing data from many different sources, in particular from comparative morphology, molecular genetics, (evolutionary) developmental biology and paleontology. None of these approaches has proven to be superior to others, although this was sometimes stated in the early enthusiasm of a newly developed field. There is constant progress in any field as well as growing knowledge of restrictions in any field. The contribution of different fields to systematics has opened up the possibility to approach questions which cannot be resolved by one field alone. The newly reported case of the occurrence of wings in derived stick insects (Phasmatodea) seems to be such a case (Whiting et al., 2003). Basal taxa of stick insects do not possess wings, but the wings of derived stick insects correspond in all details to the wings of other insects, which makes their completely convergent evolution extremely unlikely. What is responsible for this “disappearance” of wings over evolutionary time? Although this question is not yet answered, I expect that the investigation of the regulatory systems influencing wing development will provide an answer. Recent progress in developmental biology (see Davidson, 2001) has shown that regulation of gene expression during embryogenesis is extremely complex and that comparatively simple alterations such as temporal or spatial shifts in gene expression can lead to a failure in the formation of complex structures. Such “frame-shifts” do not require the complete absence of elements of the regulatory system and could probably be corrected in a comparatively simple way. One other thing that becomes clear with the increasing amount of data, especially from molecular and from developmental biology, is that one has to be very precise in formulating hypotheses of homology. Let us consider, for example, the musculature and the nervous system. Muscle cells (as epitheliomuscle cells) and Zoology 106 (2003) 4

nerve cells are present in Cnidaria and are therefore hypothesized for the ancestor of Eumetazoa (e.g. Ax, 1996). However, components of these systems, such as actin, myosin (Harrison and Vos, 1991; Kanzawa et al., 1995; Masuda et al., 1998), serotonin (Weyrer et al., 1999), acetylcholine and acetylcholine-esterase (Nielsen, 2001), are also present in sponges. Actin, for example, predates all Metazoa and is an important component of the cytoskeleton of non-metazoan organisms. As another example, let us take a look at the extracellular matrix (ECM), which is one autapomorphy of Metazoa and probably a key innovation through which cell aggregation into multicellular organisms could be achieved. ECM is composed of different types of collagen and of several further molecules among which I want to name only two: fibronectin and integrin (for a review, see Pedersen, 1991). A collagen-like molecule has also been found in fungi (Celerin et al., 1996; Grimson et al., 2000). Collagen type IV, a characteristic component of the dense layer called basal lamina which is present in Bilateria (see Ax, 1996), has also been found in sponges (Exposito et al., 1991; Boute et al., 1996), although no basal lamina is present there. Fibronectin-like molecules and molecules with an immunoreactivity against chicken integrin have been found in Eimeria (Apicomplexa) (Lopez-Bernad et al., 1996; del Cacho et al., 1997). This means that there are different levels of integration. Usually the components of a recognizable structure can be found in taxa that branched off before the structure itself evolved. Genes with high sequence similarity and therefore related to the genes coding these components (macromolecules) are often present in taxa that branched off even earlier. This of course reflects our thoughts on evolution on the molecular level, where gene duplication and diversification lead to a diversity in molecules which can then be integrated into different structural (physiological, regulatory, etc.) contexts. To sum it all up, I am convinced that there is progress in systematic biology (see also Giribet, 2002). We have something like a solid backbone, along which numerous problems exist, but conflicting hypotheses often appear in relatively restricted regions of the tree. Two “larger” systematic problems exist regarding the phylogenetic position of Arthropods and Tentaculates. In most conflicts, alternative hypotheses can be formulated and solution strategies can be focussed on. The integration of numerous data from different fields makes systematics a highly demanding, but always fascinating task.

Acknowledgements Many thanks to Gerhard Scholtz for the invitation to the symposium “Phylogeny and evolution of Metazoa” at 297

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the DZG-Tagung in Berlin, to Gonzalo Giribet and to numerous colleagues who were involved in discussions at different times.

Note added in proof In a recent paper, Boury-Esnault and colleagues (Invertebrate Biology 122: 187–202, 2003) document the existence of striated ciliary rootlets in the larva of a Plakina trilopha (Desmospongia). Therefore, striated ciliary rootlets do not support a sister-group relationship between Calcarea and Epitheliozoa, as outlined in the text.

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