Association Between Chloroplast and Mitochondrial Lineages in Oaks
S. Dumolin-Lapègue, M.-H. Pemonge, and R. J. Petit
Institut National de la Recherche Agronomique, Laboratoire de Génétique et Amélioration des Arbres Forestiers, Cestas,
France
Patterns of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) variation were studied in 378 populations
of oak trees sampled throughout the southern half of France. Six cpDNA haplotypes detected in a previous European
survey and three new cpDNA haplotypes were found in this region. Two mitochondrial polymorphisms detected
earlier by restriction analysis of PCR-amplified fragments alone, or in combination with single-strand conformation
polymorphism (SSCP), were compared with the cpDNA data. Sequencing revealed the nature of the two mitochondrial mutations: a single-base substitution and a 4-bp inversion associated with a 22-bp hairpin secondary
structure. The single-base substitution was then analyzed by allele-specific amplification. Results for the two cytoplasmic genomes were combined, which allowed the identification of 12 cpDNA-mtDNA haplotypes. The 4-bp
mtDNA inversion has appeared independently in different cpDNA lineages. Given the peculiar nature of this mtDNA
mutation, we suggest that intramolecular recombination leading to repeated inversions of the 4-bp sequence (rather
than paternal leakage of one of the two genomes) is responsible for this pattern. Furthermore, the geographic
locations of the unusual cpDNA-mtDNA associations (due to the inversion) usually do not match the zones of
contact between divergent haplotypes. In addition, in southern France, the groupings of populations based on the
mtDNA substitution were strictly congruent with those based on cpDNA. Because many populations that are polymorphic for both cpDNA and mtDNA have remained in contact since postglacial recolonization in this area without
producing any new combination of cytoplasms involving the mitochondrial substitution, we conclude that paternal
leakage is not a significant factor at this timescale. Such results confirm and expand our earlier conclusions based
on controlled crosses.
Introduction
In plants such as conifers, in which chloroplasts
and mitochondria are transmitted by different parents,
no strong association between the two genomes is expected at the intraspecific level (Schnabel and Asmussen
1989; Dong and Wagner 1994). In angiosperms, on the
contrary, chloroplasts and mitochondria are both maternally inherited in most species (Reboud and Zeyl 1994).
As a consequence, the two organellar genomes should
remain associated; that is, they should behave as if they
are completely linked (Schnabel and Asmussen 1989)
and are expected to give similar information on dispersal
and gene flow by seeds.
Occasional events of paternal leakage are difficult
to observe by conventional inheritance studies (Milligan
1992). In oaks, maternal inheritance for cpDNA and
mtDNA was demonstrated in up to 143 progenies from
several controlled crosses (Dumolin, Demesure, and Petit 1995). However, statistical considerations indicate that
paternal contributions as high as 2% cannot be excluded
with these sample sizes. In studies of organelle transmission in angiosperms, the bias toward maternal inheritance is apparently stronger for mtDNA than for
cpDNA, with more cases of biparental or paternal inheritance reported for cpDNA and very few cases of
biparental inheritance for mitochondria (Reboud and
Abbreviations: mtDNA, mitochondrial DNA; cpDNA, chloroplast
DNA.
Key words: mitochondrial DNA, chloroplast DNA, inversion,
Quercus robur complex, allele-specific amplification, postglacial recolonization.
Address for correspondence and reprints: R. J. Petit, Institut National de la Recherche Agronomique, Laboratoire de Génétique et
Amélioration des Arbres Forestiers, B.P. 45, F-33611 Gazinet Cedex,
France. E-mail: [email protected].
Mol. Biol. Evol. 15(10):1321–1331. 1998
q 1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
Zeyl 1994). However, for most species, including those
for which plastids seem to be completely absent from
the male gametes (as in oaks; Hagemann and Schröder
1989), some mitochondria from the male parent seem to
reach the egg (Chapman 1986). It therefore seems difficult to rule out a priori paternal leakage for either of
the two organelles.
Theoretical studies indicate that rare events of paternal transmission can have disproportionate importance. For example, Petit, Kremer, and Wagner (1993a)
have shown that paternal transmission rates as low as
1% in cytoplasmic genomes can have a significant effect
on their genetic structures, especially when there is an
asymmetry between pollen and seed flow, a likely situation in many plants, including oaks. According to Birky (1978): ‘‘if there are very low levels of paternal gene
transmission and recombination, these must be measured for they may become important over evolutionary
time scales even though they are negligible when we
look at the results of a single mating.’’ If paternal gene
flow is so low that it cannot be measured in controlled
crosses, indirect assessments must be proposed.
If it could be demonstrated that both organelle lineages followed a strict parallel and clonal type of evolution over a long period, the hypothesis that they are
both strictly maternally inherited would be strengthened.
Such a test should be possible by simultaneously analyzing cpDNA and mtDNA polymorphisms for numerous individuals and populations in geographic regions
where new combinations of the organelles would be
readily detected. However, to interpret such results, a
precise knowledge of the mutations involved, especially
in the case of the mtDNA genome, is necessary. Indeed,
due to the frequent and complicated rearrangements of
the genome that are known to occur during plant mtDNA evolution (Palmer 1992), recurrent mutation
events could easily compromise the interpretations.
1321
1322
Dumolin-Lapègue et al.
Simultaneous information on the polymorphism of
both organellar genomes has been obtained at a variety
of taxonomic levels for angiosperms (e.g., Berthou, Mathieu, and Vedel 1983; Holwerda, Jana, and Crosby
1986; Ishikawa et al. 1992; Ishii et al. 1993; Laurent,
Risterucci, and Lanaud 1993; Saumitou-Laprade et al.
1993; Tsunewaki 1993). However, for plant species
where both organellar genomes are at least predominantly maternally inherited, the association between the
two lineages has not been explicitly studied, even when
apparently incongruent results were observed (Timothy
et al. 1979).
In this study, we asked whether paternal transmission of cpDNA or mtDNA genomes has occurred since
recolonization of southern France by white oaks, about
9,000 years ago. To answer this question, we screened
378 populations of oaks with both cpDNA and mtDNA
markers and studied the association between the two genomes. Previous studies have identified several cpDNA
lineages in these oaks that likely originated from different glacial refugia (Dumolin-Lapègue et al. 1997). All
the main lineages meet in southern France. Furthermore,
preliminary studies indicated that some of these cpDNA
lineages also differed at two mtDNA variants. Hence,
the prospect of detecting ‘‘recombinant’’ associations of
cpDNA and mtDNA in this region seemed to be particularly good, if indeed paternal leakage of one of the two
organelles does occur at low frequencies.
Materials and Methods
Plant Material
Four or five individuals from each of 378 oak populations (a total of 1,749 samples) were sampled
throughout southern France and analyzed with both
cpDNA and mtDNA markers. About 150 samples from
the same area, which were included in a previous
cpDNA study (Dumolin-Lapègue et al. 1997), were analyzed with the mtDNA fragments and the results reported here. The four oak species studied belong to
Quercus subgenus Quercus, section Quercus (the white
oaks), in the family Fagaceae (Nixon 1993). The most
abundant species were Quercus robur L. (pedunculate
oak; 716 individuals) and Quercus pubescens Willd.
(pubescent oak; 720 individuals), followed by Quercus
petraea (Matt.) Liebl. (sessile oak; 274 individuals) and
Quercus pyrenaica Willd. (Pyrenean oak; 39 individuals). The sampled trees were separated by at least 50 m;
populations were located 20 to 30 km apart. As far as
possible, the material (acorns, buds, or leaves) was collected from old trees in ancient forests to reduce the risk
of sampling planted populations. In this paper, no additional reference will be made to the species of the trees
analyzed, because a nearly systematic local sharing of
cytoplasms between species has been documented, a
consequence of extensive interspecific hybridization and
introgression (Petit, Kremer, and Wagner 1993b; Dumolin-Lapègue et al. 1997; Petit et al. 1997).
Restriction Analysis of PCR-Amplified Fragments
Total DNA was extracted following the procedure
described by Dumolin, Demesure, and Petit (1995), ex-
cept that 1,4-dithiothreitol was used in place of b-mercaptoethanol. This DNA was used as a template in PCR
reactions involving a set of conserved primers located
in coding regions of cpDNA or mtDNA separated by
potentially more variable noncoding regions (mostly intergenic regions for cpDNA and introns for mtDNA).
Three cpDNA primer pairs, trnD/trnT (DT), psaA/trnS
(AS) (described by Demesure, Sodzi, and Petit 1995),
and trnT/trnF (TF) (described by Taberlet et al. 1991),
and two mtDNA primer pairs, nad1 exon2/nad1 exon3
(nad1-2/3) and nad4 exon1/nad4 exon2 (nad4-1/2) (Demesure, Sodzi, and Petit 1995) were used. The three
pairs of cpDNA primers were chosen because they allowed the identification of all cpDNA haplotypes that
had been detected in France in a previous study (Dumolin-Lapègue et al. 1997). The two pairs of mtDNA
primers were selected because they allowed detection of
polymorphisms in this part of the geographic range of
oaks, either by PCR-RFLP (restriction fragment length
polymorphism), for nad4-1/2, or by PCR-RFLP-SSCP,
for nad1-2/3 (Dumolin, Demesure, and Petit 1995; Dumolin-Lapègue, Bodénès, and Petit 1996). The PCR procedure was performed as described by Demesure, Sodzi,
and Petit (1995). The PCR products (5 ml) were digested
with 4-bp restriction endonucleases (5 U): TaqI and AluI
for fragment DT and HinfI for the other four fragments.
The resulting restriction fragments were separated by
electrophoresis on 8% polyacrylamide gels as described
by Dumolin, Demesure, and Petit (1995).
DNA Sequencing
Sequencing of the mtDNA variants was performed
in order to help distinguish between recurrent mutations
and true paternal contamination when new cpDNAmtDNA associations were detected. To sequence the
mtDNA fragments, new pairs of primers were synthesized by adding a 17-bp forward M13/pUC sequence at
the 59 ends of the primers nad1 exon2 and nad4 exon1,
and adding an 18-bp reverse M13/pUC sequence at the
59 ends of the primers nad1 exon3 and nad4 exon2. PCR
parameters were left unchanged, and mtDNA PCR products were purified using a QIAquick PCR purification
kit (Qiagen). DNA sequencing was carried out using a
LI-COR model 4000L automated DNA sequencer. Sequencing reactions were performed with the Sequitherm
EXCEL Long-Read DNA Sequencing Kit-LC (Epicentre Technologies), and fragments were separated on
LongRanger (FMC) or Sequagel XR (National Diagnostics) gels at a concentrations of 6.0% (for 41-cmlong gels) and 4.3% (for 66-cm- long gels). Sequencing
was performed in both directions with the M13/pUC
forward and reverse primers. Additional internal sequencing primers, designed using the OLIGO software,
version 4.1 (Rychlik and Rhoades 1989), were used (one
for nad1 and two for nad4) because of the large sizes
of the fragments.
PCR Amplification of Specific Alleles
The availability of the sequence of the nad1-2/3
intron made it possible to use another technique to
screen for the nad1-2/3 mtDNA polymorphism in the
Association Between cpDNA and mtDNA Lineages
1323
FIG. 1.—Allele-specific amplification performed to study a substitution in the nad1-2/3 fragment. The three primers used are A1, A2, and
A3. After amplification, the control and specific fragments are separated in agarose gels. The specific fragment is present only when the individual
has the nad1-2/3 b type.
oak populations. No commonly available restriction enzyme would differentiate between the two alleles; therefore, we adapted a technique of allele-specific amplification (Okayama et al. 1989; Wu et al. 1989). The solution that we selected is described in figure 1. We included a control fragment that spans the polymorphic
site in a priming competition with the specific fragment.
As in the original protocol, two primers were designed
to amplify one specific allele based on the sequence obtained for the nad1-2/3 fragment. Therefore, the first oligonucleotide (A1; fig. 1) was chosen such that the polymorphic base was at its 39 end. A second primer (A2)
was similarly designed for the other strand. Furthermore,
we included a third primer (A3), which was designed
for the same strand as A1. PCR was performed (1 cycle
of 4 min at 948C; 30 cycles of 45 s at 948C, 45 s at
588C, 45 s at 728C) using these three primers after optimization of the annealing temperature with a Robocycler Gradient 96 (Stratagene). Two fragments can
therefore be amplified: A1A2, if the 39 end of A1 matches the DNA template, and A1A3, which should always
be amplified and which acts as a PCR control (fig. 1).
Further optimizations were needed because a single-base
mismatch is not always sufficient to hinder elongation.
First, MgCl2 concentration was decreased to 1 mM. Second, because a competition was observed between primers A1 and A3 (yielding an excess of the fragment
A1A2), the concentration of A1 was decreased. The optimized concentrations were 0.20 mM for A2, 0.02 mM
for A1, and 0.18 mM for A3. PCR products were sep-
arated in 1% agarose gels. High throughput was
achieved by using large agarose gels that could be loaded up to three times with 96 samples each time (fig. 2).
However, once the conditions had been optimized and
most samples had been analyzed, a modification of the
polymerase by the manufacturer resulted in nonspecific
amplifications. Nevertheless, most trees were screened
for this mutation, as detailed below.
Screening of the Populations
Fragments DT, TF, and nad4-1/2 were studied for
all individuals (1,749) by PCR-RFLP with one restriction enzyme each, TaqI for DT and HinfI for the two
other fragments. Fragment AS was used only to distinguish between two cpDNA haplotypes (1 and 2, see Dumolin-Lapègue et al. 1997). The unusual haplotypes detected in the survey were studied with the combination
DT-AluI to check whether they had a specific point mutation that groups cpDNA haplotypes of a particular lineage (Dumolin-Lapègue et al. 1997). The substitution
located in fragment nad1-2/3 was studied by allele-specific amplification for a subset of 1,613 individuals
(92% of all individuals).
Results
cpDNA Polymorphisms and cpDNA Haplotypes
Six of the 23 cpDNA haplotypes identified in an
earlier study of cpDNA diversity in oak species throughout Europe (Dumolin-Lapègue et al. 1997) were found
1324
Dumolin-Lapègue et al.
FIG. 2.—Allele-specific amplification: separation of specific and control PCR fragments in 1% agarose gel. The picture shows one third of
the agarose gel that was loaded three times successively. The molecular marker (M) was loaded only once during the first run. The specific
fragment (A1A2) is present only when the individual has the nad1-2/3 b type, while the control fragment (A1A3) is always present.
in the region studied. In the previous study, cpDNA haplotypes were grouped into lineages (A, B, and C) on the
basis of numerous (47) cpDNA polymorphisms. Two
lineages (A and C) are supported by high bootstrap values based on parsimony analysis (99%; see fig. 3). Here,
two new polymorphisms were detected in the DT fragment, distinguishing new cpDNA haplotypes, 25 and 26,
attributed to lineages B and A, respectively. Another
cpDNA haplotype (24) belonging to lineage B was also
characterized from a new combination of fragments (table 1 and fig. 3). However, the three new cpDNA haplotypes (24, 25, and 26), in addition to cpDNA haplotype 21, are very rare compared with cpDNA haplotypes
1, 7, 10, 11, and 12. cpDNA haplotype 21 was found in
15 individuals from 5 populations, cpDNA haplotype 24
in 8 individuals from 3 populations, cpDNA haplotype
25 in 1 individual, and cpDNA haplotype 26 in 3 individuals from a single population.
Association Between the mtDNA Polymorphisms and
the cpDNA Haplotypes
Preliminary studies revealed a length variation in
nad4-1/2 digested by HinfI (Dumolin, Demesure, and
Petit 1995) and a conformation polymorphism (SSCP)
for nad1-2/3 digested by HinfI (Dumolin-Lapègue, Bodénès, and Petit 1996). For most of the 23 cpDNA haplotypes from the previous European study, one individual per haplotype had been screened for these two
mtDNA mutations (Dumolin-Lapègue, Bodénès, and
Petit 1996). To further study the association between the
cpDNA and mtDNA haplotypes, the individuals sampled in southern France and already analyzed for cpDNA variation were screened for these two mtDNA
polymorphisms. The number of individuals characterized by each cpDNA-mtDNA combination is given in
table 2. Based on results for the nad1-2/3 fragment, analyzed by allele-specific amplification, all studied indi-
viduals (663) that belong to cpDNA lineage A (i.e.,
cpDNA haplotypes 7 and 26) have the same mtDNA
haplotype (a), while all individuals that do not belong
to lineage A possess the alternative mtDNA haplotype
(b) (table 2). Similarly, all 702 individuals characterized
by cpDNA haplotypes 7 and 26 have the c mtDNA haplotype for the nad4-1/2 fragment, while all individuals
characterized by cpDNA haplotypes 12, 21, 24, and 25
possess the d mtDNA haplotype. For cpDNA haplotypes
1, 10, and 11, however, individuals have either type c
or type d (table 2). In total, 12 cpDNA-mtDNA haplotypes were detected in this study (table 1). To distinguish these haplotypes, the more abundant chloroplastmitochondrial combinations are named according to the
cpDNA haplotypes, whereas the three others are designated 1 bis, 10 bis, and 11 bis. The distribution of the
two variants for each of the two mtDNA markers is
represented on the phylogenetic tree of the cpDNA haplotypes, redrawn from Dumolin-Lapègue et al. (1997),
with the addition of the three new cpDNA haplotypes
(fig. 3a and b).
Sequence Analysis of the mtDNA Polymorphisms
The nad4-1/2 fragment was sequenced for 12 individuals, one from each of the cpDNA-mtDNA haplotypes. All sequences were 2,064 bp in length. The only
difference detected between the sequences was at positions 1096–1099, where mtDNA haplotype c has the
sequence GAAA and mtDNA haplotype d has TTTC.
The presence of a hairpin secondary structure was inferred in the polymorphic region (see fig. 4) using OLIGO, version 4.1. The hairpin stem was 22 bp in length,
which corresponds to a free- energy change (DG) of
226.5 kcal/mol, that is, a high probability of spontaneous formation. Such hairpins are likely to increase the
probability of intramolecular recombination (Kelchner
and Wendel 1996). In the case of recombination inside
Association Between cpDNA and mtDNA Lineages
1325
FIG. 3.—a, Distribution of the mitochondrial polymorphism detected in fragment nad1-2/3 (substitution) on the neighbor-joining chloroplast
tree. Numbers correspond to the 26 cpDNA haplotypes. Those underlined were found in southern France. Lineage A includes five cpDNA types
(4, 5, 6, 7, and 26), lineage B includes five (10, 11, 12, 24, and 25), and lineage C includes three (1, 2, and 3). The two highest bootstrap
values based on parsimony analysis are indicated. Allele a of fragment nad1-2/3 is represented by black squares, and allele b is rpresented by
white squares (table 1). Although the mtDNA haplotype a seems to have appeared two or three times, it must be noted that the positions of
haplotypes 20, 21, and 22 are poorly resolved, and they may therefore form a single clade with lineage A. In contrast to the other mtDNA
mutation (see b), no cpDNA haplotype is polymorphic for the two alleles. b, Distribution of the mitochondrial polymorphism detected in
fragment nad4-1/2 (inversion) on the neighbor-joining chloroplast tree. Notations are as in a, except that allele c of fragment nad4-1/2 is
represented by white circles, allele d is represented by black circles, and half-filled circles indicate that both alleles were found to be associated
with the corresponding cpDNA haplotype. It it clear that the cpDNA and the mtDNA trees are not congruent.
Table 1
Description of the cpDNA, mtDNA, and Combined cpDNA-mtDNA Haplotypes Found in this Study
CPDNAb
MTDNA
CPDNA
LINEAGEa
DT
Taq1
DT
Taq2
DT
Taq3
DT
Taq3/ac
DT
Alu1d
AS
Hinf2
TF
Hinf8
cpDNA
Haplotype
C. . . . . . . . . . . . .
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
1
2
2
2
2
1
2
2
9
9
3
3
3
2
3
3
4
3
—
—
1
2
—
—
—
—
—
—
—
—
1
1
1
1
9
9
9
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
1
2
2
1
1
7
26
10
10
24
25
11
11
12
21
A. . . . . . . . . . . . .
B. . . . . . . . . . . . .
—............
a
nad1-2/3 nad4-1/2 CP-MTDNA
PASAe
Hinf1
HAPLOTYPEf
b
b
a
a
b
b
b
b
b
b
b
b
c
d
c
c
d
c
d
d
c
d
d
d
1
1 bis
7
26
10
10 bis
24
25
11
11 bis
12
21
cpDNA lineages defined by phylogenetic analyses according to Dumolin-Lapègue et al. (1997).
The abbreviated name of the PCR fragment is followed by the name of the restriction enzyme used and the number of the polymorphic fragment, ranked from
the longest (1) to the shortest. The same principle is used to label the length variants, except that 9 indicates a point mutation. New cpDNA haplotypes and
corresponding mutations are shown in boldface.
c The largest of the two fragments generated by the point mutation in fragment DT Taq3 has two length variants.
d The point mutation detected in the DT fragment at an AluI site was used to distinguish haplotype 24 from haplotype 21, a polymorphism that distinguishes
the haplotypes of the B lineage from those of the other lineages.
e This mtDNA fragment was studied with PCR-amplification of a specific allele.
f As long as only one chloroplast-mitochondrial combination is detected, the cpDNA-mtDNA haplotype takes the name of the cpDNA haplotype. New combinations are called ‘‘bis’’ and are underlined.
b
1326
Dumolin-Lapègue et al.
Table 2
Number of Individuals Having Each mtDNA Haplotype
According to Their cpDNA Haplotype
cpDNA
Lineage Haplotype
C. . . . . . .
A. . . . . . .
B. . . . . . .
—a . . . . . .
1
7
26
10
24
25
11
12
21
nad12/3 a1
nad12/3 b1
nad41/2 c
nad41/2 d
0
660
3
0
0
0
0
0
0
142
0
0
470
8
1
225
93
11
92
699
3
4
0
0
250
0
0
51
0
0
518
8
1
7
101
15
NOTE.—Cases of polymorphic mtDNA within a cpDNA haplotype are
shown in boldface.
a A haplotype not grouped in a lineage.
the hairpin, this would lead to an inversion of the 4-bp
loop, yielding the two observed mtDNA haplotypes. Indeed, the 4-bp loop exactly matches the four polymorphic bases, and GAAA is the reverse complement of the
sequence TTTC.
The nad1-2/3 fragment was completely sequenced
for four individuals characterized by the following
cpDNA-mtDNA haplotypes: 7 (two individuals), 1, and
1 bis (one individual each). Again, the fragment had the
same length for the four individuals (i.e., 1,661 bp). The
only difference between them was a 1-bp substitution at
position 941, with an A in mtDNA haplotype a and a
T in mtDNA haplotype b.
Geographic Distribution of the Haplotypes
The geographic distribution of the 12 cpDNAmtDNA haplotypes is clearly not random (fig. 5): haplotypes from lineage B are restricted to the western part
of the studied region, while those from lineage A are in
the eastern part, and those from lineage C are in the
southeastern part. Furthermore, although both haplotypes 1 and 1 bis from lineage C are mostly restricted to
the southeastern part of France, each occupies a separate
area. The three most common haplotypes of lineage B
(i.e., 10, 11, and 12) have similar, but not identical, distributions: haplotype 11 is abundant in a region from the
mouth of the Garonne to the Massif Central and up to
the Rhone valley, whereas haplotype 12 is frequent
along the Atlantic coast, and haplotype 10 has a much
wider distribution in the western part of the studied region. Finally, haplotype 10 bis is found in two distant
populations, one in the center of the studied region and
one north of the mouth of the Garonne, and haplotype
11 bis is found in three populations located close to one
another to the north of the mouth of the Garonne. Although haplotype 7 of lineage A is very abundant in
eastern France, it was also found as far west as the Atlantic coast, in the region where lineage B is widespread.
Such an overlap between lineages A and B is also ap-
FIG. 4.—Association of the 4-bp inversion detected in the nad4-1/2 fragment with a 22-bp stem-loop hairpin. After the formation of the
hairpin, an intramolecular recombination at this level leads to the inversion of the 4 bp at the edge, which generates the two alternative
mitochondrial types c and d.
Association Between cpDNA and mtDNA Lineages
1327
FIG. 5.—Distribution of the 12 cpDNA-mtDNA haplotypes detected in this study of oaks in southern France. Each color corresponds to a
haplotype, and the size of the circle is proportional to the number of individuals analyzed. The ‘‘bis’’ haplotypes are all distinguished by stripes.
parent in the northern part of the studied area, where
haplotype 10 appears to have penetrated a region in
which haplotype 7 is abundant. Only a few exceptions
to this overall pattern were apparent, with some haplotypes of one lineage located far from their main distribution, often mixed with the presumably local haplotype. Moreover, populations fixed for haplotype 1 are
found in the northeastern part of the studied area and
around the Mediterranean coast.
Discussion
Until recently, assessment of inter- and intraspecific
cpDNA diversity has largely relied on RFLP analysis
using Southern hybridization experiments. In plant
mtDNA, intraspecific variation is detectable by RFLP
analysis, but the observed mutations likely result from
structural rearrangements. Although this variation may
be very useful for other applications, it is not appropriate for phylogenetic or other types of evolutionary studies, even at low taxonomic levels (Palmer 1992). Polymorphisms revealed by Southern hybridization are difficult to characterize further by sequencing, especially if
they fall outside of the sequence that is homologous to
the probe. Here, we used only PCR-derived techniques
combined, when necessary, with sequencing. However,
even PCR polymorphisms are difficult to find at low
taxonomic levels. Chloroplast DNA evolves four times
as slowly as the plant nuclear genome (Wolfe, Li, and
Sharp 1987), and this trend is even more pronounced in
long-lived species such as those of Fagaceae (Frascaria
et al. 1993). Nevertheless, intraspecific cpDNA variation
in angiosperms has been demonstrated for many species,
including forest trees, using more sensitive molecular
techniques (Taberlet et al. 1991; Demesure, Sodzi, and
Petit 1995, Dumolin-Lapègue et al. 1997; Soltis et al.
1997). It must be pointed out that such techniques detect
not only point mutations, but also insertion/deletions in
cpDNA, and some sites of noncoding cpDNA sequences
actually appear to be very labile to small insertion/deletion mutations (Clegg et al. 1994), which can lead to
parallel or recurrent mutation events at those sites. Nevertheless, length mutations have provided assessments
of intraspecific phylogenetic relationships that are often
concordant with those obtained via restriction site analysis (e.g., Doebley, Ma, and Renfroe 1987; Gielly and
Taberlet 1994).
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Dumolin-Lapègue et al.
FIG. 6.—Schematic distribution of the main lineages and probable directions of expansion from the inferred refugia (Spanish, Italian, and
Balkanic). The locations of the ‘‘bis’’ haplotypes corresponding to the unusual cpDNA-mtDNA combinations are represented.
Methods such as PCR-RFLP, which were successfully used for cpDNA, proved to be less useful for
mtDNA (Dumolin-Lapègue, Bodénès, and Petit 1996).
Indeed, in contrast to animal mtDNA, plant mtDNA is
very conserved in primary sequence, evolving three
times as slowly as cpDNA (Wolfe, Li, and Sharp 1987).
However, the SSCP technique can be combined with
PCR-RFLP to detect rare polymorphisms in plant
mtDNA (Dumolin-Lapègue, Bodénès, and Petit 1996).
Once detected, SSCP variants can be sequenced to allow
the subsequent design of a rapid method of allele detection.
Our sequence data confirm this low rate of evolution of plant mtDNA. The sequencing of 3,725 bp from
intervening noncoding sequences from several oak trees
belonging to different cpDNA lineages did not reveal
polymorphisms in addition to the two that had been detected previously. The two mtDNA polymorphisms appear radically different in terms of their nature and their
relationships with the cpDNA polymorphisms. In southern France, the polymorphism observed in the nad4-1/
2 fragment shows at least three cases of uncoupling with
the three common cpDNA haplotypes (table 2 and figs.
5 and 6). When plotted onto the cpDNA phylogenetic
tree, this polymorphism also appears to be quite independent of the cpDNA lineages. Although all individuals from cpDNA lineage A have the same mtDNA variant, lineages B and C differ for this mtDNA polymorphism, and four cpDNA haplotypes are polymorphic for
the mtDNA marker (fig. 3b). Such disassociation between cpDNA and mtDNA variation may result from
occasional paternal leakage in the transmission of one
or both organelles. However, the nature of the mtDNA
mutation involved can be a key to understanding this
disassociation. The mtDNA polymorphism is very likely
the result of an inversion associated with a particularly
long hairpin secondary structure. Given the length of the
hairpin, intramolecular recombination is likely to occur
repeatedly, probably even at the intraspecific level. Similar, although shorter, inversions have been identified in
the noncoding regions of cpDNA (Golenberg et al.
1993; Kelchner and Wendel 1996), but none has been
reported so far in plant mtDNA. Hence, we consider that
this observed cpDNA-mtDNA uncoupling may result
from repeated mtDNA recombination and is likely not
due to occasional events of paternal leakage.
The geographical distribution of the haplotypes
and, especially, the localization of the new cpDNAmtDNA combinations (involving the 4-bp mtDNA inversion) may provide additional insights. Indeed, if
these new combinations were systematically found in
areas of contact between complementary haplotypes, the
hypothesis of paternal leakage could nevertheless receive support.
The overall genetic structure that exists today appears to have been built up largely during the last postglacial recolonization. The most likely postglacial recolonization routes for oaks throughout Europe were inferred in a previous study (Dumolin-Lapègue et al.
1997). Three main cpDNA lineages were identified, and
these were probably initially located in different refugia
during the last glaciation before spreading over the rest
of Europe. In the present study focusing on southern
France, the probable routes of recolonization are further
refined (fig. 6). Haplotypes from lineage B have been
treated together, because they are likely to have originated from the same Iberian refugium. However, each
of the three main haplotypes (10, 11, and 12) that belong
to this lineage can dominate locally, yielding a patchy
distribution as previously described (Petit et al. 1997).
The distribution of haplotype 7 from lineage A confirms
its eastern origin (possibly in the Balkans), with a probable northern skirting of the Alps. Two extensions of
haplotype 7 farther west are also apparent, in regions
Association Between cpDNA and mtDNA Lineages
where haplotypes of lineage B are abundant. Reciprocally, haplotypes of lineage B have also extended farther
to the east (fig. 6). Haplotypes 1 and 1 bis are principally
limited to the southeastern part of France, in agreement
with the hypothesis that they originate from an Italian
refugium and that they migrated in France along the
Mediterranean coast. However, two isolated populations
fixed for haplotype 1 are found farther north, near the
Swiss border. In this case, a Swiss origin is possible,
because this haplotype has crossed the Alps to extend
northward in Europe (Dumolin-Lapègue et al. 1997).
Populations present farther west, on the other hand,
could indicate another expansion along the Mediterranean coast up to northeastern Spain, where haplotype 1
has been detected (Dumolin-Lapègue et al. 1997). However, these hypotheses should be confirmed by additional analyses in these regions and in the neighboring countries.
The localization of the new cpDNA-mtDNA combinations can be examined within this general framework (fig. 6). In our earlier work (Dumolin-Lapègue et
al. 1997), we concluded that most cpDNA mutations had
occurred before recolonization. Here, given the limited
distribution of all but one cpDNA-mtDNA haplotype
(haplotype 1 bis, also present in Italy, data not shown),
a more recent origin of the new combinations is more
likely. If these new combinations have appeared locally,
then an examination of the surrounding haplotypes in
figures 5 and 6 may give some hints as to their origins.
Oak trees characterized by haplotype 11 bis near the
mouth of the Garonne are surrounded by individuals of
haplotypes 10 and 12 that could have contributed their
mitochondria to haplotype 11 (also present in the area),
yielding this new combination. Conversely, haplotype
10 bis occurs in two disconnected areas in which no
haplotype could have contributed the new mitochondrial
haplotypes. First, in the northwestern portion of the
studied area, haplotype 10 bis is mixed with haplotype
10. The second region, in which haplotype 10 bis was
found (in the south-central portion of the studied area;
see figs. 5 and 6), is dominated by haplotype 7. Although in this case, a local origin for haplotype 10 bis
is dubious, it remains that a combination involving haplotype 10 with haplotype 7 is unlikely to have yielded
haplotype 10 bis. Indeed, this would mean that haplotype 7 had contributed only part of its mtDNA (cf. table
1). In all, there is only one out of three situations where
a mixing of preexisting haplotypes may have yielded the
new combination. Hence, based on this mtDNA inversion, it is difficult to conclude whether or not paternal
transmission or recurrent mutation is responsible for the
uncoupling of cpDNA and mtDNA haplotypes. However, the likelihood of paternal transmission seems scant
at best, given the molecular structure of the region surrounding the inversion and the apparently random geographic localization of the new combinations.
The other mtDNA fragment (the second intron of
the nad1 gene) separates cpDNA haplotypes of one lineage (A) from all other haplotypes except 18, 20, and
22 (fig. 3a). Note that the cpDNA tree is a neighborjoining tree based on a majority of insertion/deletions
1329
that are abundant and much easier to identify, although
probably more prone to recurrence than point mutations.
Hence, the groupings that are not supported by high
bootstrap values must be considered with caution (more
bootstrap values are represented in fig. 3 of DumolinLapègue et al. 1997). In southern France, no new
cpDNA-mtDNA associations involving the nad1 substitution were detected among all individuals analyzed.
This is remarkable, considering the importance of the
zone of contact between haplotypes of lineage A (shown
in blue in fig. 5) and all other haplotypes. Indeed, note
that when trees characterized by these contrasting haplotypes mate (and there is every reason to believe that
this has occurred; see Petit et al. 1997), any case of
paternal leakage involving either cpDNA or mtDNA
will necessarily result in a new haplotype. In our sample,
there were as many as 33 cases of populations mixed
with such contrasting haplotypes. In addition, this contact must have existed since recolonization took place,
approximately 9,000 years ago (Huntley and Birks
1983). Despite the cumulative effects of at least 50–100
generations of sexual reproduction, no new combinations involving the mtDNA substitution could be detected.
Conclusions
A population genetics survey of organellar genomes of plants requires techniques to detect sufficient
polymorphism to subsequently be adapted for screening
surveys. Here, polymorphisms in both organellar genomes of oaks were studied in southern France based
on an extensive population sample, and the association
of the two maternal lineages was systematically investigated. One mtDNA mutation (a 4-bp inversion located
in the first intron of the gene nad4) was partly independent of the cpDNA polymorphisms. This partial independence is likely due to recurrent inversions that result
from the secondary structure of this region of the mitochondrial genome. On the other hand, greater congruence was observed between the chloroplast and mitochondrial genomes for the second mtDNA mutation
studied (a substitution within the second intron of the
gene nad1). Overall, it appears that, at least during a
period that encompasses 50–100 generations of oaks,
despite a zone of contact between complementary haplotypes that spans several hundred kilometers, no case
of well-established paternal leakage of either organelle
could be demonstrated. This strengthens preliminary results from controlled crosses that indicate that both cytoplasmic genomes were, without exception, maternally
inherited. However, simultaneous studies of both mtDNA and cpDNA from other angiosperm species may
give contrasting results, because inheritance patterns can
vary among species. As expected, the distinctive features of plants characterized by two extranuclear genomes can provide specific evolutionary insights absent
from studies of other eukaryotes characterized by a single extranuclear genome.
1330
Dumolin-Lapègue et al.
Acknowledgments
We particularly thank the following people, who
participated in the collection of oak material: E. Bertocchi, S. Dumas, A. Ducousso, V. Le Corre, J.-M. Louvet,
R. Roubeyrie, G. Roussel, and A. Zanetto. Comments
on an earlier version of the manuscript from A. Gillies,
J. Cottrell, L. Johnson, and one anonymous reviewer
were greatly appreciated. The research was supported by
the EC research program FAIR1-CT950297 and the Action Concertée Coordonnée (ACC), Sciences du Vivant,
number 7, from the Ministère de l’Education Nationale,
de l’Enseignement Supérieur, de la Recherche et de
l’Insertion Professionnelle.
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PAMELA SOLTIS, reviewing editor
Accepted June 29, 1998