Comparative Analysis of Mitochondrial Genomes in Bombina(Anura; Bombinatoridae)

Maciej Pabijan Æ Christina Spolsky ÆThomas Uzzell Æ Jacek M. Szymura

Received: 31 August 2007 / Accepted: 9 May 2008 / Published online: 12 August 2008

� Springer Science+Business Media, LLC 2008

Abstract The complete mitochondrial genomes of two

basal anurans, Bombina bombina and B. variegata (Anura;

Bombinatoridae), were sequenced. The gene order of their

mitochondrial DNA (mtDNA) is identical to that of

canonical vertebrate mtDNA. In contrast, we show that

there are structural differences in regulatory regions and

protein coding genes between the mtDNA of these two

closely related species. Corrected sequence divergence

between the mtDNA of B. bombina and B. variegata

amounts to 8.7% (2.3% divergence in amino acids).

Comparisons with two East Asian congeners show that the

control region contains two repeat regions, LV1 and LV2,

present in all species except for B. bombina, in which LV2

has been secondarily lost. The rRNAs and tRNAs are

characterized by low nucleotide divergence. The protein

coding genes are considerably more disparate, although

functional constraint is high but variable among genes, as

evidenced by dN/dS ratios. A mtDNA phylogeny

established the distribution of autapomorphic nonsynon-

omous substitutions in the mitogenomes of B. bombina and

B. variegata. Nine of 98 nonsynonomous substitutions led

to radical amino acid replacements that may alter mito-

chondrial protein function. Most radical substitutions were

found in ND2, ND4, or ND5, encoding mitochondrial

subunits of complex I of the electron transport system.

The extensive divergence between the mitogenomes of

B. bombina and B. variegata is discussed in terms of its

possible role in impeding gene flow in natural hybrid zones

between these two species.

Keywords mtDNA � Bombina � Divergence � Control

region � ETS complex I � Hybrid zone

Introduction

In eukaryotes, transmembrane proteins imbedded in the

inner membrane of the mitochondria are the sites of energy

conversion and cellular respiration. These proteins consist

of subunits of the electron transport system (ETS) and are

encoded by either nuclear DNA (nDNA) or mitochondrial

DNA (mtDNA). The products of both genomes and their

precise interactions are essential for aerobic metabolism. A

plethora of disorders in humans linked to sequence varia-

tion in mtDNA or mitochondrially expressed nDNA

illustrates the evolutionary constraint imposed on genes of

both genomes (Larsson and Clayton 1995; Schapira 2006).

Furthermore, specific mtDNA haplotypes have been shown

to influence individual fitness in humans, mice, Drosoph-

ila, and copepods (reviewed by Ballard and Whitlock 2004;

Gemmell et al. 2004; Ballard and Rand 2005). It has been

estimated that the ETS is comprised of approximately 90

different respiratory chain subunits, the majority of which

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00239-008-9123-3) contains supplementarymaterial, which is available to authorized users.

M. Pabijan (&) � J. M. Szymura

Department of Comparative Anatomy, Jagiellonian University,

Ingardena 6, 30-060 Krakow, Poland

e-mail: maciej.pabijan@uj.edu.pl

J. M. Szymura

e-mail: jacek.m.szymura@uj.edu.pl

C. Spolsky � T. Uzzell

Center for Systematic Biology and Evolution, Academy

of Natural Sciences, 1900 Benjamin Franklin Parkway,

Philadelphia, PA 19103, USA

e-mail: cspolsky@verizon.net

T. Uzzell

e-mail: uzzell@ANSP.org

123

J Mol Evol (2008) 67:246–256

DOI 10.1007/s00239-008-9123-3

are transcribed in the nucleus, assembled in the cytoplasm,

and transported into the mitochondrion (Larsson and

Clayton 1995). The mitogenome encodes only 13 of these

protein subunits and 24 RNA components (12S and 16S

rRNA molecules, 22 tRNAs) necessary for mitochondrial

protein synthesis. Despite this small proportion, the high

mutation rate of mtDNA may drive the evolution of

mitochondrial-nuclear interactions by contributing ‘new’

amino acids for the scrutiny of natural selection (Wu et al.

2000; Schmidt et al. 2001; Willet and Burton 2004). These

results have prompted a reevaluation of the neutrality of

mtDNA and show the need for further research in this area.

The physical and functional contiguity of the nuclear

and mitochondrial proteins in the enzymatic complexes of

the ETS leads to coadaptation of the two genomes (Blier

et al. 2001; Ballard and Whitlock 2004; Ballard and Rand

2005). Moreover, interactions between nuclear-encoded

mtRNA polymerases and their promoters located in

mtDNA control regions have been shown to be species-

specific (Gaspari et al. 2004). The polymerases, and pos-

sibly additional transcription factors, may not bind to the

mtDNA as precisely if presented with a divergent, foreign

promoter (Burton et al. 2006). Hence, mitochondrial genes

cannot be exempt from the epistasis evolving between

geographically delimited sets of nuclear alleles. The dis-

association of these interactions in interpopulation or

interspecies hybrids may cause inferior mitochondrial

function (McKenzie et al. 2003; Ellison and Burton 2006;

Burton et al. 2006).

The hybridizing European fire-bellied and yellow-bel-

lied toads, Bombina bombina and B. variegata, have

evolved in different environments (Szymura 1993). The

lowland form, B. bombina, inhabits larger, permanent

ponds, while the mountain type, B. variegata, prefers

smaller transient ponds and puddles. The toads are

divergent at the molecular level, and each exhibits a suite

of phenotypic traits in morphology, life history, ecology,

and behavior, regarded as adaptations to their respective

environments (Szymura 1993). Hybrid zones between the

toads occur in areas of altitudinal transition along their

parapatric contact in central Europe (Szymura and Barton

1991; Yanchukov et al. 2006). Selection against hybrids

results from intrinsic hybrid dysfunction (Szymura and

Barton 1986; Kruuk et al. 1999). Despite extensive

hybridization, no introgression of mtDNA has taken place

outside of the narrow zones (Szymura et al. 2000; Yan-

chukov et al. 2006; Hofman et al. 2007; Hofman and

Szymura 2007). Clines in mtDNA are no wider, and are

sometimes narrower, than clines at multiple unlinked al-

lozyme loci and morphological traits (Hofman and

Szymura 2007). Postzygotic reproductive barriers between

B. bombina and B. variegata have thus been attributed to

substantial genetic divergence in the genomes of the toads

and the genetic incompatibility that ensues (Szymura

et al. 1985; Nurnberger et al. 2003; Spolsky et al. 2006),

with differences in mtDNA hypothesized to contribute to

the negative epistasis in recombined hybrids (Hofman and

Szymura 2007).

In this paper we describe the complete mtDNA

sequences of the two hybridizing European species, B.

bombina and B. variegata. We quantify divergence

between the two genomes and their functional components,

including the distinctive organization of their control

regions (Spolsky et al. 2006), and study the distribution of

substitutions across the mitogenomes. Using a phylogeny

based on four species of Bombina and five outgroup spe-

cies, we identify regions in the mtDNA of B. bombina and

B. variegata carrying nonsynonymous and radical amino

acid substitutions and, conversely, conservative nucleotide

and amino acid domains. Apart from augmenting our

understanding of postzygotic reproductive barriers in

Bombina, our results should be useful for elucidating the

mechanisms of molecular evolution of mtDNA at low

levels of divergence, and contribute to the growing number

of described mitogenomes of the most basal group of

anuran amphibians (archeobatrachians), whose mtDNA is

known to evolve particularly slowly (Igawa et al. 2008).

Materials and Methods

Laboratory Methods

A single individual of B. variegata from Radziszow near

Krakow, Poland (49�570 N, 19�480 E) and a B. bombina

from Tyniec, Poland (50�020 N, 19�480 E) were used as

sources of mtDNA. The mtDNAs were purified as by

Szymura et al. (1985, 2000). Aliquots of B. variegata

mtDNA were partially digested with either HindIII or XbaI

and subsequently cloned into compatible pUC18 sites

(Boehringer Mannheim). MtDNA was radioactively

labeled and used as a probe to screen for clones containing

mtDNA fragments. The fragments were sized to compare

with mitochondrial maps (Spolsky et al. 2006) and inserts

were sequenced with universal M13 (forward and reverse)

primers and additional walking primers. Bombina specific

primers amplifying overlapping fragments spanning the

entire mtDNA molecule on both strands were then

designed on the basis of the cloned B. variegata mtDNA

for both PCR and sequencing in B. bombina (PCR/

sequencing strategy and primers listed in Electronic Sup-

plementary Material, S1 and S2). Two sequencing

chemistries were used as appropriate for a Beckman CEQ

sequencer (B. variegata mtDNA clones) and Applied

Biosystem Analyzer (most B. bombina mtDNA

amplicons).

J Mol Evol (2008) 67:246–256 247

123

Molecular Analyses

The mtDNA sequences were edited and assembled using

SeqMan version 5.05. After alignment of the entire gen-

omes in ClustalW, the two rRNAs, 22 tRNAs, and 13

protein coding genes were identified by comparisons to the

mtDNA genomes of B. orientalis (AY585338 [San Mauro

et al. 2004a]) and B. fortinuptialis (AY458591 [Zhang

et al. 2005], incorrectly listed in the NCBI database as B.

bombina [cf. Spolsky et al. 2006]). Molecular evolutionary

analyses including base composition, sequence divergence,

numbers of transitions and transversions, and numbers of

synonymous and nonsynonymous substitutions were car-

ried out using MEGA v.3.1 (Kumar et al. 2004) and DnaSP

(Rozas et al. 2003). We also performed a sliding window

analysis of nucleotide divergence in the four Bombina

mtDNA genomes. We identified radical replacement

changes in the protein coding genes, i.e. the replacement of

one amino acid by another that belongs to a different class

defined by the properties of lateral chains (e.g., Doiron

et al. 2002). Because all mitochondrially encoded peptides

exhibit transmembrane domains, we used a mutation

matrix specifically designed for transmembrane proteins

for scoring (Jones et al. 1994). We then mapped the auta-

pomorphic and radical amino acid substitutions onto the

putative topologies of the mtDNA-encoded proteins by

applying the hidden Markov method for sequence feature

prediction implemented in PolyPhobius (Kall et al. 2005)

and TMHMM (Krogh et al. 2001). Because homologues

are likely to share the same secondary structures, PolyPh-

obius also uses existing information from homologous

sequences in protein databases.

Phylogenetic Analyses

Besides the Bombina genomes listed above, we also used the

closest living relatives of Bombina from which entire mito-

chondrial genomes are available, i.e., Discoglossus galganoi

(AY585339) and Alytes obstetricans pertinax (AY585337).

Mitochondrial genomes from three other representatives of

Archaeobatrachia (Frost et al. 2006; Roelants et al. 2007)

were used as more distant outgroups: Ascaphus truei

(AJ871087), Xenopus tropicalis (AY789013), and Pelobates

cultripes (AJ871086). Phylogenetic analyses were per-

formed on 12 mitochondrial H-strand protein-coding genes.

The L-strand-encoded ND6 gene was excluded because of

differences in base composition. The mitochondrial protein

coding sequences of these 12 genes were translated, con-

catenated to form a single chain, and aligned using ClustalW

as implemented in MEGA v.3.1.

Multiple substitutions at the same site confound phylo-

genetic inference and obscure evolutionary relationships.

In order to avoid this complication, we first plotted the ratio

of transitions to transversions to assess substitution satu-

ration (Felsenstein 2004) against divergence times in

Bombina and related genera. The divergence times, taken

from Roelants et al. (2007) and Fromhage et al. (2004), are

meant to show the general trends in our analysis, so mea-

sures of uncertainty were not considered. The analysis

(Fig. 1) clearly showed that nucleotide substitutions were

not saturated in Bombina (Fig. 1, points A and B). We next

tested whether particular codon positions contained multi-

ple substitutions at the same sites by plotting the corrected

sequence divergence measured as maximum likelihood

(ML) distances against the numbers of transitions and

transversions at first, second, and third codon positions

(S3–S6). The general time-reversible (GTR + I + G;

I = 0.42, G = 0.81) model of DNA evolution was chosen

as the best-fitting model following the Akaike information

criterion and hierarchical likelihood ratio tests in Modeltest

v3.7 (Posada and Crandall 1998). Transitions at third codon

positions were saturated (S3, S4), so we only used first and

second codon positions in the phylogenetic analysis (S5).

For the amino acid data, uncorrected pairwise distances

were plotted against ML distances incorporating the

mtREV + I + G (I = 0.56, G = 0.30) model of protein

evolution, which best fit the amino acid data according to

the AIC criterion in ProtTest v1.3 (Abascal et al. 2005).

The amino acid sequences were not saturated (S6).

The final alignments used for the phylogenetic analyses

for the nine basal anuran taxa encompassed either 7184

nucleotide positions or 3592 amino acid residues. All

alignments are available from the corresponding author.

The best-fitting model of DNA evolution for the first and

second codon position dataset (unpartitioned) was the

GTR + I + G model (I = 0.6, G = 1.36). In a separate

Fig. 1 Ratio of number of transitions (TS) to number of transversions

(TV) between (A) B. bombina and B. variegata; (B) B. orientalis and

(B. bombina + B. variegata); (C) Alytes and Discoglossus; (D)

(Alytes + Discoglossus) and Bombina; (E) Discoglossidae and

(Xenopus + Pelobates); and (F) Ascaphus and (Discoglossi-

dae + Xenopus + Pelobates). Divergence times are estimates

without confidence intervals from Roelants et al. (2007) for points

A–E and from Fromhage et al. (2004) for point F

248 J Mol Evol (2008) 67:246–256

123

analysis, we partitioned the mtDNA genes into three

functional groups (protein coding genes, rRNAs, tRNAs)

and assigned models to each through Modeltest. For the

rRNAs and for the tRNAs, Modeltest suggested values of

GTR + G (G = 0.32) and GTR + I + G (I = 0.42,

G = 1.18), respectively. ML analyses of the unpartitioned

first and second codon position dataset and the amino acid

dataset were conducted in PHYML v2.4.4 (Guindon and

Gascuel 2003), starting with the BIONJ trees. Nonpara-

metric bootstrapping was used to test the reliabilities of the

ML trees (1000 pseudoreplicates). Bayesian inference (BI)

of the phylogeny for all three datasets was carried out using

MrBayes v3.04b (Huelsenbeck and Ronquist 2001) with

the previously specified models of sequence evolution; all

other priors were set to default values. One cold and three

heated chains were run for 5 million generations, with trees

sampled every 100 generations. Generations sampled

before the chains reached stationarity (20,000), as judged

by examining the log-likelihoods of the cold chains and

plots of the generation vs. log-likelihood values of the data,

were discarded as burn-in. At least two independent runs

were carried out for each dataset in the Bayesian analyses.

Results

Genome Organization and Nucleotide Composition

The complete mtDNA genomes have been deposited at the

GenBank database under accession numbers EU115993 (B.

bombina) and AY971143 (B. variegata). Within the genus,

the B. variegata mtDNA genome was the largest, at

18,551 bp, followed by B. fortinuptialis (17,575 bp), B.

orientalis (17,173 bp), and B. bombina (17,154 bp). The

differences in mtDNA genome size can be accounted for

by size variation in the control region. The mtDNA gen-

omes of Bombina exhibit the organization and gene content

of the canonical mtDNA genome of other vertebrates

(Table 1; Fig. 3) (Boore 1999). The gene arrangement and

start and stop codons in the Bombina mtDNA genomes are

identical in all studied species (Table 1). The putative

origin of L-strand replication (OL) in the Bombina mitog-

enomes was located within the WANCY tRNA cluster,

between the tRNAAsn and the tRNACys genes. The lengths

of the protein coding genes are also similar, with the

exception of the third codon in ND1 (ACT, threonine in B.

variegata), which is absent in B. bombina. The nucleotide

frequencies of the L-strands are very similar in all four

Bombina species (Table 1) and skewed against guanine

because of bias against this nucleotide at the second and

third codon positions. The transition-to-transversion ratio

was quite variable among genes, but always skewed toward

transitions (Table 2).

Control Region Structure

We compared the control regions in all four sequenced

Bombina mtDNAs (Fig. 2A). The size of the control region

varies nearly twofold in Bombina, consisting of 3072 bp in

B. variegata, 2373 bp in B. orientalis, 1990 bp in B. for-

tinuptialis, and 1675 bp in B. bombina (Fig. 2A). The B.

fortinuptialis control region is incomplete because of an

unsequenced stretch at the 30-end of the first tandem repeat

(Zhang et al. 2005). The latter, termed LV1 by Spolsky

et al. (2006), consists of 4–12 serial repeats 70–77 bp long

(Fig. 2B). A highly variable nucleotide sequence is found

downstream of LV1, with approximately the first 150 bp

representing incomplete repeat units. Three conserved

sequence blocks (CSB1–3) are localized downstream of

LV1. The region –180 bp relative to CSB1 is alignable in

all Bombina mitogenomes studied and encompasses a

poly(T) block, the pyrimidine-rich region, PP-1, of San

Table 1 Lengths, in base pairs, of structural features in the BombinamtDNA genomes and nucleotide compositions of mitochondrial L-strands

Feature Bbom Bvar Bori Bfor Start; stop

Total length 17,154 18,551 17,173 17,575

% A 30.0% 30.0% 30.0% 30.5%

% C 26.5% 26.1% 26.7% 25.4%

% G 15.8% 16.0% 15.5% 15.3%

% T 27.7% 27.9% 27.8% 28.8%

12S rRNA 933 933 933 929

16S rRNA 1599 1601 1599 1599

ATP6 683 683 683 683 ATG; TAA

ATP8 168 168 168 168 ATG; TAA

Cytb 1141 1141 1141 1141 ATG; T–

COI 1554 1554 1554 1554 GTG; TAA

COII 688 688 688 688 ATG; T–

COIII 784 784 784 784 ATG; T–

ND1 959 962 962 962 ATG; TAG

ND2 1045 1045 1045 1045 ATG; T–

ND3 343 343 343 343 ATG; T–

ND4 1378 1378 1378 1378 ATG; T–

ND4L 297 297 297 297 ATG; TAA

ND5 1809 1809 1809 1809 ATG; TAA

ND6 510 510 510 510 ATG; AGA

Control region 1675 3072 2373 1990

Intergenic DNA 42 41 42 37

Note: Initiation and termination codons (start; stop) are given in the

last column. Incomplete stop codons (T–) presumably become func-

tional by subsequent polyadenylation of the respective mRNAs. B.orientalis mtDNA sequence from San Mauro et al. (2004a;

AY585338). B. fortinuptialis mtDNA sequence from Zhang et al.

(2005; AY458591; incorrectly listed in the NCBI database as B.bombina [cf. Spolsky et al. 2006]). Bbom, B. bombina; Bvar, B.variegata; Bori, B.orientalis; Bfor, B. fortinuptialis

J Mol Evol (2008) 67:246–256 249

123

Mauro et al. (2004a). Between CSB-1 and CSB-2 lies a

poly-C block (PP-2 [cf. San Mauro et al. 2004a]). PP-1 and

PP-2 may be involved in H-strand replication (San Mauro

et al. 2004b). The second repeat motif (LV2; Fig. 2C) is

present in only three of the species, being secondarily lost

in B. bombina. This 62- to 66-bp motif, repeated up to

13 times in B. variegata, is characterized by a conserved

50-end, AT and GT dinucleotide repeats, and a poly(T) tail

present in the three species. Additional incomplete repeats

flank LV2, and a homologous but nonrepetitive sequence is

found at the 30-end of the control region in B. bombina.

Divergence in Bombina mtDNA

We calculated the pairwise divergence among the four

Bombina mitochondrial genomes, taking into account all

rRNAs, tRNAs, and protein coding DNA sequences and, in

separate calculations, divergence in amino acid sequences,

tRNAs and rRNAs (Table 3). Uncorrected nucleotide

divergence between the hybridizing B. bombina and B.

variegata was lowest, at 8.1% or 8.7% (Kimura two-

parameter distance; K2P). The highest divergence was

between the mtDNA of the two East Asian species, B.

orientalis and B. fortinuptialis, at 14.3% (16.2% K2P).

The various functional regions of Bombina mtDNA

have evolved at different rates. The rRNA and tRNA genes

were least diverged (Fig. 3), implying functional con-

straint. Genes encoding ETS complexes III, IV, and V

always showed the lowest number of nonsynonymous

substitutions (Tables 2 and 3). In contrast, the protein

coding genes of ETS complex I were the most variable,

particularly certain regions of ND2, ND5, and ND4

(Fig. 3), and had the highest numbers of nonsynonymous

substitutions (Table 2).

Phylogenetic Relationships

Both ML and BI analyses reconstructed similar tree

topologies for nucleotide and amino acid datasets (Fig. 4).

The partitioned nucleotide dataset gave essentially the

same results as the unpartitioned nucleotide dataset; we

therefore report only the latter. Most nodes were well

supported, and the intrageneric relationships of Bombina

were fully resolved. The sister group relationship between

B. bombina and B. variegata was confirmed (Hofman et al.

2007). The East Asian B. orientalis formed a well-sup-

ported clade with the European species, to the exclusion of

B. fortinuptialis, which has a basal placement in the genus

Bombina. The only discrepancy observed between the

nucleotide and the amino acid analyses was the placement

of Alytes. In the amino acid analyses, an Alytes-Bombina

clade was recovered, while in the first and second codon

position dataset an Alytes-Discoglossus clade was apparent.

The position of Ascaphus was left unresolved by the amino

acid data.

Patterns of Substitution in Mitochondrial Protein

Coding Genes of European Bombina

The number of nucleotide substitutions calculated for all 13

mitochondrial protein coding genes between B. bombina

and B. variegata was 1130 (Table 2), of which 1032 (91%)

were silent. The uncorrected percent divergence for par-

ticular genes varied between 7.5% (COIII) and 11.5%

(ND2). The percentage of synonymous substitutions in

synonymous sites between the mitochondrial protein cod-

ing genes of B. bombina and B. variegata varied between

30% and 40% in particular genes, much higher than the

percentage of nonsynonymous substitutions in nonsyn-

onymous sites, which ranged from 0 to 2.4% (Table 2).

The number of amino acid differences in the mtDNA-

encoded peptides between B. bombina and B. variegata

ranged from 0 in COII to 29 in ND5 (Table 2) and was

positively correlated with the number of base pairs in each

gene (R2 = 0.425, p = 0.015). More revealing, the dN/dS

ratios (Table 2), a measure of the stringency of structural

and functional constraint acting on the protein coding

Table 2 Nucleotide substitutions in mitochondrial genes of B.bombina and B. variegata

Gene Nn (%) TS/TV NN (dN) NS (dS) dN/dS

12S rRNA 27 (2.9) 4.4 – – –

16S rRNA 53 (3.3) 7.8 – – –

ATP6 78 (11.4) 4.6 7 (0.014) 71 (0.572) 0.024

ATP8 13 (7.7) 3.3 1 (0.008) 12 (0.387) 0.021

Cytb 99 (8.7) 6.1 7 (0.008) 92 (0.440) 0.018

COI 153 (9.8) 9.9 1 (0.001) 152 (0.554) 0.002

COII 57 (8.3) 10.4 0 57 (0.487) 0

COIII 59 (7.5) 9.2 2 (0.005) 57 (0.375) 0.009

ND1 102 (10.6) 4.6 7 (0.010) 95 (0.541) 0.018

ND2 121 (11.5) 6.1 17 (0.022) 104 (0.535) 0.041

ND3 34 (9.9) 7.7 6 (0.023) 28 (0.416) 0.055

ND4 151 (10.9) 5.6 15 (0.015) 136 (0.546) 0.027

ND4L 33 (11.1) 7.2 1 (0.005) 32 (0.558) 0.008

ND5 174 (9.6) 9.3 29 (0.022) 145 (0.430) 0.051

ND6 56 (10.9) 6.0 5 (0.014) 51 (0.486) 0.028

R 1130 98 1032

Note: Nn, total number of nucleotide differences (percentage differ-

ences in total number of sites); TS/TV, transition-to-transversion ratio;

NN, number of nonsynonymous substitutions; dN, number of

nonsynonymous substitutions in total number of nonsynonymous

sites; NS, number of synonymous substitutions; dS, number of syn-

onymous substitutions in total number of synonymous sites; dN/dS,

ratio of number of nonsynonymous substitutions per nonsynonymous

site to number of synonymous substitutions per synonymous site

250 J Mol Evol (2008) 67:246–256

123

genes, shows that the strength of selection against nonsyn-

onymous change in the Bombina protein coding genes is

strong. According to this measure, the protein coding genes

can be arranged in the following order, from most conser-

vative to most variable: COII \ COI \ ND4L \\COIII\ Cytb \ ND1 \ ATP8 \ ATP6 \ ND4 \ ND6 \\ND2 \ ND5 \ ND3. Of the total of 98 nonsynonymous

substitutions between the mtDNA of B. bombina and B.

variegata, the majority (80) were found in ETS complex I of

the electron transport system (Table 3). Only seven

nonsynonymous substitutions were found in ETS complex

III, three in complex IV, and eight in complex V (Table 3).

Functional Significance of the Amino Acid

Substitutions

On the basis of the phylogenetic relationships among the

mtDNA genomes of the basal anurans (Fig. 4), we inferred

the derived state (either synapomorphic, autapomorphic, or

homoplasious) for all replacement substitutions observed

?

B. bombina

B. orientalis

B. fortinuptialis

5'

5'

5'

5'

3'

3'

3'

3'

B.variegataB.bombinaB.orientalisB.fortinuptialis

------------A TATTA TA-- AGGAC C T CTT TCC T AC T TTATATGC----A T--TA TG-- GGGGT C C CAT --- T TC -

CTATATATTT--A C--TA TAT- GGAC- C T CTT TCC T AT ------TATTTAGT TTT-- CATA G-AC- A C ATA TCC C AC T

TA ATG TA TAT TATGTATAAT GAGCATTCATCT T TCA GAATATCC ATA ATG TA TAT TATGTATAAT GAGCATTCATCT T TCA GAATATCC ATA ATG TA TAT TATGTATAAT GAGCATTCATCT T TCA GAATATCC ATA ATG TA TAT TATGTATAAT GAGCATTCATCT T TCA GAATATCC A

TAS

B.variegata

B.fortinuptialis

T T GG T CCAATATG GAAT TTA-- T A CATATGAC- A

AGCAGGAAC CC G CG ATA AA T TATA AT TAT TTTTT TAGCAGGAAC CC G CG ATA AA T TATA AT TAT TTTTT T

C A AA T CCATAAGG ATTC GGTG- T G GTG------ T T A GA C ATTTGTGA AC-C GGTG- C G GTGTAA--- TB.orientalis

AGCAGGAAC CC G CG ATA AA T TATA AT TAT TTTTT T

2VL1VL

(A)

(B)

(C)

Fig. 2 A comparison of control region structure in European and

East Asian Bombina species. (A) Large vertical bars represent

tandemly arranged repeat units within LV1 and LV2. Filled circles

represent conserved sequence blocks (CSB); small vertical bars

among the CSBs are pyrimidine-rich regions (PP-1 and PP-2). Length

is proportional to the number of nucleotides in each species. The

question mark in B. fortinuptialis marks an unsequenced fragment

after four repeats. (B) Alignment of the repeat unit LV1. Horizontal

bar denotes the putative termination associated sequence (TAS). (C)

Alignment of the repeat unit LV2. Conservative sites are in boldface

Table 3 Pairwise comparisons of divergence in functional units among Bombina mtDNA genomes

Total rRNA tRNA AA ETS complex I ETS complex III ETS complex IV ETS complex V

Bbom-Bvar 8.1 (8.7) 80 (3.1) 37 (2.4) 80.0 7.0 3.0 8.0 2.3

Bbom-Bori 12.4 (13.9) 152 (6.0) 62 (4.0) 146.8 16.0 10.0 16.5 4.4

Bbom-Bfor 13.8 (15.5) 160 (6.3) 116 (7.6) 215.6 18.0 20.0 16.0 5.7

Bvar-Bori 12.5 (14.0) 149 (5.9) 73 (4.8) 146.0 19.5 9.0 21.0 4.4

Bvar-Bfor 13.9 (15.7) 172 (6.8) 112 (7.3) 222.3 18.5 19.0 16.0 5.7

Bori-Bfor 14.3 (16.2) 178 (7.0) 111 (7.3) 216.0 18.5 23.0 25.0 6.2

Note: Bbom, B. bombina; Bvar, B. variegata; Bori, B.orientalis; Bfor, B. fortinuptialis. Total: the percentage of different nucleotides and Kimura

two-parameter corrected distances (K2P; percentages) in complete mtDNA genomes (excluding control regions). RNA: nucleotide differences in

mitochondrial RNA-encoding genes pooled for the 2 rRNAs and 22 tRNAs. Numbers in parentheses are uncorrected divergences given as

percentages. ETS complex: The numbers of nonsynonymous substitutions, calculated using Nei and Gojobori’s method (1986), for peptides in

subunits of the oxidative phosphorylation complex. ETS complex I: ND1–6, 4L. ETS complex III: Cytb. ETS complex IV: COI, COII, COIII.ETS complex V: ATP6, ATP8. AA: Poisson-corrected amino acid divergence for the concatenated mtDNA-encoded proteins, as a percentage

J Mol Evol (2008) 67:246–256 251

123

between the mitochondrially encoded peptides of the two

European species and the two East Asian congeners used as

outgroups. A total of 32 and 33 autapomorphic amino acid

replacements were identified in the mitochondrially enco-

ded peptides of the B. bombina and B. variegata lineages,

respectively. Character assignment was ambiguous in 19

additional homoplasious amino acid replacements in

which each of the outgroup species shared an amino acid

with one of the ingroup species. Of the autapomorphic

replacement changes that had a negative mutability score

according to a matrix for transmembrane proteins, B.

bombina possessed three, while B. variegata had six

(Table 4). ETS complex I harbored all but one (found in

ETS complex V) of these nonconservative amino acid

replacements (Table 4).

The predicted topology of the four proteins (ATP6,

ND2, ND4, and ND5) in Bombina in which radical amino

acid replacements occurred is shown in Supplementary

Material S7–S10. TMHMM and PolyPhobius predictions

Fig. 3 Sliding window analysis of uncorrected percentage diver-

gence in four Bombina mt genomes (window size, 100 bp; step size,

25 bp). Genes encoded by the L-strand—ND6, tRNA-Q, tRNA-A,

tRNA-N, tRNA-C, tRNA-Y, tRNA-S(UCN), tRNA-E, and tRNA-P are

underlined. Vertical gray bars highlight the tRNA genes (tRNA letter

abbreviations above bars). Tick marks along the X axis delimit the

rRNA and protein-coding genes

Fig. 4 Consensus phylogram reconstructed for the first and second

codon position dataset of 12 H-strand mitochondrial protein-coding

genes using maximum likelihood. Numbers represent PHYML

bootstrap support for the first and second codon position dataset

and the amino acid dataset (first line) and Bayesian posterior

probabilities for the two datasets (second line). Only support values

[50% are shown. Branch length is proportional to the number of

substitutions per site

Table 4 Autopomorphic and radical amino acid (aa) substitutions in

B. variegata and B. bombina and their location on the peptide chain

and topology of the protein

aa position Substitution Score Domain

B. variegata

ATP6 64 L-P -1 Matrix

ND2 310 P-S -1 Inter

ND4 20 S-Q -1 Inter

ND5 23 L-S -2 Trans

ND5 516 S-P -1 Inter

ND5 530 N-Y -2 Inter

B. bombina

ND4 445 A-M -1 Trans

ND5 451 S-F -1 Inter

ND5 512 P-S -1 Inter

Note: Scoring according to the amino acid mutation matrix for

transmembrane proteins proposed by Jones et al. (1994). The domain

category refers to the topology of the transmembrane proteins; the

substitution occurs in either the hydrophobic transmembrane domains

(trans), the mitochondrial matrix domains (matrix), or the mito-

chondrial intermembrane domains (inter)

252 J Mol Evol (2008) 67:246–256

123

were generally congruent, although the algorithm used in

the latter application consistently identified a higher num-

ber of transmembrane helices. In addition, the orientation

of the proteins was often contradictory. The locations of

the autapomorphic, radical amino acid substitutions in the

mitochondrial peptides of B. bombina and B. variegata are

given in Table 3 and Supplementary Material S7–S10.

The ATP6 subunit is part of the F0 segment of the ATP

synthase and takes part in the channeling of protons

through the inner membrane of the mitochondrion. The

functionally important amino acids of the ATP6 subunit,

including the polar residues comprising the proton channel

and residues in contact with nuclear subunits, are quite

conserved between humans and E. coli (Schon et al. 2001)

and Bombina (data not shown). The L-P substitution in B.

variegata, however, is clearly located outside of the

transmembrane helices and does not participate in nuclear-

mitochondrial interactions (Table 3, Supplementary Mate-

rial S7 [cf. Schon et al. 2001]), thus it probably does not

affect the functioning of the ATP synthase.

The remaining radical amino acid replacements

occurred in the ND proteins of ETS complex I. This

large, L-shaped enzyme is composed of 46 subunits, 7 of

which are hydrophobic central subunits encoded by

mtDNA (Brandt 2006). The molecular structure of this

complex remains unresolved, and how amino acid varia-

tion in specific subunits affects the functioning of the

complex is poorly known. The transmembrane domain

predictors applied to the Bombina complex I proteins

(Supplementary Material S8–S10) were often at odds as

to which amino acid residues constitute transmembrane

helices. Nonetheless, the domains in which the radical

replacement changes occurred were always well defined

and, thus, can be used as rough estimates of their loca-

tion. A single radical substitution, invoking a change from

a hydrophobic to a hydrophilic amino acid (L-S), was

detected in a transmembrane helix of ND5 in B. varieg-

ata. Other amino acid differences were located in

domains putatively outside of the transmembrane seg-

ments. Three involved proline, an amino acid with a

cyclic structure known to occur in the turns of polypep-

tide chains, and could therefore alter the secondary

structure of these surface domains. A total of three radical

substitutions occurred in a large (ca. 76 residues, positions

502–586; Supplementary Material S10) extramembrane

domain of the C-terminus of ND5.

Discussion

Despite the high degree of nucleotide divergence between

the Bombina mitochondrial genomes (up to 14% between

the two East Asian species) and their closest relatives (26%

between Discoglossus/Bombina and Alytes/Bombina), the

mitochondrial genomes of these amphibians retained

identical gene order and content and similar nucleotide

composition over the immense expanse of evolutionary

time since their divergence (during the late Triassic/early

Jurassic according to recent molecular estimates [Roelants

et al. 2007; San Mauro et al. 2004a]). The most divergent

mtDNA of the four species compared was that of B. for-

tinuptialis, which, together with B. maxima and B.

microdeladigitora, belongs to the group of large-bodied

Bombina species of Southeast Asia (Grobina). The ancient

split between the large-bodied and the small-bodied Bom-

bina species (B. bombina, B. variegata, and B. orientalis) is

supported by the grouping of B. orientalis with the Euro-

pean species in our mtDNA phylogeny, as well as by

karyotypic differences, i.e., the large-bodied species bear a

diploid chromosome complement of 2N = 28, while the

small-bodied species contain 2N = 24 (Szymura and Pas-

sakas-Szymczak 1988). B. orientalis, despite having a

distribution that is geographically proximate to the range of

B. fortinuptialis, is phylogenetically closer to the Western

Palearctic Bombina species than to its East Asian relatives.

Analysis by Yu et al. (2007) of DNA sequences from

several mitochondrial genes for Grobina and several

European and Asian Bombina species also supports this

grouping.

The average nucleotide divergence in mtDNA between

the two hybridizing species, B. bombina and B. variegata,

was 8.1 (8.7% K2P). This figure is congruent with earlier

estimates based on restriction enzymes (9.4% ± 1%

[Szymura et al. 1985], 6.0–8.1% [Szymura et al. 2000]). In

a different study, sequence divergence in Cytb haplotypes

between 62 individuals of both species amounted to

9.2% ± 0.2% K2P (Hofman and Szymura 2007), a value

slightly above the average for the entire mitogenome. The

nuclear sequence divergence between B. bombina and B.

variegata is lower than this and has been estimated as 0–

8% (Nurnberger et al. 2003) for several homologous,

putatively noncoding loci, 3.7% (3.8% K2P) for a partial

coding fragment of histone H3a (Frost et al. 2006; Gen-

Bank accession numbers DQ284275 and DQ284274 for B.

bombina and B. variegata, respectively), and 0.03%

(0.03% K2P) in exon 1 of the rhodopsin gene (Frost et al.

2006; DQ283920 and DQ283919 for B. bombina and B.

variegata, respectively). The divergence in 29 allozyme

loci between B. bombina and representatives of the major

subgroups of B. variegata, measured as Nei’s D distance,

ranged between 0.37 and 0.59 (Szymura 1993).

The Bombina control region contains highly conserved

regions, such as the CSBs and pyrimidine-rich regions, and

also length-variable regions (LV1 and LV2) comprised of

tandemly arranged repeat units. The conserved sequence

blocks (CSBs) and pyrimidine-rich stretches (PPs) possibly

J Mol Evol (2008) 67:246–256 253

123

play a role in transcription of mtDNA-encoded proteins

and H-strand replication in amphibians (San Mauro et al.

2004b). In Xenopus laevis, bidirectional promotors and

transcription factor binding sites have been localized

upstream of CSB2 and CSB3 (Antoshechkin and Bogen-

hagen 1995). These regions of high sequence conservation

are preceded by the first repeat region, LV1, consisting of

repeat units that are alignable in Bombina (Fig. 2B) and,

also, among archeobatrachians (San Mauro et al. 2004a),

suggesting homology. This sequence similarity indicates

functional constraint that may result from the presence of a

putative termination-associated sequence (TAS) in the LV1

repeat unit. TASs terminate H-strand synthesis and thus

produce the D-loop-containing form of mtDNA. The

presence of repeats that contain TAS sequences and pos-

sibly other regulatory elements downstream of LV1 raises

the possibility of a fine-tuning of mitochondrial metabo-

lism (through the regulation of mtDNA replication/

transcription) by the number of the repeat units in the

control regions, as suggested for lagomorph mtDNA (Ca-

sane et al. 1997). In contrast, the LV2 unit, tandemly

repeated at the 30 end of the Bombina control region, is

restricted to the genus Bombina and has been secondarily

lost in B. bombina. Apparently, the LV2 region in Bombina

is under less selective constraint than LV1, as evidenced by

the lack of sequence similarity to other archeobatrachian 30

repeat motifs (San Mauro et al. 2004a), its absence in B.

bombina, and no known regulatory function.

The varying dN/dS ratios observed among the protein

coding mitochondrial genes in Bombina suggest generally

high but, nonetheless, unequal levels of stringency of

structural and functional constraint. Overall, our results are

consistent with previous studies examining substitution

patterns among mitochondrial protein coding genes, in

which genes encoding proteins of ETS complexes III, IV,

and V were always much more conserved than those in

ETS complex I (e.g., Pesole et al. 1999; Doiron et al.

2002). Exceptional are perhaps the high dN/dS ratio in ND3

and relatively low ratios for ATP6 and ATP8 in Bombina.

Low ratios for ATP8 have also been documented in several

fish species (Roques et al. 2006 and references therein).

Closer inspection of the species-specific, nonsynony-

mous substitutions between the hybridizing European

Bombina species revealed that the majority can be

considered functionally neutral, yet 9 of the 98 nonsyn-

onymous substitutions between B. bombina and B.

variegata may have functional consequences. All but one

occur in ETS complex I, for which only a limited amount

of structural information is available. Moreover, the radical

replacement substitutions observed in ND4 and ND5

coincide with elevated KA/KS ratios. Both radical replace-

ments and high dN/dS ratios have been regarded as the

hallmarks of coadaptation between mitochondrial and

nuclear ETS subunits (Wu et al. 2000; Schmidt et al.

2001; Doiron et al. 2002) or evidence for the action of

positive selection (Mishmar et al. 2003; 2006). A possi-

ble alternative explanation is that the elevated rate of

amino acid substitution results from relaxed selective

constraint on sites at which the need for amino acid

conservation is lower (Elson et al 2004; Ingman and

Gyllensten 2007).

Mitochondrially encoded and nDNA-encoded proteins

build functional units constituting the mitochondrial ETS,

whereas species-specific promotor regions in mtDNA

probably coevolve with nDNA-encoded peptides. Muta-

tions in either genomic component influence the evolution

of the other, producing coadapted complexes (Blier et al.

2001). These associations are particularly prone to dis-

ruption in species hybrids (Sackton et al. 2003; Zeyl et al.

2005; Ellison and Burton 2008). Despite ongoing hybrid-

ization in spatially and temporally variable hybrid zones,

Hofman et al. (2007) have found no evidence for either

past or present mtDNA introgression between European

Bombina, which is consistent with the idea that later-gen-

eration hybrids are less fit because of cytonuclear

incompatibility leading to the disruption of mitochondrial

function (Burton et al. 2006). In this paper we have shown

that there are ample differences between the mtDNA

genomes of B. bombina and B. variegata that may have

functional consequences affecting mitochondrial oxidative

phosphorylation in hybrids. Further studies should incor-

porate experimental verification of our results through, e.g.,

in vitro assays of the efficiency of ETS complexes in

hybrid and nonhybrid individuals.

Acknowledgments The study was supported by grants from the

American Philosophical Society to J.M.S. and KBN Grant 2PO4C

087 29 to J.M.S. and M.P.

References

Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit

models of protein evolution. Bioinformatics 12:2104–2105

Antoshechkin I, Bogenhagen DF (1995) Distinct roles for two purified

factors in transcription of Xenopus mitochondrial DNA. Mol

Cell Biol 15:7032–7042

Ballard JWO, Rand DM (2005) The population biology of mitochon-

drial DNA and its phylogenetic implications. Annu Rev Ecol

Evol 36:621–642

Ballard JWO, Whitlock MC (2004) The incomplete natural history of

mitochondria. Mol Ecol 13:729–744

Blier PU, Dufresne F, Burton R (2001) Natural selection and the

evolution of mtDNA-encoded peptides: insights from studies of

protein function and cytonuclear coadaptation. Trends Genet

17:400–406

Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res

27:1767–1780

Brandt U (2006) Energy converting NADH: quinone oxidoreductase

(Complex I). Annu Rev Biochem 75:69–92

254 J Mol Evol (2008) 67:246–256

123

Burton RS, Ellison CK, Harrison JS (2006) The sorry state of F2

hybrids: consequences of rapid mitochondrial DNA evolution in

allopatric populations. Am Nat 168:S14–S24

Casane D, Dennebouy N, de Rochambeau H, Mounolou JC,

Monnerot M (1997) Nonneutral evolution of tandem repeats in

the mitochondrial DNA control region of Lagomorphs. Mol Biol

Evol 14:779–789

Doiron SL, Bernatchez L, Blier PU (2002) A comparative mitoge-

nomic analysis of the potential adaptive value of arctic charr

mtDNA introgression in brook charr populations (Salvelinusfontinalis Mitchill). Mol Biol Evol 19:1902–1909

Ellison CK, Burton RS (2006) Disruption of mitochondrial function

in interpopulation hybrids of Tigriopus californicus. Evolution

60:1382–1391

Ellison CK, Burton RS (2008) Interpopulation hybrid breakdown

maps to the mitochondrial genome. Evolution 62–63:631–638

Elson JL, Turnbull DM, Howell N (2004) Comparative genomics and

the evolution of human mitochondrial DNA: assessing the

effects of selection. Am J Hum Genet 74:229–238

Felsenstein J (2004) Inferring phylogenies. Sinauer Associates,

Sunderland, MA

Fromhage L, Vences M, Veith M (2004) Testing alternative

vicariance scenarios in Western Mediterranean discoglossid

frogs. Mol Phylogenet Evol 31:308–322

Frost DR, Grant T, Faivovich J, Bain RH, Haas A, Haddad CFB, De

Sa RO, Channing A, Wilkinson M, Donnelan SC, Raxworthy CJ,

Campbell JA, Blotto BL, Moler P, Drewes RC, Nussbaum RA,

Lynch JD, Green DM, Wheeler WC (2006) The amphibian tree

of life. Bull Am Mus Nat His 297:1–370

Gaspari M, Falkenberg M, Larsson N-G, Gustafsson CM (2004) The

mitochondrial RNA polymerase contributes critically to pro-

moter specificity in mammalian cells. EMBO J 23:4606–4614

Gemmell NJ, Metcalf VJ, Allendorf FW (2004) Mother’s curse: the

effect of mtDNA on individual fitness and population viability.

Trends Ecol Evol 19:238–244

Guindon S, Gascuel O (2003) PHYML—a simple, fast, and accurate

algorithm to estimate large phylogenies by maximum likelihood.

Syst Biol 52:696–704

Hofman S, Szymura JM (2007) Limited mtDNA introgression in a

Bombina hybrid zone. Biol J Linn Soc 91:295–306

Hofman S, Spolsky C, Uzzell T, Cogalniceanu D, Babik W, Szymura

JM (2007) Phylogeography of the fire-bellied toads, Bombina:

independent Pleistocene histories inferred from mitochondrial

genomes. Mol Ecol 16:2301–2316

Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of

phylogeny. Bioinformatics 17:754–755

Igawa T, Kurabayashi A, Usuki C, Fujii T, Sumida M (2008)

Complete mitochondrial genomes of three neobatrachian anu-

rans: a case study of divergence time estimation using different

data and calibration settings. Gene 407:116–129

Ingman M, Gyllensten U (2007) Rate variation between mitochon-

drial domains and adaptive evolution in humans. Hum Mol

Genet 16:2281–2287

Jones DT, Taylor WR, Thornton JM (1994) A mutation data matrix

for transmembrane proteins. FEBS Lett 339:269–275

Kall L, Krogh A, Sonnhammer ELL (2005) An HMM posterior

decoder for sequence feature prediction that includes homology

information. Bioinformatics 21:i251–i257

Krogh A, Larsson B, von Heijne G, Sonnhammer ELL (2001)

Predicting transmembrane protein topology with a hidden Markov

model: application to complete genomes. J Mol Biol 305:567–580

Kruuk LEB, Gilchrist JS, Barton NH (1999) Hybrid dysfunction in

fire-bellied toads (Bombina). Evolution 53:1611–1616

Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for

molecular evolutionary genetics analysis and sequence align-

ment. Brief Bioinform 5:150–163

Larsson NG, Clayton DA (1995) Molecular genetic aspects of human

mitochondrial disorders. Annu Rev Genet 29:151–178

McKenzie M, Chiotis M, Pinkert CA, Trounce IA (2003) Functional

respiratory chain analyses in murid xenomitochondrial cybrids

expose coevolutionary constraints of cytochrome b and nuclear

subunits of complex III. Mol Biol Evol 20:1117–1124

Mishmar D, Ruiz-Pesini E, Golik P, Macaulay V, Clark AG, Hosseini

S, Brandon M, Easley K, Chen E, Brown MD, Sukernik RI,

Olckers A, Wallace DC (2003) Natural selection shaped regional

mtDNA variation in humans. Proc Natl Acad Sci USA 100:

171–176

Mishmar D, Ruiz-Pesini E, Mondragon-Palomino M, Procaccio V,

Gaut B, Wallace DC (2006) Adaptive selection of mitochondrial

complex I subunits during primate radiation. Gene 378:11–18

Nei M, Gojobori T (1986) Simple methods for estimating the numbers

of synonymous and nonsynonymous nucleotide substitutions.

Mol Biol Evol 3:418–426

Nurnberger B, Hofman S, Forg-Brey G, Praetzel G, Maclean A,

Szymura JM, Abbott CM, Barton NH (2003) A linkage map for

the hybridising toads Bombina bombina and B. variegata (Anura:

Discoglossidae). Heredity 91:136–142

Pesole G, Gissi C, De Chirico A, Saccone C (1999) Nucleotide

substitution rate of mammalian mitochondrial genomes. J Mol

Evol 48:427–434

Posada D, Crandall KA (1998) Modeltest: testing the model of DNA

substitution. Bioinformatics 14:817–818

Roelants K, Gower DJ, Wilkinson M, Loader SP, Biju SD, Guillaume

K, Moriau L, Bossuyt F (2007) Global patterns of diversification

in the history of modern amphibians. Proc Natl Acad Sci USA

104:887–892

Roques S, Fox CJ, Villasana MI, Rico C (2006) The complete

mitochondrial genome of the whiting, Merlangius merlangus and

the haddock, Melanogrammus aeglefinus: a detailed genomic

comparison among closely related species of the Gadidae family.

Gene 383:12–23

Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003)

DnaSP, DNA polymorphism analyses by the coalescent and

other methods. Bioinformatics 19:2496–2497

Sackton TB, Haney RA, Rand DM (2003) Cytonuclear coadaptation

in Drosophila: disruption of cytochrome c oxidase activity in

backcrossgenotypes. Evolution 57:2315–2325

San Mauro D, Garcıa-Parıs M, Zardoya R (2004a) Phylogenetic

relationships of discoglossid frogs (Amphibia: Anura: Disco-

glossidae) based on complete mitochondrial genomes and

nuclear genes. Gene 343:357–366

San Mauro D, Gower DJ, Oommen OV, Wilkinson M, Zardoya R

(2004b) Phylogeny of caecilian amphibians (Gymnophiona)

based on complete mitochondrial genomes and nuclear RAG1.

Mol Phylogenet Evol 33:413–427

Schapira AHV (2006) Mitochondrial disease. Lancet 368:70–82

Schmidt TR, Wu W, Goodman M, Grossman LI (2001) Evolution of

nuclear- and mitochondrial-encoded subunit interaction in cyto-

chrome c oxidase. Mol Biol Evol 18:563–569

Schon EA, Santra S, Pallotti F, Girvin ME (2001) Pathogenesis of

primary defects in mitochondrial ATP synthesis. Cell Dev Biol

12:441–448

Spolsky CM, Szymura JM, Uzzell T (2006) Mapping Bombinamitochondrial genomes: the conundrum of Carpathian Bombinavariegata (Anura: Discoglossidae). J Zool Syst Evol Res

44:100–104

Szymura JM (1993) Analysis of hybrid zones with Bombina. In:

Harrison R (ed) Hybrid zones and the evolutionary process.

Oxford University Press, New York

Szymura JM, Barton NH (1986) Genetic analysis of a hybrid zone

between fire-bellied toads, Bombina bombina and B. variegata,

near Cracow in southern Poland. Evolution 40:1141–1159

J Mol Evol (2008) 67:246–256 255

123

Szymura JM, Barton NH (1991) The genetic structure of the hybrid

zone between the fire-bellied toads Bombina bombina and B.variegata: comparisons between transects and between loci.

Evolution 45:237–261

Szymura JM, Passakas-Szymczak T (1988) A new chromosome

number for Bombina (Anura, Discoglossidae). Experientia

44:521–523

Szymura JM, Spolsky C, Uzzell T (1985) Concordant change in

mitochondrial and nuclear genes in a hybrid zone between two

frog species (genus Bombina). Experientia 41:1469–1470

Szymura JM, Uzzell T, Spolsky C (2000) Mitochondrial DNA

variation in the hybridizing fire-bellied toads, Bombina bombinaand B. variegata. Mol Ecol 9:891–899

Wu W, Schmidt TR, Goodman M, Grossman LI (2000) Molecular

evolution of cytochrome c oxidase subunit I in primates: is there

coevolution between mitochondrial and nuclear genomes? Mol

Phylogenet Evol 17:294–304

Willet CS, Burton RS (2004) Evolution of interacting proteins in the

mitochondrial electron transport system in a marine copepod.

Mol Biol Evol 21:443–453

Yanchukov AW, Hofman S, Szymura JM, Mezhzherin SV, Morozov-

Leonov SY, Barton NH, Nurnberger B (2006) Hybridization of

Bombina bombina and B. variegata (Anura, Discoglossidae) at a

sharp ecotone in western Ukraine: comparisons across transects

and over time. Evolution 60:583–600

Yu G, Yang J, Zhang M, Rao D (2007) Phylogenetic and systematic

study of the genus Bombina (Amphibia: Anura: Bombinatori-

dae): new insights from molecular data. J Herpetol 41:365–377

Zeyl C, Andreson B, Weninck E (2005) Nuclear-mitochondrial

epistasis for fitness in Saccharomyces cerevisiae. Evolution

59:910–914

Zhang P, Zhou H, Chen Y-Q, Kiu Y-F, Qu L-H (2005) Mitogenomic

perspectives on the origin and phylogeny of living amphibians.

Syst Biol 54:391–400

256 J Mol Evol (2008) 67:246–256

123