The Mitochondrial Genome of a Monotreme The Platypus (Ornithorhynchus anatinus)

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1 J Mol Evol (1996) 42: Springer-Verlag New York Inc 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 Pääbo 1 1 Zoologisches Institut, Universität München, P.O. Box , D München, Germany 2 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 trna Leu (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 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

2 154 and marsupials are monophyletic and thus support the Marsupionta hypothesis. 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) 5 -cctcgatgttggatcagg-3 and (H3918) 5 -gtatgggcccgatagctt-3 were designed. Numbers within parentheses indicate the position of their 3 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 trna Leu (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 trna Leu was amplified with the primers (L2661) 5 -attaaggtgacagagacc-3 and (H2701) 5 -tattaaggagaggatttg-3, and the conserved intergenic sequence was amplified with the primers (L2736) 5 -cctgctactgcccacagg- 3 and (H2789) 5 -aattaagggaagctttta-3. Subsequently, these amplification products were used to generate probes by primer extension (Sambrook et al. 1989) in the presence of -[ 32 P]-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 trna Leu (UUR) gene were hybridized to the membranes at 60 C overnight in 6 SSPE (Sambrook et al. 1989). The membranes were washed 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. 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 trna Lys gene (Janke et al. 1994) nor the unconventional anticodon of the trna Asp 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 trna Pro in placentals (Foran et al. 1988), is found also in the platypus mitochondrial control region.

3 155 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 trna Leu (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 5 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 trna Leu (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 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 trna Leu (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 trna Leu (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 trna Leu (UUR) nor the ND1 transcript but exists as a defined RNA in about the same amounts as the trna Leu (UUR). No transcript carrying the sequence of the complementary strand could be detected either for the intergenic sequence nor for the trna Leu (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 2 -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 <

4 156 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 ( ) and using the observed average base compositions as the equilibrium frequencies. Dots denote the values observed from the data ). 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

5 157 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 (Adachi and Hasegawa 1992) a All positions Class change L t L ML pboot L t L ML pboot (X,(G,((D,P),((R,M),(H,(Ph,(Bo,Ba))))))) ML ML MARSUPIONTA (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)))))))) 7.3 +/ / (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)))))))) / / THERIA a ML indicates the log likelihood of the best tree. The differences of log-likelihood values of alterantive trees (L t ) from that of the best tree (L ML ) with their standard deviations ( ) 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

6 158 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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