J Mol Evol (1996) 42:153–159
© Springer-Verlag New York Inc. 1996
The Mitochondrial Genome of a Monotreme—The Platypus
(Ornithorhynchus anatinus)
Axel Janke,1* Neil J. Gemmell,2** Gertaud Feldmaier-Fuchs,1 Arndt von Haeseler,1 Svante Pääbo1
1
2
Zoologisches Institut, Universität München, P.O. Box 202136, D-80021 München, Germany
Department of Genetics and Human Variation, La Trobe University, Bundoora 3083, Australia
Received: 4 February 1995 / Accepted: 1 August 1995
Abstract. The complete nucleotide sequence of the
mitochondrial genome of a platypus (Ornithorhynchus
anatinus) was determined. Its overall genomic organization is similar to that of placental mammals, Xenopus
laevis, and fishes. However, it contains an apparently
noncoding sequence of 88 base pairs located between the
genes for tRNALeu(UUR) and ND1. The base composition of this sequence and its conservation among
monotremes, as well as the existence of a transcript from
one of the strands, indicate that it may have a hithertounknown function. When the protein-coding sequences
are used to reconstruct a phylogeny of mammals, the data
suggest that monotremes and marsupials are sister
groups and thus that placental mammals represent the
most ancient divergence among mammals.
Key words: Mitochondrial DNA — Monotremes —
Mammalian phylogeny
Introduction
Extant mammals (Mammalia) fall into three groups: placentals (Eutheria), with approximately 3,000 species,
* Present address: Institute of Genetics, Sölvegatan 29, S-22362 Lund,
Sweden
** Present address: Department of Genetics, University of Cambridge,
Cambridge CB2 3EH, United Kingdom
Correspondence to: A. Janke
marsupials (Metatheria), with 249 species (Kirsch and
Calaby 1977), and monotremes (Prototheria), with three
species. Whereas the monophyly of these three groups is
well accepted, the relationships of the groups to each
other remain enigmatic. A case in point is the
monotremes. While the existence of, for example, hair,
suckling of the young, and maintenance of a constant
body temperature support their inclusion within mammals, the paucity of extant monotreme taxa and a poor
fossil record (Carroll 1988; Zeller 1993) make their relationship to the other mammalian groups difficult to
analyze by traditional means.
The generally held hypotheses concerning the origin
of monotremes assume a dichotomy of mammals between Theria (placentals and marsupials) and Prototheria
(monotremes) (Crompton and Jenkins 1979; Carroll
1988; Kielan-Jaworowska 1979; Kermack and Kermack
1984; Marshall 1979). In contrast, an alternative but
widely disregarded hypothesis holds that monotremes
and marsupials, the so-called ‘‘Marsupionta,’’ are sister
groups (Gregory 1947; Kühne 1973, 1975). Molecular
studies have been unable to resolve this issue. Whereas a
study of the protamine P1 gene (Retief et al. 1993) seems
to support the view that the monotremes diverged early
in mammalian evolution, studies of myoglobin sequences (Goodman et al. 1985) and of the mitochondrial
12S rRNA gene (Gemmell and Westerman 1994) fail to
resolve the issue. Here, we present the sequence of the
entire mitochondrial genome of a monotreme, the platypus (Ornithorhynchus anatinus) and compare it to the
mitochondrial genomes of a bird, a frog, a marsupial, and
six placentals. The results indicate that the monotremes
154
and marsupials are monophyletic and thus support the
‘‘Marsupionta’’ hypothesis.
briefly at room temperature in 2×SSC/0.1% SDS and subsequently
washed twice at 60°C in 0.2×SSC/0.1% SDS for 10 min. Autoradiography was performed at −70°C for 24 h using Amersham Hyperfilm
and intensifying screens.
Materials and Methods
Mitochondrial DNA (mtDNA) from the liver of a platypus (Ornithorhynchus anatinus) was isolated and cloned into EMBL3 as described
(Gemmell et al. 1994). From the clone pPmt1, which contained almost
the entire mitochondrial genome, four HindIII fragments (6.9 kb, 5.3
kb, 2.6 kb, and 1 kb) were subcloned into the phagemid pBluescript
SK+. From the 5.3-kb insert three BglI fragments were further subcloned into pBluescript SK+. Exo III deletions from the inserts were
sequenced by the dideoxy-chain termination technique. Regions not
determined from both strands by the sequences of the deletion clones
were sequenced by primer walking.
Approximately 500 bp of the ND1 gene were missing from pPmt1.
Therefore, the primers (L2411) 58-cctcgatgttggatcagg-38 and (H3918)
58-gtatgggcccgatagctt-38 were designed. Numbers within parentheses
indicate the position of their 38 nucleotide in the sequence; H and L
refer to the light and heavy strands, respectively. They were used to
amplify this segment as well as surrounding sequences that encompass
part of the 16S rRNA gene, the tRNALeu(UUR) gene, a conserved
intergenic sequence, and the complete ND1 gene. The same primers
were also used to amplify the homologous sequences from Tachyglossus aculeatus and Zaglossus bruijni. The gene for the tRNALeu was
amplified with the primers (L2661) 58-attaaggtgacagagacc-38 and
(H2701) 58-tattaaggagaggatttg-38, and the conserved intergenic sequence was amplified with the primers (L2736) 58-cctgctactgcccacagg38 and (H2789) 58-aattaagggaagctttta-38. Subsequently, these amplification products were used to generate probes by primer extension
(Sambrook et al. 1989) in the presence of a-[32P]-dCTP using the four
primers used for amplification. The double-stranded PCR products
were purified with Gene-clean (Bionova, La Jolla, CA) and directly
sequenced (Bachmann et al. 1990).
For the phylogenetic analyses, the mitochondrial protein-coding
genes of the following species were used: human (Anderson et al.
1981), mouse (Bibb et al. 1981), cow (Anderson et al. 1982), rat (Gadaleta et al. 1989), fin whale (Arnason et al. 1991), harbor seal (Arnason
and Johnson 1992), opossum (Janke et al. 1994), frog (Roe et al. 1985),
and chicken (Desjardins and Morais 1990). The protein-coding genes
were concatenated and aligned by eye using ESEE (Cabot and Beckenbach 1989). Positions with gaps and sequences where the alignment
was ambiguous were excluded from analyses as was the gene for ND
6. Phylogenetic analyses were performed using the PHYLIP (Felsenstein 1989), and the PROTML (Adachi and Hasegawa 1992) program
packages as well as programs by A.v.H. The sequence of the platypus
mitochondrial genome is available from the EMBL data base (accession number: X83427). The alignment is available from the Munich
www server.
For northern blot analyses, total RNA was prepared (Chomcynski
and Sacchi 1987) from a platypus cell line. Five micrograms of total
RNA was separated on a 2% agarose gel containing 7% formaldehyde.
As a control for the transfer and hybridization, 0.5 ng of the 16S/ND1
region, amplified as described above, was loaded next to each sample.
After electrophoresis, the gel was washed twice for 15 min in desalted
water, once for 10 min in 100 mM ammonium acetate, and stained 10
min in 10 mM ethidium bromide solution. After visualization of the
nucleic acids on a UV-transilluminator, the gel was washed and incubated twice for 15 min in 20×SSC prior to transfer onto nitrocellulose
membrane. Four sets of samples and controls were run on the same gel
to achieve similar conditions. Subsequently, these were separated by
cutting the membrane after transfer. The membrane was dried for 2 h
at 80°C. Four strand-specific probes (see above) for the putative L- and
H-strand transcripts of the conserved intergenic sequence and the
tRNALeu(UUR) gene were hybridized to the membranes at 60°C overnight in 6×SSPE (Sambrook et al. 1989). The membranes were washed
Results and Discussion
General Features of the Genome
The mitochondrial genome of the platypus is 17,019 base
pairs long and encodes 22 tRNAs, 13 proteins, and 2
rRNAs. As in other vertebrates, 12 of the proteins are
encoded on the L-strand. In six of the ten cases where the
stop codon TAA is used, the codons are inferred to be
incomplete and are presumably completed posttranscriptionally by polyadenylation. The stop codons TAG and
AGG seem to be used twice and once, respectively,
whereas the stop codon AGA is not used. The start codon
ATG is utilized in seven genes, and ATT is used four and
ATA two times. The genes are organized as in fish
(Chang et al. 1994; Tzeng et al. 1992), the frog, and
placentals. Thus, the monotreme lacks the gene rearrangements that have been described in birds (Desjardins
and Morais 1990) and marsupials (Pääbo et al. 1991).
The transfer RNA genes can be folded into structures
similar to those of other vertebrates (not shown) and
share conserved sequence positions with other vertebrate
tRNA genes. Thus, the platypus genome exhibits neither
the unusual structure of the tRNALys gene (Janke et al.
1994) nor the unconventional anticodon of the tRNAAsp
gene (Janke and Pääbo 1993) seen in the marsupial Didelphis virginiana. Furthermore, the tRNA gene for
serine (UCN), which in placentals and marsupials differs
from other vertebrates in having six instead of five base
pairs in the anticodon stem and only one base between
the acceptor stem and the DHU stem (Yokogawa et al.
1991; Janke et al. 1994), lacks both these unusual features in the monotreme. The structures of the tRNA
genes do therefore not give reasons to assume that RNA
editing or other unusual processes affect the gene products in monotremes.
In the control region, three conserved sequence blocks
(CSBs), which are involved in the initiation of H-strand
synthesis, have been identified in placentals (Walberg
and Clayton 1981; Bennet and Clayton 1990). CSB I and
a putative CSB II exist in the platypus whereas the CSB
III is absent, as is the case in the cow (Anderson et al.
1982) and whales (Southern et al. 1988, Dillon and
Wright 1993). Three further short conserved sequences
that exist in placentals (Saccone et al. 1991) and the
opossum (Janke et al. 1994) are partially present also in
the platypus. The termination associated sequence
(TAS), which is involved in the termination of mitochondrial DNA replication and is located near the tRNAPro in
placentals (Foran et al. 1988), is found also in the platypus mitochondrial control region.
155
Fig. 1. Alignment of the conserved intergenic sequence of the three monotreme species, the platypus (Ornithorhynchus anatinus) and the two
echidnas, Tachyglossus aculeatus and Zaglossus bruijni.
Fig. 2. Northern blot analysis of the conserved intergenic sequence
(CIS) showing a transcript corresponding to the L-strand sequence to be
present in roughly the same amounts as the tRNALeu(UUR). A DNA
fragment (c) was used to control for the efficiency of hybridization.
As in several other vertebrates, the control region of
the platypus contains repeated sequence motifs. The sequence TTTGAAAAA is repeated seven times and has
two to three additional T residues added to the 58 end of
the first three repeats. The second motif comprises the
core sequence GAG(G/A)A(T/A)AAAACTATTTT
which is repeated 20 times. In another individual, the
motif is repeated 25 times (Gemmell et al. 1994). Thus,
this sequence repeat differs in copy number between individual animals.
A Conserved Intergenic Sequence
In general, intergenic sequences in metazoan mitochondrial genomes are of short length. This is the case also in
the platypus mitochondrial genome, where zero to three
bases are generally found between genes. In contrast, the
genes for tRNALeu(UUR) and ND1 are separated by 88
base pairs. In order to find out if this sequence is present
in other monotremes, the homologous region was amplified and sequenced from the echidnas Tachyglossus aculeatus and Zaglossus bruijni, which are believed to
share a common ancestor with the platypus 30–50 million years ago (Gemmell and Westerman 1994; Retief et
al. 1993; Hope et al. 1990). Figure 1 shows that this
sequence exists in all three extant monotremes and that
the sequences are similar enough to be aligned.
The intergenic sequence exhibits no obvious similarity to the adjacent tRNALeu(UUR) nor to any other region of the platypus mitochondrial genome. It is thus not
obviously the result of a duplication of a mitochondrial
sequence (Cantatore et al. 1987). Although the platypus
intergenic sequence contains an open reading frame of
24 codons, insertions and deletions in the other monotremes suggest that the sequence does not encode a polypeptide. No significant similarity to sequences in data
banks exists and the sequence cannot be folded into any
stable secondary structure.
When the nucleotide composition of the intergenic
sequence is compared to those of other parts of the genome, it is found to lack the strong bias against G residues that is typical of third codon positions and noncoding sequences. Rather, its nucleotide composition is
similar, for example, to that of ribosomal RNA genes.
This, in conjunction with the conservation of the sequence among monotremes, suggests that the intergenic
sequence encodes some hitherto-unknown function.
In order to elucidate whether the intergenic sequence
gives rise to a stable transcript, RNA from a platypus cell
line was analyzed by northern blot analysis using probes
specific for two strands of the intergenic sequence as
well as the two strands of the tRNALeu(UUR). Figure 2
shows that a transcript carrying the L-strand sequence of
the intergenic sequence exists. The length of the transcript indicates that it is neither an extension of the
tRNALeu(UUR) nor the ND1 transcript but exists as a
defined RNA in about the same amounts as the
tRNALeu(UUR). No transcript carrying the sequence of
the complementary strand could be detected either for
the intergenic sequence nor for the tRNALeu(UUR).
Since the rates of formation of tRNAs, rather than their
rates of transcription, are proportional to their steadystate levels (King and Attardi 1993), the intergenic sequence is similar to the adjacent tRNA gene also in that
a processed transcript of one of the strands is formed at
a much higher copy number than the other. Further work
will have to clarify the function this sequence performs
in monotremes.
Base Composition, Multiple Substitutions, and
Tree Reconstruction
The protein-coding sequences, with the exception of the
gene for ND 6, which is encoded on the L-strand and
differs in nucleotide composition from the other genes,
were aligned. After exclusion of regions where the alignment was ambiguous, 9,840 nucleotide positions remained. The aligned sequences were tested for homogeneity of their nucleotide composition by a x2-test (von
Haeseler et al. 1993) for each codon position separately.
The second codon positions were homogeneous (P =
0.74) whereas the first codon positions were not (P <
156
Fig. 4. A phylogenetic tree based on the inferred amino acid sequence of 12 mitochondrial proteins. The tree is constructed by a
maximum likelihood algorithm for protein sequences (Adachi and Hasegawa 1992).
Fig. 3. Plots of expected observed transversions (Tv) vs observed
transitions (Ts) calculated according to Hasegawa et al. (1985) assuming various transition–transversion ratios (a) and using the observed
average base compositions as the equilibrium frequencies. Dots denote
the values observed from the data.
0.001). This is due to the six leucine codons (CUN,
UUR), which allow a synonymous transition in the first
codon position. At these positions the chicken and human sequences tend to prefer cytosine over thymine residues. When transitions at the first codon position of
leucine codons were excluded, the base composition was
homogeneous also at first codon position (P = 0.44). At
third codon positions, differences in base composition
are drastic. Several species, most conspicuously the
chicken and human, show preference for cytosine over
thymine residues.
Figure 3 shows the observed numbers of transversions
and transitions for each of the 45 comparisons among the
ten taxa used as well as the theoretical predictions for
these numbers using the model of sequence evolution
proposed by Hasegawa et al. (1985), and various transition–transversion ratios. If the numbers of transitions are
small, the curves representing observed numbers of transitions increase rapidly until a maximum number of transitions are reached, after which the curves decline toward
the points of randomization. While the data points for
first and second codon positions are located in the regions where all curves show an upward trend, the observed numbers of substitutions at third codon positions
are in a region of the graph where the predicted numbers
of observed transitions are declining, indicating that multiple substitutions will obscure the phylogenetic information (Janke et al. 1994). Based on these observations and
the fact that transitions at the first codon position of
leucine codons are silent, transitions at the first positions
of leucine codons and at third positions of all codons
were excluded from the phylogenetic analyses.
When this data set was used to reconstruct a phylogeny of mammals, a tree with the branching structure
shown in Fig. 4 was obtained. In this tree, the
monotremes and marsupials are sister taxa and consequently the first divergence among mammals leads to
placentals on the one hand and the ‘‘Marsupionta’’ on
the other. This is supported by bootstrap replications of
both the tree generated by neighbor-joining (94.4%) and
maximum parsimony (96.3%) procedures. The result
remained robust when different methods of correcting
157
Table 1. ML analysis of the complete aligned amino acid sequence (3,280 positions) and of the sequences that show a class change (989 positions)
based on the JTT-F model, using PROTML, version 2.1.1 (Adachi and Hasegawa 1992)a
All positions
(X,(G,((D,P),((R,M),(H,(Ph,(Bo,Ba)))))))
(X,(G,((D,P),((R,M),(H,(Ba,(Ph,Bo)))))))
(X,(G,((D,P),((H,(R,M)),(Ph,(Bo,Ba))))))
(X,(G,((D,P),(H,((R,M),(Ph,(Bo,Ba)))))))
(X,(G,(D,P),((H,(R,M)),(Ba,(Ph,Bo)))))))
(X,(G,(D,(P,((R,M),(H,(Ph,(Bo,Ba))))))))
(X,(G,(D,(P,((R,M),(H,(Ba,(Ph,Bo))))))))
(X,(G,(D,(P,((H,(R,M)),(Ph,(Ba,Bo)))))))
(X,(G,(P,(D,((R,M),(H,(Ph,(Bo,Ba))))))))
Class change
Lt − LML
s
pBoot
Lt − LML
s
pBoot
ML
−17.5
−28.5
−39.2
−49.0
−7.3
−23.6
−36.3
−26.0
+/−21.1
+/−19.7
+/−18.5
+/−28.5
+/−16.7
+/−26.4
+/−25.4
+/−14.0
0.474
0.121
0.050
0.003
0.006
0.247
0.061
0.015
0.007
ML
−14.9
−12.8
−14.0
−28.5
−11.4
−25.1
−23.9
−14.5
+/−12.3
+/−8.5
+/−8.1
+−15.0
+/−7.5
+/−14.2
+/−10.9
+/−6.2
0.762
0.079
0.044
0.024
0.004
0.075
0.004
0.001
0.000
‘‘MARSUPIONTA’’
‘‘THERIA’’
a
ML indicates the log likelihood of the best tree. The differences of
log-likelihood values of alterantive trees (Lt) from that of the best tree
(LML) with their standard deviations (s) following estimated by the
formula of Kishino and Hasegawa (1989) are shown. pBoot indicates
the estimated bootstrap probability (Kishino et al. 1990) among 50
near-optimal trees that are chosen from 2,027,025 possible trees. Only
those trees that in either analysis have a bootstrap probability over 0.5%
are shown. Opossum and platypus are in bold. The first five trees
represent the Marsupionta hypothesis, followed by three trees that have
placentals and monotremes as sister taxa and the tree that represents the
Theria hypothesis.
Ba = Balaenoptera (whale), Bo = Bos (cow), D = Didelphis (opossum),
G = Gallus (bird), H = Homo (human), M = Mus (mouse), P = platypus,
Ph = Phoca (seal), R = Rattus (rat), X = Xenopus (frog)
for multiple substitutions (Jukes and Cantor 1969;
Kimura 1980; Felsenstein 1989) were used. When a
maximum likelihood tree was constructed (DNAML,
version 3.5; Felsenstein 1989), the sister-group status of
monotremes and marsupials was again supported even if
the alternative hypothesis that marsupials and placentals
are sister taxa could not be rejected at the 5% level of
significance. However, irrespective of which combinations of outgroups and of which transition–transversion
ratios (T = 2–16) were used, the tree in Fig. 4 remained
the best.
When the analysis was confined to the first codon
positions, the topology and level of statistical support
remained similar to those for the entire data set. In contrast, when second codon positions were used, different
topologies, all with little statistical support, were found
depending on the methods used. This is most likely due
to the limited number of substitutions at these positions.
Similarly, when alignable parts of rRNA and tRNA sequences (1,501 and 1,042 nucleotides, respectively) were
analyzed separately or in combination, they could not
clarify the phylogenetic position of the monotremes.
Thus, almost all information supporting the sister-group
status of monotremes and marsupials comes from the
first codon positions.
Base compositional and mutational effects as well as
the paucity of character states at the nucleotide sequence
level may influence analyses performed at the nucleotide
sequence level and may make the analysis of amino acid
sequences preferable (Cao et al. 1994). Thus, a maximum likelihood tree was constructed using the complete
data set, the JTT amino acid transition matrix (Jones et
al. 1992), and the observed average frequencies of amino
acids as the equilibrium frequencies. The most likely tree
is shown in Fig. 4. The log likelihood of the best tree,
which pairs marsupials and placentals as sister groups, is
1.86 standard deviations worse than that of the tree in
Fig. 4 (Table 1). Thus, also at the amino acid sequence
level, the sister-group status of monotremes and marsupials is supported although the alternative could not be
rejected at the 5% level of significance.
The JTT transition matrix assumes that all positions in
an amino acid sequence are free to vary according to the
matrix. However, an inspection of the alignment of the
proteins indicates that this is unlikely to be the case. Out
of 3,280 aligned amino acid positions, 1,396 show only
nonpolar amino acids (Ala, Ile, Leu, Met, Val, Phe, Pro),
630 only polar amino acids (Asp, Cys, Gln, Gly, Ser,
Thr, Trp, Tyr), 155 only positively charged amino acids
(Arg, His, Lys), and 110 only negatively charged amino
acids (Asp, Glu). Only 989 positions show changes that
result in amino acid replacements between at least two of
the chemical classes indicated. Thus, the latter codons
are more likely not to dramatically violate a model which
assumes that every position has the same transitional
probability.
The analysis was confined to the 989 amino acid positions that in this data set change at least once between
chemical classes of amino acid. This obviously violates
the model in that only positions where substitutions are
observed are used. However, this may be a less-serious
model violation than the assumption that all positions
evolve according to the same transition matrix. In this
analysis, the best tree again supports the ‘‘Marsupionta’’
hypothesis, and this time the likelihood of the alternative
tree, where the marsupials and placentals are sister
groups, is 2.46 standard deviations lower than the best
tree (Table 1). Thus, when the analysis is confined to the
amino acid positions that allow at least some replacements to change the gross chemical properties of the
158
amino acid, the traditional view that placentals and marsupials are sister taxa becomes even more unlikely.
Conclusion
The mitochondrial data strongly suggest that marsupials
and monotremes are sister taxa. If this is correct, 19
morphological features interpreted as shared derived features between placentals and marsupials (Marshall 1979)
have either arisen independently in placentals and marsupials or been lost on the lineage to monotremes. On the
other hand, some dental traits support the monophyly of
marsupials and monotremes. For example, marsupials
and monotremes both have three premolars and four molars, and they replace only the last premolar during development (Kühne 1973, 1975). Further molecular data
are expected to resolve this issue in a definitive way.
Acknowledgments. We are indebted to Drs. F. Catzeflis, M. Hasegawa, M. Westerman, and especially Dr. J.A. Marshall Graves for suggestions and discussion and to the DFG and Genzentrum Munich for
financial support.
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