Complete Sequence of the Mitochondrial DNA of the Primitive Opisthobranch Gastropod Pupa strigosa: Systematic Implication of the Genome Organization

Atsushi Kurabayashi* and Rei UeshimaGo,{dagger}{ddagger}

*Institute of Biological Sciences, University of Tsukuba, Tsukuba, Japan;
{dagger}Department of Biological Science, University of Tokyo, Tokyo, Japan; and
{ddagger}PRESTO, Japan Science and Technology Corporation


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
The complete sequence (14,189 bp) of the mitochondrial DNA of the opisthobranch gastropod Pupa strigosa was determined. The genome contains 13 protein, 2 rRNA, and 22 tRNA genes typical of metazoan mtDNA. The Pupa mitochondrial genome is highly compact and shows the following unusual features, like pulmonate land snails: (1) extremely small genome size, (2) absence of lengthy noncoding regions (with the largest intergenic spacer being only 46 nt), (3) size reduction of encoded genes, and (4) many overlapping genes. Several tRNA genes exhibit bizarre secondary structures with reduced T or D stems, and many tRNA genes have unstable acceptor stems that might be corrected by posttranscriptional RNA editing. The Pupa mitochondrial gene arrangement is almost identical to those of pulmonate land snails but is radically divergent from those of the prosobranch gastropod Littorina saxatilis and other molluscs. Our finding that the unique gene arrangement and highly compact genome organization are shared between opisthobranch and pulmonate gastropods strongly suggests their close phylogenetic affinity.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Metazoan mitochondrial (mt) DNA is a closed circular molecule with an approximate genome size of 16 kbp (Wolstenholme 1992Citation ). This small organelle genome normally encodes genes for 2 ribosomal subunit RNAs (small and large rRNA [srRNA and lrRNA]), 22 tRNAs, and 13 protein subunits (cytochrome c oxidase subunits I–III [COIIII], cytochrome b apoenzyme [Cytb], ATP synthase subunits 6 and 8 [ATPase6 and ATPase8], and NADH dehydrogenase subunits 1–6 and 4L [ND16, 4L]). In addition, metazoan mtDNA usually contains at least one lengthy noncoding sequence which regulates and initiates mtDNA replication and transcription (control region; Wolstenholme 1992Citation ).

In coelomate animals, mitochondrial gene arrangements are generally conserved within each phylum. For example, in vertebrates, all 37 genes are arranged in the same relative order from teleost fishes through amphibians to eutherian mammals (e.g., Anderson et al. 1981Citation ; Roe et al. 1985Citation ; Tzeng et al. 1992Citation ; Boore 1999Citation ). Although minor rearrangements have been reported for marsupials (Pääbo et al. 1991Citation ), birds (Desjardins and Morais 1990Citation ), reptiles (Kumazawa and Nishida 1995Citation ), and a lamprey (Lee and Kocher 1995Citation ), all of these cases involve only a few translocations of several genes and/or the control region. Likewise, gene arrangements are very similar even in the largest metazoan phylum, Arthropoda: mtDNAs of insects, crustaceans, and chelicerates show almost the same gene arrangements except for minor translocations of some tRNA genes and several rearrangements in ticks (Clary and Wolstenholme 1985Citation ; Valverde et al. 1994Citation ; Staton, Daehler, and Brown 1997Citation ; Black and Roehrdanz 1998Citation ; Boore 1999Citation ; Campbell and Barker 1999Citation ).

However, phylum Mollusca is the marked exception to the rule, and the mitochondrial genomic structure is unusually variable. Complete mitochondrial gene organizations have been determined for five molluscan species (representing three different classes): the blue mussel Mytilus edulis, of the class Bivalvia (Hoffmann, Boore, and Brown 1992Citation ); the black chiton Katharina tunicata, of the class Polyplacophora (Boore and Brown 1994aCitation ); and three species of land snails, Albinaria coerulea (Hatzoglou, George, and Lecanidou 1995Citation ), Cepaea nemoralis (Terrett, Miles, and Thomas 1996Citation ), and Euhadra herklotsi (Yamazaki et al. 1997Citation ), of the class Gastropoda. Mitochondrial gene arrangements of these three molluscan classes are highly divergent from each other, with only a few gene boundaries shared among them (Yamazaki et al. 1997Citation ). Mitochondrial gene contents, as well as gene arrangements, are also variable within these molluscs. The Mytilus mtDNA lacks the ATPase8, which is normally encoded in the metazoan mitochondrial genome. Furthermore, Mytilus and Katharina mtDNAs contain supernumerary tRNA genes in addition to the standard set of 22 tRNAs. Since mitochondrial gene content has been considered more conservative than the gene arrangements (Wolstenholme 1992Citation ), such deviations from the typical metazoan pattern are surprising.

Recently, Wilding, Mill, and Grahame (1999)Citation determined partial mtDNA sequences of the prosobranch gastropod Littorina saxatilis, and they demonstrated that mitochondrial gene arrangements are highly variable not only among different molluscan classes, but also within the class Gastropoda. The mitochondrial gene arrangements of Littorina are entirely different from those of another gastropod, Cepaea, except for only one gene boundary. Such a radical change in the mitochondrial gene arrangement within a closely related taxon has never been observed in other coelomate phyla.

Gastropods are the largest class of the phylum Mollusca and exhibit the highest diversity in morphology and ecology. However, phylogenetic relationships of gastropods have been controversial, and there are many gastropodan subgroups whose systematic positions are still ambiguous (Salvini-Plawen and Steiner 1996Citation ; Ponder and Lindberg 1996, 1997Citation ). In recent years, mitchondrial gene arrangements have attracted the attention of evolutionary biologists as novel phylogenetic markers (Smith et al. 1993Citation ; Boore and Brown 1994b, 1998Citation ; Boore et al. 1995Citation ; Kumazawa and Nishida 1995Citation ; Boore 1999Citation ; Dowton 1999Citation ). Gastropodan mitochondrial gene arrangements exhibiting high levels of variability are apparently rich in characters and may provide invaluable information for phylogenetic reconstruction. In the traditional classification system, the class Gastropoda has been divided into three subclasses, Prosobranchia, Pulmonata, and Opisthobranchia (Vaught 1989Citation ; Brusca and Brusca 1990Citation ). Although either complete or partial mtDNA sequences have been determined for a prosobranch (Wilding, Mill, and Grahame 1999Citation ) and three pulmonate gastropods (Hatzoglou, George, and Lecanidou 1995Citation ; Terrett, Miles, and Thomas 1996Citation ; Yamazaki et al. 1997Citation ), there has been no available information on mitochondrial genomic organization for the other major subgroup, Opisthobranchia. In the present study, we determined the complete mtDNA sequence of an opisthobranch gastropod, Pupa strigosa, a representative of the superfamily Acteonoidea. Acteonoid snails have been regarded as one of the most primitive extant opisthobranchs (Hyman 1967Citation ; Salvini-Plawen and Steiner 1996Citation ). In this paper, we present the first data on an opisthobranch mtDNA and describe notable features of the genome. Pupa mtDNA shows many unique features that are shared with pulmonate land snails. We will discuss the systematic implications of our findings and the phylogenetic utility of mitochondrial genomic structures.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
An opisthobranch snail P. strigosa, was collected from off Kisami, Shimoda, Shizuoka pref., Japan, at a depth of 30 m by dredging of the sandy bottom. A single adult snail was used for the following experiments.

In the present study, the standard protocols for the DNA experiments were carried out as described in Sambrook, Fritsch, and Maniatis (1989)Citation , with minor modifications. The total genomic DNA was extracted from the whole soft part and then purified with the CTAB method as described by Shahjahan (1995)Citation .

As described below, a total length of Pupa mtDNA was amplified by polymerase chain reaction (PCR). In brief, we first amplified a partial mitochondrial segment and then amplified the remaining mtDNA region. A partial mitochondrial segment containing the Cytb was amplified from Pupa total DNA by PCR using the primers 5'-TGRGGNGCNACNGTNATYACNAAYYT-3' and 5'-RAARTAYCAYTCNGGYTGRATRTG-3'. PCR reactions consisted of 30 cycles of denaturation at 94°C for 30 s, annealing at 40°C for 30 s, and extension at 72°C for 1 min. The amplified PCR product was then cloned into Escherichia coli pUC 19 vector, and the nucleotide sequences were determined from multiple clones as described below. The remaining mtDNA fragment was then amplified from the total DNA by the long PCR method (Cheng et al. 1994Citation ) using the LA PCR kit, version 2 (Takara). Long PCR was carried out with the following primers, designed based on the Cytb sequence determined as above: 5'-GGATCAACAAACCCTTTAGGGGATTTAAATCATGT-3' and 5'-GGAATAGGATATGTGTAGAGCACTAAGTCCTGCAATT-3'. The long PCR reactions consisted of 30 cycles of denaturation at 98°C for 20 s, and both annealing and extension at 68°C for 15 min. The amplified DNA fragment of approximately 14 kbp was then electrophoresed in 1.0% low-melting-point agarose gel (FMC BioProducts), and the DNA was purified from the excised gel using GELase (Epicenter Technologies). The amplified mtDNA fragment was then digested by the restriction enzyme HindIII, and the resultant fragments were cloned into plasmid vectors pUC18 or pUC19. Series of unidirectionally deleted subclones were made of each plasmid clone by using Exonuclease III and mung bean nuclease. Clones and subclones were amplified after transformation into the transformation-competent DH5{alpha} strain of E. coli, and the plasmid DNA was extracted by the alkaline lysis method. DNA sequence determination was performed by the dideoxy chain termination method (Sanger, Nicklen, and Coulson 1977Citation ) using the Ready Reaction kit with ampliTaq DNA polymerase, CS+, (Perkin Elmer) and the automated sequencer ABI-373A or ABI-377 (Perkin Elmer).

Sequence data were analyzed by the GENETIX program package (Software Development). Protein and rRNA genes were assigned by comparison with their counterparts in the fruit fly Drosophila yakuba (Clary and Wolstenholme 1985Citation ), the black chiton K. tunicata (Boore and Brown 1994aCitation ), and the land snail A. coerulea (Hatzoglou, George, and Lecanidou 1995Citation ). The tRNA genes were identified by their potential to form their characteristic secondary structures. Nucleotide and amino acid sequences of the protein genes were aligned with their counterparts of other molluscs by the CLUSTAL W program (Thompson, Higgins, and Gibson 1994Citation ), and the alignment was corrected by visual observation. Phylogenetic trees based on mt-encoded protein gene sequences were performed with the neighbor-joining (NJ) and maximum-likelihood (ML) methods using PHYLIP, version 3.5c (Felsenstein 1996Citation ), and with the maximum-parsimony (MP) method using PAUP, version 3.1. (Swofford 1996Citation ). In this analysis, we employed concatenated nucleotide sequences of COI (partial), COII, ATPase 8, ATPase 6, ND1, ND6, and Cytb (partial), because sequence data on the prosobranch gastropod L. saxatilis are only available for these genes (Wilding, Mill, and Grahame 1999Citation ). Gaps and ambiguous parts of the alignment were excluded for the analysis. We also excluded third codon positions because of the fast substitution rate. The total number of nucleotides used for the phylogenetic analysis was 2,304, which corresponds to 1,152 codons. The sequence data of Pupa mtDNA have been deposited in the GenBank/EMBL/DDBJ database under accession number AB028237.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Genome Composition and Gene Organization of Pupa mtDNA
The total length of Pupa mtDNA is only 14,189 bp. This genome size is quite small for metazoan mtDNAs. Pupa mtDNA is only 395 bp larger than the mtDNA of the pseudocoelomate nematode Caenorhabditis elegans, the smallest metazoan mitochondrial genome known to date (Okimoto et al. 1992Citation ). Among coelomate phyla, such an extremely small mtDNA is unusual, and similar genome size is comparable only with those of pulmonate land snails (14,130 bp in Albinaria [Hatzoglou, George, and Lecanidou 1995Citation ] and 14,100 bp in Cepaea [Terrett, Miles, and Thomas 1996Citation ]). The extremely small genome size of Pupa mtDNA is the consequence of highly compact genome organization and marked reduction of both encoded gene size and intergenic sequences (see below).

The A+T content of Pupa mtDNA is 61.1%. In comparison with other molluscan mtDNAs, this A+T content is similar to that of Cepaea (59.8%; Terrett, Miles, and Thomas 1996Citation ) and Mytilus (62%; Hoffmann, Boore, and Brown 1992Citation ), but not as high as those in Albinaria (70.7%; Hatzoglou, George, and Lecanidou 1995Citation ) and Katharina (69.0%; Boore and Brown 1994aCitation ).

Although some molluscan mtDNAs show unusual gene contents, such as Mytilus mtDNA lacking the ATPase8 (Hoffmann, Boore, and Brown 1992Citation ) and both Mytilus and Katharina mtDNA with additional tRNA genes (Hoffmann, Boore, and Brown 1992Citation ; Boore and Brown 1994aCitation ), the Pupa mtDNA contains all 37 genes (13 protein genes: COIIII, ND16 and 4L, ATPase6, ATPase8, Cytb, 2 rRNA genes, and 22 tRNA genes) typical of metazoan mtDNA. As shown in figure 1 , 24 of these genes are encoded in one strand, which is called the "major strand" here, according to Hatzoglou, George, and Lecanidou (1995)Citation , and the remaining 13 genes are encoded in the opposite strand, the "minor strand."



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Fig. 1.—A partly schematic representation of the mtDNA sequence of Pupa strigosa. Genes are abbreviated as in the text. Numbers within parentheses indicate omitted nucleotides. The predicted amino acid sequences of protein genes are shown by single-letter codes. The unorthodox initiation codon, which is presumed to be translated with formyl-methionine regardless of the codon, is shown in parenthesies. Asterisks denote stop codons and abbreviated stop codons. A dart (>) marks the last nucleotide of each gene and indicates the direction of transcription. The anticodon of each tRNA gene is underlined. A 25-nt palindromic inverted repeat and a 31-nt sequence with the potential to form a hairpin structure within noncoding regions are indicated by a double underline and a number sign (#), respectively

 
One of the remarkable features of Pupa mtDNA is the highly compact genome organization. As shown in figure 1 , most of the Pupa mitochondrial genes either abut directly or have very small numbers of nucleotides separating them. In addition, there are six overlaps between adjacent genes. In five of those cases, the overlapping genes are encoded on the same strand: two cases are overlaps between a tRNA and a protein gene (tRNAPro-ND6, 1 nt; tRNALys-COI, 6 nt), and three cases are overlaps of tRNA genes (tRNATyr-tRNATrp, 6 nt; tRNAGly-tRNAHis, 5 nt; tRNALeu(UUR)-tRNAGln, 5 nt). In the other case, the overlapping genes are encoded in the opposite strands: 4 nt overlap between tRNACys and ATPase6. Although overlaps of a few genes are general features of metazoan mtDNAs (Wolstenholme 1992Citation ), the occurrence of so many overlaps in Pupa mtDNA is unique. It should be noted that pulmonate land snails also have many overlapping genes (Hatzoglou, George, and Lecanidou 1995Citation ; Yamazaki et al. 1997Citation ).

Another notable feature of Pupa mtDNA is the presence of three consecutive protein-protein gene junctions (ND6-ND5, ND5-ND1, and ND4L-Cytb) that are not separated by an intervening tRNA gene (figs. 1 and 2 ). It has been suggested that the secondary structure of a tRNA gene between a pair of protein genes functions as a signal for the precise cleavage of the polycistronic primary transcript (Ojala et al. 1980Citation ; Ojala, Montoya, and Attardi 1981Citation ). In accordance with this hypothesis, many cases of protein-protein gene borders with no intervening tRNAs have the potential to form hairpin structures (Bibb et al. 1981Citation ; Clary and Wolstenholme 1985Citation ; Okimoto et al. 1992Citation ; Boore and Brown 1994aCitation ; Hatzoglou, George, and Lecanidou 1995Citation ). However, in Pupa mtDNA, we could not find any such stable hairpinlike secondary structures at the protein-protein gene boundaries.

Finally, the gene arrangement of Pupa mtDNA is highly derived. It bears almost no similarity to any other metazoans except some pulmonate land snails. In comparison with other gastropods, the mitochondrial gene arrangement of Pupa is also radically different from that of the prosobranch Littorina (fig. 2 ). Pupa and Littorina mtDNAs share only two gene boundaries, tRNAPro-ND6 and tRNAVal-lrRNA, but the remaining gene arrangements are totally different from each other. On the other hand, the Pupa mitochondrial gene arrangement shows a remarkable similarity with those of pulmonate gastropods. Except for translocations of tRNATyr, tRNATrp, tRNACys, and tRNAAla, the gene arrangements of Pupa mtDNA are identical to those of the pulmonate Albinaria. Phylogenetic significance of the gene order variation among gastropods is discussed below.



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Fig. 2.—Comparison of mitochondrial gene arrangements among Pupa strigosa, Albinaria coerulea (Hatzoglou, George, and Lecanidou 1995Citation ), Littorina saxatilis (Wilding, Mill, and Grahame 1999), and Katharina tunicata (Boore and Brown 1994a). All genes are transcribed from left to right, but the underlined genes are transcribed in the opposite direction. Arrows connecting homologous genes or blocks of genes indicate translocations (inversions are indicated by arrows with a circular arrow). Numerous translocations of tRNA genes are not depicted, except those occurreding between P. strigosa and A. coerulea. Gene designations are as in figure 1

 
Noncoding Regions
Metazoan mtDNAs usually have lengthy noncoding regions that vary in size from 121 nt to >20 kbp (Jacobs et al. 1988Citation ; Boyce, Zwick, and Aquadro 1989Citation ). However, one of the remarkable features of Pupa mtDNA is that it contains virtually no such noncoding region of significant length; the longest noncoding region is only 46 nt. A similar situation is reported only for pulmonate land snails; the largest noncoding region of Albinaria mtDNA is 42 nt (Hatzoglou, George, and Lecanidou 1995Citation ).

In most metazoan mtDNAs, the largest noncoding region is thought to contain the signals for transcription and replication, and hence is referred to as the control region (Wolstenholme 1992Citation ). In Pupa mtDNA, the second largest noncoding region, 43 nt located between tRNAIle and COIII (fig. 1 ), is a possible candidate for a control region. The most notable feature of this region is the existence of a 25-nt palindromic inverted repeat. Such an inverted repeat sequence is occasionally found in putative control regions of various metazoan mtDNAs (Wolstenholme 1992Citation ), and such an inverted repeat has been implicated to function as a bidirectional promoter (L'Abbe et al. 1991Citation ). The exact location of this sequence coincides with the boundary between the oppositely transcribed portions of the genome (fig. 1 ), the appropriate location for a bidirectional promoter. Interestingly, land snail mtDNAs also have a noncoding region at the same position between COIII and tRNAIle. Although these possible homologous regions in land snails show low sequence similarity to Pupa, the corresponding region in Albinaria mtDNA also contains a 20-nt perfect palindrome like that found in Pupa (Hatzoglou, George, and Lecanidou 1995Citation ). Finally, this 43-nt region shows the highest A+T content (79.1%) for Pupa mtDNA, like the AT-rich (control) region of Drosophila mtDNA (Clary and Wolstenholme 1985Citation ).

Another notable noncoding region in Pupa mtDNA is the 46-nt sequence located between ND2 and tRNALys (fig. 1 ). This is the largest noncoding sequence in the Pupa mtDNA and contains a 31-nt sequence which can be folded in a hairpin structure with a stem of 9 bp and a loop of 13 nt, including a tract of five T's (fig. 1 ). It is particularly interesting that such a hairpin structure with a T-rich loop has been supposed to function as the origin of the second strand in a variety of metazoan mtDNAs (Wolstenholme 1992Citation ).

Codon Usage and Codon Bias
The genetic code of Pupa mtDNA (table 1 ) appears to be the same as those of protostomian phyla including other molluscs (Clary and Wolstenholme 1985Citation ; Hoffmann, Boore, and Brown 1992Citation ; Valverde et al. 1994Citation ; Boore and Brown 1994a, 1995Citation ; Hatzoglou, George, and Lecanidou 1995Citation ; Terrett, Miles, and Thomas 1996Citation ; Wilding, Mill, and Grahame 1999Citation ). It differs from the universal code in that ATA codon for methionine, TGA for tryptophan, and AGR for serine.


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Table 1 Codon Usage of Pupa mtDNA Encoded Proteins

 
Metazoan mtDNAs occasionally show some kind of codon bias. In Pupa mtDNA, the codons ending in A or T (65.7%) are more frequent than those ending in G or C (table 1 ). This pattern of codon bias apparently reflects a high A+T content for Pupa mtDNA, and such codon bias is a common feature of protostomian mtDNAs (e.g., Clary and Wolstenholme 1985Citation ; Crozier and Crozier 1993Citation ; Boore and Brown 1994a, 1995Citation ). Strand-specific codon bias, resulting from differences in nucleotide composition between DNA strands, has been reported for some metazoan mtDNAs; codons ending in T or G are preferentially used more in the one strand (H strand) than in the other strand (L strand), and codons ending in A or C show the opposite tendency (Asakawa et al. 1991Citation ). Pupa mtDNA does not show prominent strand-specific codon bias: genes encoded by either the major strand or the minor strand use codons ending in G or T with similar frequencies (54.5% and 50.9%, respectively).

Protein Genes
Five of Pupa's 13 protein genes start with the orthodox initiation codon ATG, two start with ATA (ND3 and ND6), three start with GTG (COII, ND4, and ND5), and three start with TTG (Cytb, ND1, and ND4L) (table 2 ). TTG is not used as the initiation codon in most coelomates, but it is used occasionally in pulmonate land snails (Hatzoglou, George, and Lecanidou 1995Citation ) and frequently in pseudocoelomate nematodes (Okimoto et al. 1992Citation ). Seven Pupa protein genes terminate with the stop codons TAA or TAG, and the remaining six genes are inferred to end in abbreviated stop codons, T or TA (table 2 ). The transcripts of the latter genes would be modified to form a complete termination signal UAA by polyadenylation after cleavage of the polycistronic RNA, as demonstrated for other metazoan mtDNAs (Ojala et al. 1980Citation ; Ojala, Montoya, and Attardi 1981Citation ).


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Table 2 Initiation and Termination Codons and Comparison of Amino Acid Lengths of Pupa, Albinaria, Littorina, Katharina, and Drosophila

 
Pupa protein genes are generally short among metazoans (table 2 ; see also table II of Wolstenholme 1992Citation ). The Pupa ND5, which consists of only 526 amino acids, is the shortest observed among metazoans. In addition, the length of the Pupa ND2 is the shortest among coelomate animals. In comparison with other molluscs, most Pupa protein genes are shorter than their counterparts in the prosobranch gastropod Littorina and the polyplacophoran Katharina. It should be noted that the pulmonate gastropoda Albinaria also has similarly short mt-encoded proteins, like Pupa.

Ribosomal RNA Genes
In Pupa mtDNA, srRNA and lrRNA are located between tRNAGlu and tRNAMet, and between tRNAVal and tRNALeu(CUN), respectively (figs. 1 and 2 ). If we assume that the rRNA genes occupy all of the available space between adjacent genes, the gene lengths can be estimated as 729 and 1,069 nt for srRNA and lrRNA, respectively. These are among the shortest of metazoan mitochondrial rRNA genes (see table IV of Wolstenholme 1992Citation ). Such extremely small mitochondrial rRNA genes are only comparable with those of land snails (srRNA/lrRNA: 759/1,035 nt in Albinaria [Hatzoglou, George, and Lecanidou 1995Citation ]; 710/1,216 nt in Cepaea [Terrett, Miles, and Thomas 1996Citation ]; 770/1,024 nt in Euhadra [Yamazaki, personal communication]) and pseudocoelomate nematodes (693/953 nt and 701/960 nt in Caenorhabditis elegans and Ascaris suum, respectively [Okimoto et al. 1992Citation ]).

Transfer RNA Genes
The Pupa mitochondrial genome encodes for the 22 tRNA genes typical of metazoans. Anticodons of Pupa mitochondrial tRNAs are identical to those of other molluscs (Hoffmann, Boore, and Brown 1992Citation ; Boore and Brown 1994aCitation ; Hatzoglou, George, and Lecanidou 1995Citation ; Yamazaki et al. 1997Citation ).

Secondary Structures of tRNAs
As shown in figure 3 , most (but not all) Pupa mitochondrial tRNA genes can be folded into normal cloverleaf secondary structures. All Pupa mitochondrial tRNA genes have seven-member aminoacyl stems, five-member anticodon stems, and seven-member anticodon loops. Most Pupa mitochondrial tRNAs have a variable loop being constructed by 4–5 nt, a dihydrouridine (D) arm with a paired stem of 3–4 bp and a loop of 3–10 nt, and a T{Psi}C (T) arm with a stem of 3–7 bp and a loop of 3–11 nt.



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Fig. 3.—Putative secondary structures of the 22 mitochondrial tRNA genes of Pupa strigosa. Watson-Crick base-pairing is indicated by solid lines, and G-T pairs are indicated with plus signs. The nucleotides overlapping with adjacent downstream genes on the same strand are boxed

 
On the other hand, several Pupa tRNA genes show abnormal secondary structures. In both tRNASer(AGN) and tRNASer(UCN), the D arms are unpaired. Although tRNASer(AGN) with an unpaired D arm is a common feature for metazoan mtDNAs (Wolstenholme 1992Citation ), tRNASer(UCN) lacking a paired D arm is unusual. Furthermore, in Pupa tRNAVal, the T arm and variable loop are replaced by a single loop (the TV-replacement loop), as reported for nematode mitochondrial tRNA genes (Okimoto et al. 1992Citation ). Pupa tRNAPhe also has an unusual T arm whose stem is extremely reduced to only 2 bp (fig. 3 ). In comparison with other gastropods, such bizarre mitochondrial tRNA genes do not appear in the prosobranch Littorina (Wilding, Mill, and Grahame 1999Citation ) and the pulmonate Albinaria (Hatzoglou, George, and Lecanidou 1995Citation ); all mitochondrial tRNAs, excluding tRNASer(AGN), can be folded into standard cloverleaf secondary structures in these gastropods. However, two other pulmonates, Euhadra and Cepaea, also have many tRNA genes with reduced T stems and tRNASer(UCN) lacking a D stem like Pupa (Yamazaki et al. 1997Citation ). It is very interesting that the secondary structures of mitochondrial tRNA genes are highly variable not only between the different gastropodan subclasses, but also within the subclass Pulmonata.

In the Pupa tRNASer(AGN), as many as 9 bp could potentially form for an anticodon stem. The potential of forming a long (9- or 8-bp) anticodon stem of tRNASer(AGN) is conserved in all studied molluscs (Hatzoglou, George, and Lecanidou 1995Citation ; Yamazaki et al. 1997Citation ; see also fig. 3 of Hoffmann, Boore, and Brown [1992Citation ] and fig. 5 of Boore and Brown [1994a]Citation ).



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Fig. 5.—Gastropodan phylogeny estimated from 2,304 nt (first and second codon positions) of mt-encoding protein genes. The topology shown here is a strict consensus tree combining the results of maximum-parsimony and maximum-likelihood analyses and neighbor-joining analysis based on the Kimura two-parameter distance. The numbers above and below the branches indicate the bootstrap percentages based on 1,000 replicates of maximum-parsimony and neighbor-joining analyses, respectively

 
Overlapping tRNA Genes and the Possible Occurrence of RNA Editing
Five Pupa tRNA genes are overlapped by downstream genes encoded in the same strand (figs. 1 and 3 ). It has been suggested that respective transcripts of such overlapping genes are resolved by alternative processing in animal mtDNA (Ojala, Montoya, and Attardi 1981Citation ). This explanation can apply to respective transcripts of Pupa tRNALeu(UUR) and tRNAPro. However, in the cases of tRNAGly, tRNALys, and tRNATyr, overlapping cannot be simply resolved by this mechanism. Because these tRNA genes have many mismatches in their overlapping nucleotides at the 3' parts of aminoacyl stems (fig. 3 ), unstable aminoacyl stems would remain even after the alternative processing.

Recently, Yokobori and Pääbo (1995)Citation demonstrated that in some mitochondrial tRNA genes of the land snail Euhadra, such mismatches can be corrected by a unique RNA editing mechanism after transcription. In this system, the RNA editing alters all overlapping nucleotides in the 3' regions of aminoacyl stems into the adenine residues. As a result, mismatches are replaced by normal Watson-Crick base pairs in the mature transcript. Although a detailed mechanism for this unique tRNA editing is not clear, the possible molecular process has been inferred as follows: a primary transcript is precisely processed such that downstream encoded gene product emerges intact, while an upstream tRNA lacks the overlapping nucleotides at its 3' end, and the lacking nucleotides are filled in by a reaction related to polyadenylation (Yokobori and Pääbo 1995, 1997Citation ; Börner et al. 1997Citation ). We believe the same editing mechanism to be operating in the Pupa tRNAGly, tRNALys, and tRNATyr for the following reasons: (1) The 5' ends of the aminoacyl stems in these genes are almost invariably composed of T residues, such that stable stems will be formed (fig. 3 ) if the overlapping nucleotides are altered to A residues like in Euhadra. (2) These genes are from exactly the same tRNA species of Euhadra whose transcripts are demonstrated to be edited (Yokobori and Pääbo 1995Citation ).

The Pupa tRNAVal also has a mismatched base pair in the aminoacyl stem. Interestingly, the first four nucleotides of this stem are T's, as in the cases mentioned above. Although this tRNA is not demonstrated to be overlapped with the downstream gene, the 5' end of the downstream gene, lrRNA, is ambiguous. It is therefore possible that the tRNAVal would represent another case of overlap with the mismatched aminoacyl stem repaired by polyadenylation. Determination of sequence for the mature tRNAVal and the exact end of lrRNA are needed.

Systematic Implication of the Pupa Mitochondrial Gene Arrangement
Our data demonstrated that the mitochondrial gene arrangement of the opisthobranch Pupa is radically different from that of the prosobranch Littorina but shows a close similarity to those of pulmonate land snails (fig. 2 ). Comparing the gastropodan mitochondrial gene arrangements with those of other metazoans, the Littorina mitochondrial gene arrangement shows a marked similarity to that of the polyplacophora Katharina (Wilding, Mill, and Grahame 1999Citation ). Only two major rearrangements, inversion of a large mitochondrial segment and translocation of a tRNA gene cluster, and transpositions of three individual tRNA genes are necessary for interconversion between them (fig. 2 ). Furthermore, if we ignore frequent translocation of tRNA genes, the Littorina gene orders, such as COI-COII-ATPase8-ATPase6, srRNA-lrRNA-ND1, and ND6-Cytb, are shared not only with other molluscs, but also with different phyla such as arthropods and vertebrates (e.g., Anderson et al. 1981Citation ; Clary and Wolstenholme 1985Citation ). On the other hand, the gene arrangements common to Pupa and pulmonates are unique and show no significant similarity to any other metazoans. It is thus clear that Littorina mtDNA retains an ancestral gene arrangement that may have acquired in a common ancestor of molluscs, while the opisthobranch Pupa and pulmonate land snails have apomorphic gene arrangements derived from the ancestral type.

Mitochondrial gene arrangement is a very complex character set. Because of the enormous number (more than 2 x 1052) of possible gene arrangements for 37 genes, the possibility that the same gene order would arise independently is very small. In recent years, several examples of convergent rearrangements have been discovered (Boore and Brown 1998Citation ; Mindell, Sorenson, and Dimcheff 1998Citation ). All such exceptional cases, although very rare, involve only a single transposition event occurring within a presumed "hot spot" for rearrangements. However, in the present case, parallel evolution of the highly apomorphic gene arrangements seems unlikely, because the genomic rearrangements involve numerous transpositions and inversions. The most parsimonious explanation for the gastropodan mitochondrial gene arrangement data is that the unique gene orders shared among euthyneuran (pulmonates + opisthobranchs) snails evolved in their most recent common ancestor after it split from the prosobranch lineage (fig. 4 ). Thus, the highly derived gene arrangement can be regarded as strong synapomorphies supporting the monophyly of a gastropodan lineage leading to euthyneurans.



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Fig. 4.—Evolution of gastropodan mitochondrial gene arrangements. Some diagnostic gene arrangements informative for gastropodan phylogeny are boxed. The prosobranch gastropod Littorina retains the primitive gene arrangement which is shared with a nongastropodan mollusc and different phyla. On the other hand, opisthobranch and pulmonate gastropods share unique gene arrangements derived from the primitive ones, suggesting their most recent common ancestry. The tree topology outside gastropods is based on the generally accepted view of protostomian phylogeny (e.g., Brusca and Brusca 1990Citation ). A large arrow indicates the occurrence of radical mitochondrial genomic rearrangements during gastropodan evolution. Gene abbreviations and symbols showing gene translocations are as in figure 2

 
To test the reliability of gene order data as a phylogenetic marker, we performed a standard phylogenetic analysis based on sequence data of mt-encoded protein genes. We estimated phylogenetic relationships among gastropods by using the polyplacophora K. tunicata as an outgroup. As shown in figure 5 , phylogenetic trees reconstructed by the MP, NJ, and ML methods show identical topologies, indicating a close phylogenetic affinity of euthyneuran gastropods. Monophyly of the euthyneuran clade is strongly supported by high (100%) bootstrap confidence in both MP and NJ analyses and by significant differences in log-likelihoods of alternative trees from that of the ML tree (data not shown). In accordance with these results, monophyly of euthyneuran gastropods has been supported in the recent phylogenetic analyses based on morphological data and 18S rRNA sequences (Ponder and Lindberg 1996, 1997Citation ; Winnepenninckx et al. 1998Citation ). Therefore, all of the available data are consistent with the single origin of the unique gene arrangements of euthyneuran gastropods (fig. 4 ), supporting the phylogenetic utility of mitochondrial gene arrangement data.

Phylogenetic Significance of Other Mitochondrial Features
Several Unique Features Associated with Genome Minimization
In addition to the unique gene arrangements, the opisthobranch Pupa and pulmonate land snails share several unusual features, such as very small genome size, lack of lengthy noncoding regions, marked size reduction of encoded genes, and occurrence of many overlapping genes (Hatzoglou, George, and Lecanidou 1995Citation ). All of these features are apparently associated with extremely compact genome organization. Since metazoan mtDNA generally lacks introns and lengthy intergenic spacers (Wolstenholme 1992Citation ), pressure for a compact mitochondrial genome may be universally operating to some extent. However, none of these unusual phenotypes have been observed in other coelomate animals, including the prosobranch Littorina and other molluscs (Hoffmann, Boore, and Brown 1992Citation ; Boore and Brown 1994a; Wilding, Mill, and Grahame 1999Citation ), suggesting that the selection pressure is unusually high in these gastropods. Thus, the extremely compact mitochondrial genome associated with many unusual features can be regarded as an another unique synapomorphy of euthyneuran gastropods.

tRNA with Unusual Secondary Structure
Although metazoan mitochondrial tRNAs, except for tRNASer(AGN), normally show the cloverleaf secondary structure (Wolstenholme 1992Citation ), some gastropodan mitochondrial tRNA genes exhibit unusual secondary structures with reduced T or D stems (Yamazaki et al. 1997Citation ). In the present study, we found such bizarre mitochondrial tRNA genes in the opisthobranch Pupa, as well as some pulmonates. Wolstenholme (1992)Citation mentioned that the presence of such bizarre tRNA genes can provide some important clues for phylogenetic reconstruction. However, in gastropods, mitochondrial tRNA secondary structure is highly variable, even within a closely related species (Yamazaki et al. 1997Citation ). Furthermore, the tRNA genes with unusual secondary structures are occasionally different among gastropodan taxa, indicating that the bizarre mitochondrial tRNA has evolved independently in gastropods.

Why has the reduction of either the D stem or the T stem occurred convergently in gastropodan mitochondrial tRNA genes? Macey et al. (1997)Citation also found parallel loss of D stem in reptilian cysteine mitochondrial tRNA, and they proposed a model for the modification of tRNA secondary structure by replication slippage. However, this hypothesis is not applicable to the gastropods, because we cannot find the direct or noncontiguous repeated sequences that Macey et al. (1997)Citation observed in reptilian tRNA. As an alternative explanation, Yamazaki et al. (1997Citation ) argued that the reduction of either tRNA stem would be caused by a strong pressure for mitochondrial genome minimization. In accordance with this hypothesis, the mtDNAs carrying many bizarre tRNA genes are always associated with extremely small genome size (ca. 14-kbp mtDNAs in helicoidean pulmonate snails [Yamazaki et al. 1997Citation ] and some nematodes [Okimoto et al. 1992Citation ]). Since the loss of either stem is always associated with total size reduction of tRNA itself, it is evident that genome minimization pressure and subsequent size reduction of the tRNA gene may change the tRNA secondary structure. In line with this view, the parallel loss or reduction of either tRNA stem is likely to have occurred in euthyneuran gastropodan mtDNAs on which extensive genome minimization pressure operates. Interestingly, several euthyneuran snails, Pupa and Albinaria, have small mitochondrial-genome-like helicoidean snails, but most of the mitochondrial tRNAs retain the standard cloverleaf secondary structures. Such a variation of tRNA secondary structure within euthyneuran snails may suggest that the functional constraint on mitochondrial tRNA would differ among gastropodan taxa and that the normal secondary structure, a presumed ancestral state, can be maintained in certain taxa, even under strong pressure for genome minimization.

tRNA Editing
Yokobori and Pääbo (1995)Citation discovered a unique RNA editing system operating in some Euhadra mitochondrial tRNAs. This type of tRNA editing is directly demonstrated by cDNA sequencing only for this pulmonate, but the possible occurrence of the same system has also been suggested for other pulmonates (Hatzoglou, George, and Lecanidou 1995Citation ; Yamazaki et al. 1997Citation ) and a prosobranch gastropod, Nordotis gigantea (unpublished data). In the present study, we also found the characteristic mismatches of the aminoacyl stem that can be resolved by the editing system in some mitochondrial tRNA genes of the opisthobranch Pupa. Thus, the Euhadra-type tRNA editing phenomenon may be a general feature common in gastropodan mitochondria.

In other molluscan classes, the bivalve Mytilus and the polyplacophoran Katharina have no mitochondrial tRNA genes with such unstable aminoacyl stems (Hoffmann, Boore, and Brown 1992Citation ; Boore and Brown 1994aCitation ), and the editing system may be lacking in these molluscs. Recently, Tomita, Ueda, and Watanabe (1996)Citation found the same type of RNA editing to be operating in the cephalopod Loligo bleekeri. It should be noted that the class Cephalopoda is the suggested candidate for the sister group of the class Gastropoda (Salvini-Plawen 1980Citation ; Salvini-Plawen and Steiner 1996Citation ). Thus, the unique mitochondrial tRNA editing mechanism shared between gastropods and cephalopods seems to have occurred in the common ancestor of the clade Visceroconcha (gastropods + cephalopods) and may suggest the potential utility of the RNA editing system as a phylogenetic marker for molluscan evolution. To elucidate the systematic significance of the mitochondrial tRNA editing system, many more data on various molluscan classes, such as Caudofoveata, Solenogastres, Monoplacophora, and Scaphopoda, are needed.

Perspective
In this study, we showed that mitochondrial genomic structures are radically different between the prosobranch Littorina and euthyneuran gastropods. As discussed above, Littorina mtDNA retains the presumed primitive gene arrangement and less compact genome structure shared with the polyplacophora Katharina and other phyla, while euthyneuran mtDNAs show the unique and highly apomorphic features. Radical changes in the gene arrangements and extensive genome minimization associated with many unusual features must have occurred exclusively in the common ancestral lineage leading to euthyneurans after it diverged from prosobranch lineages (fig. 4 ). The rate of genomic rearrangement is apparently accelerated in the former gastropodan lineage.

In the most recent classification of gastropods (Ponder and Lindberg 1997Citation ; Ponder 1998Citation ), opisthobranchs and pulmonates are placed in the clade Heterobranchia, together with some minor superfamilies. The clade Heterobranchia, in turn, becomes the sister group of a prosobranch clade, Caenogastropoda, which includes Littorina. At present, it is not clear whether the unique mitochondrial genome organization was acquired in the common ancestor of all heterobranch gastropods or in the common ancestor of a much higher lineage of heterobranchs, such as the clade Euthyneura. It is also uncertain how the highly specialized gene arrangements of euthyneuran type have evolved from the presumed ancestral type like Littorina or Katharina. Investigation of many more gastropodan mtDNAs, especially mtDNAs of lower heterobranch gastropods, will provide invaluable information for understanding the detailed evolutionary process of the gastropodan mitochondrial genome.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
We thank Professor Toshiki Makioka and Dr. Osamu Numata of Institute of Biological Sciences, University of Tsukuba, for valuable suggestions and encouragement on the study, and Mr. Hitoshi Ueda and the staff of the Shimoda Marine Research Center, University of Tsukuba, for help with collection of the material. We also thank two anonymous reviewers for helpful comments on the manuscript. This work was supported by a Grant-in-Aid for Science Research for the Ministry Education, Science, Sports and Culture of Japan and Toray Science Foundation.


    Footnotes
 
Naruya Saitou,

1 Keywords: mitochondrial genome gastropoda gene arrangement opisthobranch phylogeny, tRNA Back

2 Address for correspondence and reprints: Rei Ueshima, Department of Biological Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: rueshima{at}biol.s.u-tokyo.ac.jp Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 

    Anderson, S., A. T. Bankier, B. G. Barrell, M. H. L. De Bruijn, A. R. Coulson, I. C. Eperon, F. Sanger, and I. G. Young. 1981. Sequence and organization of the human mitochondrial genome. Nature 290:457–465.

    Asakawa, S., Y. Kumazawa, T. Araki, H. Himeno, K. Miura, and K. Watanabe. 1991. Strand-specific nucleotide bias in echinoderm and vertebrate mitochondrial genomes. J. Mol. Evol. 32:511–520.[ISI][Medline]

    Bibb, M. H., R. A. Van Etten, C. T. Wright, W. M. Walberg, and D. A. Clayton. 1981. Sequence and genome organization of mouse mitochondrial DNA. Cell 26:167–180.

    Black, W. C. IV, and R. L. Roehrdanz. 1998. Mitochondrial gene order is not conserved in Arthropoda: prostriate and metastriate tick mitochondrial genomes. Mol. Biol. Evol. 15:1772–1785.[Abstract/Free Full Text]

    Boore, J. L. 1999. Animal mitochondrial genomes. Nucleic Acids Res. 27:1767–1780.[Abstract/Free Full Text]

    Boore, J. L., and W. M. Brown. 1994a. Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicata. Genetics 138:423–443.

    ———. 1994b. Mitochondrial genomes and the phylogeny of molluscs. Nautilus 108(Suppl. 2):61–78.

    ———. 1995. Complete sequence of the mitochondrial DNA of the annelid worm Lumbricus terrestris. Genetics 141:305–319.

    ———. 1998. Big trees from little genomes: mitochondrial gene order as a phylogenetic tool. Curr. Opin. Genet. Dev. 8:668–674.[ISI][Medline]

    Boore, J. L., T. M. Collins, D. Stanton, L. L. Daehler, and W. M. Brown. 1995. Deducing the pattern of arthropod phylogeny from mitochondrial DNA rearrangements. Nature 376:163–165.

    Börner, G. V., S. Yokobori, M. Mörl, M. Dörner, and S. Pääbo. 1997. RNA editing in metazoan mitochondria: staying fit without sex. FEBS Lett. 409:320–324.[ISI][Medline]

    Boyce, T. H., M. E. Zwick, and C. F. Aquadro. 1989. Mitochondrial DNA in the bark weevils: Size, structure and heteroplasmy. Genetics 123:825–836.

    Brusca, R. C., and G. J. Brusca. 1990. Invertebrates. Sinauer, Sunderland, Mass.

    Campbell, N. J. H., and S. C. Barker. 1999. The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: fivefold tandem repetition of a coding region. Mol. Biol. Evol. 16:732–740.[Abstract]

    Cheng, S., S.-Y. Chang, P. Gravitt, and R. Respess. 1994. Long PCR. Nature 369:684–685.

    Clary, D. O., and D. R. Wolstenholme. 1985. The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization and genetic code. J. Mol. Evol. 22:252–271.[ISI][Medline]

    Crozier, R. H., and Y. C. Crozier. 1993. The mitochondrial genome of honeybee Apis mellifera: Complete sequence and genome organization. Genetics 133:97–117.

    Desjardins, P., and R. Morais. 1990. Sequence and gene organization of chicken mitochondrial genome: a novel gene order in higher vertebrates. J. Mol. Biol. 212:599–634.[ISI][Medline]

    Dowton, M. 1999. Relationships among the cyclostome braconid (Hymenoptera: Braconidae) subfamilies inferred from a mitochondrial tRNA gene rearrangement. Mol. Phylogenet. Evol. 11:283–287.[ISI][Medline]

    Felsenstein, J. 1996. PHYLIP: phylogeny inference package. Version 3.5c. Distributed by the author, Department of Genetics, University of Washington, Seattle.

    Hatzoglou, H., C. George, and R. Lecanidou. 1995. Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea. Genetics 140:1353–1366.

    Hoffmann, R. J., J. L. Boore, and W. M. Brown. 1992. A novel mitochondrial genome organization for the blue mussel, Mytilus edulis. Genetics 131:397–412.

    Hyman, L. H. 1967. The invertebrates, Vol. 6. Mollusca. 1. McGraw-Hill, New York.

    Jacobs, H. T., D. J. Elliott, V. B. Math, and A. Farquharson. 1988. Nucleotide sequence and gene organization of sea urchin mitochondrial DNA. J. Mol. Biol. 202:185–217.[ISI][Medline]

    Kumazawa, Y., and M. Nishida. 1995. Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers. Mol. Biol. Evol. 12:759–772.[Abstract]

    L'Abbe, D., J. F. Duhaime, B. F. Lang, and R. Morais. 1991. The transcription of DNA in chicken mitochondria initiates from one major bidirectional promoter. J. Biol. Chem. 266:10844–10850.[Abstract/Free Full Text]

    Lee, W.-J., and T. D. Kocher. 1995. Complete sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome: early establishment of the vertebrate genome organization. Genetics 139:873–887.

    Macey, R. J., A. Larson, N. B. Ananjeva, and T. J. Papenfuss. 1997. Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs. Mol. Biol. Evol. 14:30–39.[Abstract]

    Mindell, D. P., M. D. Sorenson, and D. E. Dimcheff. 1998. Multiple independent origins of mitochondrial gene order in birds. Proc. Natl. Acad. Sci. USA 95:10693–10697.

    Ojala, D., C. Merkel, R. Gelfand, and G. Attardi. 1980. The tRNA genes punctuate the reading of genetic information in human mitochondrial DNA. Cell 22:393–403.

    Ojala, D., J. Montoya, and G. Attardi. 1981. tRNA punctuation model of RNA processing in human mitochondria. Nature 290:470–474.

    Okimoto, R., J. L. Macfarlane, D. O. Clary, and D. R. Wolstenholme. 1992. The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum. Genetics 130:471–498.

    Pääbo, S., W. K. Thomas, K. M. Whitfield, Y. Kumazawa, and A. C. Wilson. 1991. Rearrangements of mitochondrial transfer RNA genes in marsupials. J. Mol. Evol. 33:426–430.[ISI][Medline]

    Ponder, W. F. 1998. Classification. P. 607 in P. L. Beesley, G. J. B. Ross, and A. Wells, eds. Mollusca: the southern synthesis. Fauna of Australia. Vol. 5, Part B. CSIRO Publishing, Melbourne, Australia.

    Ponder, W. F., and D. R. Lindberg. 1996. Gastropodan phylogeny—challenges for the 90s. Pp. 135–155 in T. D. Taylor, ed. Origin and evolutionary radiation of the Mollusca. Oxford Scientific, Oxford, England.

    ———. 1997. Towards a phylogeny of gastropod molluscs: an analysis using morphological characters. Zool. J. Linn. Soc. 119:83–265.[ISI]

    Roe, B. A., D.-P. Ma, R. K. Wilson, and J. F.-H. Wong. 1985. The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. J. Biol. Chem. 260:9759–9774.[Abstract/Free Full Text]

    Salvini-Plawen, L. v. 1980. A reconsideration of systematics in the Mollusca (phylogeny and higher classification). Malacologia 19:249–278.

    Salvini-Plawen, L. v., and G. Steiner. 1996. Synapomorphies and plesiomorphies in higher classification of Mollusca. Pp. 29–53 in T. D. Taylor, ed. Origin and evolutionary radiation of the Mollusca. Oxford Scientific, Oxford, England.

    Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

    Shahjahan, R. M. 1995. Lower incubation temperature increases yield of insect genomic DNA isolated by the CTAB method. Biotechniques 19:332–334.

    Smith, M. J., A. Arndt, S. Gorski, and E. Fajber. 1993. The phylogeny of echinoderm classes based on mitochondrial gene arrangements. J. Mol. Evol. 36:545–554.[ISI][Medline]

    Staton, J. L., L. L. Daehler, and W. M. Brown. 1997. Mitochondrial gene arrangement of the horseshoe crab Limulus polyphemus: conservation of major features among arthropod classes. Mol. Biol. Evol. 14:867–874.[Abstract]

    Swofford, D. L. 1996. PAUP. National Museum of Natural History, Smithsonian Institution, Washington, D.C.

    Terrett, J. A., S. Miles, and R. H. Thomas. 1996. Complete DNA sequence of Cepaea nemoralis (Gastropoda: Stylommatophora). J. Mol. Evol. 42:160–168.[ISI][Medline]

    Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680.[Abstract]

    Tomita, K., T. Ueda, and K. Watanabe. 1996. RNA editing in the acceptor stem of squid mitochondrial tRNA(Tyr). Nucleic Acids Res. 24:4987–4991.[Abstract/Free Full Text]

    Tzeng, C.-S., C.-F. Hui, S.-C. Shen, and P. C. Huang. 1992. The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genome: conservation and variations among vertebrates. Nucleic Acids Res. 20:4853–4858.[Abstract]

    Valverde, J. R., B. Batuecas, C. Moratilla, and R. Garesse. 1994. The complete mitochondrial DNA sequence of the crustacean Artemia franciscana. J. Mol. Evol. 39:400–408.[ISI][Medline]

    Vaught, K. C. 1989. A classification of the living Mollusca. American Malacologists, Melbourne, Fla.

    Wilding C. S., P. J. Mill, and J. Grahame. 1999. Partial sequence of the mitochondrial genome of Littorina saxatilis: relevance to gastropod phylogenetics. J. Mol. Evol. 48:348–359.[ISI][Medline]

    Winnepenninckx, B., G. Steiner, T. Backeljau, and R. D. Wachter. 1998. Details of gastropod phylogeny inferred from 18s rRNA sequences. Mol. Phylogenet. Evol. 9:55–63.[ISI][Medline]

    Wolstenholme, D. R. 1992. Animal mitochondria DNA: structure and evolution. Int. Rev. Cytol. 141:173–216.[ISI][Medline]

    Yamazaki, N., R. Ueshima, J. A. Terrett et al. (12 co-authors). 1997. Evolution of pulmonate gastropod mitochondrial genomes: comparisons of gene organizations of Euhadra, Cepaea and Albinaria and implications of unusual tRNA secondary structures. Genetics 145:749–758.

    Yokobori, S., and S. Pääbo. 1995. Transfer RNA editing in land snail mitochondria. Proc. Natl. Acad. Sci. USA 92:10432–10435.

    ———. 1997. Polyadenylation creates the discriminator nucleotide of chicken mitochondrial tRNA Tyr. J. Mol. Biol. 265:95–99.[ISI][Medline]

Accepted for publication November 1, 1999.