* Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain
Departamento de Biología, Facultad de Ciencias del Mar, Universidad de Cádiz, Cádiz, Spain
Correspondence: E-mail: cristina{at}mncn.csic.es.
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Abstract |
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Key Words: Opisthobranchia Pulmonata indels gene arrangement mtDNA
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Introduction |
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In cases where phylogenetic analyses based on sequence substitutions render conflicting results, rare genomic changes (RGCs) may be very helpful in discerning among alternative phylogenetic hypotheses (see Rokas and Holland [2000] for a review). Insertion/deletion (indel) events within well-conserved regions of the genome and gene rearrangements are two examples of RGCs that have been successfully used to resolve deep phylogenetic relationships (Rivera and Lake 1992; Macey et al. 1997; Boore and Brown 1998; Keeling and Palmer 2000; Venkatesh, Erdmann, and Brenner 2001).
The potential of RGCs for phylogenetic reconstruction has been tested in gastropods, a group of mollusks known to have high sequence substitution rates (Thomaz, Guiller, and Clarke 1996; Davison 2002). For instance, the absence or reduction of entire stem/loop structures in several domains of the secondary structure of the mitochondrial large subunit rRNA has been shown to be a molecular synapomorphy of derived gastropods (Lydeard et al. 2000, 2002b). Moreover, mitochondrial DNA gene rearrangements have been useful in identifying phylogenetic affinities among several gastropod groups at different taxonomic levels (Kurabayashi and Ueshima 2000b; Rawlings, Collins, and Bieler 2001; Lydeard et al. 2002a).
Gastropods were traditionally classified into three main subclasses, Prosobranchia, Pulmonata, and Opisthobranchia (Thiele 1931). However, recent morphological studies rejected the monophyly of Prosobranchia (Haszprunar 1988) and failed to recover the monophyly of Opisthobranchia (Salvini-Plawen and Steiner 1996; Ponder and Lindberg 1997; Dayrat and Tillier 2002; fig. 1A). According to new morphological data, the monophyletic Pulmonata (Salvini-Plawen 1970; Tillier 1984; Haszprunar 1985; Haszprunar and Huber 1990; Nordsieck 1992; Dayrat and Tillier 2002) and the paraphyletic Opisthobranchia (Boettger 1955; Haszprunar 1988; Salvini-Plawen and Steiner 1996; Ponder and Lindberg 1997) are clearly distinct from the remaining gastropods and are grouped together in the clade Euthyneura Spengel 1881. The monophyly of Euthyneura is generally accepted and is supported by several morphological synapomorphies (Gosliner 1981; Haszprunar 1988; Salvini-Plawen and Steiner 1996; but see Dayrat and Tillier 2002). Euthyneura together with the paraphyletic group Heterostropha (pyramidelids and other related groups) define the clade Heterobranchia (Haszprunar 1985).
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Most phylogenetic analyses based on nuclear 18S and 28S rRNA gene sequences rejected the monophyly of both Opisthobranchia and Pulmonata (Tillier and Ponder 1992; Rosenberg et al. 1994; Tillier et al. 1994; Tillier, Masselot, and Tillier 1996; Winnepenninckx et al. 1998; Wollscheid and Wägele 1999; Yoon and Kim 2000; Dayrat et al. 2001; Wollscheid et al. 2001). However, these analyses rendered conflicting results, and their conclusions lacked sufficient support because of the low resolution of nuclear rRNA genes at this taxonomic level. In contrast, a recent study (Wade and Mordan 2000) based on a nuclear fragment including partial 5.8S rDNA, complete ITS-2, and partial large subunit rDNA sequences supported the monophyly of the above-mentioned gastropod groups. Thollesson (1999) proposed a phylogenetic hypothesis for Euthyneura based on a small fragment (480 bp) of the mitochondrial rrnL gene and rejected the monophyly of opisthobranchs (fig. 1B). In contrast, Grande et al. (2002) tentatively concluded that opisthobranchs are monophyletic based on the phylogenetic analyses of the mitochondrial cox1, rrnL, nad6, and nad5 genes from several species representing five different orders of opisthobranchs. These conflicting results may be clarified and resolved if more representatives of each gastropod group are included in the phylogenetic analyses, if large sequence data sets are compiled, and if the potential phylogenetic utility of RGCs is explored, as suggested by Grande et al. (2002).
To further understand phylogenetic relationships within derived gastropods, we have determined partial sequences of mitochondrial cox1 and nad5 genes and the complete sequences of mitochondrial trnV, rrnL, trnL(cun), trnA, trnP, and nad6 genes for representative species of the main orders of Pulmonata and Opisthobranchia, as well as the heterostrophan Pyramidella dolabrata. The new sequences were analyzed with current methods of phylogenetic inference and screened for RGCs.
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Materials and Methods |
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Molecular and Phylogenetic Analyses
Gene boundaries were determined by comparison with other gastropod mitochondrial genomes. Protein-coding genes were delimited by their start and stop codons. Deduced tRNAs were folded into their corresponding cloverleaf secondary structure.
Nucleotide (for the mitochondrial rrnL gene) and deduced amino-acid sequences (for the mitochondrial cox1, nad6, and nad5 genes) were aligned using ClustalX version 1.62b (Thompson et al. 1997) followed by refinement by eye. Ambiguous alignments and gaps were excluded from the analysis using Gblocks 0.73b (Castresana 2000). Modeltest version 3.06 (Posada and Crandall 1998) was used to estimate the evolutionary model that best fit the nucleotide (mitochondrial rrnL gene) data set. The Akaike information criteria (AIC) implemented in Modeltest selected the GTR + I + (Rodriguez et al. 1990) evolutionary model.
Bayesian inferences (BI) of gastropod phylogeny were performed with MrBayes 3.0b3 (Huelsenbeck and Ronquist 2001) by Metropolis coupled Markov chain Monte Carlo (MCMCMC) sampling for 1 million generations (four simultaneous MC chains; sample frequency 100; burn-in 1,000 generations). Bayesian analyses were run independently at least twice, beginning with different starting trees (Huelsenbeck and Bollback 2001). We used GTR + I + (for the rrnL nucleotide sequence data) and mtREV (Adachi and Hasegawa 1996) (for the cox1, nad6, and nad5 amino-acid sequence data) as evolutionary models. Support for tree nodes was determined based on the values of Bayesian posterior probability (BPP) obtained from a majority-rule consensus tree with MrBayes 3.0b3.
To further confirm the gastropod trees reconstructed by the Bayesian method of inference, the deduced amino-acid sequences of mitochondrial protein-coding genes (cox1, nad6, and nad5) were combined into a single data set that was subjected to maximum-parsimony (MP) and minimum evolution (ME) methods of phylogenetic inference. The MP analyses were performed with PAUP* 4.0b10 (Swofford 2002) using heuristic searches (TBR branch swapping; MulTrees option in effect) with 10 random additions of taxa. The ME analyses (Rzhetsky and Nei 1992) were carried out with PAUP* 4.0b10 using mean character distances. Robustness of MP and ME analyses was tested by bootstrapping with 1,000 pseudoreplicates each (Felsenstein 1985).
The sequences reported in this article have been deposited in the GenBank database (accession nos. AY345014AY345055).
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Results |
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Both the MP and the ME phylogenetic inferences based on a 15-taxon data set that combined the deduced amino acid sequences of mitochondrial cox1, nad6, and nad5 genes and with Loligo as outgroup recovered congruent trees that also supported the Opisthobranchia + S. pectinata clade (97% and 100% bootstrap values, respectively) as well as the basal position of O. celtica and P. dolabrata (83% and 98% bootstrap values, respectively) with respect to Opisthobranchia + S. pectinata. The same two nodes were also supported with high bootstrap values when the deduced amino acid sequences of mitochondrial cox1 and nad6 genes and the same ingroup taxa were analyzed, with Littorina as outgroup (not shown).
The new gastropod phylogeny was further confirmed by a Bayesian inference based on an extended 47-taxon sequence data set. The majority-rule consensus tree resulting from the Bayesian inference under the GTR + I + (rrnL gene) and mtREV (cox1 gene) substitution models is presented in figure 4. The recovered phylogeny was in agreement with that based on the 15-taxon data set. Euthyneura are not monophyletic because of the relative position of the heterostrophan P. dolabrata. Pulmonata is polyphyletic with basommatophoran, systelommatophoran, and stylommatophoran lineages recovered in different positions of the tree. The basommatophoran S. pectinata was included within the Opisthobranchia with high statistical support (100% BPP). The systelommatophoran pulmonate O. celtica together with P. dolabrata were placed as the sister group of the Opisthobranchia + S. pectinata clade (90% BPP). Stylommatophoran pulmonates are monophyletic (100% BPP), and together with the marine ellobioideid pulmonate Myosotella myosotis, were placed as the most basal of the analyzed gastropod lineages. Within Opisthobranchia, the order Sacoglossa was recovered as the most basal group (fig. 4). The orders Anaspidea, Tylodinoidea, and Cephalaspidea formed a well-supported clade (100% BPP). The validity of the order Architectibranchia as an independent opisthobranch lineage was confirmed by our analysis. The order Pleurobranchoidea was located within Nudibranchia, rendering the latter paraphyletic (opisthobranch intrarelationships will be further analyzed elsewhere).
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An Indel in the Deduced Amino Acid Sequence of the cox1 Gene
The deduced amino acid sequences of the cox1, nad6, and nad5 genes of the same 14 species of derived gastropods were screened for indels. A single informative amino-acid indel was found at position 46 of the mitochondrial Cox1 protein (numbered according to the Roboastra europaea Cox1 amino acid sequence). The indel in the Cox1 protein was further analyzed in a larger data set that included 47 gastropod species. All analyzed opisthobranchs, as well as the pulmonate Siphonaria pectinata, had a Glycine at that position (fig. 5). The remaining pulmonates and the heterostrophan Pyramidella dolabrata had a deletion (fig. 5). The caenogastropodan Littorina saxatilis had a Glycine at that position, and the squid Loligo bleekeri had an Asparragine at that position. We searched GenBank for additional gastropod cox1 gene sequences and found that Caenogastropoda and related groups (formerly included in Prosobranchia), as well as the heterostrophan Cornirostra pellucida, shared a Glycine at position 46 of the mitochondrial Cox1 protein. In contrast, all mitochondrial Cox1 amino acid sequences of Pulmonata found in GenBank (except Siphonaria zelandica) had a deletion at the above-mentioned position (fig. 5).
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Discussion |
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The placement of the systelommatophoran Onchidella in a basal position relative to opisthobranchs independently rejected the monophyly of pulmonates (figs. 3 and 4). Different morphologists have considered systelommatophorans as either opisthobranchs (Boettger 1955), pulmonates (Haszprunar 1988; Haszprunar and Huber 1990; Tillier and Ponder 1992) or even as an independent group closely related to opisthobranchs (Salvini-Plawen 1970).
The heterostrophan Pyramidella was recovered within the Euthyneura rendering this clade paraphyletic (figs. 3 and 4). In fact, pyramidellids have been considered by many authors to be opisthobranchs (Thorson 1946; Fretter and Graham 1949; Thompson 1973) because both groups share a rhinophoral nerve, a sinistral larval shell produced by the planktotrophic veliger (heterostrophy), simultaneous hermaphroditism, and the absence of a pectinibranch gill (ctenidium), among other characters. However, some authors criticized the validity of these characters as true synapomorphies (Gosliner 1981; Robertson 1985; Haszprunar 1988).
Interestingly, we found two mitochondrial RGCs, the relative position of the trnP gene and an indel in the Cox1 protein, that can be used as a valuable independent source to confirm and strengthen phylogenetic relationships within Heterobranchia (Euthyneura and heterostrophans) recovered from primary sequence data. The mitochondrial gene order is highly conserved in Heterobranchia with few tRNA gene translocations (Kurabayashi and Ueshima 2000b; Grande et al. 2002). Hence, the distinct relative position of the trnP gene in different taxa (fig. 3) seems a very promising phylogenetic marker. According to our results, the mitochondrial gene order rrnL, trnL(cun), trnA, trnP, nad6, and nad5 is associated with the Opisthobranchia + Onchidella + Pyramidella clade, and it might represent a molecular synapomorphy of these taxa. However, it is also likely that this gene order may be the ancestral state of Heterobranchia. The relative position of the mitochondrial trnP gene would need to be determined in more heterostrophans and pulmonates to discern between these two competing hypotheses. According to our results, a Glycine in position 46 of the Cox1 protein was present in Caenogastropoda and related basal gastropods (formerly included in Prosobranchia) and was further deleted in the ancestor of Euthyneura + Pyramidella. A reversal (or a convergence) due to structural constraints to the ancestral condition in gastropods (i.e., presence of Glycine) may have occurred in the ancestor of Opisthobranchia + Siphonaria. Alternatively, although less parsimonious, several independent deletions of the Glycine in different lineages of pulmonates (except Siphonaria) and the heterostrophan Pyramidella may also explain the pattern found with equal likelihood.
The phylogenetic hypothesis presented here corroborates the close relationships among all lineages of opisthobranchs (their monophyly is only rejected because of the relative position of Siphonaria), as previously suggested (Thiele 1931; Grande et al. 2002); yet it strongly rejects the validity of pulmonates as a natural group (against most morphological studies; e.g., Haszprunar and Huber [1990]; Dayrat and Tillier [2002]). These results stress the need of a thorough re-evaluation of the morphological characters that were used to define the monophyly of pulmonates, and they support the independent and recurrent evolution of the lung as the respiratory surface in gastropods. The recovered phylogeny provides a robust phylogenetic framework for many comparative studies involving this group and may allow a better understanding of evolutionary trends within gastropods.
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Acknowledgements |
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Footnotes |
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