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Details of Gastropod Phylogeny Inferred from 18S rRNA Sequences
MOLECULAR PHYLOGENETICS AND EVOLUTION
Vol. 9, No. 1, February, pp. 55–63, 1998 ARTICLE NO. FY970439
Details of Gastropod Phylogeny Inferred from 18S rRNA Sequences Birgitta Winnepenninckx,*,1 Gerhard Steiner,† Thierry Backeljau,‡ and Rupert De Wachter* *Departement Biochemie, Universiteit Antwerpen (UIA), Universiteitsplein 1, B-2610 Antwerpen, Belgium; †Institute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria; and ‡Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussel, Belgium Received February 4, 1997; revised June 6, 1997
molecular data (e.g., Tillier et al., 1992, 1994, 1996; Rosenberg et al., 1994; Winnepenninckx et al., 1996). A recent 18S rRNA analysis of molluscan relationships suggested that this molecule might be suitable to resolve phylogenetic problems at infraclass levels (Winnepenninckx et al., 1996). In the present paper we further explore this issue by analyzing a number of generally accepted ideas on the infraclass phylogeny of Gastropoda using 11 new and 7 published (Winnepenninckx et al., 1992, 1994, 1996) complete gastropod 18S rRNA sequences. The points dealt with are the position and suggested paraphyly of Prosobranchia and Archaeogastropoda, as well as the monophyly of taxa such as Caenogastropoda, Neotaenioglossa, Muricacea, Euthyneura, Pulmonata, and Stylommatophora. In this context, particular attention will be paid to the monophyly and position of the Systellommatophora, a group which includes the families Veronicellidae, Onchidiidae, and Rathousiidae, and according to some authors also the Rhodopidae (von Salvini-Plawen, 1970) and the Smeagolidae (Climo, 1980; Tillier and Ponder, 1992). Systellommatophora are considered to be either pulmonates (e.g., Van Mol, 1974; Solem, 1979; Tillier, 1984; Haszprunar, 1988b; Haszprunar and Huber, 1990; Tillier and Ponder, 1992) or opisthobranchs (e.g., Boettger, 1955), although von Salvini-Plawen (1970) considered them as a proper subclass, the Gymnomorpha, related to the opisthobranchs. Their status as a separate group was confirmed by von Salvini-Plawen and Steiner (1996), who related them to the pulmonates. However, systellommatophoran monophyly (e.g., von Salvini-Plawen, 1970) is still debated (e.g., Climo, 1980; Tillier, 1984; Haszprunar and Huber, 1990).
Some generally accepted viewpoints on the phylogenetic relationships within the molluscan class Gastropoda are reassessed by comparing complete 18S rRNA sequences. Phylogenetic analyses were performed using the neighbor-joining and maximum parsimony methods. The previously suggested basal position of Archaeogastropoda, including Neritimorpha and Vetigastropoda, in the gastropod clade is confirmed. The present study also provides new molecular evidence for the monophyly of both Caenogastropoda and Euthyneura (Pulmonata and Opisthobranchia), making Prosobranchia paraphyletic. The relationships within Caenogastropoda and Euthyneura data turn out to be very unstable on the basis of the present 18S rRNA sequences. The present 18S rRNA data question, but are insufficient to decide on, muricacean (Neogastropoda), neotaenioglossan, pulmonate, or stylommatophoran monophyly. The analyses also focus on two systellommatophoran families, namely, Veronicellidae and Onchidiidae. It is suggested that Systellommatophora are not a monophyletic unit but, due to the lack of stability in the euthyneuran clade, their affinity to either Opisthobranchia or Pulmonata could not be determined. r 1998 Academic Press
INTRODUCTION Gastropoda is the largest molluscan class and includes the common terrestrial, freshwater, and marine snails and slugs. It has an excellent fossil record going back to 550 MYA (Runnegar and Pojeta, 1985). The class is traditionally divided into three subclasses: Prosobranchia (Streptoneura), Opisthobranchia, and Pulmonata (together the latter constitute the Euthyneura). The phylogenetic relationships between and within these subclasses are longstanding problems (e.g., Ponder and Lindberg, 1996, 1997; von SalviniPlawen and Steiner, 1996; for a review of earlier work see Bieler, 1992), which are increasingly studied with
MATERIALS AND METHODS Amplification and Sequencing of the 18S rRNA Genes The taxonomy of the gastropod species used in this study is given in Table 1. Sampling locations are listed in Table 2. The species were frozen alive. After dissection, DNA was extracted (Winnepenninckx et al., 1993) from the tissues indicated in Table 2. The 18S rRNA genes were PCR-amplified, cloned, and sequenced as
1 Present address: Royal Belgian Institute for Natural Sciences, Vautierstraat 29, B-1000 Brussel, Belgium.
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1055-7903/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Taxonomy of the Gastropod Species Used in This Study Subclass Prosobranchia a
Superorder
Order
Archaeogastropoda a Caenogastropoda
Stylommatophora
Mesurethra Sigmurethra
Basommatophora Systellommatorphora Opisthobranchia
Family
Genus
Vetigastropoda Neritimorpha
Trochidae Neritidae Buccinidae Nassariidae Fasciolariidae Muricidae Littorinidae Bursidae Calyptraeidae Clausiliidae Achatinidae Succineidae Helicidae Siphonariidae Onchidiidae Veronicellidae Aplysidae
Monodontab Nerita Pisaniab Nassariusb Fasciolariab Thais Littorinab Bursab Crepidulab Baleab Limicolaria Oxylomab Helix Siphonaria Onchidella Laevicaulisb Aplysiab
Neogastropoda
Neotaenioglossa
Pulmonata
Suborder
Onchidiacea Soleolifera Anaspidea
Discopoda
Holopodopes Aulocopoda Holopoda
Note. The taxonomy of the Archaeogastropoda is based on Haszprunar (1988b, Table 5, p. 428), except for its ranking as superorder; the Caenogastropoda are classified according to Ponder and Ware´n (1988). The classification of the Neogastropoda is based on Ponder (1973); Pulmonata are classified according to Solem (1979), except for the placement of Siphonaria, which follows von Salvini-Plawen (1970). a Currently considered nonmonophyletic. b Sequence determined in this study.
described by Winnepenninckx et al. (1995), using the primers published in Winnepenninckx et al. (1994) and two M13 universal primers. Data Analysis The new gastropod 18S rRNA sequences were added to the alignment of Van de Peer et al. (1996a) using the computer program DCSE (De Rijk and De Wachter, 1993), which considers primary as well as secondary TABLE 2 Sources and Tissue Type of Gastropod Species Used for This Study Species Aplysia sp. Balea biplicata Bursa rana
Sampling location Hong Kong Mortsel (Belgium) Hong Kong
Tissue Digestive gland Complete organism Bucal mass 1 foot muscle Albumen gland Albumen gland
Crepidula adunca Vancouver (Canada) Fasciolaria lignaria Bahar Ic-Cagnaq (Malta) Laevicaulis alte Laboratory bred Digestive gland (Go¨rlitz, Germany) Littorina obtusata Oosterschelde (Nether- Albumen gland 1 lands) muscle tissue Monodonta labio Hong Kong Digestive 1 reproductive gland Nassarius singuin- Hong Kong Penis 1 foot muscles jorensis Oxyloma sp. Go¨rlitz (Germany) Digestive gland Pisania striata Bahar Ic-Cagnaq Digestive 1 reproduc(Malta) tive gland
structure similarity. If necessary, manual adjustments were made with the same program. The secondary structure model of Van de Peer et al. (1996a) was used. Sequence regions corresponding to the amplification primer at the 58 end of the gene were removed prior to reconstruction of phylogenetic trees. The 18S rRNA sequences were analyzed using neighbor-joining (NJ) and maximum parsimony (MP) methods. The program TREECON (Van de Peer and De Wachter, 1993) was used to construct NJ trees based on the formulas of Jukes and Cantor (1969), Kimura (1980), or Van de Peer et al. (1996b). Gaps were not taken into account. Tree stability was assessed via bootstrapping over 1000 replicates. MP trees were constructed on the phylogenetically informative sites using either the heuristic or the exhaustive search option of PAUP (Swofford, 1993). Stability of MP trees was assessed via bootstrapping over 1000 replicates and the calculation of decay indices (Bremer, 1988; Donoghue et al., 1992). As until now there is no consensus about the minimal bootstrap value necessary to regard a cluster as firmly supported, bootstrap values were arbitrarily considered to reflect strong support if they exceeded 70% (Hillis and Bull, 1993). RESULTS New gastropod 18S rRNA sequences were submitted to the EMBL sequence data library and have the following accession nos.: Aplysia sp., X94268; Balea biplicata, X94278; Bursa rana, X94269; Crepidula
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adunca, X94277; Fasciolaria lignaria, X94275; Laevicaulis alte, X94273; Littorina obtusata, X94274; Monodonta labio, X94271; Nassarius singuinjorensis, X94273; Oxyloma sp., X94276; Pisania striata, X94272. Figure 1A shows the NJ tree obtained on the basis of the Jukes and Cantor (1969) distances of an alignment of complete 18S rRNA sequences of 18 gastropods. The bivalve Galeomma takii was arbitrarily chosen as outgroup. The same topology was obtained with Kimura (1980) distances. The data suggest that Archaeogastropoda are paraphyletic and give rise to the Apogastropoda, i.e., the caenogastropods and Euthyneura. Consequently, Prosobranchia appear as a paraphyletic group. All Caenogastropoda belong to a single cluster. Yet, since C. adunca and F. lignaria have a strongly supported common origin, which is well supported by bootstrap resampling, neither Neotaenioglossa (partim
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Discopoda) nor Muricacea (represented by Buccinidae, Fasciolariidae, Muricidae, and Nassariidae) form monophyletic groups. There is 100% bootstrap support for the monophyly of Euthyneura, which form two unsupported clades consisting of (1) the three stylommatophorans, Helix aspersa, Ba. biplicata, and Oxyloma sp., and (2) the five remaining euthyneurans. Surprisingly, the achatinid Limicolaria kambeul belongs to this latter clade instead of to the first. Both systellommatophorans have a common origin, but this result was not supported by bootstrap analysis. A tree based on a distance matrix calculated using the equation of Van de Peer et al. (1996b) showed small topological shifts of nodes with low bootstrap values (indicated by dots in Fig. 1A). Onchidella celtica becomes a sister group to a Siphonaria–Limicolaria– Laevicaulis–Aplysia cluster.
FIG. 1. (A) NJ tree computed on the basis of Jukes and Cantor (1969) distances calculated from an alignment of 18 gastropod 18S rRNA sequences. The bivalve Galeomma takii was used as an outgroup. Numbers at a node indicate percentage bootstrap values higher than 70%. Dots mark nodes that change when distances are computed using the equation of Van de Peer et al. (1996b). (B) Majority rule consensus tree of the NJ trees obtained when replacing G. takii 20 times by another mollusc (Table 3). The following shortened taxon names are used: Bas., Basommatophora; Styl., Stylommatophora; Syst., Systellommatophora; Ng., Neogastropoda; Nt., Neotaenioglossa; Vet., Vetigastropoda; and Ner., Neritimorpha.
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TABLE 3 Species Used as Outgroups in Fig. 1B, and the Molluscan Class to Which They Belong Species
Class
Argopecten irradians Barbatia virescens Cardita canaliculata Barbatia virescens Chlamys islandica Corbula crassa Crassostrea virginica Galeomma takii Glycymeris glycymeris Mactromeris polynyma Mulinia lateralis Mytilus edulis Placopecten magellanicus Pholas dactylus Spisula solida Tresus capax Tridacna sp. Scutopus ventrolineatus Acanthopleura japonica Lepidochitona sp. Antalis vulgaris
Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Caudofoveata Polyplacophora Polyplacophora Scaphopoda
Replacing the outgroup G. takii by each of 20 other molluscs (Table 3), using the computer program TREECON, yielded the majority rule consensus tree shown in Fig. 1B. It supports the previous results (Fig. 1A), except that the majority of outgroups does not support systellomatophoran monophyly. The stability of the NJ tree (Fig. 1A) was further assessed by excluding the ingroup species one by one. Exclusion of any of the euthyneuran taxa caused changes in the
poorly supported nodes of their cluster (not shown). Removing L. obtusata from the caenogastropod cluster also caused a shift in the euthyneuran cluster. A heuristic search on the 266 parsimony informative sites in the alignment of the 18 gastropods, with the bivalve G. takii as outgroup, yielded a single MP tree of 738 steps, which is shown in Fig. 2. Changing input orders 100 times had no influence on this result. The MP tree differs from the NJ tree (Fig. 1A) only in branching points with low bootstrap values, so that the conclusions of Fig. 1A are confirmed. Prosobranchia and Archaeogastropoda are both paraphyletic groups. The paraphyly of the Prosobranchia is strongly supported by bootstrap sampling, but that of the Archaeogastropoda is not. Within the Caenogastropoda, neither Neotaenioglossa (partim Discopoda) nor Muricacea are monophyletic groups. Yet, Muricacea polyphyly is not significantly supported. In the euthyneuran clade, none of the nodes receives high bootstrap values. When G. takii was replaced by another molluscan outgroup 20 times (Table 3) or when an ingroup taxon was removed, nodes with high bootstrap and decay indices were not affected. The poorly supported nodes within the caenogastropod cluster showed no ingroup effect either, and only a slight outgroup effect. On the contrary, in the euthyneuran cluster, branching points strongly depended on the outgroup choice (Fig. 2) and the ingroup taxa included. Removing Aplysia sp., Oxyloma sp., Lim. kambeul, L. obtusata, or Siphonaria pectinata caused either a collapse or a shift of the topology of the euthyneuran clade (not shown). As the use of too-distant outgroups may cause an increase of homoplasy between in- and outgroup species, we focused on the caenogastropods and euthy-
FIG. 2. MP tree found on the basis of the 266 informative sites of an alignment of 18 gastropod 18S rRNA sequences with Galeomma takii as outgroup. Bootstrap values higher than 70% are indicated above the nodes. Figures below branching points indicate how many additional steps were necessary for this clade not to be unequivocally supported (decay index); those at the right of a branching point indicate how many of 20 different outgroups (Table 3) supported this branching point. Abbreviations of taxon names as in Fig. 1.
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neurans separately. Figure 3A shows the results of the Euthyneura NJ tree, with the caenogastropod Littorina littorea as outgroup. In comparison to the complete tree (Fig. 1A), this yielded small topological shifts. In the tree of Fig. 3A, the polyphyly of the Sigmurethra and the Systellommatophora appears to be strongly supported by bootstrap resampling. Figure 3B shows the results of the NJ analysis of the caenogastropods, with the pulmonate S. pectinata as outgroup. The tree confirms the polyphyly of the Neotaenioglossa (partim Discopoda) and Muricacea. It shows that the Fasciolaria–Nassarius–Crepidula cluster and the monophyly of the genus Littorina are well supported by bootstrap analysis. An exhaustive search on the 83 phylogenetically informative sites of the alignment of the euthyneuran
sequences yielded three MP trees of 211 steps. Their strict consensus topology, which is shown in Fig. 4A, has only three nodes in common with the equivalent NJ tree (Fig. 3A), but does not contradict the MP tree based on all sequences (Fig. 2). An exhaustive search on the 62 phylogenetically informative sites of the caenogastropod sequences yielded two MP trees of 142 steps. In the strict consensus tree, which is shown in Fig. 4B, only the monophyly of the genus Littorina is significantly supported by bootstrap resampling. DISCUSSION The present 18S rRNA analyses confirm the hypothesis that the archaeogastropods, namely Neritimorpha and Vetigastropoda, have a basal position within the
FIG. 3. NJ tree of the Jukes and Cantor (1969) distances between (A) eight euthyneuran 18S rRNA sequences with the prosobranch Littorina littorea as outgroup and (B) eight caenogastropod 18S rRNA sequences with the pulmonate Siphonaria pectinata as outgroup. Only bootstrap values higher than 70% are indicated. Abbreviations of taxon names as in Fig. 1.
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FIG. 4. Strict consensus tree (A) of the three MP trees (L 5 211) found on the basis of the 83 informative sites of an alignment of eight euthyneuran 18S rRNA sequences with the prosobranch Littorina littorea as outgroup and (B) of the two MP trees (L 5 142) found on the basis of the 62 informative sites of an alignment of eight caenogastropod 18S rRNA sequences with the pulmonate Siphonaria pectinata as outgroup. Only bootstrap values higher than 70% are indicated. Dots indicate nodes in common with the NJ trees of Fig. 3. Abbreviations of taxon names as in Fig. 1.
Gastropoda. On the basis of, for example, the reduction of the right ctenidium, incorporation of the right kidney in the genital duct, and the mode of fertilization, Neritimorpha were considered to be closely related to the Caenogastropoda (e.g., Naef, 1911). Yet, the lack of skeletal rods in the ctenidium, the presence of juvenile coiling, and the deep mantle cavity suggest that Neritimorpha are an early, highly specialized archaeogastropod offshoot (e.g., von Salvini-Plawen and Haszprunar, 1987; Haszprunar, 1988a,b; Hickman, 1988; Healy,
1988). The similarities between Neritimorpha and Caenogastropoda were therefore ascribed to convergence. The present 18S rRNA data also refute a close relationship between Neritimorpha and Caenogastropoda and consistently suggest that Archaeogastropoda, including both Neritimorpha and Vetigastropoda, is a basal grade within the Gastropoda (e.g., Hickman, 1988; Haszprunar, 1993; von Salvini-Plawen and Steiner, 1996; Ponder and Lindberg, 1997). Our result is also congruent with the results of a study using about 150 nucleotides
GASTROPOD PHYLOGENY
of the 28S rRNA (Rosenberg et al., 1994) and the paper by Harasewych et al. (1997) on the basis of 450 nucleotides of the 18S rRNA. The early divergence of the Vetigastropoda is supported by several gastropod plesiomorphies such as two excretory organs, fringed head tentacles, and the eye type (e.g., Haszprunar, 1988b). The 18S rRNA data suggest the monophyly of the Apogastropoda (5Caenogastropoda and Euthyneura) and consequently prosobranch paraphyly. This result is in accordance with findings on the basis of 28S rRNA (Emberton et al., 1990; Tillier et al., 1992, 1994; Rosenberg et al., 1994) and partial 18S rRNA (Harasewych et al., 1997). Previously, Caenogastropoda and Euthyneura had already been synapomorphically linked by the way in which the pleural ganglia make contact with the pedal ganglia (e.g., von Salvini-Plawen and Haszprunar, 1987; Haszprunar, 1988b; Bieler, 1992; Ponder and Lindberg, 1997). Caenogastropod monophyly was accepted by most authors on the basis of the special type of osphradium (e.g., von Salvini-Plawen, 1980; Haszprunar, 1985b, 1988b), as well as by sequence comparisons of 28S rRNA (Tillier et al., 1992, 1994; Emberton et al., 1990), but was questioned by Rosenberg et al. (1994). The value of the osphradium as an indication of caenogastropod synapomorphy is supported by our 18S rRNA analyses, which consistently suggest a single origin for the caenogastropods. Consequently, our 18S rRNA data reject the hypothesis that Neogastropoda originated from the Archaeogastropoda (Ponder, 1973), but support the view that neogastropods are derived from neotaenioglossans (5mesogastropods) (Haszprunar, 1988a,b; Healy, 1988; Taylor and Morris, 1988). An extensive discussion of the anatomical features supporting the two contradictory hypotheses was given by Taylor and Morris (1988). As our analyses included only one of the three neogastropod superfamilies (Ponder, 1973), namely the Muricacea, not much can be concluded with respect to neogastropod monophyly, which was accepted on the basis of several morphological features (e.g., Ponder, 1973; Taylor and Morris, 1988; Kantor, 1996; Ponder and Lindberg, 1997), chromosome numbers (Patterson and Burch, 1978), DNA content (Hinegardner, 1974), and the 28S rRNA analyses of Rosenberg et al. (1994). However, the present 18S rRNA analyses consistently suggest that at least Muricacea and Neotaenioglossa (partim Discopoda) are not monophyletic. A lack of support for the monophyly of the neogastropods, also represented by the Muricacea, was also reported by Tillier et al. (1992, 1994) with 28S rRNA data. The close relationship between the neogastropod families Fasciolariidae (F. lignaria) and Nassariidae (N. singuinjorensis), as suggested by Ponder (1973), is confirmed by the 18S rRNA data. Yet, although Fasciolariidae, Nassariidae, and Buccinidae are sometimes considered one unit (e.g., Ponder, 1973; Ponder and Ware´n, 1988), in
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our trees the families Fasciolariidae and Nassariidae do not show a close relationship to the Buccinidae (P. striata), but have a common origin with the neotaenioglossan family Calyptraeidae (C. adunca). In accordance with the 28S rRNA analyses of Tillier et al. (1992, 1994), Littorinidae forms the most basal branch in the caenogastropod cluster. The present 18S rRNA study supports the monophyly of Euthyneura, as generally accepted on the basis of the existence of parietal ganglia, the development of a head shield with special sense organs, a pallial caecum, and the type of the gills, circulatory system, and genital system (e.g., Haszprunar, 1985a, 1988a). Mainly because of the presence of a ‘‘lung,’’ pulmonate monophyly was broadly accepted (e.g., von SalviniPlawen, 1970; Tillier, 1984; Haszprunar, 1985a). Yet the distinction between pulmonates and opisthobranchs was previously questioned by Boettger (1955), who suggested that Opisthobranchia were paraphyletic (see Haszprunar, 1988b for a review). Recently, Ponder and Lindberg (1997) also found support neither for opisthobranch nor for pulmonate monophyly. Our 18S rRNA data fail to reveal pulmonate monophyly. However, due to unstable results, it cannot be concluded that the Pulmonata are para- or polyphyletic, either. The 28S rRNA analyses of Tillier et al. (1992, 1994) and the partial 18S rRNA study of Harasewych et al. (1997) yielded the same ambiguous result on this issue. Their data also strongly supported the Pulmonata–Aplysia monophyly, but there was no stability in the clade. More recently, 28S rRNA data suggested that Aplysia is the sister group of the pulmonates excluding Amphibolidae (Tillier et al., 1996). Emberton et al. (1990) and Rosenberg et al. (1994) reported that on the basis of 28S rRNA data, Pulmonata were monophyletic. Yet, since their study did not include any opisthobranch sequence, this hypothesis was premature. Due to the instability of the branching pattern within the pulmonate-opisthobranch clade (Figs. 1, 2, 3A, and 4A), we cannot decide on the status and relationships of the Stylommatophora and Systellommatophora. Most of our analyses suggest that Systellommatophora are not monophyletic and that Onchidiidae is the most basal branch in the Euthyneura. This result is supported by the congruence of both MP and NJ trees (Figs. 1B, 2, 3A, and 4A) and the moderate bootstrap support in the NJ tree (Fig. 3A). In addition, Climo (1980) interpreted the systellommatophorans as a polyphyletic assemblage at the base of the euthyneurans. This hypothesis implies that the morphological similarities between the Veronicellidae and the Onchidiidae (e.g., Tillier, 1984) are convergent features. Finally, the present study does not support a relationship of the Succineidae (represented by Oxyloma sp.) to the Opisthobranchia, as suggested on the basis of comparisons of the alimentary and reproductive system (Rigby, 1965). Indeed, all 18S rRNA analyses support a relationship between Helix,
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Balea, and Oxyloma, but not between Oxyloma and the opisthobranch Aplysia. The unstable results within the Euthyneura may be due (1) to the explosive way in which they arose (Climo, 1980) and/or (2) to the limited number of representatives available for 18S rRNA analysis. In conclusion, although the present 18S rRNA data provide new support for some well-established gastropod relationships, they are insufficient to unambiguously resolve some of the current debates. Whether this is due to a taxon sampling problem or to the molecule remains to be investigated. ACKNOWLEDGMENTS We are indebted to Dr. B. Morton (Hong Kong), Dr. P. Schembri (Malta), and Dr. H. Reise (Germany) for helping us with the collection of parts of the material. Two anonymous referees provided helpful and constructive comments. B.W. has held an IWT scholarship. This work was funded by grants from the National Fund for Scientific Research.
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Haszprunar, G. (1993). The archaeogastropoda—A clade, a grade or what else? Am. Malacol. Bull. 10: 165–177. Healy, J. M. (1988). Sperm morphology and its systematic importance in the Gastropoda. Malac. Rev. Suppl. 4: 251–267. Hickman, C. S. (1988). Archaeogastropod evolution, phylogeny and systematics: A reevaluation. Malac. Rev. Suppl. 4: 17–35. Hillis, D. M., and Bull, J. J. (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42: 182–192. Hinegardner, R. (1974). Cellular DNA content of the Mollusca. Comp. Biochem. Physiol. 47A: 447–460. Jukes, T. H., and Cantor, C. R. (1969). Evolution of protein molecules. In ‘‘Mammalian Protein Metabolism’’ (H. N. Munro, Ed.), pp. 21–132, Academic Press, New York. Kantor, Y. I. (1996). Phylogeny and relationships of Neogastropoda. In ‘‘Origin and Evolutionary Radiation of the Mollusca’’ (J. D. Taylor, Ed.), pp. 221–231, Oxford Sci., Oxford. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111–120. Naef, A. (1911). Studien zur generellen Morphologie der Mollusken. 1. Teil: uber Torsion und Asymmetrie der Gastropoden. Ergeb. Forstschr. Zool. 3: 73–164. Pattersen, C. M., and Burch, J. B. (1978). Chromosomes of pulmonate molluscs. In ‘‘Pulmonates,’’ Vol. 2A, ‘‘Systematics, Evolution and Ecology’’ (V. Fretter and J. Peake, Eds.), pp. 171–217, Academic Press, London. Ponder, W. F. (1973). The origin and evolution of the Neogastropoda. Malacologia 12: 295–338. Ponder, W. F., and Ware´n, A. (1988). Classification of the Caenogastropoda and Heterostropha—A list of the family group names and higher taxa. Malac. Rev. Suppl. 4: 288–328. Ponder, W. F., and Lindberg, D. R. (1996). Gastropod phylogeny— Challenges for the 90s. In ‘‘Origin and Evolutionary Radiation of the Mollusca’’ (J. D. Taylor, Ed.), pp. 135–155, Oxford Sci., Oxford. Ponder, W. F., and Lindberg, D. R. (1997). Towards a phylogeny of gastropod molluscs: An analysis using morphological characters. Zool. J. Linn. Soc. 119: 83–265. Rigby, J. E. (1965). Succinea putris: A terrestrial opisthobranch mollusc. Proc. Zool. Soc. London 144: 445–486. Rosenberg, G., Kuncio, G. S., Davis, G. M., and Harasewych, M. G. (1994). Preliminary ribosomal RNA phylogeny of gastropod and unionidean bivalve mollusks. Nautilus Suppl. 2: 111–121. Runnegar, B., and Pojeta, J., Jr. (1985). Origin and diversification of the Mollusca. In ‘‘The Mollusca,’’ Vol. 10, ‘‘Evolution’’ (M. R. Clarke and E. R. Trueman, Eds.), pp. 1–57, Academic Press, London. Solem, A. (1979). A theory of land snail biogeographic patterns through time. In ‘‘Pathways in Malacology’’ (S. Van Der Spoel, A. C. Van Bruggen, and J. Lever, Eds.), pp. 225–249, Bohn, Scheltema & Holtema, Utrecht, dr. W. Junk b.v., The Hague. Swofford, D. L. (1993). PAUP: Phylogenetic analysis using parsimony, Version 3.1.1. Illinois Natural History Survey, Champaign, IL. Taylor, J. D., and Morris, N. J. (1988). Relationships of the neogastropods. Malac. Rev. Suppl. 4: 167–180. Tillier, S. (1984). Relationships of gymnomorph gastropods (Mollusca: Gastropoda). Zool. J. Linn. Soc. 82: 345–362. Tillier, S., and Ponder, W. F. (1992). New species of Smeagol from Australia and New Zealand, with a discussion of the affinities of the genus (Gastropoda: Pulmonata). J. Moll. Stud. 58: 135–155. Tillier, S., Masselot, M., Philippe, H., and Tillier, A. (1992). Phyloge´nie mole´culaire des Gastropoda (Mollusca) fonde´e sur le se´quencage partiel de l’ ARN ribosomique 28S. C. R. Acad. Sci. Paris Se´r. III 314: 79–85. Tillier, S., Masselot, M., Guerdoux, J., and Tillier, A. (1994). Mono-
GASTROPOD PHYLOGENY phyly of the major gastropod taxa tested from partial 28S rRNA sequences, with emphasis on Euthyneura and hot-vent limpets Peltospiroidea. Nautilus Suppl. 2: 122–140. Tillier, S., Masselot, M., and Tillier, A. (1996). Phylogenetic relationships of the pulmonate gastropods from rRNA sequences, and tempo, and age of the stylommatophoran radiation. In ‘‘Origin and Evolutionary Radiation of the Mollusca’’ (J. D. Taylor, Ed.), pp. 267–285, Oxford Sci., Oxford. Van de Peer, Y., and De Wachter, R. (1993). TREECON: A software package for the construction and drawing of evolutionary trees. Comput. Appl. Biosci. 9: 177–182. Van De Peer, Y., Van Den Broeck, I., De Rijk, P., and De Wachter, R. (1996a). Database on the structure of small ribosomal subunit RNA. Nucleic Acids Res. 24: 86–91. Van De Peer, Y., Van Der Auwera, G., and De Wachter, R. (1996b). The evolution of Stramenopiles and Alveolates as derived by ‘‘substitution rate calibration’’ of small ribosomal subunit RNA. J. Mol. Evol. 42: 201–210. Van Mol, J.-J. (1974). Evolution phyloge´ne´tique du ganglion ce´re´broide chez les Gaste´ropodes Pulmone´s. Haliotis 4: 77–86. von Salvini-Plawen, L. (1970). Zur systematischen Stellung von Soleolifera und Rhodope (Gastropoda, Euthyneura). Zool. Jb. Syst. Bd. 97 8: 285–299. von Salvini-Plawen, L. (1980). A reconsideration of systematics in the
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Mollusca (phylogeny and higher classification). Malacologia 19: 249–278. von Salvini-Plawen, L., and Haszprunar, G. (1987). The Vetigastropoda and the systematics of streptoneurous Gastropoda (Mollusca). J. Zool. London 211: 747–770. von Salvini-Plawen, L., and Steiner, G. (1996). Synapomorphies and plesiomorphies in higher classification of Mollusca. In ‘‘Origin and Evolutionary Radiation of the Mollusca’’ (J. D. Taylor, Ed.), pp. 29–53, Oxford Sci., Oxford. Winnepenninckx, B., Backeljau, T., Van de Peer, Y., and De Wachter, R. (1992). Structure of the small ribosomal subunit RNA of the pulmonate snail, Limicolaria kambeul, and phylogenetic analysis of the Metazoa. FEBS Lett. 309: 123–126. Winnepenninckx, B., Backeljau, T., and De Wachter, R. (1993). Extraction of high molecular weight DNA from molluscs. Trends Genet. 9: 407. Winnepenninckx, B., Backeljau, T., and De Wachter, R. (1994). Small ribosomal subunit RNA and the phylogeny of Mollusca. Nautilus Suppl. 2: 98–110. Winnepenninckx, B., Backeljau, T., and De Wachter, R. (1995). Phylogeny of protostome worms derived from 18S rRNA sequences. Mol. Biol. Evol. 12: 641–649. Winnepenninckx, B., Backeljau, T., and De Wachter, R. (1996). Investigation of molluscan phylogeny on the basis of 18S rRNA sequences. Mol. Biol. Evol. 13: 1306–1317.
Vol. 9, No. 1, February, pp. 55–63, 1998 ARTICLE NO. FY970439
Details of Gastropod Phylogeny Inferred from 18S rRNA Sequences Birgitta Winnepenninckx,*,1 Gerhard Steiner,† Thierry Backeljau,‡ and Rupert De Wachter* *Departement Biochemie, Universiteit Antwerpen (UIA), Universiteitsplein 1, B-2610 Antwerpen, Belgium; †Institute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria; and ‡Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussel, Belgium Received February 4, 1997; revised June 6, 1997
molecular data (e.g., Tillier et al., 1992, 1994, 1996; Rosenberg et al., 1994; Winnepenninckx et al., 1996). A recent 18S rRNA analysis of molluscan relationships suggested that this molecule might be suitable to resolve phylogenetic problems at infraclass levels (Winnepenninckx et al., 1996). In the present paper we further explore this issue by analyzing a number of generally accepted ideas on the infraclass phylogeny of Gastropoda using 11 new and 7 published (Winnepenninckx et al., 1992, 1994, 1996) complete gastropod 18S rRNA sequences. The points dealt with are the position and suggested paraphyly of Prosobranchia and Archaeogastropoda, as well as the monophyly of taxa such as Caenogastropoda, Neotaenioglossa, Muricacea, Euthyneura, Pulmonata, and Stylommatophora. In this context, particular attention will be paid to the monophyly and position of the Systellommatophora, a group which includes the families Veronicellidae, Onchidiidae, and Rathousiidae, and according to some authors also the Rhodopidae (von Salvini-Plawen, 1970) and the Smeagolidae (Climo, 1980; Tillier and Ponder, 1992). Systellommatophora are considered to be either pulmonates (e.g., Van Mol, 1974; Solem, 1979; Tillier, 1984; Haszprunar, 1988b; Haszprunar and Huber, 1990; Tillier and Ponder, 1992) or opisthobranchs (e.g., Boettger, 1955), although von Salvini-Plawen (1970) considered them as a proper subclass, the Gymnomorpha, related to the opisthobranchs. Their status as a separate group was confirmed by von Salvini-Plawen and Steiner (1996), who related them to the pulmonates. However, systellommatophoran monophyly (e.g., von Salvini-Plawen, 1970) is still debated (e.g., Climo, 1980; Tillier, 1984; Haszprunar and Huber, 1990).
Some generally accepted viewpoints on the phylogenetic relationships within the molluscan class Gastropoda are reassessed by comparing complete 18S rRNA sequences. Phylogenetic analyses were performed using the neighbor-joining and maximum parsimony methods. The previously suggested basal position of Archaeogastropoda, including Neritimorpha and Vetigastropoda, in the gastropod clade is confirmed. The present study also provides new molecular evidence for the monophyly of both Caenogastropoda and Euthyneura (Pulmonata and Opisthobranchia), making Prosobranchia paraphyletic. The relationships within Caenogastropoda and Euthyneura data turn out to be very unstable on the basis of the present 18S rRNA sequences. The present 18S rRNA data question, but are insufficient to decide on, muricacean (Neogastropoda), neotaenioglossan, pulmonate, or stylommatophoran monophyly. The analyses also focus on two systellommatophoran families, namely, Veronicellidae and Onchidiidae. It is suggested that Systellommatophora are not a monophyletic unit but, due to the lack of stability in the euthyneuran clade, their affinity to either Opisthobranchia or Pulmonata could not be determined. r 1998 Academic Press
INTRODUCTION Gastropoda is the largest molluscan class and includes the common terrestrial, freshwater, and marine snails and slugs. It has an excellent fossil record going back to 550 MYA (Runnegar and Pojeta, 1985). The class is traditionally divided into three subclasses: Prosobranchia (Streptoneura), Opisthobranchia, and Pulmonata (together the latter constitute the Euthyneura). The phylogenetic relationships between and within these subclasses are longstanding problems (e.g., Ponder and Lindberg, 1996, 1997; von SalviniPlawen and Steiner, 1996; for a review of earlier work see Bieler, 1992), which are increasingly studied with
MATERIALS AND METHODS Amplification and Sequencing of the 18S rRNA Genes The taxonomy of the gastropod species used in this study is given in Table 1. Sampling locations are listed in Table 2. The species were frozen alive. After dissection, DNA was extracted (Winnepenninckx et al., 1993) from the tissues indicated in Table 2. The 18S rRNA genes were PCR-amplified, cloned, and sequenced as
1 Present address: Royal Belgian Institute for Natural Sciences, Vautierstraat 29, B-1000 Brussel, Belgium.
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1055-7903/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Taxonomy of the Gastropod Species Used in This Study Subclass Prosobranchia a
Superorder
Order
Archaeogastropoda a Caenogastropoda
Stylommatophora
Mesurethra Sigmurethra
Basommatophora Systellommatorphora Opisthobranchia
Family
Genus
Vetigastropoda Neritimorpha
Trochidae Neritidae Buccinidae Nassariidae Fasciolariidae Muricidae Littorinidae Bursidae Calyptraeidae Clausiliidae Achatinidae Succineidae Helicidae Siphonariidae Onchidiidae Veronicellidae Aplysidae
Monodontab Nerita Pisaniab Nassariusb Fasciolariab Thais Littorinab Bursab Crepidulab Baleab Limicolaria Oxylomab Helix Siphonaria Onchidella Laevicaulisb Aplysiab
Neogastropoda
Neotaenioglossa
Pulmonata
Suborder
Onchidiacea Soleolifera Anaspidea
Discopoda
Holopodopes Aulocopoda Holopoda
Note. The taxonomy of the Archaeogastropoda is based on Haszprunar (1988b, Table 5, p. 428), except for its ranking as superorder; the Caenogastropoda are classified according to Ponder and Ware´n (1988). The classification of the Neogastropoda is based on Ponder (1973); Pulmonata are classified according to Solem (1979), except for the placement of Siphonaria, which follows von Salvini-Plawen (1970). a Currently considered nonmonophyletic. b Sequence determined in this study.
described by Winnepenninckx et al. (1995), using the primers published in Winnepenninckx et al. (1994) and two M13 universal primers. Data Analysis The new gastropod 18S rRNA sequences were added to the alignment of Van de Peer et al. (1996a) using the computer program DCSE (De Rijk and De Wachter, 1993), which considers primary as well as secondary TABLE 2 Sources and Tissue Type of Gastropod Species Used for This Study Species Aplysia sp. Balea biplicata Bursa rana
Sampling location Hong Kong Mortsel (Belgium) Hong Kong
Tissue Digestive gland Complete organism Bucal mass 1 foot muscle Albumen gland Albumen gland
Crepidula adunca Vancouver (Canada) Fasciolaria lignaria Bahar Ic-Cagnaq (Malta) Laevicaulis alte Laboratory bred Digestive gland (Go¨rlitz, Germany) Littorina obtusata Oosterschelde (Nether- Albumen gland 1 lands) muscle tissue Monodonta labio Hong Kong Digestive 1 reproductive gland Nassarius singuin- Hong Kong Penis 1 foot muscles jorensis Oxyloma sp. Go¨rlitz (Germany) Digestive gland Pisania striata Bahar Ic-Cagnaq Digestive 1 reproduc(Malta) tive gland
structure similarity. If necessary, manual adjustments were made with the same program. The secondary structure model of Van de Peer et al. (1996a) was used. Sequence regions corresponding to the amplification primer at the 58 end of the gene were removed prior to reconstruction of phylogenetic trees. The 18S rRNA sequences were analyzed using neighbor-joining (NJ) and maximum parsimony (MP) methods. The program TREECON (Van de Peer and De Wachter, 1993) was used to construct NJ trees based on the formulas of Jukes and Cantor (1969), Kimura (1980), or Van de Peer et al. (1996b). Gaps were not taken into account. Tree stability was assessed via bootstrapping over 1000 replicates. MP trees were constructed on the phylogenetically informative sites using either the heuristic or the exhaustive search option of PAUP (Swofford, 1993). Stability of MP trees was assessed via bootstrapping over 1000 replicates and the calculation of decay indices (Bremer, 1988; Donoghue et al., 1992). As until now there is no consensus about the minimal bootstrap value necessary to regard a cluster as firmly supported, bootstrap values were arbitrarily considered to reflect strong support if they exceeded 70% (Hillis and Bull, 1993). RESULTS New gastropod 18S rRNA sequences were submitted to the EMBL sequence data library and have the following accession nos.: Aplysia sp., X94268; Balea biplicata, X94278; Bursa rana, X94269; Crepidula
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adunca, X94277; Fasciolaria lignaria, X94275; Laevicaulis alte, X94273; Littorina obtusata, X94274; Monodonta labio, X94271; Nassarius singuinjorensis, X94273; Oxyloma sp., X94276; Pisania striata, X94272. Figure 1A shows the NJ tree obtained on the basis of the Jukes and Cantor (1969) distances of an alignment of complete 18S rRNA sequences of 18 gastropods. The bivalve Galeomma takii was arbitrarily chosen as outgroup. The same topology was obtained with Kimura (1980) distances. The data suggest that Archaeogastropoda are paraphyletic and give rise to the Apogastropoda, i.e., the caenogastropods and Euthyneura. Consequently, Prosobranchia appear as a paraphyletic group. All Caenogastropoda belong to a single cluster. Yet, since C. adunca and F. lignaria have a strongly supported common origin, which is well supported by bootstrap resampling, neither Neotaenioglossa (partim
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Discopoda) nor Muricacea (represented by Buccinidae, Fasciolariidae, Muricidae, and Nassariidae) form monophyletic groups. There is 100% bootstrap support for the monophyly of Euthyneura, which form two unsupported clades consisting of (1) the three stylommatophorans, Helix aspersa, Ba. biplicata, and Oxyloma sp., and (2) the five remaining euthyneurans. Surprisingly, the achatinid Limicolaria kambeul belongs to this latter clade instead of to the first. Both systellommatophorans have a common origin, but this result was not supported by bootstrap analysis. A tree based on a distance matrix calculated using the equation of Van de Peer et al. (1996b) showed small topological shifts of nodes with low bootstrap values (indicated by dots in Fig. 1A). Onchidella celtica becomes a sister group to a Siphonaria–Limicolaria– Laevicaulis–Aplysia cluster.
FIG. 1. (A) NJ tree computed on the basis of Jukes and Cantor (1969) distances calculated from an alignment of 18 gastropod 18S rRNA sequences. The bivalve Galeomma takii was used as an outgroup. Numbers at a node indicate percentage bootstrap values higher than 70%. Dots mark nodes that change when distances are computed using the equation of Van de Peer et al. (1996b). (B) Majority rule consensus tree of the NJ trees obtained when replacing G. takii 20 times by another mollusc (Table 3). The following shortened taxon names are used: Bas., Basommatophora; Styl., Stylommatophora; Syst., Systellommatophora; Ng., Neogastropoda; Nt., Neotaenioglossa; Vet., Vetigastropoda; and Ner., Neritimorpha.
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TABLE 3 Species Used as Outgroups in Fig. 1B, and the Molluscan Class to Which They Belong Species
Class
Argopecten irradians Barbatia virescens Cardita canaliculata Barbatia virescens Chlamys islandica Corbula crassa Crassostrea virginica Galeomma takii Glycymeris glycymeris Mactromeris polynyma Mulinia lateralis Mytilus edulis Placopecten magellanicus Pholas dactylus Spisula solida Tresus capax Tridacna sp. Scutopus ventrolineatus Acanthopleura japonica Lepidochitona sp. Antalis vulgaris
Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Bivalvia Caudofoveata Polyplacophora Polyplacophora Scaphopoda
Replacing the outgroup G. takii by each of 20 other molluscs (Table 3), using the computer program TREECON, yielded the majority rule consensus tree shown in Fig. 1B. It supports the previous results (Fig. 1A), except that the majority of outgroups does not support systellomatophoran monophyly. The stability of the NJ tree (Fig. 1A) was further assessed by excluding the ingroup species one by one. Exclusion of any of the euthyneuran taxa caused changes in the
poorly supported nodes of their cluster (not shown). Removing L. obtusata from the caenogastropod cluster also caused a shift in the euthyneuran cluster. A heuristic search on the 266 parsimony informative sites in the alignment of the 18 gastropods, with the bivalve G. takii as outgroup, yielded a single MP tree of 738 steps, which is shown in Fig. 2. Changing input orders 100 times had no influence on this result. The MP tree differs from the NJ tree (Fig. 1A) only in branching points with low bootstrap values, so that the conclusions of Fig. 1A are confirmed. Prosobranchia and Archaeogastropoda are both paraphyletic groups. The paraphyly of the Prosobranchia is strongly supported by bootstrap sampling, but that of the Archaeogastropoda is not. Within the Caenogastropoda, neither Neotaenioglossa (partim Discopoda) nor Muricacea are monophyletic groups. Yet, Muricacea polyphyly is not significantly supported. In the euthyneuran clade, none of the nodes receives high bootstrap values. When G. takii was replaced by another molluscan outgroup 20 times (Table 3) or when an ingroup taxon was removed, nodes with high bootstrap and decay indices were not affected. The poorly supported nodes within the caenogastropod cluster showed no ingroup effect either, and only a slight outgroup effect. On the contrary, in the euthyneuran cluster, branching points strongly depended on the outgroup choice (Fig. 2) and the ingroup taxa included. Removing Aplysia sp., Oxyloma sp., Lim. kambeul, L. obtusata, or Siphonaria pectinata caused either a collapse or a shift of the topology of the euthyneuran clade (not shown). As the use of too-distant outgroups may cause an increase of homoplasy between in- and outgroup species, we focused on the caenogastropods and euthy-
FIG. 2. MP tree found on the basis of the 266 informative sites of an alignment of 18 gastropod 18S rRNA sequences with Galeomma takii as outgroup. Bootstrap values higher than 70% are indicated above the nodes. Figures below branching points indicate how many additional steps were necessary for this clade not to be unequivocally supported (decay index); those at the right of a branching point indicate how many of 20 different outgroups (Table 3) supported this branching point. Abbreviations of taxon names as in Fig. 1.
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neurans separately. Figure 3A shows the results of the Euthyneura NJ tree, with the caenogastropod Littorina littorea as outgroup. In comparison to the complete tree (Fig. 1A), this yielded small topological shifts. In the tree of Fig. 3A, the polyphyly of the Sigmurethra and the Systellommatophora appears to be strongly supported by bootstrap resampling. Figure 3B shows the results of the NJ analysis of the caenogastropods, with the pulmonate S. pectinata as outgroup. The tree confirms the polyphyly of the Neotaenioglossa (partim Discopoda) and Muricacea. It shows that the Fasciolaria–Nassarius–Crepidula cluster and the monophyly of the genus Littorina are well supported by bootstrap analysis. An exhaustive search on the 83 phylogenetically informative sites of the alignment of the euthyneuran
sequences yielded three MP trees of 211 steps. Their strict consensus topology, which is shown in Fig. 4A, has only three nodes in common with the equivalent NJ tree (Fig. 3A), but does not contradict the MP tree based on all sequences (Fig. 2). An exhaustive search on the 62 phylogenetically informative sites of the caenogastropod sequences yielded two MP trees of 142 steps. In the strict consensus tree, which is shown in Fig. 4B, only the monophyly of the genus Littorina is significantly supported by bootstrap resampling. DISCUSSION The present 18S rRNA analyses confirm the hypothesis that the archaeogastropods, namely Neritimorpha and Vetigastropoda, have a basal position within the
FIG. 3. NJ tree of the Jukes and Cantor (1969) distances between (A) eight euthyneuran 18S rRNA sequences with the prosobranch Littorina littorea as outgroup and (B) eight caenogastropod 18S rRNA sequences with the pulmonate Siphonaria pectinata as outgroup. Only bootstrap values higher than 70% are indicated. Abbreviations of taxon names as in Fig. 1.
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FIG. 4. Strict consensus tree (A) of the three MP trees (L 5 211) found on the basis of the 83 informative sites of an alignment of eight euthyneuran 18S rRNA sequences with the prosobranch Littorina littorea as outgroup and (B) of the two MP trees (L 5 142) found on the basis of the 62 informative sites of an alignment of eight caenogastropod 18S rRNA sequences with the pulmonate Siphonaria pectinata as outgroup. Only bootstrap values higher than 70% are indicated. Dots indicate nodes in common with the NJ trees of Fig. 3. Abbreviations of taxon names as in Fig. 1.
Gastropoda. On the basis of, for example, the reduction of the right ctenidium, incorporation of the right kidney in the genital duct, and the mode of fertilization, Neritimorpha were considered to be closely related to the Caenogastropoda (e.g., Naef, 1911). Yet, the lack of skeletal rods in the ctenidium, the presence of juvenile coiling, and the deep mantle cavity suggest that Neritimorpha are an early, highly specialized archaeogastropod offshoot (e.g., von Salvini-Plawen and Haszprunar, 1987; Haszprunar, 1988a,b; Hickman, 1988; Healy,
1988). The similarities between Neritimorpha and Caenogastropoda were therefore ascribed to convergence. The present 18S rRNA data also refute a close relationship between Neritimorpha and Caenogastropoda and consistently suggest that Archaeogastropoda, including both Neritimorpha and Vetigastropoda, is a basal grade within the Gastropoda (e.g., Hickman, 1988; Haszprunar, 1993; von Salvini-Plawen and Steiner, 1996; Ponder and Lindberg, 1997). Our result is also congruent with the results of a study using about 150 nucleotides
GASTROPOD PHYLOGENY
of the 28S rRNA (Rosenberg et al., 1994) and the paper by Harasewych et al. (1997) on the basis of 450 nucleotides of the 18S rRNA. The early divergence of the Vetigastropoda is supported by several gastropod plesiomorphies such as two excretory organs, fringed head tentacles, and the eye type (e.g., Haszprunar, 1988b). The 18S rRNA data suggest the monophyly of the Apogastropoda (5Caenogastropoda and Euthyneura) and consequently prosobranch paraphyly. This result is in accordance with findings on the basis of 28S rRNA (Emberton et al., 1990; Tillier et al., 1992, 1994; Rosenberg et al., 1994) and partial 18S rRNA (Harasewych et al., 1997). Previously, Caenogastropoda and Euthyneura had already been synapomorphically linked by the way in which the pleural ganglia make contact with the pedal ganglia (e.g., von Salvini-Plawen and Haszprunar, 1987; Haszprunar, 1988b; Bieler, 1992; Ponder and Lindberg, 1997). Caenogastropod monophyly was accepted by most authors on the basis of the special type of osphradium (e.g., von Salvini-Plawen, 1980; Haszprunar, 1985b, 1988b), as well as by sequence comparisons of 28S rRNA (Tillier et al., 1992, 1994; Emberton et al., 1990), but was questioned by Rosenberg et al. (1994). The value of the osphradium as an indication of caenogastropod synapomorphy is supported by our 18S rRNA analyses, which consistently suggest a single origin for the caenogastropods. Consequently, our 18S rRNA data reject the hypothesis that Neogastropoda originated from the Archaeogastropoda (Ponder, 1973), but support the view that neogastropods are derived from neotaenioglossans (5mesogastropods) (Haszprunar, 1988a,b; Healy, 1988; Taylor and Morris, 1988). An extensive discussion of the anatomical features supporting the two contradictory hypotheses was given by Taylor and Morris (1988). As our analyses included only one of the three neogastropod superfamilies (Ponder, 1973), namely the Muricacea, not much can be concluded with respect to neogastropod monophyly, which was accepted on the basis of several morphological features (e.g., Ponder, 1973; Taylor and Morris, 1988; Kantor, 1996; Ponder and Lindberg, 1997), chromosome numbers (Patterson and Burch, 1978), DNA content (Hinegardner, 1974), and the 28S rRNA analyses of Rosenberg et al. (1994). However, the present 18S rRNA analyses consistently suggest that at least Muricacea and Neotaenioglossa (partim Discopoda) are not monophyletic. A lack of support for the monophyly of the neogastropods, also represented by the Muricacea, was also reported by Tillier et al. (1992, 1994) with 28S rRNA data. The close relationship between the neogastropod families Fasciolariidae (F. lignaria) and Nassariidae (N. singuinjorensis), as suggested by Ponder (1973), is confirmed by the 18S rRNA data. Yet, although Fasciolariidae, Nassariidae, and Buccinidae are sometimes considered one unit (e.g., Ponder, 1973; Ponder and Ware´n, 1988), in
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our trees the families Fasciolariidae and Nassariidae do not show a close relationship to the Buccinidae (P. striata), but have a common origin with the neotaenioglossan family Calyptraeidae (C. adunca). In accordance with the 28S rRNA analyses of Tillier et al. (1992, 1994), Littorinidae forms the most basal branch in the caenogastropod cluster. The present 18S rRNA study supports the monophyly of Euthyneura, as generally accepted on the basis of the existence of parietal ganglia, the development of a head shield with special sense organs, a pallial caecum, and the type of the gills, circulatory system, and genital system (e.g., Haszprunar, 1985a, 1988a). Mainly because of the presence of a ‘‘lung,’’ pulmonate monophyly was broadly accepted (e.g., von SalviniPlawen, 1970; Tillier, 1984; Haszprunar, 1985a). Yet the distinction between pulmonates and opisthobranchs was previously questioned by Boettger (1955), who suggested that Opisthobranchia were paraphyletic (see Haszprunar, 1988b for a review). Recently, Ponder and Lindberg (1997) also found support neither for opisthobranch nor for pulmonate monophyly. Our 18S rRNA data fail to reveal pulmonate monophyly. However, due to unstable results, it cannot be concluded that the Pulmonata are para- or polyphyletic, either. The 28S rRNA analyses of Tillier et al. (1992, 1994) and the partial 18S rRNA study of Harasewych et al. (1997) yielded the same ambiguous result on this issue. Their data also strongly supported the Pulmonata–Aplysia monophyly, but there was no stability in the clade. More recently, 28S rRNA data suggested that Aplysia is the sister group of the pulmonates excluding Amphibolidae (Tillier et al., 1996). Emberton et al. (1990) and Rosenberg et al. (1994) reported that on the basis of 28S rRNA data, Pulmonata were monophyletic. Yet, since their study did not include any opisthobranch sequence, this hypothesis was premature. Due to the instability of the branching pattern within the pulmonate-opisthobranch clade (Figs. 1, 2, 3A, and 4A), we cannot decide on the status and relationships of the Stylommatophora and Systellommatophora. Most of our analyses suggest that Systellommatophora are not monophyletic and that Onchidiidae is the most basal branch in the Euthyneura. This result is supported by the congruence of both MP and NJ trees (Figs. 1B, 2, 3A, and 4A) and the moderate bootstrap support in the NJ tree (Fig. 3A). In addition, Climo (1980) interpreted the systellommatophorans as a polyphyletic assemblage at the base of the euthyneurans. This hypothesis implies that the morphological similarities between the Veronicellidae and the Onchidiidae (e.g., Tillier, 1984) are convergent features. Finally, the present study does not support a relationship of the Succineidae (represented by Oxyloma sp.) to the Opisthobranchia, as suggested on the basis of comparisons of the alimentary and reproductive system (Rigby, 1965). Indeed, all 18S rRNA analyses support a relationship between Helix,
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Balea, and Oxyloma, but not between Oxyloma and the opisthobranch Aplysia. The unstable results within the Euthyneura may be due (1) to the explosive way in which they arose (Climo, 1980) and/or (2) to the limited number of representatives available for 18S rRNA analysis. In conclusion, although the present 18S rRNA data provide new support for some well-established gastropod relationships, they are insufficient to unambiguously resolve some of the current debates. Whether this is due to a taxon sampling problem or to the molecule remains to be investigated. ACKNOWLEDGMENTS We are indebted to Dr. B. Morton (Hong Kong), Dr. P. Schembri (Malta), and Dr. H. Reise (Germany) for helping us with the collection of parts of the material. Two anonymous referees provided helpful and constructive comments. B.W. has held an IWT scholarship. This work was funded by grants from the National Fund for Scientific Research.
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