The Complete Maternal and Paternal Mitochondrial Genomes of the
Mediterranean Mussel Mytilus galloprovincialis: Implications for the Doubly
Uniparental Inheritance Mode of mtDNA
Athanasia Mizi,* Eleftherios Zouros, Nicholas Moschonas, and George C. Rodakis*
*Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimioupolis,
Athens, Greece; and Department of Biology, University of Crete, Heraklion, Crete, Greece
The maternal (F) and paternal (M) mitochondrial genomes of the mussel Mytilus galloprovincialis have diverged by about
20% in nucleotide sequence but retained identical gene content and gene arrangement and similar nucleotide composition
and codon usage bias. Both lack the ATPase8 subunit gene, have two tRNAs for methionine and a longer open-reading
frame for cox3 than seen in other mollusks. Between the F and M genomes, tRNAs are most conserved followed by rRNAs
and protein-coding genes, even though the degree of divergence varies considerably among the latter. Divergence at nad3
is exceptionally low most likely because this gene includes the origin of transcription of the lagging strand (OL). Noncoding
regions are the least conserved with the notable exception of the central domain of the main control region and a segment of
another noncoding region immediately following nad3. The amino acid divergence (14%) of the two genomes is smaller
than in two other pairs of conspecific genomes that are available in GenBank, that of the clam Venerupis philippinarum
(34%) and of the fresh water mussel Inversidens japanensis (50%), suggesting that doubly uniparental inheritance of
mtDNA emerged at different times in the three species or that there has been a relatively recent replacement of the male
genome by the female in the Mytilus line. The latter hypothesis is supported from phylogenetic and population studies of
Mytilidae. That the M genome contains a full complement of genes with no premature termination codons argues against it
being a selfish element that rides with the sperm. It is shorter than the F by 118 bp, which apparently cannot account for the
postulated replicative advantage of this genome over the F in male gonads. The high similarity of the two genomes explains
why the F genome may assume the role of the M genome, but it does not exclude the possibility that for this to happen some
M-specific sequences must be transferred on to the F genome by means of recombination. If such sequences exist they
would most likely be located in noncoding regions.
Introduction
Several bivalve species are known to have two highly
differentiated mitochondrial genomes (Skibinski, Gallagher,
and Beynon 1994a, 1994b; Zouros et al. 1994a, 1994b; Liu,
Mitton, and Wu 1996; Hoeh et al. 1997; Passamonti and Skali
2001; Curole and Kocher 2002; Hoeh, Stewart, and Guttman
2002; Serb and Lydeard 2003) one of which follows the
standard maternal inheritance (and is known as type F for
female-transmitted) and the other is transmitted through
the sperm (and is known as M for male-transmitted). Thus,
each genome obeys the rule of uniparental transmission.
The phenomenon, known as doubly uniparental inheritance
(DUI, Zouros et al. 1994a), has been studied more extensively in the blue mussel genus Mytilus, where it was first
noted (Skibinski, Gallagher, and Beynon 1994a, 1994b;
Zouros et al. 1994a, 1994b).
Even before the discovery of DUI, it was known from
the work of Hoffmann, Boore, and Brown (1992) that the
Mytilus mtDNA possesses characteristics not commonly
found among metazoan mitochondrial genomes. It lacks
the ATPase8 gene, has two tRNAs for methionine, and
most notably, its gene arrangement is very different from
other known animal mtDNAs. It was subsequently determined that the genome examined by Hoffmann, Boore,
and Brown (1992) was maternally transmitted (Skibinski,
Gallagher, and Beynon 1994b; Rawson and Hilbish
Key words: Mytilus, mitochondrial genome, maternally or paternally
inherited mtDNA.
E-mail: [email protected].
Mol. Biol. Evol. 22(4):952–967. 2005
doi:10.1093/molbev/msi079
Advance Access publication January 12, 2005
1995; Stewart et al. 1995). Comparison of partial sequences
from both genomes suggested that the M genome evolves
faster than the F (Skibinski, Gallagher, and Beynon 1994b;
Rawson and Hilbish 1995; Stewart et al. 1995; Quesada,
Skibinski, and Skibinski 1996; Hoeh et al. 1997; Quesada,
Warren, and Skibinski 1998). Further studies of DUI in
mussels suggested that the F genome may occasionally
invade the sperm-transmission route and assume the role
of the M genome (Hoeh et al. 1997; Saavedra, Reyero,
and Zouros 1997; Quesada, Wenne, and Skibinski 1999).
Also it has been observed that F and M genomes may
recombine in the male gonad, where they are found together
(Ladoukakis and Zouros 2001).
For a better understanding of the mechanism and evolution of DUI we need the complete sequences of the two
genomes. At present, the complete sequences of two F
genomes have been published, those of the blue Mytilus
edulis (Hoffmann, Boore, and Brown 1992; Boore, Medina,
and Rosenberg 2004) and of the fresh water mussel Lampsilis ornata (Serb and Lydeard 2003). The sequences of
the F and M genomes of the venerid clam Venerupis
philippinarum and the fresh water mussel Inversidens japanensis are available in GenBank (accession numbers
AB065375, AB065374, AB055625, AB055624, respectively; M. Okazaki and R. Ueshima, personal communication). This paper is the first to present together the complete
sequence of F and M genomes of a species, namely, the
Mediterranean mussel Mytilus galloprovincialis. In particular, the M. galloprovincialis M genome is the first paternally transmitted mitochondrial genome whose complete
sequence is presented and discussed.
In addition to presenting these sequences, we use them
to obtain answers to the following specific questions. (1) Do
Molecular Biology and Evolution vol. 22 no. 4 Ó Society for Molecular Biology and Evolution 2005; all rights reserved.
Mussel Maternal and Paternal Mitochondrial Genomes 953
the two genomes differ in gene content and gene arrangement? Such differences might be expected from two genomes
that have different patterns of transmission and distribution in
male and female tissues and whose sequences have diverged,
on evidence from partial sequences, by more than 20%. (2)
Do different parts of the genome diverge at different rates? (3)
Are there common patterns of divergence among F and M
pairs from different species with DUI?
Materials and Methods
Adult mussels were collected from the port of Heraklion in Crete and from Nea Peramos in Saronicos Gulf,
Greece. Mussels were sexed by microscopic examination
for presence of eggs or sperm in gonad tissue. For the F
genome, total DNA was extracted from the gonad tissue
of females. The M genome is present predominantly in
the gonads of males, whose somatic cells contain the F
genome. In order to minimize the contamination of the
preparation of the M genome by the F, we used the male’s
sperm after induction of spawning.
PROMEGA Taq polymerase was used in all polymerase chain reaction (PCR) reactions. All primer pairs used are
available as Supplementary Material online. To obtain the F
genome, the mtDNA of a female individual was amplified by
long PCR in two fragments. Long PCR amplifications were
carried out in 50-ll reaction volumes containing 50–100 ng
of template DNA, 0.3 mM of each primer, 0.5 mM deoxynucleoside triphosphate, 3.5 mM MgCl2, and 0.75 ll of
enzyme mix (Roche ‘‘Expand Long Template PCR System’’) in buffer 2 supplied by the company. Reaction conditions were in accordance to supplier’s recommendations.
The first pair of primers, Lola1 and LCOIIIr, amplified a fragment of 6.7 kb while the second, LCOIIIf and Lola2, a fragment of 10 kb. These two large fragments served as templates
in PCR reactions to obtain products of 500–1,200 bp. Given
that the majority of the F mitochondrial sequence was known
for M. edulis (Hoffmann, Boore, and Brown 1992), several
sets of primers were designed using this sequence.
The M genome of one male individual was amplified by
long PCR in two fragments using the same reaction
conditions and two sets of primers. The first, 16M-Sl-f (with
a recognition site for SalI) and C3M-Sc-r (with a recognition
site for SacI), amplified a fragment of 6.7 kb. The second,
C3M-f-X and 16M-r-X (both with sites for XbaI), amplified
a fragment of 11 kb. The product of the first reaction was
digested with SacI and SalI and three fragments of length
1.2, 2, and 3.5 kb were obtained. The digestion of the product
of the second reaction with SacI and XbaI gave four fragments
of length 1.1, 1.2, 1.7, and 7 kb. The 7-kb fragment was subsequently used as template in PCR reactions to obtain three
smaller fragments of 1.7, 2, and 3.5 kb length. All fragments
were cloned into plasmid vector pBluesript II KS (Stratagene) in Escherichia coli DH5a cells. Conjunctive fragments
between sequential clones were amplified by PCR reactions.
Sequencing was performed by an ABI 377 automated
sequencer with the ABI Prism BigDye Terminator Cycle
Sequencing Ready Reaction kit (PE Biosystems), in
addition to the standard dideoxynucleotide chain termination method (Sanger, Nicklen, and Coulson 1977) or the
use of a commercial outlet (MWG Company, Germany). Primer walking was applied when cloned fragments were
greater than ;1.2 kb.
Sequences were characterized after alignment and
comparison with data from the National Center for Biotechnology Information databank, using the Blast network
service (Altschul et al. 1990), or from cited publications.
Alignment of DNA and protein sequences was performed
by ClustalX version 1.83 (Thompson et al. 1997) after selection of optimal parameters (opening and extension gap
penalties). Corrections were made manually aiming at maximizing sequence similarity. tRNA genes were identified by
examining regions known to code for tRNA genes in the
genomes of M. edulis (Hoffmann, Boore, and Brown
1992) and of Mytilus californianus (Beagley, Okimoto,
and Wolstenholme 1999). Sequences that have the potential
to form the characteristic mitochondrial tRNA cloverleaf
structure were found only in these regions of the M.
galloprovincialis genome. To examine whether nucleotide
differences or indels between the F and M genomes accumulate at different rates in different regions of the tRNA
secondary structure (e.g., stems versus loops), nucleotide
differences at the same region of the tRNA structure were
counted over all tRNAs and compared among regions
with a chi-square test. The two methionine tRNAs in
each of the two mitochondrial genomes of M. galloprovincialis (this study) and of V. philippinarum (GenBank
accession numbers AB065375, regions 10602–10669 and
10676–10741, and AB065374, regions 9275–9340 and
9339–9409, respectively; M. Okazaki and R. Ueshima, personal communication) were aligned using the program
ClustalX version 1.83 (Thompson et al. 1997) with equal
‘‘pairwise’’ and ‘‘multiple’’ alignment parameter values
(gap opening and extension penalties 5 3.00), and compared
phylogenetically by a neighbor-joining tree constructed
using the program MEGA version 2.1 (Kumar et al. 2001)
with Jukes-Cantor correction. Potential secondary structures
near or at the 5#-end of protein genes have been produced by
the RNA ‘‘mfold’’ program 3.1 (Zuker 2003). Sequences of
the main control region (CR) were aligned and divided in
domains according to Cao et al. (2004).
Amino acid sequences for protein-coding genes were
obtained using the genetic code of Drosophila mtDNA
(Hoffmann, Boore, and Brown 1992), and nucleotide
alignments were subsequently adjusted to correspond to
amino acid alignments. Zerofold-, twofold-, and fourfolddegenerated positions were identified using software DnaSP
version 3.53 (J. Rozas and R. Rozas 1999). This program was
also used to estimate codon usage and to construct sliding
window plots. Estimation of genetic distances (K) was based
on Kimura’s two-parameter model (1980) using the software
MEGA version 2.1 (Kumar et al. 2001). The divergence of
protein genes in synonymous (Ks) and nonsynonymous (Ka)
sites was calculated by the modified Nei-Gojobori method
with Jukes-Cantor correction, and the p distance at the amino
acid level was calculated using the computer program MEGA
version 2.1 (Kumar et al. 2001). The four-cluster analysis
method under minimum evolution (Rzhetsky, Kumar, and
Nei 1995) was used to examine whether the F and M mitochondrial genomes from different species cluster according
to their mode of transmission or according to species of
954 Mizi et al.
FIG. 1.—Gene maps of the mitochondrial genomes of Mytilus galloprovincialis. All genes are transcribed clockwise. The one-letter amino acid code
is used for tRNA designation. L1, L2, M1, M2, S1, and S2 designate tRNAs recognizing codons CUN, UUR, AUA, AUG, AGN, and UCN, respectively.
Black areas indicate noncoding regions; CR, putative CR; UR1–UR6, unassigned regions 1 to 6. Noncoding sequences of less than 10 bp are not shown.
atp6, ATP synthase subunit 6; cox1–3, cytochrome c oxidase subunits I, II, and III; cob, cytochrome b apoenzyme; nad1–6 and nad4L, nicotinamide
adenine dinucleotide dehydrogenase subunits 1–6 and 4L; rrnaS and rrnaL, small and large subunits of ribosomal RNA. The dotted line shows the
position of a 1,045-bp insertion that was found in the sequenced M genome. ND, corresponds to the first 11 bp of the normal trnN; DCR, corresponds
to bases 77–902 of the CR; Y, is a full copy of the trnY; cobD, corresponds to the first 88 bp of cob; DG, corresponds to the last 56 bp of trnG. Numbers
inside the circle indicate the size of the genome.
origin. For each tetrad of compared sequences, the analysis
produces a ‘‘complement probability’’ (CP) which denotes
how much more probable the produced topology is over any
other alternative topology. CP values larger than 0.95 indicate that the topology is significantly better than any other
topology. For this analysis the amino acid sequences of
any given protein gene from the maternal and the paternal
mitochondrial genomes of three species that are known to
have DUI (M. galloprovincialis, this study; V. philippinarum
and I. japanensis, GenBank accession numbers AB065375,
AB065374, AB055625, AB055624, respectively, M. Okazaki
and R. Ueshima, personal communication) were aligned by
the program ClustalX version 1.83 (Thompson et al. 1997)
using the default parameters, and all alternative topologies
were tested using the program PHYLTEST version 2.0
(Kumar 1996). This was done separately for each protein
gene and for the concatenated alignments of all protein genes.
Sequence data from this article have been deposited
to GenBank under the accession numbers AY497292,
AY363687, AY496974, and AY496975.
Results and Discussion
Genome Size, Nucleotide Composition, and Gene Number
and Order
The F genome of M. galloprovincialis was found to
be virtually identical to the F genome of M. edulis, as pre-
sented in the incomplete form by Hoffmann, Boore, and
Brown (1992) and in the complete form by Boore,
Medina, and Rosenberg (2004). Mytilus edulis and M.
galloprovincialis are members of the M. edulis species
complex (which also includes Mytilus trossulus) they
hybridize in the areas of sympatry, and their taxonomic
status as distinct species is debatable (Gossling 1992).
The sequence of the F genome of M. galloprovincialis
we report here is 16,744 bp, only four nucleotides longer
from the M. edulis F genome assembled by Boore,
Medina, and Rosenberg (2004). Its gene order (fig. 1) is
also identical to that of the M. edulis F genome and the
overall nucleotide divergence, K, is 0.009 (standard
error, SE 5 0.001), which is within the range expected
for conspecific mtDNA genomes. For comparison, the
overall mean divergence of four human mitochondrial
genomes (Cambridge Reference Sequence [NC_001807],
a Swedish [X93334], an African [D38112], and a
Japanese [AB055387]) is 0.005 (SE 5 0.000) and the
mean divergence for the 26 complete genomes of
Drosophila simulans (Ballard 2004) is 0.016 (SE 5 0.001).
The M genome of M. galloprovincialis has the same
gene order as the F genome including noncoding regions
(fig. 1). The most prominent difference of the M genome
from the F is the presence of a 1,045-bp insertion, which
consists of the first 11 bp of trnN and the last 56 bp of trnG
separated by a continuous length of 978 bp that contains part
of the main CR, a full copy of the trnY and the first 88 bp of
Mussel Maternal and Paternal Mitochondrial Genomes 955
cob. Examination of 19 M genomes, each extracted from a
different individual, failed to identify the presence of this
segment. In all these genomes the PCR product obtained
after using primers 12S-f-X and 16M-r-X (see Supplementary Material online), was approximately 1 kb shorter than
the product from the sequenced genome. Two of the short
products were reamplified using the internal primers
SMALL-f-X and LARGE-r-X and found to be ;800 bp,
instead of 1,853 bp which was the size of the product from
the sequenced genome. Part of the two 800 bp products was
sequenced and found to match the corresponding part of the
sequenced genome after the excision of the 1,045-bp segment (GenBank accession numbers AY496974 and
AY496975). This suggests that this segment represents
an insertion that is present in the specific genome we have
sequenced and is not typical of the M. galloprovincialis M
genome. A detailed model of how this insertion was produced will be published elsewhere. The net size of the typical M genome is 16,626 bp, shorter by 118 bp from the F
genome. The difference is accounted mostly by the main CR
and specifically its first and third domain (Cao et al. 2004).
The gene arrangement of both M. galloprovincialis
genomes and the M. edulis F genome are identical to each
other, but is remarkably different from that known in other
fully sequenced metazoan mtDNAs (Hoffmann, Boore,
and Brown 1992; Boore 1999). No overlapping genes are
observed. Both genomes of M. galloprovincialis contain
the full complement of genes of the metazoan mtDNA and
an extra tRNA for methionine, but lack the ATPase8 subunit
gene. There are seven noncoding sequences larger than 10 bp.
Of these the largest (shown as CR in fig. 1) is apparently the
main CR for replication and transcription (Cao et al. 2004).
As noted by Hoffmann, Boore, and Brown (1992), all
genes of the mussel mtDNA are coded by the same strand.
Coding of all genes by the same DNA strand is a feature
of all marine bivalves whose full mtDNA sequence is known
(Crassostrea gigas, NC_001276, S.-H. Kim, E.-Y. Je, and
D.-W. Park, personal communication; V. philippinarum,
AB065374 and AB065375, M. Okazaki and R. Ueshima,
personal communication) but not in the two freshwater species L. ornata (Serb and Lydeard 2003) and I. japanensis
(AB055624, AB055625, M. Okazaki and R. Ueshima, personal communication). In other mollusks a relatively small
number of mitochondrial genes are transcribed from the second strand. The scaphopods Graptacme eborea and Siphonodentalium lobatum are an exception, with about an equal
number of genes encoded by each strand (Boore, Medina,
and Rosenberg 2004; Dreyer and Steiner 2004). The occurrence of all genes in the same strand is a relatively rare phenomenon in metazoans and, in addition to bivalves, has been
reported in some annelids (Lumbricus terrestris, Boore and
Brown 1995; Platynereis dumerilii, Boore and Brown 2000)
and brachiopods (Terebratulina retusa, Stechmann and
Schlegel 1999; Terebratalia transversa, Helfenbein, Brown,
and Boore 2001; Laqueus rubellus, Noguchi et al. 2000).
The nucleotide compositions of the two genomes are
summarized in table 1. The G 1 T content of the F and M
coding strand is 58.1% and 56.8%, respectively, and thus
the sense strand can be considered as the heavy (H) strand
of the molecule. The A 1 T content of the H strand is also
relatively high (61.8%, F; 63.0%, M). Similar values have
been reported in L. ornata (62%, Serb and Lydeard 2003),
Pupa strigosa (61.1%, Kurabayashi and Ueshima 2000),
and Cepaea nemoralis (59.8%, Yamazaki et al. 1997), but
in other mollusks the A 1 T content is much higher (Albinaria
coerulea, 70.7%, Hatzoglou, Rodakis, and Lecanidou 1995;
Katharina tunicata, 69.0%, Boore and Brown 1994; G.
eborea, 74.1%, Boore, Medina, and Rosenberg 2004). This
variation in A 1 T content is among the highest observed
within a phylum and reflects the high heterogeneity of molluscan mtDNA (Boore 1999).
There is a marked bias in favor of T against C, which is
not restricted to any particular class of genes and does not
differ between the two genomes. The GC and AT asymmetry
between the two mitochondrial DNA strands can be
expressed in terms of GC skew and AT skew calculated
according to Perna and Kocher (1995): GC skew 5 (G ÿ
C)/(G 1 C) and AT skew 5 (A ÿ T)/(A 1 T), where G,
C, A, and T are the occurrences of the four bases in the H
strand. In M. galloprovincialis F and M mitochondrial
genomes, the GC skew and the AT skew are F: 10.24
and ÿ0.17, and M: 10.28 and ÿ0.16, respectively. Similar
calculations for fourfold synonymous sites produce a
remarkably high value for GC skew (10.45, F; 10.42,
M), indicating strong bias against codons ending in C. It
is common for animal mitochondrial genomes to deviate
from random usage of nucleotides. The deviation has been
attributed to several factors: unequal presentation of specific
nucleotides in the nucleotide pool; preference of mitochondrial gamma DNA polymerase for specific nucleotides;
a higher incidence of mutation due to longer exposure
of the leading strand during replication (Sueoka 1962;
Asakawa et al. 1991; Jermiin and Crozier 1994; Jermiin
et al. 1994, 1996). L. Cao, E. Kenchington, A. Mizi,
G. C. Rodakis, and E. Zouros (unpublished data) have
obtained evidence for the involvement of the last factor.
The amount of divergence varies along the genome
(fig. 2). For most of the length of the genome the divergence
remains similar to the average of 0.20 (table 1). There are
three regions of high and two regions of low divergence.
The first region of high divergence maps at the major CR
and specifically its first domain (variable domain 1, VD1).
It is followed by a region of low divergence, which maps
in the second domain (conserved domain, CD) of the major
CR. The high divergence of VD1 and the conservation of CD
have been discussed by Cao et al. (2004). The second region
of high divergence corresponds to another noncoding region
(UR1, fig. 1). The second conserved region contains the
nad3 and the adjacent segment of UR4 and most likely corresponds to OL, the origin of the transcription of the lagging
strand (L. Cao, E. Kenchington, A. Mizi, G. C. Rodakis, and
E. Zouros, unpublished data). This conserved region is followed immediately by the third highly divergent point,
which corresponds to the remaining part of UR4.
Ribosomal RNA Genes
The definition of boundaries of rrnaS was based on
the assumption that it occupies all the space between
trnF and trnG. For rrnaL we assumed that the first base
at the 5#-end comes immediately after trnD, but the 3#end of the gene cannot be decided objectively because it is
956 Mizi et al.
Table 1
Length, Base Composition, and Sequence Divergence of F and M Genes and Noncoding Regions
Base Composition (%)
Noncoding
mtDNA Type
Length
T
C
A
G
K
Ks
Ka
CR-VD1
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
654
472
364
359
139
75
115
103
162
150
275
434
1,749
1,650
1,517
1,520
1,244
1,241
947
949
2,191
2,190
717
717
1,665
1,653
729
729
936
936
1,194
1,209
916
916
948
948
351
351
1,308
1,308
282
282
1,705
1,687
477
477
11,228
11,213
16744
16626
29.1
28.0
32.2
32.3
14.4
9.3
33.9
31.1
35.2
34.7
30.5
31.3
29.8
29.6
34.1
34.4
34.3
31.4
32.7
30.2
32.7
30.9
38.8
37.5
33.8
33.4
34.0
34.3
34.9
35.6
36.0
36.8
37.3
37.4
31.5
35.6
38.2
39.1
33.7
35.2
39.4
37.3
35.5
36.3
39.2
39.2
35.3
36.0
34.3
34.5
14.9
17.4
14.3
14.8
13.0
17.3
10.4
18.4
16.0
15.3
11.6
12.2
13.7
15.0
14.0
14.1
14.5
15.1
13.2
14.1
13.2
14.7
14.2
14.0
15.5
17.0
15.2
16.4
16.6
16.5
18.1
18.0
15.0
15.3
13.9
12.0
12.9
11.2
14.9
13.4
12.9
14.7
14.9
14.2
9.1
10.1
14.9
14.8
14.5
14.7
26.8
35.6
37.4
37.1
42.5
41.3
33.0
31.1
24.7
21.3
29.5
34.1
31.0
34.6
32.6
32.7
27.5
33.7
32.3
33.9
32.3
33.8
22.8
22.8
26.7
24.4
26.9
25.6
23.2
22.4
23.8
24.5
22.4
21.3
27.6
26.8
20.7
22.4
25.7
26.4
24.4
26.9
26.6
25.5
25.3
28.5
25.2
25.9
27,5
28.5
29.2
19.1
16.2
15.9
30.2
32.0
22.6
19.4
24.1
28.7
28.4
22.4
25.4
20.8
19.3
18.8
23.8
19.9
21.8
21.7
21.8
20.7
24.2
25.6
24.0
25.3
24.0
23.7
25.3
25.5
22.2
20.6
25.3
28.7
27.0
25.7
28.2
27.3
25.8
25.0
23.3
21.1
23.0
24.0
26.4
22.2
24.5
23.3
23.8
22.3
0.459 (0.047)
NA
NA
0.049 (0.012)
NA
NA
0.459 (0.114)
NA
NA
0.458 (0.099)
NA
NA
0.406 (0.073)
NA
NA
0.239 (0.036)
NA
NA
0.268 (0.017)
NA
NA
0.113 (0.009)
NA
NA
0.156 (0.013)
NA
NA
0.151 (0.014)
NA
NA
0.154 (0.009)
NA
NA
0.216 (0.021)
0.832 (0.101)
0.081 (0.013)
0.192 (0.012)
0.815 (0.066)
0.033 (0.005)
0.218 (0.021)
0.982 (0.127)
0.059 (0.011)
0.223 (0.019)
0.901 (0.099)
0.072 (0.011)
0.213 (0.016)
0.736 (0.070)
0.085 (0.011)
0.233 (0.020)
0.828 (0.090)
0.091 (0.013)
0.282 (0.021)
0.981 (0.108)
0.152 (0.017)
0.127 (0.021)
0.417 (0.079)
0.030 (0.011)
0.276 (0.018)
0.934 (0.085)
0.102 (0.011)
0.253 (0.037)
1.048 (0.214)
0.128 (0.028)
0.276 (0.016)
0.800 (0.063)
0.122 (0.011)
0.258 (0.029)
0.418 (0.068)
0.219 (0.029)
0.235 (0.006)
0.815 (0.025)
0.091 (0.004)
0.215 (0.004)
NA
NA
CR-CD
CR-VD2
UR1
UR2
UR4
CR1UR1-6
tRNA Genes
rRNA Genes
All tRNA genes
rrnaL
rrnaS
All rRNA genes
Protein genes
Divergence (SE)
Gene/Region
atp6
cox1
cox2
cox3
cob
nad1
nad2
nad3
nad4
nad4L
nad5
nad6
All proteins
Complete
NOTE.—Length of protein genes includes the initiation and stop codons. NA, not applicable.
followed by a noncoding sequence (CR). For consistency, we
adopted the endpoint suggested by Hoffmann, Boore, and
Brown (1992). The location, the length, and the nucleotide
content of the two ribosomal RNA genes do not differ in the
two M. galloprovincialis genomes. The divergence between
the genomes is the same for the two rRNA genes and smaller
than the average for the genome (fig. 1 and table 1).
Transfer RNA Genes
The identification of tRNA genes was based on their
potential to form cloverleaf structures (fig. 3). All tRNA
genes exist in the F and M genomes of M. galloprovincialis.
Beagley, Okimoto, and Wolstenholme (1999) have shown
that the M. californianus F genome possesses a trnS2 gene
which does not contain a dihydrouridine arm (DHU arm)
and is located between cox3 and trnM1. Absence of a
DHU arm is not unusual in mitochondrial serine tRNAs,
whether they recognize the UCN or the AGN codon
(Tomita et al. 2002; Serb and Lydeard 2003). We have
found that the trnS2 gene of both the F and M genomes
of M. galloprovincialis is also located between cox3 and
trnM1 (fig. 1). Hoffmann, Boore, and Brown (1992)
suggested that trnS2 was located between nad3 and cox1
Mussel Maternal and Paternal Mitochondrial Genomes 957
FIG. 2.—Degree of divergence between F and M genomes. The genome was linearized at the first nucleotide position of CR (fig. 1) and scanned with
a sliding window of 150 bp moved in steps of 50 bp. The two highly conserved regions (horizontal arrows) correspond to: (A) central domain (CD) of the
putative CR; and (B) nad3 plus the adjacent 100 bp of UR4. The three highly diverged regions (downward arrows) correspond to: (C) first variable domain
(VD1) of CR; (D) UR2; and (E) the remaining part of UR4.
in the M. edulis F genome, but Beagley, Okimoto, and
Wolstenholme (1999) presented direct evidence that the
transcript of the region containing this sequence remains
linked to the transcript of cox1 and is in fact a pseudogene.
The sequence proposed by Hoffmann, Boore, and Brown
(1992) as trnS2 was also found in the UR4 of the F genome
of M. galloprovincialis (nucleotide positions 8880–8944)
and can be folded into a typical tRNA secondary structure,
but with a CGA rather than a TGA anticodon, which is the
anticodon in all molluscan mtDNAs. In the M genome of
M. galloprovincialis, this sequence (region UR4, nucleotide positions from 8614 to 8678) cannot form a tRNA-like
structure with a serine anticodon. Further evidence that this
sequence is not a functional tRNA comes from the sequence’s divergence between the two genomes. The Kimura
distance is 0.455 (SE 5 0.116) compared to 0.088 (SE 5
0.041) for trnS2 that is located between cox3 and trnM1.
The 0.455 value is outside the range for homologous tRNAs,
which varies from 0.067 to 0.169.
Both F and M genomes contain 23 tRNA genes, one
more than is typical for the metazoan mtDNA. The additional tRNA has the anticodon TAT, thus the extra tRNA
codes for methionine. Nine out of the 12 protein genes of
the Mytilus genome start with the ATG codon and two with
the ATA (table 2). From this and also from the fact that in
all known metazoan mtDNAs the most common initiation
codon is ATG, we may conclude that the methionine tRNA
with the CAT anticodon represents the ancestral form and
that, as suggested by Hoffmann, Boore, and Brown (1992),
the second methionine tRNA has arisen by duplication. The
F and M genomes of the venerid V. philippinarum also have
two tRNA genes for methionine, but both have the ‘‘ancestral’’ CAT anticodon. The F genome of L. ornata, both F
and M genomes of I. japanensis, and the genome of the
oyster Crassostrea gigas, a species that apparently does
not have DUI (Curole and Kocher 2002), contain only
one tRNA for methionine, again with the CAT anticodon.
Therefore, presence of two methionine tRNA genes is not a
feature of bivalves in general or of bivalve species with DUI
in particular.
The Mytilidae and Ostreidae belong to subclass Pteriomorphia, while the Veneridae belong to subclass Heteroconchia (Carter, Campbell, and Campbell 2000). This and the
fact that the four methionine tRNAs of M. galloprovincialis
and the four tRNAs of V. philippinarum form two distinct
clusters in phylogenetic trees (fig. 4) make the hypothesis
of two independent duplication events more likely than the
alternative hypothesis before the separation of Mytilidae
and Veneridae. The presence of the duplication in both F
and M genomes of mytilids M. edulis–M. galloprovincialis
and of the venerid V. philippinarum may suggest that the
duplication predates the split of the two genomes and thus
the emergence of DUI in these species. But this explanation
would be inconsistent with the absence of the duplication
from the two unionid species, L. ornata and I. japanensis
which have DUI, unless we assumed that DUI emerged independently in each of the three families (Mytilidae, Unionidae,
and Veneridae) in which it is known to occur. A more likely
explanation is that separate duplication events occurred in an
F genome of the M. edulis/galloprovincialis line and in an F
genome of the V. philippinarum line and that these F genomes
replaced their respective M genomes through the phenomenon of ‘‘mtDNA masculinization’’ or ‘‘sex reversal.’’
One feature of DUI in mytilids is that occasionally an F
genome may ride with the sperm and become maternally
inherited from that point onward (Zouros et al. 1994b;
Hoeh et al. 1996, 1997; Saavedra, Reyero, and Zouros
1997; Quesada, Wenne, and Skibinski 1999). Such ‘‘masculinized’’ F genomes are found in high frequencies in
several populations together with the standard M genome
(Ladoukakis et al. 2002) and, on evidence from phylogenetic trees, have replaced the original M lineage and reset
the F/M divergence to zero in several taxa (Hoeh et al.
1997; Quesada, Wenne, and Skibinski 1999). To explain
the presence of the TAT anticodon in one of the duplicates
in the M. edulis–M. galloprovincialis genomes, one would
958 Mizi et al.
FIG. 3.—Putative cloverleaf structures for the 23 tRNA genes of the F Mytilus galloprovincialis genome. Circled nucleotides indicate substitutions in
the M genome. Arrows indicate nucleotides that are present in the M genome but missing from the F. Watson-Crick pairing is shown by solid lines and GT pairs by dots.
need to make the further assumption that a C-to-T mutation
had occurred in the anticodon of one of the duplicate genes of
the F genome before its masculinization. This hypothesis is
fully consistent with the nucleotide divergences of the four
methionine tRNA genes (fig. 4) which join the genes according to their anticodon rather than according to the genome in
which they are found.
The tRNAs are the most conserved genes between
the two M. galloprovincialis mitochondrial genomes
(table 1). Substitutions are unequally distributed among
the various structural parts of the tRNA. Even though
substitutions are more common in loops than in stems, there
are large differences among the four types of stems (P 5
0.001, from the chi-square test) and among the three types
Table 2
Number of Amino Acids and Initiation and Termination Codons in Completely Sequenced Molluscan mtDNAs
Protein Gene
Species
atp8
cox1
cox2
cox3
cob
nad1
nad2
nad3
nad4
nad4L
nad5
nad6
214
213
186
230
225
231
235
220
230
235
234
219
222
211
245
245
238
238
238
ATG/Ta
ATT/Ta
ATT/Ta
ATG/TAA
ATG/TAA
ATG/TAA
ATG/TAG
ATG/TAG
ATG/TAA
ATG/TAG
ATT/TAG
ATG/TAA
ATG/TAG
ATT/Ta
ATA/TAG
GTG/TAG
ATG/TAG
ATG/TAG
ATG/TAA
55
35
53
—
53
55
—
—
53
70
51
53
54
27
—
—
—
—
—
ATG/TAG
ATA/Ta
ATG/TAG
—
ATG/TAA
ATG/TAA
—
—
ATG/TAG
ATG/TAG
ATG/TAG
ATG/TAA
ATG/TAA
ATA/Ta
—
—
—
—
—
509
508
496
505
512
513
512
532b
513
513
510
509
510
509
532
535
551
554
550
TTG/TAa
TTG/TAA
TAT/TAA
ATG/TAG
GTG/TAA
ATG/TAA
GTG/TAA
ATT/b
ATG/Ta
TTG/TAN
ATG/TAA
ATG/TAA
ATG/TAG
ATG/Ta
GTT/TAG
ATA/TAA
ATG/TAA
ATA/TAA
ATA/TAA
224
215
217
233
223
231
226
283
229
226
230
228
224
208
656
515
242
242
242
ATG/TAA
ATA/TAG
ATT/TAG
ATG/TAA
ATG/Ta
ATG/TAA
ATG/TAA
ATG/Ta
ATG/TAG
ATG/TAG
ATG/TAA
GTG/TAA
ATG/TAG
ATG/TAA
ATT/TAA
ATT/TAG
ATG/TAG
ATG/TAG
ATG/TAA
259
262
271
244
259
259
264
262b
259
259
259
263
258
278
291
297
264
311
311
ATG/TAA
ATA/Ta
ATA/Ta
ATA/TAG
ATG/TAA
ATG/TAA
ATA/TAA
b
/TAA
ATG/TAA
ATG/TAG
ATG/TAA
ATG/Ta
ATG/Ta
ATT/Ta
GTT/TAA
GTG/TAG
ATG/Ta
ATG/TAA
ATG/TAA
367
366
380
372
379
379
383
383
379
382
379
375
373
359
415
440
397
397
402
ATA/Aa
ATT/TAA
ATA/TAA
ATG/TAG
ATG/Ta
ATG/TAG
ATT/TAA
GTG/TAA
ATG/TAA
ATC/TAG
ATG/TAA
TTG/Ta
ATG/TAA
ATG/Ta
ATG/TAA
ATG/TAG
ATG/TAA
ATG/TAA
ATG/TAG
299
307
294
340
294
315
298
292
316
300
313
302
297
295
309
305
305
305
305
ATG/TAA
ATG/TAA
ATA/Ta
ATA/TAA
ATT/Ta
ATA/TAA
ATC/TAG
ATA/TAA
ATG/TAA
ATC/TAA
ATA/TAA
TTG/TA
ATT/Ta
ATA/TAA
ATA/TAA
ATA/TAA
GTG/TAa
GTG/Ta
GTG/Ta
307
302
315
332
331
361
319
331
338
321
346
296
311
315
338
338
314
315
315
ATG/TAA
ATT/Ta
ATG/Ta
ATG/TAG
ATG/Ta
ATG/TAA
ATG/TAA
ATA/TAA
GTG/TAG
ATG/TAA
ATG/TAA
ATG/TAG
ATG/TAA
ATA/TAA
ATA/TAG
ATG/TAG
ATG/TAG
ATG/TAG
ATG/TAA
117
115
134
116
116
117
126
119
120
118
124
116
126
108
134
119
116
116
116
ATA/Ta
ATA/TAA
ATT/TAG
ATG/TAG
ATG/TAA
ATG/TAA
ATG/TAA
ATG/TAG
ATG/TAA
ATG/TAG
ATA/TAA
ATA/Ta
ATA/Ta
ATA/TAG
GTG/TAA
ATA/TAA
ATG/TAA
ATG/TAA
ATG/TAA
437
433
417
451
443
430
457
453
442
448
454
454
478
415
452
427
435
435
435
ATG/TAA
ATT/Ta
ATA/Ta
ATA/TAA
ATT/TAA
ATG/TAG
GTG/TAA
ATT/TAG
ATA/Ta
ATT/TAA
ATT/TAA
ATA/TAG
GTG/TAA
ATG/Ta
ATG/TAA
ATA/TAA
ATG/TAA
ATG/TAA
ATG/TAA
99
99
79
94
96
99
98
102
100
98
98
95
93
93
135
95
93
93
93
ATG/Ta
ATA/TAA
ATA/Ta
ATG/Ta
ATT/TAA
ATG/TAG
GTG/TAG
ATA/TAG
ATG/TAG
GTG/TAG
ATG/TAG
TTG/Ta
TTG/Ta
ATA/Ta
ATG/TAG
ATA/TAG
ATG/TAA
ATG/TAA
ATG/TAA
545
545
561
565
559
580
567
588
571
578
566
526
549
553
541
562
568
568
562
ATT/TAG
ATA/TAG
ATG/TAG
ATG/TAG
ATG/Ta
ATA/TAA
GTG/TAG
ATA/TAA
ATG/Ta
ATG/TAG
ATA/TAG
GTG/TAA
TTG/TAG
ATG/TAA
ATA/TAA
GTT/TAG
ATA/TAa
ATA/Ta
ATA/Ta
155
153
164
158
163
168
162
175
166
162
168
161
157
157
163
163
158
158
158
ATG/TAA
ATA/TAA
ATT/Ta
ATG/TAG
ATG/TAG
ATG/TAG
ATC/TAA
ATA/TAG
ATG/Ta
ATT/TAA
ATG/TAG
ATA/TAA
TTG/TAA
ATG/Ta
ATA/TAA
ATG/TAG
ATG/TAA
ATG/TAA
ATG/TAA
NOTE.—Aco, Albinaria coerulea (Hatzoglou, Rodakis, and Lecanidou 1995); Bgl, Biomphalaria glabrata (DeJong, Emery, and Adema 2004); Cne, Cepaea nemoralis (Yamazaki et al. 1997); Cgi, Crassostrea gigas (GenBank accession
number NC_001276, S.-H. Kim, E.-Y. Je, and D.-W. Park, personal communication); Geb, Graptacme eborea (Boore, Medina, and Rosenberg 2004); Hru, Haliotis rubra (GenBank accession number NC_005940, B. T. Maynard, L. J. Kerr,
J. M. McKiernan, E. S. Jansen, and P. J. Hanna, personal communication); Ija_F and Ija_M, Inversidens japanensis F- and M-type genomes practically complete (GenBank accession numbers AB055625 and AB055624, M. Okazaki and
R. Ueshima, personal communication); Ktu, Katharina tunicata (Boore and Brown 1994a); Lor, Lampsilis ornata (Serb and Lydeard 2003); Lbl, Loligo bleekeri (Tomita et al. 2002); Pst, Pupa strigosa (Kurabayashi and Ueshima 2000); Reu,
Roboastra europaea (Grande et al. 2002); Slo, Siphonodentalium lobatum (Dreyer and Steiner 2004); Vph_F and Vph_M, Venerupis (Ruditapes) philippinarum F- and M-type genomes (GenBank accession numbers AB065375 and AB065374,
M. Okazaki and R. Ueshima, personal communication); Med_F, Mytilus edulis F-type genome (Boore, Medina, and Rosenberg 2004); Mga_F, and Mga_M, Mytilus galloprovincialis F- and M-type genomes (this study).
a
TAA stop codon is completed by addition of 3# A residues to the mRNA.
b
Incomplete data.
Mussel Maternal and Paternal Mitochondrial Genomes 959
Aco
Bgl
Cne
Cgi
Geb
Hru
Ija_F
Ija_M
Ktu
Lor
Lbl
Pst
Reu
Slo
Vph_F
Vph_M
Med_F
Mga_F
Mga_M
Initiation and termination codons Aco
Bgl
Cne
Cgi
Geb
Hru
Ija_F
Ija_M
Ktu
Lor
Lbl
Pst
Reu
Slo
Vph_F
Vph_M
Med_F
Mga_F
Mga_M
Number of amino acids
atp6
21 (91.3)
NA
NA
NA
2 (8.7)
23
a
For the two serine tRNAs all bases between amino acid acceptor stem and anticodon stem were considered as the DHU loop. NA, not applicable.
70 (59.3)
NA
NA
NA
48 (40.7)
118
219 (95.2) 159 (98.8) 87 (89.7) 181 (91.0)
6 (2.6)
NA
NA
15 (7.5)
2 (0.9)
NA
NA
1 (0.5)
3 (1.3)
NA
NA
2 (1.0)
NA
2 (1.2) 10 (10.3)
NA
230
161
97
199
16 (76.2)
NA
NA
NA
5 (23.8)
21
Total
Extra
arm
One Base Anticodon Anticodon
After DHU
Stem
Loop
DHU
loopa
DHU
stem
181 (97.3) 112 (76.7)
3 (1.6)
NA
1 (0.5)
NA
1 (0.5)
NA
NA
34 (23.3)
186
146
42 (100.0)
NA
NA
NA
0
42
286 (88.8)
28 (8.7)
0 (0.0)
8 (0.5)
NA
322
Identical nucleotides in the two genomes
Substitutions that do not affect base pairing
Substitutions in stems where no pairing occurs
Substitutions that affect pairing
Substitutions and indels in loops
Total
Ojala, Montoya, and Attardi (1981) suggested that
the secondary structure of a tRNA gene between a pair of protein genes is responsible for the precise cleavage of the polycistronic primary transcript. In the absence of an intervening
tRNA, this role can be played by a stem-loop structure, the 5#end part of the gene itself, or a combination of the two. Potential hairpin structures at protein-protein gene junctions with
no intervening tRNA have been reported in several studies
(e.g., Bibb et al. 1981; Clary and Wolstenholme 1985;
Okimoto et al. 1992; Boore and Brown 1994). Nineteen of
the 23 tRNA genes of the M. galloprovincialis genome are
clustered in four groups of two to seven members (fig. 1).
Of the remaining four, one (trnY) lies immediately before
TWC-stem TWC-loop
Discriminator
Base
Secondary Structures at Junctions Between
Protein-Coding Genes
Amino acid
Two Bases
Acceptor Stem Before DHU
of loops (P 5 6.5 3 10ÿ11, from the chi-square test).
Excepting the two bases before DHU which are fully conserved, we may recognize four classes according to the
degree of conservation (table 3). One class consists of
the DHU stem, the anticodon stem, and the anticodon loop
all of which are equally and highly conserved (P 5 0.129,
from the chi-square test) with an average of 3% substitution
rate. The amino acid acceptor stem, the extra arm, the TWC
stem, and the discriminator base form the second class with
a mean substitution rate of 10% (P 5 0.879, from the chisquare test). The DHU loop at 23% and the TWC loop at
40% form the two most variable classes. In total, only 8.2%
of nucleotide differences between the two genomes are in
sites that affect base pairing; 59.1% of the substitutions are
located in loops. Another 30.4% are located in stem positions but have not affected base pairing. The remaining
2.3% of substitutions are located in positions where no pairing is observed. The overall small degree of divergence of
tRNAs can therefore be attributed to the small number of
sites that do not affect tRNA function. Yet, even nucleotides that are located in loops cannot be considered as fully
neutral because several of them participate in the formation
of the tertiary structure of the molecule (Dirheimaer et al.
1995).
Table 3
Number of Nucleotide Substitutions (percent in parenthesis) in Different Regions of the tRNA Secondary Structure Summed Over All tRNA Genes
FIG. 4.—Neighbor joining tree of the methionine tRNAs of Mytilus
galloprovincialis and Venerupis philippinarum mitochondrial genomes. F
and M denote gender-specific genomes. Numbers 1 and 2 denote orthology. trnM1 of M. galloprovincialis are located between trnS2 and
nad2 and have the TAT anticodon, whereas trnM2 are located between
genes trnK and trnL1 and have the CAT anticodon. trnM(1) and trnM(2)
of V. philippinarum are found in tandem between trnY and trnD in both
genomes and both have the CAT anticodon. Numbers indicate percentage
of bootstrap support from 1,000 replicates.
1,374 (88.9)
52 (3.4)
4 (0.3)
14 (0.9)
101 (6.5)
1,545
960 Mizi et al.
Mussel Maternal and Paternal Mitochondrial Genomes 961
FIG. 5.—Putative secondary structures preceding or included in the 5#-end of protein-coding genes that are not preceded by a tRNA. UR1, UR2, and
UR4 precede cox2, nad1, and cox1, respectively. For cox3, atp6, nad5, and nad6 the putative structure includes the initiation codon (enclosed). Numbers
indicate nucleotide positions or length in bp between two structures. Solid lines indicate Watson-Crick pairing and dots G-T pairs.
the 5#-end of cob and the other three (trnV, trnT, trnF) occupy
positions between protein-coding genes. Thus, 5 of the 12
protein-coding genes (cob, nad4, nad2, nad3, and nad4L)
have a tRNA preceding their 5#-end. Four other genes
(cox2, nad1, cox1, and nad5) have a noncoding sequence preceding their 5#-end that is capable of forming a stem and loop
structure (fig. 5). In cox1 this structure corresponds to the
pseudo-trnS2, which Beagley, Okimoto, and Wolstenholme
(1999) have shown is included in the cox1 mRNA (discussed
above). This leaves only three protein-coding genes (cox3,
atp6, and nad6) without a tRNA or a noncoding sequence
at the 5# end. In all these three genes, a putative stem-and-loop
structure that includes the translation initiation codon can be
formed downstream from the 5#-end part of the gene (fig. 5).
Protein Genes
The ATPase8 subunit gene, that is normally present
in the metazoan mitochondrial genome, is missing from
the mussel mtDNA, thus reducing the protein genes to 12.
Atp8 is also missing from the mtDNAs of other bivalves
(Crassostrea, Venerupis, and Inversidens), nematodes
(Okimoto et al. 1992), platyhelminths (Le et al. 2000), and
chaetognaths (unpublished data, cited in Boore, Medina,
and Rosenberg 2004) but, interestingly, is present in the mitochondrial F genome of the unionid bivalve L. ornata (Serb
and Lydeard 2003).
The protein-encoding genes are of the same length in
the two genomes, except for cox1, cob, and nad5 in which
the length difference is 4, 5, and 6 amino acids, respectively
(table 2). An ambiguity arises concerning the termination
point of cox3. The size of this gene is relatively conserved
in metazoans (normally below 300 codons; see examples
in table 2), but in both M. galloprovincialis genomes this
gene has a length of 311 codons. Hoffmann, Boore, and
Brown (1992) were the first to note the increased number
of codons in the F genome of M. edulis and suggested that
the polypeptide may consist of 311 amino acids or that transcription is terminated before the end of the open-reading
frame. Based on the sequence similarity to other known
cox3 genes, Boore, Medina, and Rosenberg (2004) suggested that the gene in M. edulis may have an incomplete
transcription termination codon resulting in a transcript of
264 codons. This hypothesis is supported by the fact that
the additional 47 amino acid sequence bears no resemblance to any known cox3 genes. Alternatively, one may
962 Mizi et al.
FIG. 6.—Amino acid divergence (p distance) of three pairs of conspecific genomes for the 12 protein genes. Black bars: Mytilus galloprovincialis; gray bars: Venerupis philippinarum; blank bars: Inversidens
japanensis.
use the conventional rule that preference for the end of the
coding part of a gene must be given to a complete termination codon, as long as it appears before the first nucleotide of the next gene. According to this convention, the gene
must be assumed to consist of 311 amino acids. Our comparison of the two genomes is based on the assumption that
the extra length of 47 codons belongs to the protein-coding
part of the genome.
Synonymous (Ks) and non-synonymous (Ka) values
between the two genomes vary among protein genes (table
1). Ka is particularly low for cox1, but Ks is not, suggesting
that this gene is under selective constraint. The conservation of cox1 is common in animal mtDNA (Pesole et al.
1999; Saccone et al. 1999). Ks and Ka deviate from average
in nad6, but in opposite ways. In nad3 both K values are
lower than average. Similar comparisons can be made for
the V. philippinarum and I. japanensis genomes. In both
pairs the cox1 has the lowest divergence and the nad6 divergence is among the largest, but in neither pair is the nad3
divergence particularly low (fig. 6). The low rate of evolution of nad3 relative to other protein genes seems to be a
characteristic property of the M. edulis species complex. A
broad survey of sequenced animal mitochondrial genomes
(table 4) shows that a rate of evolution at the nad3 gene as
low as in Mytilidae is found in Petromyzoniformes, but this
low rate is shared by all protein-coding genes of Petromyzoniformes (see ‘‘ratio(b)/(a)’’ in table 4). The explanation
for the conservation of the nad3 gene in the M. edulis species complex may lie in the fact that nad3 contains the origin of the replication of the lagging stand (L. Cao, E.
Kenchington, A. Mizi, G. C. Rodakis, and E. Zouros,
unpublished data).
Codon Usage
All codons occur in both M. galloprovincialis mitochondrial genomes (table 5). TTT (phenylalanine) is the most
frequent codon followed by TTA (leucine). TTT is also the
most frequent codon in L. ornata (Serb and Lydeard 2003)
and in C. nemoralis (Terrett, Miles, and Thomas 1996),
whereas TTA is most common in A. coerulea (Hatzoglou,
Rodakis, and Lecanidou 1995), P. strigosa (Kurabayashi
and Ueshima 2000), Roboastra europaea (Grande et al.
2002), G. eborea (Boore, Medina, and Rosenberg 2004),
and K. tunicata (Boore and Brown 1994). These two codons
are the most frequently used codons in other invertebrate
mtDNAs (Garesse 1988; Cantatore et al. 1989; Okimoto
et al. 1992; Asakawa et al. 1995; De Giorgi et al. 1996;
Helfenbein, Brown, and Boore 2001). TTT is also very
Table 4
Average Intrataxon Diversity (p distances) of Mitochondrial Protein Genes of Completely Sequenced Metazoan mtDNAs
Divergence (SE)
Taxon
Class: Actinopterygii (157)
Class: Amphibia (7)
Phylum: Annelida (2)
Phylum: Arthropoda (45)
Phylum: Brachiopoda (3)
Subphylum: Chephalochordata (2)
Class: Chondrichthyes (6)
Phylum: Cnidaria (2)
Class: Dipnoi (3)
Phylum: Echinodermata (7)
Suborder: Petromyzoniformes (2)
Class: Myxini (2)
Class: Mammalia (99)
Phylum: Mollusca (11)
Phylum: Nematoda (10)
Phylum: Platyhelminthes (10)
Sauropsida (37)
Subphylum: Urochordata (3)
Mytilus galloprovincialis F/M
Venerupis philippinarum F/M
Inversidens japanensis F/M
All Proteins
0.188
0.310
0.444
0.480
0.467
0.111
0.168
0.228
0.260
0.333
0.068
0.178
0.237
0.572
0.492
0.504
0.220
0.494
0.146
0.381
0.531
(0.014)
(0.020)
(0.030)
(0.018)
(0.026)
(0.015)
(0.016)
(0.025)
(0.022)
(0.020)
(0.015)
(0.023)
(0.016)
(0.018)
(0.018)
(0.020)
(0.015)
(0.022)
(0.022)
(0.028)
(0.031)
(a) All Proteins Excluding nad3
0.186
0.310
0.442
0.476
0.466
0.111
0.167
0.226
0.258
0.332
0.071
0.178
0.236
0.568
0.489
0.504
0.254
0.487
0.155
0.388
0.524
(0.013)
(0.018)
(0.029)
(0.017)
(0.024)
(0.015)
(0.015)
(0.024)
(0.021)
(0.019)
(0.014)
(0.022)
(0.015)
(0.017)
(0.017)
(0.019)
(0.014)
(0.021)
(0.022)
(0.026)
(0.030)
(b) nad3
0.206
0.304
0.465
0.522
0.479
0.108
0.184
0.237
0.283
0.353
0.043
0.174
0.251
0.615
0.519
0.511
0.253
0.564
0.043
0.303
0.602
(0.027)
(0.034)
(0.046)
(0.028)
(0.038)
(0.023)
(0.026)
(0.038)
(0.038)
(0.033)
(0.019)
(0.034)
(0.026)
(0.028)
(0.029)
(0.031)
(0.025)
(0.035)
(0.019)
(0.042)
(0.045)
Ratio (b)/(a)
1.108
0.981
1.052
1.097
1.028
0.973
1.102
1.049
1.097
1.063
0.606
0.978
1.064
1.083
1.061
1.014
0.996
1.158
0.277
0.781
1.149
NOTE.—Number in parenthesis under ‘‘Taxon’’ is the number of complete mtDNA sequences included in the alignments (alignment source: NCBI\ENTREZ\).
Mussel Maternal and Paternal Mitochondrial Genomes 963
Table 5
Codon Usage in F and M Genomes
aa
Phe
Codon
N
%
aa
Codon
N
%
aa
Codon
N
%
aa
Codon
N
%
TTT
219
245
75
59
180
191
102
97
67
73
18
20
93
95
50
32
153
148
51
48
145
170
78
69
96
102
33
33
133
131
127
115
5.85
6.55
2.00
1.58
4.80
5.11
2.72
2.59
1.79
1.95
0.48
0.53
2.48
2.54
1.33
0.85
4.09
3.96
1.36
1.28
3.87
4.55
2.08
1.84
2.56
2.73
0.88
0.88
3.55
3.50
3.39
3.07
Ser
TCT
74
74
11
22
31
28
28
18
70
67
14
19
26
31
27
19
58
54
14
14
38
44
14
21
79
77
25
30
67
52
21
20
1.98
1.98
0.29
0.59
0.83
0.75
0.75
0.48
1.87
1.79
0.37
0.51
0.69
0.83
0.72
0.51
1.55
1.44
0.37
0.37
1.01
1.17
0.37
0.56
2.11
2.06
0.67
0.80
1.79
1.39
0.56
0.53
Tyr
TAT
100
96
60
60
9
11
3
1
50
48
24
24
38
34
19
22
70
79
44
37
71
86
46
39
40
39
31
29
46
54
57
47
2.67
2.57
1.60
1.60
0.24
0.29
0.08
0.03
1.33
1.28
0.64
0.64
1.01
0.91
0.51
0.53
1.87
2.11
1.18
0.99
1.90
2.30
1.23
1.04
1.07
1.04
0.83
0.78
1.23
1.44
1.52
1.25
Cys
TGT
49
50
27
25
45
55
57
54
27
29
12
15
24
20
19
18
58
58
27
30
86
82
78
76
66
79
37
37
65
67
142
120
1.31
1.34
0.72
0.67
1.20
1.47
1.52
1.44
0.72
0.78
0.32
0.40
0.64
0.53
0.51
0.48
1.55
1.55
0.72
0.80
2.30
2.19
2.08
2.03
1.76
2.11
0.99
0.99
1.74
1.79
3.79
3.21
TTC
Leu
TTA
TTG
Leu
CTT
CTC
CTA
CTG
Ile
ATT
ATC
Met
ATA
ATG
Val
GTT
GTC
GTA
GTG
TCC
TCA
TCG
Pro
CCT
CCC
CCA
CCG
Thr
ACT
ACC
ACA
ACG
Ala
GCT
GCC
GCA
GCG
TAC
Ter
TAA
TAG
His
CAT
CAC
Gln
CAA
CAG
Asn
AAT
AAC
Lys
AAA
AAG
Asp
GAT
GAC
Glu
GAA
GAG
TGC
Trp
TGA
TGG
Arg
CGT
CGC
CGA
CGG
Ser
AGT
AGC
AGA
AGG
Gly
GGT
GGC
GGA
GGG
NOTE.—In each codon the upper value corresponds to F and the lower to M genome. Incomplete termination codons were not excluded. Codons that match the corresponding tRNA anticodon are underlined; aa, amino acid; N, number of occurrences of the codon in the genome.
frequent in primitive chordates (like amphioxus, Branchiostoma lanceolatum, Spruyt et al. 1998), but not in most of vertebrates, where CTA (e.g., Cyprinus, Chang, Huang, and Lo
1994; Homo sapiens, Ingman et al. 2000) or ATT (e.g.,
Xenopus laevis, Roe et al. 1985; Danio rerio, Broughton,
Milam, and Roe 2001) are the most frequently used codons.
The four least used codons in M. galloprovincialis mtDNA
are TCC, CGC, ACC, and ACG. Of these, CGC is also among
the least common in the mtDNA of other mollusks.
Synonymous codons, whether fourfold (4FD) or twofold (2FD) degenerate, are recognized by the same tRNA
(table 5), with the exception of the methionine codons
which are recognized by a different tRNA (see above).
In nine 2FD and seven 4FD families the most frequently
used codon does not match the tRNA’s anticodon. This
has been observed in other metazoan mtDNA as well
(Roe et al. 1985; Wolstenholme 1992, Rand and Kann
1998; Crease 1999; Broughton, Milam, and Roe 2001)
and suggests that strict codon-anticodon complementarity
has not affected the codon composition of the genome.
Deviations from equal frequency of the four nucleotides in 4FD sites are common in the animal mtDNA
and have been attributed to several factors, such as unequal
presence of the four nucleotides in the nucleotide pool,
preference of the mitochondrial gamma DNA polymerase
for specific nucleotides, or asymmetrical mutation rate
owing to different duration of exposure of the lagging
strand during replication (Sueoka 1962; Asakawa et al.
1991; Jermiin and Crozier 1994; Jermiin et al. 1994,
1996). L. Cao, E. Kenchington, A. Mizi, G. C. Rodakis,
and E. Zouros (unpublished data) have noted that 4FD sites
with longer exposure have a higher probability to be occupied by T and a lower probability to be occupied by G. This
observation implies that the frequency with which the four
codons of an amino acid are used depends on the distribution of the amino acid’s residues along the genome. Yet,
these correlations account for less than a third of the bias
in codon usage and cannot explain the bias in 2FD codon
families. Thus, other factors must also affect codon bias.
One among these may be the degree with which tRNAs recognize their different synonymous codons. If this degree
varies among codons, the tRNA itself may act as a selection
factor for synonymous mutations and may thus determine
the frequency of synonymous codons in the genome.
Implications for DUI of mtDNA
The two mitochondrial genomes of M. galloprovincialis are remarkably similar in gene content, gene arrangement, nucleotide composition, and codon usage in spite
the fact that their primary DNA sequence has diverged
by 20% and, more importantly, that their mode of transmission and distribution among female and male tissues is dramatically different. This is not true for the F and M genomes
964 Mizi et al.
FIG. 7.—A treelike presentation of the results of four-cluster analysis
based on the concatenated amino acid sequence of all 12 protein-coding
genes of the F and M genomes of Mytilus galloprovincialis, Venerupis
philippinarum, and Inversidens japanensis. CP: complement probability.
The p distance for the concatenated amino acid sequences between the F
and M genomes of M. galloprovincialis, V. philippinarum and I. japanensis is 0.139 (SE 5 0.006), 0.343 (SE 5 0.008) and 0.506 (SE 5 0.008),
respectively. Branch length is proportional to p distance.
of two other species (V. philippinarum and I. japanensis)
whose complete sequences are available in GenBank
(accession numbers, AB065375, AB065374, AB055625,
AB055624, respectively; M. Okazaki and R. Ueshima, personal communication). Figure 6 illustrates the amino acid
divergence at all 12 protein genes between the F and M
genomes of M. galloprovincialis, V. philippinarum, and
I. japanensis. In all 12 comparisons the divergence is smallest in M. galloprovincialis and largest in I. japanensis, with
the exception of cox2 and nad4L, where the divergence in
V. philippinarum is higher than in I. japanensis. In order to
examine the phylogenetic status of the F and M mitochondrial genomes among the three species, we applied the fourcluster analysis to all combinations of four sequences drawn
from two different species. Given the large phylogenetic
divergence among the three families, only amino acid
sequences were used for this analysis. When genes were
used individually, the sequences paired according to their
species rather than according to their mode of transmission
for all 12 genes. Figure 7 shows the result of the four-cluster
analysis based on the concatenated alignment of all protein
genes. The CP for any combination is one, suggesting that
the topology joining the sequences according to species is
much better than any alternative topology.
One explanation for the result of figure 7 is that DUI
emerged independently in the lines leading to the three
species. The question of multiple origins of DUI was first
addressed by Hoeh et al. (1996, 1997) on the basis of partial
sequences of F and M genomes from different species of
Mytilidae and Unionidae families. These authors rejected
the hypothesis of multiple origins of DUI in favor of
the hypothesis of ‘‘masculinization’’ or ‘‘role reversal’’
of the F genome and subsequent replacement of the
‘‘old’’ M with the newly masculinized F. This phenomenon
has been observed in pair-matings of M. galloprovincialis
(Saavedra, Reyero, and Zouros 1997) and in natural populations of M. trossulus (Stewart et al. 1995) and M. galloprovincialis (Ladoukakis et al. 2002) where males were
found with two types of F genomes, one in the soma and the
other in the gonad. Role reversal also provides the most
likely explanation for the F and M genomes of M. trossulus
in the Baltic Sea (Quesada, Wenne, and Skibinski 1999).
This explanation suggests that the history of species with
DUI is punctuated with waves of invasion and replacement
of the M line by the F. It remains a matter of speculation
whether the replacement is stochastic or driven by selection.
If we assumed a relatively constant rate, the whole-genome
data comparison we present here indicates that the last wave
in M. edulis–M. galloprovincialis occurred much more
recently than in V. philippinarum or I. japanensis.
Mitochondrial gene arrangement in mollusks is known
to be very different from typical metazoan mtDNAs and to
vary extensively among molluscan species (Boore 1999).
Serb and Lydeard (2003) have discussed this feature in
bivalves from the point of its phylogenetic utility. Concentrating on the three F/M pairs, we see again a marked
difference between M. galloprovincialis, where the gene
arrangement is identical, and I. japanensis, where there
must have been at least two gene-order inversions in the
light strand and one in the heavy strand. Also two tRNAs
(trnD and trnV) are coded by opposite stands. Interestingly,
the gene order of the M genome of I. japanensis is more
similar to the gene order of the L. ornata F genome published by Serb and Lydeard (2003) than to its conspecific
F genome, an observation that raises the question of
whether gene arrangement can be a reliable taxonomic
character for species with DUI. In V. philippinarum the differences are confined to a gene duplication of cox2 in the F
genome and an extra trnM in the M genome.
The large divergence between the F and M genomes of
I. japanensis is typical of all unionid species that have been
examined so far. Hoeh, Stewart, and Guttman (2002) analyzed that cox1 sequences form a collection of unionid species from seven different genera. All species appeared to
possess the DUI mode of inheritance and, remarkably, F
and M types formed two distinct clusters, suggesting that
no masculinization event has occurred in unionids for
the last 200 Myr, the estimated time of divergence between
the most distantly related species in the collection. Curole
and Kocher (2002) observed that in the unionids Lampsilis
teres, Quadrula quadrula, and Quadrula refulgens, cox2 is
longer in the M genome than in the F and speculated that
this difference may render the F genome unsuitable to function as M. A similar argument can be based on differences
in gene arrangement between F and M genomes if the large
differences observed in I. japanensis, the only unionid species whose complete sequences of both genomes are available, turned out to be typical for unionids.
The small number of mitochondria carried by the
sperm and the preponderance of the M genome in the male
gonad of Mytilidae forced early studies of DUI to suggest
that the M genome enjoys a replication advantage over the
F during male gametogenesis (Skibinski, Gallagher, and
Beynon 1994b; Zouros et al. 1994b; Saavedra, Reyero,
and Zouros 1997). It is known that replication is faster
in mitochondrial genomes with large deletions (e.g., Diaz
et al. 2002 and references therein). As noted, the M genome
of M. galloprovincialis is smaller than the F by 118 bp.
Assuming a linear relation between molecule length and
rate of replication, it would mean that the relative replicative advantage of M will be about 1%, hardly sufficient to
Mussel Maternal and Paternal Mitochondrial Genomes 965
explain this genome’s domination in the male gonad. A
more likely hypothesis is that male primordial cells start
with a mitochondrial population in which egg and sperm
mitochondria are not in the same proportion to the frequencies in the fertilized egg (Cao, Kenchington, and Zouros
2004) or that egg mitochondria are actively eliminated from
the developing gonad.
The M genome occurs almost exclusively in the male
gonad and sperm where the F genome is absent or occurs in
hardly detectable amounts (Garrido-Ramos et al. 1998).
This in combination with this genome’s faster rate of evolution prompted the speculation that the M genome may
simply be a selfish element whose only function is to secure
a ride with the sperm (Hurst and Hoekstra 1994). An opposite suggestion has also been made, i.e., that far from being
nonfunctional the M genome may evolve under pressure to
meet the specific needs of sperm (Skibinski, Gallagher, and
Beynon 1994b). Our results do not address the second
hypothesis but argue against the first. That no premature
termination codons occur in any protein gene of the M
genome is unexpected from a molecule devoid of normal
function for 5 Myr, which is the time of split of the M from
the F genome in the M. edulis species group suggested by
Rawson and Hilbish (1995) on the basis of rrnaS divergence. An even stronger argument can be made from the
F and M genomes of V. philippinarum and I. japanensis
whose F/M split must be much older (fig. 7) and where also
no premature stop codons occur.
The remarkable similarity between the two genomes
of M. galloprovincialis may explain the relatively high frequency of masculinization in the M. edulis species group,
but also makes more difficult to answer the question of
whether F and M genomes contain sequences that are
responsible for their different mode of transmission. The
most notable difference between the two M. galloprovincialis genomes is in the first domain of the large unassigned
region, which most likely corresponds to the major CR
of the genome (Cao et al. 2004). This domain, which is
referred to as the first variable region (VR1), is about
150 bp shorter in the M genome (25% of its length), and
its divergence between the two genomes is almost 50%
(Cao et al. 2004). Similar differences have been found
between F and M genomes of M. trossulus from the Baltic
Sea (Burzynski et al. 2003). As suggested by Burzynski
et al. (2003) and Cao et al. (2004) the CR, and more specifically its VR1 domain, is the most probable region of the
mussel mitochondrial genome to house ‘‘transmissionspecific’’ sequences, i.e., sequences that may be part of
the mechanism that determines whether the genome will
be transmitted through the egg or the sperm. If M- and
F-specific indels and nucleotide sequence differences in
the CR do actually have a transmission-specific role, then
an F genome cannot function as M unless it acquires Mspecific sequences at this part of the molecule. This acquirement might be possible through recombination. Ladoukakis
and Zouros (2001) and Burzynski et al. (2003) have shown
that recombination does indeed occur in the Mytilus
mtDNA. The question can be settled only if several typical
F, typical M, and masculinized F genomes are sequenced.
The possible involvement of recombination in masculinization suggests still another explanation why masculinization
is presently rare or absent in unionids. Given the large differences in gene arrangement between conspecific F and M
genomes, recombination in these species will likely generate nonfunctional molecules and, thus, will prevent the
transfer of critical ‘‘masculinizing’’ sequence domains
from the M genome into the F.
Supplementary Material
Table of oligonucleotides used in PCR reactions and
for sequencing of F- and M-type genomes.
Acknowledgments
This work was supported by the Greek General
Secretariat for Research and Technology (grant PENED01ED42 to A. M., E. Z. and G.C.R.) and by the University
of Athens (to G.C.R.). We are grateful to the two anonymous reviewers and particularly to one of them who made
extensive editorial suggestions on the first draft of the
paper.
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Richard Thomas, Associate Editor
Accepted January 3, 2005