:110LECliLAR PHYLOGENETICS AND EVOLUTION Vol. 9, ~o. 1, February, pp. 55--63, 1998 ART! CL E NO FY970439 Details of Gastropod Phylogeny lnferred from 18S rRNA Sequences Birgitta Winnepenninckx, *· 1 Gerhard Steiner, t Thierry Backeljau,+ and Rupert De Wachter* *Departement Biochemie, Universiteit Antwerpen (UIA), Universiteitsplein 1, B-2610 Antwerpen, Belgium; tlnstitute of Zoology, University of Vienna, Althanstrasse 14, A-1 090 Vienna, Austria; and :f:Royal Belgian lnstitute of Natura! Sciences, Vautierstraat 29, B-1 000 Brussel, Belgium Received February 4, 1997; revised June 6, 1997 molecular data (e.g., Tillier et al., 1992, 1994, 1996; Some generally accepted viewpoints on the phyloge­ Rosenberg et al., 1994; Winnepenninckx et al., 1996). A netic relationships within the molluscan class Gas­ recent 188 rRNA analysis of molluseau relationships tropoda are reassessed by comparing complete 18S suggested that this molecule might be suitable to rRNA sequences. Phylogenetic analyses were per­ resolve phylogenetic problems at infraclass levels (Win­ formed using the neighbor-joining and maximum par­ nepenninckx et al., 1996). In the present paper we simony methods. The previously suggested basal posi­ further explore this issue by analyzing a number of tion of Archaeogastropoda, including Neritimorpha generally accepted ideas on the infraclass phylogeny of and Vetigastropoda, in the gastropod clade is con­ Gastropoda using 11 new and 7 publisbed (Winnepen­ firmed. The present study also provides new molecular ninckx et al., 1992, 1994, 1996) complete gastropod 188 evidence for the monophyly of both Caenogastropoda rRNA sequences. The points dealtwithare the position and Euthyneura (Pulmonata and Opisthobranchia), and suggested paraphyly ofProsobranchia and Archaeo­ making Prosobranchia paraphyletic. The relation­ ships within Caenogastropoda and Euthyneura data gastropoda, as well as the monophyly of taxa such as turn out to he very unstable on the basis of the present Caenogastropoda, Neotaenioglossa, Muricacea, Euthy­ 18S rRNA sequences. The present 18S rRNA data ques­ neura, Pulmonata, and Stylommatophora. In this con­ tion, but are insufficient to decide on, muricacean text, particular attention willbe paid to the monophyly (Neogastropoda), neotaenioglossan, pulmonate, or sty­ and position ofthe Systellommatophora, a group which lommatophoran monophyly. The analyses also focus includes the families Veronicellidae, Onchidiidae, and on two systellommatophoran families, namely, Veroni­ Rathousiidae, and adcording to some authors also the cellidae and Onchidiidae. It is suggested that Systellom­ Rhodopidae (von Salvini-Plawen, 1970) and the matophora are not a monophyletic unit but, due to the Smeagolidae (Climo, 1980; Tillier and Ponder, 1992). lack of stability in the euthyneuran clade, their affinity Systellommatophora are considered to be either pulmo­ to either Opisthobranchia or Pulmonata could not he nates (e.g., Van Mol, 1974; Solem, 1979; Tillier, 1984; determined. r 1998 Academie Pre ss Haszprunar, 1988b; Haszprunar and Huber, 1990; Tillier and Ponder, 1992) or opisthobranchs (e.g., Boett­ ger, 1955), although von Salvini-Plawen (1970) consid­ INTRODUCTION ered them as a proper subclass, the Gymnomorpha, related to the opisthobranchs. Their status as a sepa­ Gastropoda is the largest molluseau class and in­ rate group was confirmed by von Salvini-Plawen and cludes the common terrestrial, freshwater, and marine Steiner (1996), who related them to the pulmonates. snails and slugs. It has an excellent fossil record going However, systellommatophoran monophyly (e.g., von back to 550 MYA (Runnegar and Pojeta, 1985). The Salvini-Plawen, 1970) is still debated (e.g., Climo, das~ is traditionally divided into three subclasses: 1980; Tillier, 1984; Haszprunar and_Huber,,,. 1990). Prosobranchia (Streptoneura), Opisthobranchia, and Pulmonata (together the latter constitute the Euthy­ MATERIALS AND METHODS neura). The phylogenetic relationships between and within these subclasses are longstanding problems Amplification and Sequencing ofthe 188 rRNA Genes (e.g., Ponder and Lindberg, 1996, 1997; von Salvini­ The taxonomy of the gastropod species used in this Plawen and Steiner, 1996; fora review of earlier work study is given in Table 1. Sampling locations are listed see Bieler, 1992), which are increasingly stuclied with in Table 2. The species were frozen alive. After dissec­ tion, DNA was extracted (Winnepenninckx et al., 1993) ' Present address: Royal Belgian Institute for Natura! Sciences, from the tissues indicated in Table 2. The 188 rRNA Vautierstraat 29, B-1000 Brussel, Belgium. genes were PCR-amplified, cloned, and sequenced as 55 1055-7903/98 $25.00 Copyright © 1998 by Academie Press All rights of reproduetion in any form reserved. 56 WINNEPENNINCKX ET AL. TABLE 1 Taxonomy of the Gastropod Species Used in This Study -------- Subclass Superorder Order Suborder Family Genus ----------- Prosobranchia" Archaeogastropoda a Vetigastropoda Trochidae Monodontab Neritimorpha Neritidae Nerita Caenogastropoda N eogastropoda Buccinidae Pisaniab N assariidae Nassariusb Fasciolariidae Fasciolaria6 Muricidae Thais Neotaenioglossa Discopoda Littorinidae Littorinab Bursidae Bursab Calyptraeidae Crepidula6 Pulmonaü, Stylommatophora :VIes urethra Cl a usiliidae Baleab Sigmurethra Holapodopes Achatinidae Limicolaria Aulocopoda Succineidae Oxylomab Holopoda Helicidae Helix Basommatophora Siphonariidae Sipkonaria Systellommatorphora Onchidiacea Onchidiidae Onchidella Soleolifera Veronicellidae Laevicaulisb Opisthobranchia Anaspidea Aplysidae Aplysiab 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 Warén (1988). The classification of the Neogastropoda is based on Ponder (1973); Pulmorrata are classified according to Solem I 1979), except for the placement of Siphonaria, which follows von Salvini-Plawen (1970). a Currently considered nonmonophyletic. 6 Sequence determined in this study. described by Winnepenninckx et al. (1995), using the structure similarity. If necessary, manual adjustments primers published in Winnepenninckx et al. (1994) and were made with the same program. The secondary two M13 universa! primers. structure model of Van de Peer et al. (1996a) was used. Data Analysis Sequence regions corresponding to the amplification primer at the 5' end ofthe gene were removed prior to The new gastropod 188 rRNA sequences were added reconstruction of phylogenetic trees. The 188 rRNA to the alignment of Van de Peer et al. (1996a) using the sequences were analyzed using neighbor-joining (NJ) computer program DC8E (De Rijk and De Wachter, and maximum parsimony (MP) methods. The program 1993). which considers primary as well as secondary TREECON (Van de Peer and De Wachter, 1993) was used to construct NJ trees based on the formulas of TABLE 2 Jukes and Cantor (1969), Kimura (1980), or Van de Peer et al. (1996b). Gaps were nottaken into account. Sourees and Tissue Type of Gastropod Species Used Tree stability was assessed via bootstrapping over 1000 for This Study replicates. MP trees were constructed on the phyloge­ ----- - ----·----------=-----~~---~--~ netically informative sites using either the heuristic or Species Sampling location the exhaustive search option ofPAUP (8wofford, 1993). A.plysia ,;p. HongKong Oigestive gland Stability of MP trees was assessed via bootstrapping Balea bipli,·atu :Vlortsell Belgium 1 Complete organism over 1000 replicates and the calculation of decay indi­ Bursa ronu HongKong Bucal mass + foot ces (Bremer, 1988; Donoghue et al., 1992). As until now muscle there is no consensus about the minimal bootstrap Crepidula adunca Vaneauver <Canada 1 Albumen gland Fasciolaria !i[;naria Bahar Ic-Cagnay Albumen gland value necessary toregard a cluster as firmly supported, (Malta) bootstrap values were arbitrarily considered to reflect Labaratory bred DigestJve gland strong support if they exceeded 70% (Hillis and Bull, ( Görlitz, Germany 1 1993). Littorzna ohtusata Oostersehelde iN ether­ Albumen gland ~ lands) muscle tissue lv!onodonta labio HongKong Digestive -~ reproduc­ tive gland RESULTS Nassarius srnguin­ HongKong Penis + foot musdes jorensis New gastropod 188 rRNA sequences were submitted Oxyloma sp. Görlitz (Germany) Digestive gland to the EMBL sequence data library and have the Pisania stnata Bahar Ic-Cagnaq Digestive +- reproduc­ following accession nos.: Aplysia sp., X94268; Balea (Malta) tive gland biplicata, X94278; Bursa rana, X94269; Crepidula GASTROPOD PHYLOGENY 57 adunca, X94277; Faseiataria lignaria, X94275; Laevi­ Discopoda) nor Muricacea (represented by Buccinidae, cauli:; alte, X94273; Littorina obtusata, X94274; Mo­ Fasciolariidae, Muricidae, and Nassariidae) form mono­ nodonta labio, X94271; Nassarius singuinjorensis, phyletic groups. There is 100% bootstrap support for X94273; Oxyloma sp., X94276; Pisania striata, X94272. the monophyly of Euthyneura, which form two unsup­ Figure lA shows the NJ tree obtained on the basis of ported ciades consisting of(l) the three stylommatopho­ the Jukes and Cantor (1969) distauces of an alignment rans, Helix aspersa, Ba. biplicata, and Oxyloma sp., of complete 188 rRNA sequences of 18 gastropods. The and (2) the five remaining euthyneurans. Surprisingly, bivalve Galeomma takii was arbitrarily chosen as the achatinid Limicolaria kambeul belongs to this outgroup. The same topology was obtained with Kimura latter clade