Investigation of molluscan phylogeny using large-subunit and small-subunit nuclear rRNA sequences

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Abstract

The Mollusca represent one of the most morphologically diverse animal phyla, prompting a variety of hypotheses on relationships between the major lineages within the phylum based upon morphological, developmental, and paleontological data. Analyses of small-ribosomal RNA (SSU rRNA) gene sequence have provided limited resolution of higher-level relationships within the Mollusca. Recent analyses suggest large-subunit (LSU) rRNA gene sequences are useful in resolving deep-level metazoan relationships, particularly when combined with SSU sequence. To this end, LSU (∼ 3.5 kb in length) and SSU (∼ 2 kb) sequences were collected for 33 taxa representing the major lineages within the Mollusca to improve resolution of intraphyletic relationships. Although the LSU and combined LSU + SSU datasets appear to hold potential for resolving branching order within the recognized molluscan classes, low bootstrap support was found for relationships between the major lineages within the Mollusca. LSU + SSU sequences also showed significant levels of rate heterogeneity between molluscan lineages. The Polyplacophora, Gastropoda, and Cephalopoda were each recovered as monophyletic clades with the LSU + SSU dataset. While the Bivalvia were not recovered as monophyletic clade in analyses of the SSU, LSU, or LSU + SSU, the Shimodaira–Hasegawa test showed that likelihood scores for these results did not differ significantly from topologies where the Bivalvia were monophyletic. Analyses of LSU sequences strongly contradict the widely accepted Diasoma hypotheses that bivalves and scaphopods are closely related to one another. The data are consistent with recent morphological and SSU analyses suggesting scaphopods are more closely related to gastropods and cephalopods than to bivalves. The dataset also presents the first published DNA sequences from a neomeniomorph aplacophoran, a group considered critical to our understanding of the origin and early radiation of the Mollusca.

Introduction

Recent phylogenetic research on major metazoan lineages has relied heavily on the nuclear small subunit ribosomal rRNA gene (SSU rRNA or 18S), and prompted reevaluation of traditional theories of animal evolution (e.g. Aguinaldo et al., 1997; Balavoine and Adoutte, 1998; Halanych et al., 1995). Although rate variation between sites within SSU rRNA has made the gene useful for examining relationships between organisms with varying degrees of relatedness, SSU rRNA alone has not been sufficient to resolve some higher-level relationships among metazoans. For example, major relationships within the Mollusca have proven difficult to reconstruct with SSU rRNA gene data (Steiner and Hammer, 2000; Winnepenninckx et al., 1996). Winnepenninckx et al. (1996) suggested two hypotheses to account for this lack of resolution. Rates of evolution within the gene may be inappropriate for the relationships being investigated, because changes accumulated during divergence of the molluscan classes have been subsequently masked by multiple substitutions. Alternatively, the Mollusca may have diversified rapidly, not allowing sufficient changes in SSU to permit accurate reconstruction of major relationships.

Simulations by Halanych (1998) have suggested that in such cases where SSU rRNA alone is inadequate to recover relationships, additional sequence data with similar properties may provide greater signal and thus greater resolving power. The large-subunit (LSU) rRNA gene is linked to the SSU gene in a tandem repeat, having a shared evolutionary history. Several recent studies (Mallatt and Winchell, 2002; Medina et al., 2001; Winchell et al., 2002) have investigated the utility of LSU rRNA gene sequences for higher-level relationships within the Metazoa. Each of these studies has shown combined datasets of LSU and SSU may provide greater resolution of higher-level relationships among metazoans than is achieved by analysis of SSU sequences alone. The present study investigates the ability of a combined LSU + SSU dataset to provide information regarding class level relationships within the Mollusca not available from SSU sequence alone.

The Mollusca represent one of the most diverse metazoan phyla both in terms of species number as well as in range of body plans. The diversity of the phylum is represented by seven or eight extant clades, commonly recognized as “classes.” The Neomeniomorpha and Chaetodermomorpha (often referred to collectively as the Aplacophora), along with the Polyplacophora, are believed to be basally divergent lineages of the Mollusca (Salvini-Plawen and Steiner, 1996; Wingstrand, 1985). Together the three groups are referred to as the Aculifera (Scheltema, 1993). The Conchifera, comprised of the Monoplacophora, Bivalvia, Scaphopoda, Gastropoda, and Cephalopoda, appear to have arisen from a univalved common ancestor (Wingstrand, 1985).

Although the Aculifera are widely agreed to have diverged prior to the diversification of the Conchifera, relationships between the basal molluscs have been variously interpreted. Based upon morphological data, the Chaetodermomorpha (= Caudofoveata) have been described as the earliest diverging lineage within the Mollusca (Salvini-Plawen, 1972, Salvini-Plawen, 1980, Salvini-Plawen, 1985) (Fig. 1A). Cladistic analyses of morphological datasets have evidenced the Neomeniomorpha (= Solenogastres) as the most basal of extant lineages (Haszprunar, 2000; Salvini-Plawen and Steiner, 1996) (Fig. 1B). Under either scenario the Aplacophora and Aculifera are viewed as paraphyletic grades, with the Polyplacophora branching as the sister group to the Conchifera to form the Testaria (Salvini-Plawen, 1972, Salvini-Plawen, 1980). Alternative interpretations of morphological and developmental characters have maintained the monophyly of the Aculifera, with the Neomeniomorpha and Chaetodermomorpha as members of a monophyletic Aplacophora forming the sister group to the Polyplacophora (Ivanov, 1996; Scheltema, 1993, Scheltema, 1996) (Fig. 1C).

The Conchifera has been divided into two major clades, the Diasoma containing the Bivalvia and Scaphopoda, and the Cyrtosoma (sensu lato) including the Monoplacophora, Gastropoda, and Cephalopoda (Fig. 1D). This widely accepted view (e.g. Brusca and Brusca, 1990; Meglitsch and Schram, 1991) is based primarily on paleontological evidence (Runnegar and Pojeta, 1974). The term Cyrtosoma is used herein to refer only to the Gastropoda and Cephalopoda, due to the likely paraphyly of the Monoplacophora (sensu Wingstrand, 1985). The Diasoma hypothesis, based upon inferred common origins of bivalves and scaphopods has come into question. Waller (1998) has proposed close relationship between the Scaphopoda and Cephalopoda based upon inferred developmental commonalities (Fig. 1E). A cladistic analysis by Haszprunar (2000) also contradicts the Diasoma hypothesis, finding the Scaphopoda to be the sister group to the Cyrtosoma (Fig. 1F).

To gain further understanding of molluscan diversification, we have sequenced LSU and SSU genes for all extant major lineages of the Mollusca, except monoplacophorans. Herein we evaluate the phylogenetic signal present in these rRNA genes, and their utility in resolving higher level molluscan relationships.

Section snippets

Taxon sampling

Molluscan taxa were chosen from available material to provide a broad representation of extant lineages based on traditional expectations. Genomic DNA was isolated from 32 mollusk and 1 outgroup taxa (Table 1) using the DNeasy Tissue Kit (Qiagen), with an additional two sequences obtained from GenBank. DNA extractions of molluscan samples were taken from mantle or muscle tissue, with the exception of Chaetoderma sp. and Helicoradomenia sp. where, due to size, whole animals were used. (Due to

Alignment and base composition

Total lengths of the alignments, number of unambiguously aligned characters included in analyses, number of variable characters, and number of parsimony informative characters for the SSU, LSU, and LSU + SSU data are shown in Table 3.

Stationarity of base frequencies is an assumption of parsimony and likelihood based methods of phylogenetic reconstruction (Swofford et al., 1996). Therefore, the relative nucleotide composition of the datasets was evaluated using the “basefreqs” command in PAUP (

Discussion

The LSU + SSU data provided high bootstrap support for some relationships within the major molluscan clades, but showed limited ability to confidently recover relationships between these clades. Recent studies employing LSU + SSU datasets to investigate metazoan phylogenetics (Mallatt and Winchell, 2002; Medina et al., 2001; Winchell et al., 2002), have suggested the utility of LSU, particularly when combined with SSU, in elucidating major events in metazoan diversification. In each of these cases

Acknowledgements

Akiko Okusu kindly provided samples of Cryptoplax japonica, Dentalium octangulatum, Ischnochiton comptus, and Nordotis discus. Janet Voight kindly provided samples of Arboliopsis sp., Benthoctopus yaquinae, Graneledone pacifica, Histioteuthis sp., and Vampyroteuthis infernalis from the collection of the Field Museum of Natural History. Monica Medina provided samples, unpublished primer sequences and LSU sequence for Diaulula, as well as expertise on long PCR techniques. Hilary Morrison provided

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