Download Complete Sequence of the Mitochondrial DNA of

Complete Sequence of the Mitochondrial DNA of the Primitive
Opisthobranch Gastropod Pupa strigosa: Systematic Implication of the
Genome Organization
Atsushi Kurabayashi* and Rei Ueshima†‡
*Institute of Biological Sciences, University of Tsukuba, Tsukuba, Japan; †Department of Biological Science,
University of Tokyo, Tokyo, Japan; and ‡PRESTO, Japan Science and Technology Corporation
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
Metazoan mitochondrial (mt) DNA is a closed circular molecule with an approximate genome size of 16
kbp (Wolstenholme 1992). 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 [COI–III], cytochrome b apoenzyme [Cytb], ATP
synthase subunits 6 and 8 [ATPase6 and ATPase8], and
NADH dehydrogenase subunits 1–6 and 4L [ND1–6,
4L]). In addition, metazoan mtDNA usually contains at
least one lengthy noncoding sequence which regulates
and initiates mtDNA replication and transcription (control region; Wolstenholme 1992).
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. 1981;
Roe et al. 1985; Tzeng et al. 1992; Boore 1999). Although minor rearrangements have been reported for
marsupials (Pääbo et al. 1991), birds (Desjardins and
Morais 1990), reptiles (Kumazawa and Nishida 1995),
and a lamprey (Lee and Kocher 1995), 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
1985; Valverde et al. 1994; Staton, Daehler, and Brown
1997; Black and Roehrdanz 1998; Boore 1999; Campbell and Barker 1999).
Key words: mitochondrial genome, gastropoda, gene arrangement, opisthobranch, phylogeny, tRNA.
Address for correspondence and reprints: Rei Ueshima, Department of Biological Science, University of Tokyo, Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan. E-mail: [email protected].
Mol. Biol. Evol. 17(2):266–277. 2000
q 2000 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
266
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 1992); the black chiton Katharina
tunicata, of the class Polyplacophora (Boore and Brown
1994a); and three species of land snails, Albinaria coerulea (Hatzoglou, George, and Lecanidou 1995), Cepaea nemoralis (Terrett, Miles, and Thomas 1996), and
Euhadra herklotsi (Yamazaki et al. 1997), 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. 1997). 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
1992), such deviations from the typical metazoan pattern
are surprising.
Recently, Wilding, Mill, and Grahame (1999) 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
Opisthobranch Mitochondrial Genome
gastropods have been controversial, and there are many
gastropodan subgroups whose systematic positions are
still ambiguous (Salvini-Plawen and Steiner 1996; Ponder and Lindberg 1996, 1997). In recent years, mitchondrial gene arrangements have attracted the attention of
evolutionary biologists as novel phylogenetic markers
(Smith et al. 1993; Boore and Brown 1994b, 1998;
Boore et al. 1995; Kumazawa and Nishida 1995; Boore
1999; Dowton 1999). 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 1989; Brusca and
Brusca 1990). Although either complete or partial
mtDNA sequences have been determined for a prosobranch (Wilding, Mill, and Grahame 1999) and three
pulmonate gastropods (Hatzoglou, George, and Lecanidou 1995; Terrett, Miles, and Thomas 1996; Yamazaki
et al. 1997), 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 1967; Salvini-Plawen and Steiner 1996).
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
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), 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).
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 59-TGRGGNGCNACNGTNATYACNAAYYT-39 and 59-RAARTAYCAYTCNGGYTGRATRTG-39. PCR reactions consisted of 30 cycles of denaturation at 948C for 30 s, annealing at 408C for 30
s, and extension at 728C 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 remain-
267
ing mtDNA fragment was then amplified from the total
DNA by the long PCR method (Cheng et al. 1994)
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: 59GGATCAACAAACCCTTTAGGGGATTTAAATCATGT-39 and 59-GGAATAGGATATGTGTAGAGC
ACTAAGTCC-TGCAATT-39. The long PCR reactions
consisted of 30 cycles of denaturation at 988C for 20
s, and both annealing and extension at 688C 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 DH5a 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 1977) using the Ready Reaction kit with ampliTaq DNA polymerase, CS1, (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 1985), the black chiton K. tunicata
(Boore and Brown 1994a), and the land snail A. coerulea (Hatzoglou, George, and Lecanidou 1995). 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
1994), and the alignment was corrected by visual observation. Phylogenetic trees based on mt-encoded protein gene sequences were performed with the neighborjoining (NJ) and maximum-likelihood (ML) methods using PHYLIP, version 3.5c (Felsenstein 1996), and with
the maximum-parsimony (MP) method using PAUP,
version 3.1. (Swofford 1996). 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 1999). 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.
268
Kurabayashi and Ueshima
Results and Discussion
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. 1992). 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 1995] and 14,100
bp in Cepaea [Terrett, Miles, and Thomas 1996]). 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 A1T content of Pupa mtDNA is 61.1%. In
comparison with other molluscan mtDNAs, this A1T
content is similar to that of Cepaea (59.8%; Terrett,
Miles, and Thomas 1996) and Mytilus (62%; Hoffmann,
Boore, and Brown 1992), but not as high as those in
Albinaria (70.7%; Hatzoglou, George, and Lecanidou
1995) and Katharina (69.0%; Boore and Brown 1994a).
Although some molluscan mtDNAs show unusual
gene contents, such as Mytilus mtDNA lacking the
ATPase8 (Hoffmann, Boore, and Brown 1992) and both
Mytilus and Katharina mtDNA with additional tRNA
genes (Hoffmann, Boore, and Brown 1992; Boore and
Brown 1994a), the Pupa mtDNA contains all 37 genes
(13 protein genes: COI–III, ND1–6 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), and the remaining 13
genes are encoded in the opposite strand, the ‘‘minor
strand.’’
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 1992),
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 1995; Yamazaki et al. 1997).
Another notable feature of Pupa mtDNA is the
presence of three consecutive protein-protein gene junc-
tions (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. 1980; Ojala, Montoya,
and Attardi 1981). 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. 1981; Clary and Wolstenholme
1985; Okimoto et al. 1992; Boore and Brown 1994a;
Hatzoglou, George, and Lecanidou 1995). 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.
Noncoding Regions
Metazoan mtDNAs usually have lengthy noncoding regions that vary in size from 121 nt to .20 kbp
(Jacobs et al. 1988; Boyce, Zwick, and Aquadro 1989).
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 1995).
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 1992). 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 1992), and such an inverted repeat has
been implicated to function as a bidirectional promoter
(L’Abbe et al. 1991). 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
Opisthobranch Mitochondrial Genome
269
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.
270
Kurabayashi and Ueshima
FIG. 2.—Comparison of mitochondrial gene arrangements among Pupa strigosa, Albinaria coerulea (Hatzoglou, George, and Lecanidou
1995), 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.
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 1995). Finally, this 43-nt region shows
the highest A1T content (79.1%) for Pupa mtDNA, like
the AT-rich (control) region of Drosophila mtDNA
(Clary and Wolstenholme 1985).
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 1992).
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 A1T content for Pupa mtDNA, and such
codon bias is a common feature of protostomian
mtDNAs (e.g., Clary and Wolstenholme 1985; Crozier
and Crozier 1993; Boore and Brown 1994a, 1995).
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. 1991). 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).
Codon Usage and Codon Bias
Protein Genes
The genetic code of Pupa mtDNA (table 1) appears
to be the same as those of protostomian phyla including
other molluscs (Clary and Wolstenholme 1985; Hoffmann, Boore, and Brown 1992; Valverde et al. 1994;
Boore and Brown 1994a, 1995; Hatzoglou, George, and
Lecanidou 1995; Terrett, Miles, and Thomas 1996;
Wilding, Mill, and Grahame 1999). It differs from the
universal code in that ATA codon for methionine, TGA
for tryptophan, and AGR for serine.
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 1995) and
frequently in pseudocoelomate nematodes (Okimoto et
al. 1992). Seven Pupa protein genes terminate with the
Opisthobranch Mitochondrial Genome
271
Table 1
Codon Usage of Pupa mtDNA Encoded Proteins
AA
Codon
Phe . .
Leu . .
TTT
TTC
TTA
TTG
CTT
CTC
CTA
CTG
Ile . . . . ATT
ATC
Met . . ATA
ATG
Val . . . GTT
GTC
GTA
GTG
N
%
AA
Codon
N
%
AA
Codon
N
%
AA
Codon
N
%
196
69
219
96
99
32
99
47
177
45
116
63
94
40
87
71
5.5
1.9
6.1
2.7
2.8
0.9
2.8
1.3
4.9
1.3
3.2
1.8
2.6
1.1
2.4
2.0
Ser . . . .
TCT
TCC
TCA
TCG
CCT
CCC
CCA
CCG
ACT
ACC
ACA
ACG
GCT
GCC
GCA
GCG
71
17
69
33
49
34
41
20
79
28
60
16
99
45
77
35
2.0
0.5
1.9
0.9
1.4
1.0
1.1
0.6
2.2
0.8
1.7
0.5
2.8
1.3
2.2
1.0
Tyr . . . . . .
TAT
TAC
TAA
TAG
CAT
CAC
CAA
CAG
AAT
AAC
AAA
AAG
GAT
GAC
GAA
GAG
77
54
6
1
49
32
27
29
57
44
49
23
39
30
46
38
2.2
1.5
0.2
0.0
1.4
0.9
0.8
0.8
1.6
1.2
1.4
0.6
1.1
0.8
1.3
1.1
Cys . . . .
TGT
TGC
TGA
TGG
CGT
CGC
CGA
CGG
AGT
AGC
AGA
AGG
GGT
GGC
GGA
GGG
37
15
54
43
20
6
17
13
53
27
62
49
62
41
66
93
1.0
0.4
1.5
1.2
0.6
0.2
0.5
0.4
1.5
0.8
1.7
1.4
1.7
1.1
1.8
2.6
Pro . . . .
Thr . . . .
Ala . . . .
Term . . . . .
His . . . . . .
Gln . . . . . .
Asn . . . . .
Lys . . . . . .
Asp . . . . .
Glu . . . . . .
Trp . . . .
Arg . . . .
Ser . . . .
Gly . . . .
NOTE.—AA 5 amino acid; N 5 total number of particular codon in all proteins; Term 5 termination codons. The total number of codons was 3,582. Incomplete
termination codons were excluded.
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. 1980; Ojala, Montoya, and Attardi 1981).
Pupa protein genes are generally short among
metazoans (table 2; see also table II of Wolstenholme
1992). 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 1992). 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 1995]; 710/1,216 nt in
Cepaea [Terrett, Miles, and Thomas 1996]; 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. 1992]).
Table 2
Initiation and Termination Codons and Comparison of Amino Acid Lengths of Pupa, Albinaria, Littorina, Katharina,
and Drosophila
NO.
PUTATIVE INITIATION
PROTEIN
ATPase6 . . . .
ATPase8 . . . .
COI . . . . . . . .
COII . . . . . . .
COIII . . . . . .
Cytb . . . . . . .
ND1 . . . . . . .
ND2 . . . . . . .
ND3 . . . . . . .
ND4 . . . . . . .
ND4L . . . . . .
ND5 . . . . . . .
ND6 . . . . . . .
Pupa
ATG
ATG
ATG
GTG
ATG
TTG
TTG
ATG
ATA
GTG
TTG
GTG
ATA
TAA
TAA
TAA
TAA
Taa
Taa
TAa
TAG
Taa
Taa
Taa
TAA
TAA
Albinaria
ATG
ATG
TTG
ATG
ATG
ATA
ATG
ATG
ATA
ATG
ATG
ATT
ATG
Taa
TAG
TAa
TAA
TAA
TAa
TAA
TAA
Taa
TAA
Taa
TAG
TAA
AND
TERMINATION CODONS
Littorina
ATG
ATG
—
ATG
—
ATG
ATG
—
—
—
—
—
ATG
TAG
TAG
TAA
TAA
—
—
TAA
—
—
—
—
—
TAG
OF
Katharina
Drosophila
Pupa
Albinaria
ATG
ATG
ATG
ATG
ATG
ATG
ATG
GTG
ATG
ATA
ATG
ATG
ATG
ATG
ATT
ATAA
ATG
ATG
ATG
ATA
ATT
ATT
ATG
ATG
ATT
ATT
219
53
509
228
259
375
302
296
116
436
95
526
161
214
55
509
224
259
367
299
307
117
437
99
545
155
TAA
TAG
Taa
TAG
TAA
TAA
TAA
TAG
TAA
Taa
TAG
Taa
Taa
TAa
TAA
TAA
Taa
TAA
TAA
TAA
Taa
TAA
Taa
TAa
Taa
TAA
AMINO ACIDS
Litto- Katha- Drorina
rina sophila
231
52
P
228
—
P
312
—
—
—
—
—
170
230
53
513
229
259
379
316
338
120
442
100
571
166
224
53
512
228
262
378
324
341
117
446
96
573
174
NOTE.—Codons with lowercase a’s are incomplete termination codons, presumably completed by polyadenylation at an ‘‘a’’ site. P denotes partially sequenced
Littorina proteins.
272
Kurabayashi and Ueshima
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.
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 1992; Boore
and Brown 1994a; Hatzoglou, George, and Lecanidou
1995; Yamazaki et al. 1997).
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, fivemember 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 TCC (T) arm with a stem of 3–7 bp and a
loop of 3–11 nt.
On the other hand, several Pupa tRNA genes show
abnormal secondary structures. In both tRNASer(AGN) and
tRNA Ser(UCN) , the D arms are unpaired. Although
tRNASer(AGN) with an unpaired D arm is a common feature for metazoan mtDNAs (Wolstenholme 1992),
tRNASer(UCN) lacking a paired D arm is unusual. Fur-
Opisthobranch Mitochondrial Genome
thermore, 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. 1992). 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
1999) and the pulmonate Albinaria (Hatzoglou, George,
and Lecanidou 1995); 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. 1997). 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 1995; Yamazaki et al.
1997; see also fig. 3 of Hoffmann, Boore, and Brown
[1992] and fig. 5 of Boore and Brown [1994a]).
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 1981).
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 39 parts of aminoacyl stems (fig. 3),
unstable aminoacyl stems would remain even after the
alternative processing.
Recently, Yokobori and Pääbo (1995) 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 39 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 39 end, and the lacking
nucleotides are filled in by a reaction related to polyadenylation (Yokobori and Pääbo 1995, 1997; Börner et
al. 1997). We believe the same editing mechanism to be
operating in the Pupa tRNAGly, tRNALys, and tRNATyr for
273
the following reasons: (1) The 59 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
1995).
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 59 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 1999). 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.
1981; Clary and Wolstenholme 1985). 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 3 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 1998; Mindell, Sorenson,
and Dimcheff 1998). 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
274
Kurabayashi and Ueshima
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 1990). 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.
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 1 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.
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, 1997;
Winnepenninckx et al. 1998). 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
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 neighborjoining 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.
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
1995). All of these features are apparently associated
with extremely compact genome organization. Since
metazoan mtDNA generally lacks introns and lengthy
intergenic spacers (Wolstenholme 1992), pressure for a
Opisthobranch Mitochondrial Genome
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 1992; Boore
and Brown 1994a; Wilding, Mill, and Grahame 1999),
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 1992), some gastropodan
mitochondrial tRNA genes exhibit unusual secondary
structures with reduced T or D stems (Yamazaki et al.
1997). In the present study, we found such bizarre mitochondrial tRNA genes in the opisthobranch Pupa, as
well as some pulmonates. Wolstenholme (1992) 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. 1997). 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) 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) observed in reptilian tRNA. As an alternative explanation,
Yamazaki et al. (1997) 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. 1997] and some nematodes [Okimoto et al. 1992]). 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 second-
275
ary 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) 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 1995; Yamazaki et al.
1997) 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 1992; Boore and Brown
1994a), and the editing system may be lacking in these
molluscs. Recently, Tomita, Ueda, and Watanabe (1996)
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
1980; Salvini-Plawen and Steiner 1996). 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 1 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.
276
Kurabayashi and Ueshima
In the most recent classification of gastropods
(Ponder and Lindberg 1997; Ponder 1998), 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.
Acknowledgments
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.
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NARUYA SAITOU, reviewing editor
Accepted November 1, 1999