Evolution of the Mitochondrial rps3 Intron in Perennial and Annual
Angiosperms and Homology to nad5 Intron 1
Jérôme Laroche and Jean Bousquet
Centre de Recherche en Biologie Forestière, Université Laval, Québec, Canada
The plant mitochondrial rps3 intron was analyzed for substitution and indel rate variation among 15 monocot and
dicot angiosperms from 10 genera, including perennial and annual taxa. Overall, the intron sequence was very
conserved among angiosperms. Based on length polymorphism, 10 different alleles were identified among the 10
genera. These allelic differences were mainly attributable to large indels. An insertion of 133 nucleotides, observed
in the Alnus intron, was partially or completely absent in the other lineages of the family Betulaceae. This insertion
was located within domain IV of the secondary-structure model of this group IIA intron. A mobile element of 47
nucleotides that showed homology to sequences located in rice rps3 intron and in intergenic plant mitochondrial
genomes was found within this insertion. Both substitution and indel rates were low among the Betulaceae sequences, but substitution rates were increasingly larger than indel rates in comparisons involving more distantly related
taxa. From a secondary-structure model, regions involved in helical structures were shown to be well preserved
from indels as compared to substitutions, but compensatory changes were not observed among the angiosperm
sequences analyzed. Using approximate divergence times based on the fossil record, substitution and indel rate
heterogeneity was observed between different pairs of annual and perennial taxa. In particular, the annual petunia
and primrose evolved more than 15 and 10 times faster, for substitution and indel rates respectively, than the
perennial birch and alder. This is the first demonstration of an evolutionary rate difference between perennial and
annual forms in noncoding DNA, lending support to neutral causes such as the generation time, population size,
and speciation rate effects to explain such rate heterogeneity. Surprisingly, the sequence from the rps3 intron had
a high identity with the sequence of intron 1 from the angiosperm mitochondrial nad5 gene, suggesting a common
origin of these two group IIA introns.
Introduction
Autocatalytic introns are of central interest in genetics because several of them are mobile elements that
may insert into intronless alleles. They are also related
to ribozymes, by which they direct and catalyze the
splicing of the flanking exons (Michel and Ferat 1995).
These characteristics are important clues that could link
the introns to their early or late origin, a question which
remains unresolved in evolutionary genetics (Logsdon
et al. 1995; Long, Rosenberg, and Gilbert 1995). Of
most classes of autocatalytic introns, plant mitochondrial introns are those for which the least is known concerning the modes and tempo of evolution.
On the basis of secondary-structure models, autocatalytic introns are classified into groups I, II (subgroups A and B), and III (Michel and Dujon 1983;
Christopher and Hallick 1989). Group I introns are the
most widespread and have been found in all eucaryotic
genomes, as well as in eubacterial and bacteriophage
genomes (Lambowitz and Belfort 1993). Group II introns have been found in fungi and plant organellar genomes and in some cyanobacterial and proteobacterial
genomes, which are probable ancestors of mitochondria
and chloroplasts (Ferat and Michel 1993; Lambowitz
and Belfort 1993; Michel and Ferat 1995). This type of
intron shares structural and catalytic characteristics with
nuclear pre-mRNA introns and the small nuclear RNA
components of the spliceosome (Cech 1986). Group III
Key words: Betulaceae, intron secondary structure, mobile element, indel, substitution, rate heterogeneity.
Address for correspondence and reprints: Jean Bousquet, Pavillon
Marchand, Université Laval, Sainte-Foy, Canada G1K 7P4. E-mail:
[email protected].
Mol. Biol. Evol. 16(4):441–452. 1999
q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
introns are found in lower euglenoid taxa and are known
to form a mixed group II/group III twintron in the chloroplast genome (Copertino, Christopher, and Hallick
1991).
Group II introns are characterized by the folding of
the RNA sequence into six double-helical domains radiating from a central wheel (Michel and Dujon 1983;
Michel, Umesono, and Ozeki 1989). Also, tertiary interactions have been identified between structural domains of group I and II introns (Jacquier and Michel
1987; Jaeger, Westhof, and Michel 1993; Michel and
Ferat 1995). These secondary and tertiary interactions
are likely to stabilize the folding of the catalytic core of
introns. Hence, the helical regions of the secondary
structure appear more conserved than the loops. For the
chloroplastic group II introns, it has been suggested that
some domains are more involved in the stability of the
secondary structure because very different substitution
rates have been found among the different domains
(Learn et al. 1992). Thus, owing to the importance of
accurate splicing, mutations that disrupt secondary and
tertiary interactions are likely to be eliminated by strong
selective pressure (Michel and Ferat 1995). The analyses
of substitution and indel rates in plant mitochondrial intron sequences have also shown that numbers of indels
are correlated with numbers of substitutions, but the latter seem to accumulate more readily as the taxonomical
distance increases between taxa compared (Laroche et
al. 1997). Such a saturation in the number of indels detected could be due to multiple events at the same site
or to the fact that indels are tolerated only at a limited
number of sites, mainly located in the loops of the secondary structure. Hence, for both substitutions and indels in intron sequences, functional constraints are likely
441
442
Laroche and Bousquet
to be imposed by the secondary and tertiary interactions
(Learn et al. 1992).
In higher-plant mitochondrial genomes, the rps3
gene is clustered with the rpl16 gene in an overlapping
operon sequence, and it contains one intron (Leblanc et
al. 1995). With regard to nucleotide substitutions, the
mitochondrial rps3 intron was the most variable among
six mitochondrial introns sampled in a set of angiosperm
taxa (Laroche et al. 1997) and thus, it might be most
informative regarding the modes and tempo of evolution
of intron mitochondrial DNA. In lower-plant mitochondrial genomes, such as in the Bryophyte Marchantia
polymorpha, which contains 32 introns of group I and
II, no introns are observed in its rps3 gene sequence
(Oda et al. 1992). The primitive mitochondrial genomes
of the rhodophyte Chondrus crispus (Leblanc et al.
1995) and the protozoan Reclinomonas (Lang et al.
1997) lack the majority of introns including the one in
the rps3 gene. The rps3 gene is also present in the chloroplast genome of higher plants, but it is not interrupted
by an intron. However, the chloroplast rps3 gene of Euglena gracilis contains a mixed group II/group III twintron of only 409 nucleotides (Copertino, Christopher,
and Hallick 1991), and the alga Chlamydomonas contains an extra-long coding region instead of an intron
(Turmel and Otis 1994).
In this study, the complete nucleotide sequence of
the mitochondrial rps3 intron and its secondary structure
were determined for a set of taxa representing distant
monocot and dicot families as well as taxa from the
family Betulaceae, in order to analyze rate variation and
to study the distribution of substitutions and indels with
regard to the secondary structure of the rps3 intron for
distantly related groups and among closely related taxa
within a single family. Betulaceae is a small family of
perennial angiosperms well described at the morphological and molecular levels (Crane 1989; Bousquet,
Strauss, and Li 1992; Savard, Michaud, and Bousquet
1993). Significantly slower rates of substitution for both
chloroplast and mitochondrial gene-coding sequences
were observed in the Betulaceae when compared to annual dicots and monocots (Bousquet et al. 1992; Laroche et al. 1997). We also demonstrate a rate heterogeneity pattern between annual and perennial plant sequences for this intron, which parallels that observed for
chloroplast, nuclear, and mitochondrial gene-coding regions (Bousquet et al. 1992; Gaut et al. 1992; EyreWalker and Gaut 1997; Laroche et al. 1997).
Materials and Methods
DNA Extraction and Amplification
To estimate the amount of sequence variation in the
mitochondrial rps3 intron among angiosperm taxa, the
following sequences were retrieved from Genbank (with
accession numbers): within the monocots, Oryza sativa
(D21251) and Zea mays (U96618), Cyperales, Poaceae;
within the dicots, Petunia hybrida (X67028), Asteridae,
Solanales and Oenothera berteriana (X69140), Rosidae,
Myrtales. The complete nucleotide sequence for the rps3
intron was obtained for 10 species representing the ma-
jor generic and subgeneric taxonomical subdivisions
within the family Betulaceae (dicots, Hamamelidae, Fagales): within the subfamily Betuleae, Alnus glutinosa
(AF080076) from subgenus Alnus, Alnus maritima
(AF080077) from subgenus Clethropsis, Betula alleghaniensis (AF080078) from section Costatae, Betula
glandulosa (AF080079) from section Humiles, and Betula pendula (AF080081) from section Betulae; within
the subfamily Coryleae, Carpinus caroliniana
(AF080083), Corylus avellana (AF080084), Corylus
colurna (AF080085), Corylus cornuta (AF080086), and
Ostrya virginiana (AF080087). The outgroup Quercus
rubra (AF080088) was selected from the closely related
family Fagaceae (Hamamelidae, Fagales) (Maggia and
Bousquet 1994). Total genomic DNA from all species
was extracted by a CTAB method (Bousquet, Simon,
and Lalonde 1990). The mitochondrial rps3 intron was
amplified by PCR for 45 cycles (948C for 30 s, 558C
for 1 min, and 728C for 1 min, 30 s), followed by 10
min at 728C, using the forward primer 59-ATCTGAATCGTAGTTCAGAT-39 and the reverse primer 59CAAAGGTGAGTMTCGTAGGT-39, located in exons 1
and 2, respectively. The forward and the reverse strands
from each taxa were cycle-sequenced using the original
primers and several internal primers: upstream, with 59GATGAGACTAAGCAGCCACC-39 and 59-TCTTATTCATTCAGGGTGCT-39; downstream, with 59-GCCGAG C A C C C T G A A T G AAT-39 and 59-CTC C T T C C CTTCCACTGCAT-39. Oligonucleotides were synthesized
with a 394 DNA/RNA Synthesizer, and the reactions
were loaded on a 373 XL DNA Sequencer (Perkin Elmer Applied Biosystems).
Sequence Analysis
Sequence analysis was carried out with the Wisconsin Package, version 9.0 (Genetics Computer Group
[GCG], Madison, Wisc.). Sequence alignment was conducted with PILEUP and corrected by eye with LINEUP.
Database searching for similarity between nucleotide sequences was conducted with BLAST. Values of minimum free energy for some regions were calculated with
MFOLD. Secondary-structure model determination of
the A. maritima rps3 intron was carried out according
to a previously determined model (Michel, Umesono,
and Ozeki 1989), and with the program STEMLOOP.
This approach was preferred to the use of a probabilistic
model (Muse 1995) because a secondary structure was
already inferred for the group IIA nad5 intron (Michel,
Umesono, and Ozeki 1989) for which a high sequence
identity was found with the rps3 intron (see results below). Numbers of substitutions per site (rates) among
the angiosperm sequences were calculated for each domain. The procedure used here avoids the circularity of
identifying sequence segments on the basis of maximum
conservation and then comparing rates of evolution
among different domains (Golenberg et al. 1993). This
also allowed the study of the evolution of secondary
structure itself and the strength of secondary-structure
models previously reported in the literature. Overall
numbers of substitutions per site (K0) were calculated
according to the two-parameter model of Kimura (1980)
Evolution of Angiosperm rps3 Intron
with MEGA, version 1.0 (Kumar, Tamura, and Nei
1993). No attempt was made at correcting for compensatory changes occurring in stem regions since no such
changes were detected in this study.
The numbers of indels per site are usually estimated by summing up all indels in each pairwise taxa comparison and dividing by the number of available sites,
because each indel is considered to be the result of a
single mutational event (Aldrich et al. 1988; Saitou and
Ueda 1994; Laroche et al. 1997). However, because the
number of sites in indels can be large, this procedure
can overestimate the total number of sites and underestimate the indel rate per site. Therefore, the rate of
indel per site between two nucleotide sequences was obtained by the following formula:
I 5 N/(L 2 D 1 N)
where I 5 indel rate, N 5 total number of indels, L 5
total number of sites, and D 5 number of sites involved
in indels. In this equation, the number of sites involved
in all indels between two sequences is subtracted from
the total number of sites, and the total number of indels
is added to the total number of sites to recover the sites
where the indels occurred. This should allow for a more
realistic estimation of numbers of indels per site.
Results
Primary and Secondary Structures of the rps3 Intron
The beginning and end of the rps3 intron was determined from previously published monocot and dicot
sequences. With a range of 1475 base pairs (bp) to 1847
bp, 10 distinct rps3 intron-length variants were observed
among angiosperm taxa, five of which were observed
among the five Betulaceae genera sampled (table 1, diagonal). At the intrageneric level, a total of three substitutions were found among the sequences of B. alleghaniensis, B. glandulosa, and B. pendula; two small
indels were found between the sequences of A. maritima
and A. glutinosa; and one substitution and two small
indels were found among the sequences of C. avellana,
C. colurna, and C. cornuta. Thus, because of the very
low intragenus variability observed, further description
will be reported for only one taxon per genus for these
last three genera.
The major length polymorphism among the Betulaceae taxa was caused primarily by a large indel of 133
bp in the sequence of Alnus, as compared to the shortest
sequence of Betula (table 1, fig. 1). The sequences of
the Coryleae (Corylus, Carpinus, and Ostrya) shared 43
bp at the 59 end and 30 bp at the 39 end with the Alnus
indel. The outgroup sequence of Quercus (Fagaceae)
shared 10 bp at the 59 end and 5 bp at the 39 end of this
indel with the Betulaceae sequences. Between these two
small homologous stretches, the sequence of Quercus
contained an indel region of 92 bp for which no match
was possible with the Betulaceae sequences. This portion of the alignment was considered to be nonhomologous and was excluded from the calculation of the substitution rates between Betulaceae and Quercus sequences. This portion of the rps3 intron also appeared quite
443
variable in a preceding study, and proper alignment between Zea, Triticum, Petunia, and Oenothera could not
be achieved (Laroche et al. 1997).
Many repeats and inverted repeats were found
within the sequence of the rps3 intron. The most important ones were found within the large indel in the
sequences of Alnus and of the Coryleae (fig. 1). A long
inverted repeat (32 bp) was found within the large indel
in the Alnus sequence. This inverted repeat could form
a stable stem-loop structure with a free energy of 268.3
kcal/mol. The truncated indel in the sequences of the
Coryleae retained a part of the long inverted repeat: 24
bp in Corylus, with a free energy of 235.7 kcal/mol,
and 18 bp in Carpinus and Ostrya, with a free energy
of 222.7 kcal/mol, respectively (fig. 1). Two mutations,
in the sequences of Carpinus and Ostrya, shortened the
inverted repeat by six nucleotides (fig. 1). No strong
hairpin structure was found in the Betula sequence. A
structure with a free energy of 212.9 kcal/mol was observed in the corresponding Quercus sequence. In the
Alnus sequence, the large indel also contained four elements of three sets of overlapping direct repeats of 9
bp (no. 1, fig. 1), 13 bp (no. 2, fig. 1), and 17 bp (nos.
3 and 39, fig. 1). The elements of direct repeats nos. 1
and 2 were also present upstream from the indel in the
Alnus intron sequence, and in the other angiosperm sequences.
The complete rps3 intron sequences of A. maritima
and Q. rubra and the portions corresponding to the large
insert found in these two sequences were submitted to
a BLAST search against nonredundant database sequences. Surprisingly, in the large indel of the Alnus
sequence, a portion of 47 bp matched with noncoding
angiosperm mitochondrial sequences, and the last 20 bp
of this portion belonged to the first element of the large
inverted repeat (fig. 1). This 47-bp segment was also
found in the mitochondrial rps3 intron from Oryza sativa, at a different location in the 39 end of the intron
(data not shown). Other positive matches were with intergenic mitochondrial sequences of diverse angiosperm
taxa. For the large indel (92 bp, fig. 1) of the Quercus
sequence, matches with high scores were observed with
two regions of the mitochondrial rps3 intron in Arabidopsis thaliana and Brassica napus.
An unexpected result was that the rps3 intron sequences showed high identity with the first intron of the
plant mitochondrial nad5 gene (fig. 2). The highest
BLAST score showed more than 80% identity on a 113bp stretch. An overall secondary-structure model, which
corresponds to group IIA, was already derived for the
plant mitochondrial nad5 intron (Michel, Umesono, and
Ozeki 1989), so the segments highly similar between
nad5 and rps3 introns helped to find the overall secondary-structure model for the rps3 intron (figs. 2–3). The
most similar regions are likely involved in secondary
base pairing, characterizing the group II introns (fig. 2).
The helices of the different domains and the central
wheel were particularly conserved between the two introns analyzed (fig. 2). The loops were much more variable in length (see numbers between brackets in fig. 2),
35
0.020 6 0.003
153
0.122 6 0.010
153
0.123 6 0.010
141
0.111 6 0.010
136
0.106 6 0.009
140
0.109 6 0.009
137
0.107 6 0.009
141
0.110 6 0.009
139
0.108 6 0.009
Oryza
a
113
0.083 6 0.008
106
0.075 6 0.007
115
0.077 6 0.007
112
0.077 6 0.007
114
0.077 6 0.007
117
0.079 6 0.007
115
0.077 6 0.007
44
0.025 6 0.004
44
0.025 6 0.004
1649a
Oenothera
berteriana
98
0.072 6 0.007
99
0.071 6 0.007
105
0.076 6 0.008
100
0.072 6 0.007
103
0.075 6 0.008
101
0.073 6 0.007
47
0.031 6 0.004
44
0.029 6 0.004
35
0.024 6 0.004
1475a
Petunia
hybrida
18
0.011 6 0.003
18
0.012 6 0.003
16
0.010 6 0.003
18
0.011 6 0.003
17
0.011 6 0.003
35
0.019 6 0.003
36
0.019 6 0.003
33
0.020 6 0.003
30
0.020 6 0.004
1650a
Quercus
rubra
9
0.006 6 0.002
6
0.004 6 0.002
11
0.007 6 0.002
10
0.006 6 0.002
34
0.017 6 0.003
31
0.016 6 0.003
30
0.018 6 0.003
27
0.018 6 0.003
18
0.010 6 0.002
1734a
Alnus
maritima
NOTE.—The numbers of substitutions and indels per site were obtained from pairwise deletion of gap sites from all comparisons.
Intron length for each species.
Ostrya
Carpinus
Corylus
Betula
Alnus
Quercus
Petunia
147
0.117 6 0.010
145
0.117 6 0.010
133
0.105 6 0.009
129
0.100 6 0.009
132
0.103 6 0.009
129
0.100 6 0.009
132
0.103 6 0.009
130
0.101 6 0.009
11
0.006 6 0.002
1847a
1843a
Zea
Oenothera
Oryza
sativa
Zea
mays
9
0.006 6 0.002
9
0.006 6 0.002
8
0.005 6 0.002
37
0.022 6 0.004
34
0.020 6 0.003
30
0.019 6 0.003
28
0.019 6 0.004
16
0.010 6 0.003
4
0.002 6 0.001
1599a
Betula
alleghaniensis
7
0.004 6 0.002
4
0.002 6 0.001
34
0.018 6 0.003
32
0.017 6 0.003
31
0.019 6 0.003
26
0.018 6 0.003
18
0.011 6 0.003
5
0.003 6 0.001
6
0.004 6 0.002
1668a
Corylus
cornuta
3
0.002 6 0.001
33
0.018 6 0.003
30
0.016 6 0.003
33
0.020 6 0.003
29
0.020 6 0.004
18
0.011 6 0.003
7
0.004 6 0.002
9
0.006 6 0.002
4
0.002 6 0.001
1672a
Carpinus
caroliniana
33
0.018 6 0.003
31
0.017 6 0.003
30
0.018 6 0.003
28
0.019 6 0.004
17
0.010 6 0.002
6
0.004 6 0.001
8
0.005 6 0.002
3
0.002 6 0.001
1
0.001 6 0.001
1674a
Ostrya
virginiana
Table 1
Evolutionary Rates of Mitochondrial rps3 Intron Among Angiosperm Taxa. Numbers of Indels and Numbers of Indels per Site (above diagonal), Numbers of
Substitutions and Number of Substitutions per Site (below diagonal)
444
Laroche and Bousquet
Evolution of Angiosperm rps3 Intron
445
FIG. 1.—Sequence alignment of a portion of the loop in domain IV of the mitochondrial rps3 intron. Inverted repeats are indicated by the
large arrows. Direct-repeat elements are indicated by the small, numbered traits. The mobile element that had the highest scores in the BLAST
search is boxed with the two small direct repeats (6 bp) indicated by the dashed boxes. Note that the corresponding portion in Quercus sequence
is not homologous to the Betulaceae sequences.
but they also contained residue stretches that could participate in base-pairing interactions.
The secondary-structure model derived for the rps3
intron sequence of A. maritima also corresponds to those
of group IIA introns with six domains (I–VI) radiating
from a central wheel, regions of exon- and intron-binding sites (EBS and IBS) and g–gg9 potentially involved
in tertiary interactions (fig. 3). Although the EBS1–
EBS2 and IBS1–IBS2 regions appeared very different
between the nad5 and rps3 introns, they were located in
the same regions of domain I and at the 39 end of exon
1. According to this model, the large indel found in
Alnus, Corylus, Ostrya, and Carpinus sequences was located in the loop of domain IV. In the rps3 intron, domain IV was the largest, with 43.3% of the total sequence length, and domain II was the smallest, with
only 1.4% of the total sequence length.
Substitution and Indel Rates in rps3 Intron Sequences
Overall numbers of substitutions per site were estimated with pairwise deletion of gap sites to allow a
direct comparison with the numbers of indels per site,
which were obtained for each pairwise comparison.
These estimates varied greatly between the different an-
giosperm rps3 intron sequences compared (lower left
matrix, table 1). Within dicots, substitution rates were
similar across subclasses (Asteridae, Rosidae, and Hamamelidae), although rates between Oenothera (Onagraceae, Rosidae) and the Betulaceae (Hamamelidae)
were higher than those between Petunia (Solanaceae,
Asteridae) and these dicots. Within the Betulaceae, the
Coryleae (Corylus, Carpinus, and Ostrya) were more
similar in sequence with each other than the Betuleae
were (Betula and Alnus), although these differences
were not significant (data not shown).
A large rate heterogeneity was observed for substitutions between annual and perennial taxa. Using approximate divergence times based on the fossil record
to estimate rates per year, differences from 10- to 30fold were observed between, on one hand, the annuals
Oryza-Zea (Poaceae) and Petunia-Oenothera and, on the
other hand, the perennials Alnus-Betula and CarpinusOstrya (table 2). Errors in calibration dates could not
account for such rate heterogeneity between the various
groups compared, with the largest differences observed
between perennial and annual taxa. Using Oryza or Zea
as reference taxon in lineage relative-rate tests (Li and
Bousquet 1992), significant differences were observed
446
Laroche and Bousquet
FIG. 2.—Sequence alignment showing the most conserved regions between mitochondrial nad5-1 and rps3 introns. Numbers in brackets
indicate length of omitted segments. Small dots correspond to gaps which were introduced to increase similarity. The portions involved in
helices of domains I–VI are underlined; EBS and IBS stand for exon- and intron-binding sites, respectively; # refers to the g–g9 base pair;
*indicates the nucleotide involved in the lariat formation (see also fig. 3).
between, on one hand, the annuals Petunia-Oenothera,
and on the other hand, the perennials Alnus-Betula or
Carpinus-Ostrya (P , 0.01 in the four tests conducted).
No such test could be conducted to compare the annuals
Oryza and Zea to the perennial Betulaceae because of
the nonavailability of suitable outgroup sequences outside the angiosperms.
The numbers of indels per site were also found to
vary extensively across all angiosperm pairwise comparisons (upper right matrix, table 1). Indel rates were
similar across dicot subclasses (Asteridae, Rosidae, and
Hamamelidae), and higher rates were observed between
Oenothera and the Betulaceae than between Petunia and
these perennial dicots. Within the Betulaceae, there was
less difference in indel rates between Coryleae and Betulaeae than in substitution rates. Again here, extensive
rate heterogeneity was observed between annual and perennial taxa. Using approximate divergence times derived from the fossil record to estimate rates per year,
differences from 10- to 30-fold were also found, between, on one hand, the annuals Oryza-Zea and PetuniaOenothera and, on the other hand, the perennials AlnusBetula and Carpinus-Ostrya (table 2). Using Oryza or
Zea as reference taxon in lineage relative-rate tests (Li
and Bousquet 1992), significant differences were observed between, on one hand, the annuals Petunia-Oen-
Evolution of Angiosperm rps3 Intron
447
FIG. 2 (Continued)
othera, and, on the other hand, the perennials AlnusBetula or Carpinus-Ostrya (P , 0.01 in the four tests
conducted). For the same reason mentioned above, no
such test could be conducted between the annuals Oryza
and Zea and the perennial Betulaceae.
Indel rates seemed generally more constrained than
substitution rates. Indeed, using a substitution rates (K)
to indel rates (I) ratio (K/I), the substitution rates were
found to increase more rapidly than the indel rates as
taxonomical distance increased. The ratio varied between 1.0 and 2.7 for comparisons within the Betulaceae
family and for some comparisons between Betulaceae
taxa and Fagaceae (Quercus). The ratio values increased
to 3.0 and 4.8 for comparisons between different subclasses of the dicots (Petunia-Asteridae and Oenothera-
Rosidae), between Zea and Oryza (Poaceae), and between the monocots (Poaceae) and the dicots (Petunia
and Oenothera). The ratio values varied between 5.0 and
6.4 for most comparisons between annuals and perennials. Even if most substitutions and indels observed in
the rps3 intron between angiosperm taxa were located
within the loops of the different domains, particularly
for domains III and IV (table 3), substitutions were more
evenly distributed than indels, which could account for
the increasing substitution-to-indel rate ratio as taxonomical distances increased.
The central wheel of the secondary structure was
particularly preserved from substitutions and indels (see
nucleotide positions between main domains in fig. 2).
The main loop of domain IV was the least conserved,
448
Laroche and Bousquet
FIG. 3.—Secondary-structure model of the mitochondrial rps3 gene of Alnus maritima based on Michel et al. (1989). Regions potentially
involved in tertiary interactions are indicated: IBS1–IBS2, EBS1–EBS2, g–g9, and the bulged nucleotide A in the domain VI. The loops are
not drawn to scale.
and most substitutions and indels among angiosperm sequences were located in this region (data not shown for
indel rates, but see table 3 for substitution rates). Among
the six domains, domain II was the most conserved, with
only one substitution in the Oenothera sequence, followed by domains V, VI, III, I, and IV (table 3). In
general, there was not much difference in substitution
rates between helix regions alone and the overall domain. Surprisingly, in some pairwise comparisons, the
substitution rates were higher for the regions involved
in base-pairing interactions than for the overall domain
(table 3). However, no compensatory changes were detected.
Discussion
The rps3 intron was not an arbitrary choice as a
case study. In a preliminary screening, six mitochondrial
Table 2
Numbers of Substitutions (K) and Indels (I) per Site per Year Between Angiosperm Taxa
Divergence time
(Myr)a
Petunia vs. Oenothera
Oryza vs. Zea
Alnus vs. Betula
Carpinus vs. Ostrya
90–70
70–50
85–75
65–55
r (I)
1.32
4.40
1.47
4.60
3
3
3
3
210
10 –1.70
10211–6.16
10211–1.67
10212–5.43
r (K)
3
3
3
3
210
10
10211
10211
10212
4.63
1.42
3.33
1.38
3
3
3
3
210
10 –5.96
10210–1.99
10211–3.77
10211–1.64
3
3
3
3
10210
10210
10211
10211
a References for approximate divergence times are as follows: Petunia vs. Oenothera (divergence between Asteridae and Rosidae): Cronquist (1988, pp. 413–
415, 359–361) and Stewart and Rothwell (1993, p. 483), Oryza vs. Zea: G. L. Stebbins, cited in Wolfe et al. (1989), and Alnus vs. Betula and Carpinus vs. Ostrya:
Crane (1989).
Evolution of Angiosperm rps3 Intron
449
Table 3
Numbers of Substitutions per Site for Each Domain of Mitochondrial rps3 Intron Between Angiosperm Taxa
Oryza/Zea
Domain I (466 sites)
Helix (224 sites)
Domain II (25 sites)
Domain III (229 sites)
Helix (44 sites)
Domain IV (478 sites)
Domain V (34 sites)
Domain VI (80 sites)
Helix (49 sites)
0.015 6 0.006
0.014 6 0.008
0
0.022 6 0.010
0
0.039 6 0.009
0
0.013 6 0.013
0
Oryza/Oenothera
Alnus/Betula
6
6
6
6
6
6
6
6
6
0.004 6 0.003
0.009 6 0.006
0
0.009 6 0.006
0.023 6 0.024
0.006 6 0.004
0
0
0
0.111
0.085
0.042
0.022
0.151
0.163
0.061
0.079
0.086
0.016
0.020
0.043
0.010
0.063
0.020
0.044
0.033
0.044
Alnus/Oenothera
0.060
0.060
0.042
0.059
0.023
0.063
0.030
0.052
0.064
6
6
6
6
6
6
6
6
6
0.012
0.017
0.043
0.020
0.023
0.012
0.030
0.026
0.037
Oenothera/Petunia
0.072
0.071
0.042
0.094
0.023
0.093
0.061
0.038
0.042
6
6
6
6
6
6
6
6
6
0.013
0.019
0.043
0.021
0.023
0.015
0.044
0.022
0.030
NOTE.—See table 1 for complete name of taxa. Secondary model according to figure 3. The numbers of substitutions per site were obtained from complete
deletion of gap sites from all comparisons.
introns were tested for amplification with polymerase
chain reaction (PCR) in the family Betulaceae: cox2,
nad1, nad4, nad5ab, nad5-de, and rps3. From this
screening, five introns were successfully amplified, and
a fragment of the expected size was obtained. No intron
was found to split the coding sequence of the cox2 gene
(data not shown). This observation is consistent with
those of De Benedetto et al. (1992) and Rabbi and Wilson (1993), who found extensive variation in the occurrence of this intron among angiosperms. From the five
introns successfully amplified in the family Betulaceae,
a length polymorphism was observed only for rps3 intron. This intron was also the most variable in sequence
among a set of four annual dicots and monocots (Laroche et al. 1997). We therefore focused on this intron
for a detailed analysis of substitution and indel rates and
of patterns of sequence variation between annual and
perennial angiosperms.
Secondary Structures and Paralogy Between
Mitochondrial Introns rps3 and nad5
According to the secondary structure obtained here,
the angiosperm mitochondrial rps3 intron belongs to
group IIA like the intron 1 of the mitochondrial nad5
gene. It has been shown that domain V may be regarded
as a specific attribute of group II introns (Michel and
Ferat 1995). In this study, not only domain V but also
large parts of domains I and VI and all of domain II
appeared highly conserved between the mitochondrial
rps3 and nad5 introns. These observations indicate a
recent common origin of these two introns and support
the idea of intron spread by means of reverse-splicing
mechanisms (Malek, Brennicke, and Knoop 1997). The
gain of an intron into a coding sequence through duplication and integration into a new site has also been reported for the introns of cox1, cox3, and rrn26 in the
mitochondrial genome of Marchantia polymorpha and
for the introns nad1 and nad2 in higher plants (reported
in Schuster and Brennicke 1994, and references therein).
Between the rps3 and nad5 introns, only the 59 of the
helical part of domain III remained strongly conserved,
and sequences of domain IV could not be aligned. Also,
in the rps3 intron, domain II was the shortest and the
most conserved domain, while domain IV was the longest and the most variable. The same observation was
found for most of the mitochondrial introns analyzed
(Michel, Umesono, and Ozeki 1989). This gradient of
substitution rates observed among the six domains
would indicate different levels of constraint, with the
most variable domains being less essential for the catalytic activity even if they are presumed to be involved
in the folding of ribozymes or with protein binding
(Lambowitz and Belfort 1993).
It is rather surprising to observe relatively high
substitution rates for the helical part of each domain,
which could cause sequence mispairing, as previously
observed for rRNA genes (Rzhetsky 1995). But we
found no evidence of compensatory changes between
the two DNA strands of helical structures. These observations suggest that weak helical core structures are possible for plant mitochondrial group II introns, which
could be explained by incompleteness or absence of an
RNA-editing process in these regions (Carrillo and Bonen 1997).
Different substitution rates were also observed
among structural domains of the chloroplast trnV intron,
but domain II was longer and more variable than domain
IV (Michel, Umesono, and Ozeki 1989; Learn et al.
1992). Such differences in length and substitution rates
among domains between chloroplastic and mitochondrial introns could be the result of distinct evolutionary
modes of these genomes (Wolfe et al. 1987; Michel and
Ferat 1995). These results also show that if the central
core and the overall structure of the six domains were
very conserved, secondary and tertiary interactions
could differ among taxa following extensive evolution
by substitutions and indels. A secondary-structure model has also been inferred for the chloroplast rps3 intron
in E. gracilis (Copertino, Christopher, and Hallick
1991), but it differs greatly in its primary sequence from
the plant mitochondrial rps3 intron. Also, the former
contains a group III intron within the sequence of the
group II intron.
The occurrence of an insert of variable length in
domain IV of the rps3 intron of the Betulaceae was investigated from an evolutionary perspective (fig. 4). According to the known phylogeny of the family Betulaceae (Crane 1989; Bousquet, Strauss, and Li 1992), this
DNA fragment, absent in the outgroup sequence of
Quercus and in other angiosperm sequences, was likely
inserted before the diversification that led to the actual
450
Laroche and Bousquet
FIG. 4.—Commonly accepted phylogenetic tree of the family Betulaceae (Crane 1989; Bousquet, Strauss, and Li 1992) based on morphological characters and rbcL nucleotide sites showing gains (downward arrows) and losses (upward arrows) of the large indel in mitochondrial rps3 intron. 1—The indel was gained before the divergence
of the family. 2—Partial loss of 60 nucleotides during the Coryleae
cladogenesis. 3—Complete loss in the genus Betula. 4—Occurrence
of two nucleotide substitutions shortening the inverted repeat in the
truncated indel of Ostrya and Carpinus.
family Betulaceae (event 1). It was eventually lost, partially so (event 2) in the subfamily Coryleae (Corylus,
Carpinus, and Ostrya), and completely so (event 3) in
the genus Betula. Finally, two mutations in the OstryaCarpinus lineage shortened the inverted repeat (event 4).
The insert was conserved in its most complete form only
in the sequences of Alnus, known as the most primitive
member of the family, on the basis of fossil evidence
(see in Bousquet, Strauss, and Li 1992). We have further
shown that part of this fragment is an inverted repeat
found to be homologous to other sequences of angiosperm mitochondrial genomes, suggesting a possible
transpositional event. The presence of such repeated elements in the large and variable domain IV in the secondary structure of rps3 intron may increase the recombinational activity in the mitochondrial genome (Malek,
Brennicke, and Knoop 1997). Such recombinational activity caused by repeated sequences is a common feature
of the plant mitochondrial genome (Houchins et al.
1986; André, Levy, and Walbot 1992).
Heterogeneity of Substitution and Indel Rates
The plant mitochondrial rps3 intron appears to be
the most variable in length and in sequence among the
plant mitochondrial introns analyzed to date (Laroche et
al. 1997). Rates of substitution were fairly similar to
rates of indel in the mitochondrial rps3 intron when
comparisons were made between closely related taxa,
such as within the family Betulaceae. However, when
the comparisons involved a Betulaceae sequence and the
outgroup sequence Quercus from the Fagaceae, indel
rates did not increase as much as substitution rates. Indeed, substitution rates were, on average, 2.5 times higher than indel rates. The ratio was even more unbalanced
when taxonomically more distant comparisons were
made. This trend toward larger substitution-to-indel ratios with increasing taxonomical distance was already
reported for other mitochondrial introns (Laroche et al.
1997) and for a noncoding chloroplast DNA region (Golenberg et al. 1993). This effect could be attributable to
the secondary and tertiary structures of the rps3 intron
that would impose major constraints, eliminating more
readily indels that could disrupt the overall stability, or
to the fact that multiple indels at the same site could not
be detected. Indeed, substitutions were found to be more
evenly distributed than indels, indicating that they would
be more easily tolerated: many substitutions were observed in putatively important sites or regions involved
in secondary and tertiary interactions, and much of the
sequence variation observed, attributable to indels, was
within the loops of the different domains, particularly
domains III and IV.
In the Betulaceae, the substitution rate of the mitochondrial rps3 intron was very low. This substitution
rate compares to that observed at the rbcL locus between the same genera (Bousquet, Strauss, and Li 1992),
and it is three times lower than that for 18S rRNA between the same taxa and 30 times lower than that estimated for the intergenic spacers ITS1 and ITS2 of nuclear rDNA between the same taxa (Savard, Michaud,
and Bousquet 1993). Moreover, the lineage relative-rate
tests (Li and Bousquet 1992) conducted between Asterideae and Rosideae taxa on one hand, and the Betulaceae on the other hand, and the estimation of evolutionary rates per year in these diverse groups of plants and
in the Poaceae revealed extensive heterogeneity in rates
of molecular evolution between annual and perennial
plant taxa.
Such rate heterogeneity, observed for substitutions
as well as for indels, does not appear to result from
biased procedures of rate estimation. Refined models of
nucleotide substitution have been recently proposed to
take into account the interdependence of sites and variability of substitution rates across sequences for which
base-pairing interactions occur among distant sites
(Schöniger and von Haeseler 1994; Rzhetsky 1995; Tillier and Collins 1995). These models have been applied
to rRNA sequences of very distant taxa (Rzhetsky 1995)
and rapidly evolving mammalian mitochondrial gene sequences (Schöniger and von Haeseler 1994). Very little
difference was observed between these models and
those assuming independence of sites and homogeneous
distribution of variation across sites, such as those of
Jukes and Cantor (1969) and Kimura (1980), at high
levels of sequence identity, and significant differences
were only observed at lower sequence identity levels,
where it becomes extremely difficult to align sequences
(Schöniger and von Haeseler 1994). Thus, these methods were not required in this study of angiosperm mitochondrial intron sequences which could be easily
aligned by eye and in which the estimated numbers of
substitutions were low. In addition, the single-strand
model of Tillier and Collins (1995) could be particularly
appropriate when large numbers of compensatory
changes are detected. In this study, no compensatory
changes were observed. Thus, the large rate heterogeneity in substitutions and indels here observed between
annual and perennial plant taxa cannot be attributable to
a biased estimation of the number of substitutions due
to compensatory changes.
This new observation of rate heterogeneity between
annual and perennial taxa for a noncoding region fol-
Evolution of Angiosperm rps3 Intron
lows the trend observed for chloroplast, nuclear, and mitochondrial coding regions (Bousquet, Strauss, and Li
1992; Gaut et al. 1992; Eyre-Walker and Gaut 1997;
Laroche et al. 1997). The much slower rate of evolution
in perennial taxa such as the Betulaceae, now detected
at the level of noncoding mitochondrial DNA, lends
support to the idea that evolutionary forces affecting all
regions of the different genomes are likely to be involved, such as generation time, population size, and
speciation rate (Bousquet et al. 1992; Eyre-Walker and
Gaut 1997). Furthermore, molecular rate heterogeneity
appears to correlate with rates of morphological evolution in a growing number of taxa (Bousquet, Strauss,
and Li 1992; Bousquet et al. 1992; Omland 1997), although the main driving force behind this evolutionary
trend remains to be identified.
Conclusions
The results presented here show that the regions
essential for the folding of the angiosperm mitochondrial rps3 intron are well preserved from mutations, particularly from indels. The indel rates were more similar
to the substitution rates when closely related taxa were
compared, but the indels-to-substitutions ratio decreased
when more distant species were compared. The overall
rates of substitution of the rps3 intron were in the range
of those of synonymous rates of substitution for plant
mitochondrial exons (Laroche et al. 1997) and in the
range of that estimated from chloroplast coding regions;
hence, they are not very phylogenetically informative at
the intrafamilial level. However, substitution rate heterogeneity between annual and perennial taxa was observed
for a noncoding (mitochondrial) region, which parallels
the trend previously observed for coding regions in the
three plant genomes. The observed paralogy detected
here between introns rps3 and nad5 is significant, lending support to intron transfer between different mitochondrial genes. This observation stresses the need for
an overall phylogenetic tree of introns, organellar and
nuclear, in order to understand their complete evolutionary history.
Acknowledgments
We thank W. J. Elisens (Department of Botany,
University of Oklahoma) for kindly providing seeds of
A. maritima; J. Renaud and S. Pelletier (RSVS, Université Laval) for primer synthesis and DNA sequencing;
C. Lemieux (Département de Biochimie, Université Laval) for discussions concerning intron evolution and secondary structures; and D. J. Perry (CRBF, Université
Laval) for comments on an earlier draft of this manuscript. This work was supported by a FCAR of Québec
fellowship to J.L. and by NSERC of Canada and FCAR
grants to J.B.
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Accepted December 8, 1998