*Institute of Biological Anthropology, University of Oxford, Oxford, England;
Centro de Primatologia do Rio de Janeiro, Fundação Estadual de Engenharia do Meio Ambiente, Rio de Janeiro, Brazil;
and
Department of Biology and Center for Molecular Genetics, University of California at San Diego
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Abstract |
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Introduction |
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The greatest significance of nuclear paralogs of mitochondrial DNA, however, may be their utility in comparing molecular evolution of homologous sequences in mitochondrial and nuclear environments (Arctander 1995
; Sunnucks and Hales 1996
; Lopez et al. 1997
). Although data on comparison of modes of evolution in mitochondrial and nuclear compartments are accumulating, there is currently little information on whether there are differences in mode of evolution among different nuclear paralogs.
In this study, we report on multiple nuclear insertions of a segment of cytochrome b in callitrichine primates (marmosets and tamarins) which were discovered during a project on the molecular phylogeny of marmosets (Callithrix). These data allow the comparison of molecular evolution of mitochondrial DNA and four nuclear paralogs, along with comparison among the nuclear paralogs. We also present a novel method of phylogenetic reconstruction based on different modes of molecular evolution in the mitochondrial and nuclear compartments and use it to infer multiple independent insertion events of the nuclear paralogs.
The callitrichine primates comprise five genera (Callithrix, Cebuella, Callimico, Saguinus, and Leontopithecus) that form a sister clade with squirrel monkeys (Saimiri) and capuchins (Schneider et al. 1996
; von Dornum and Ruvolo 1999
). The marmosets consist of three species groups (Rylands et al. 1993
), the Callithrix jacchus group in the Atlantic forest and eastern Brazil (including C. jacchus, Callithrix penicillata, Callithrix kuhli, Callithrix geoffroyi, and Callithrix aurita in this study), the Callithrix argentata group in central Brazil (including C. argentata, Callithrix melanura, Callithrix humeralifer, and Callithrix mauesi in this study) and the monospecific pygmy marmoset, Cebuella, in western Amazonia.
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Materials and Methods |
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DNA Extraction, Amplification, Cloning, and Sequencing
Genomic DNA was isolated from liver, skeletal muscle, and blood using standard procedures (Sambrook 1989). Total DNA was extracted from hairs using 5% Chelex 100 (Biorad, Hercules) following the method of Garza and Woodruff (1992). Total DNA was extracted from the museum skin using a commercial kit (Qiagen Tissue Kit; Qiagen) according to the manufacturer's instructions. Polymerase chain reaction (PCR) amplifications were performed in a thermal cycler (Hybaid) in a 25-µl total volume containing 0.51.0 U Taq polymerase (PE Applied Biosystems), 1 x PCR buffer, 50 mM each dNTP, 1.5 mM MgCl2, and 25 µg bovine serum albumin (Fraction V, Sigma, St. Louis, Mo.). Cycling parameters were 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 5060°C for 60 s, and 72°C for 90 s, followed by 72°C for 10 min. Direct cloning of PCR products was carried out using TA cloning kits (Invitrogen) according to the manufacturer's instructions.
Primers and dNTPs were removed from PCR products using Geneclean (BIO 101). Sequencing was performed on both strands manually with Sequenase (USB) and 35S-dATP; for direct sequencing of PCR products, a modified protocol with Nonidet P-40 (Sigma) was used (Garza and Woodruff 1992). Products of sequencing reactions were separated on 8% polyacrylamide gels (Long Ranger, J. T. Baker). Dried gels were exposed to X-ray film (X-Omat or Biomax; Kodak Eastman) for 17 days. Sequences were read by eye. In later experiments, sequencing was performed by cycle sequencing using dRhodamine terminators (PE Applied Biosystems) and run on an ABI 377 sequencing apparatus. Where possible, sequences were verified from multiple clones. Sequences obtained by TA cloning were given a transformation number and a clone number (e.g., Cjacchus 2.16.4 represents clone 4 from transformation 16, which was of specimen C. jacchus 2).
Primers
The original primers used to amplify a portion of cytochrome b were L15375 (5'-GGCTCAAGTAACCCATCAGG-3') and H5 (5'-TACTGGTTGTCCTCCGATTC-3'). Primers used to amplify a 2.2-kb segment containing cytochrome b and control region were L14724 (5'-CGAAGCTTGATATGAAAAACCATCGTTG-3') and CCRH2 (5'-CAGAAGGCTAGGACCAAACCT-3'). Clade-specific primers were CBAF/CBAR for clade A (CBAF: 5'-TGACGCCCATAAATACCTATCCGGG-3'; CBAR: 5'-AGCTCGGTTCTCGTGAGGTTTACG-3') and CBEF/CBER for clade E (CBEF: 5'-CCATCCAACATCTCAGCATGATGAAA-3'; CBER: 5'-GCCCCTCAGAATGATATTTGTCCTCA-3').
Sequence Analysis
Sequences were unambiguously aligned by eye. As sequences obtained with different primer pairs were of slightly different lengths, the ends of some sequences were removed to create a 354-bp alignment for further analysis. Phylogenetic analyses were performed using PAUP* (Swofford 1999
). For maximum-parsimony analyses, the transition-to-transversion ratio was set to 4:1 (taken from the data set), codon position weighting varied from first : second : third of 1:1:1 or 3:12:1 (taken from the data set), and random branch addition was used. For neighbor-joining trees, Kimura two-parameter distances were used. All bootstrap resamplings were performed with 500 replicates.
The significance of the ratio of interclade pairwise distances by codon position was determined using a randomization test. In each iteration of the test, the sequences belonging to the pair of clades under consideration were randomly assigned to two new groups with the same sizes as the clades. The difference in distance ratios by codon position for interclade comparison to the maximum intraclade comparison was then compared with the same difference obtained from the actual data. Results were considered significant at the 1% level if the difference from the randomization exceeded the difference from the data on 1% or fewer of the iterations (one-tailed test). All randomizations were performed using 500 iterations.
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Results and Discussion |
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Across-species comparisons showed that the sequences fell into five different groups, AE. In order to increase the number of sequences obtained from each individual, primers specific to sequences from groups A and E were designed and used to obtain group A and E sequences from most individuals, although in most cases these sequences were still determined by cloning of PCR products (except for group A sequences from Cpenicillata 4 and Ckuhli 3). In total, two to five cytochrome blike sequences were obtained from each individual marmoset sample (fig. 1 ), with five sequences being obtained from Chumeralifer 3. Relative to published primate cytochrome b sequences (e.g., those included in fig. 2 ), all sequences in group D have two deletions of 1 and 2 bp and all sequences in group E have independent deletions of 3 and 4 bp. Inspection of the sequences showed that none of the five groups of sequences could be explained by in vitro recombination of the other sequences during the PCR.
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Identification of Clade A as the Mitochondrial Clade
The ratio of the mean number of substitutions for codon position 3 to codon position 2 within clades is much greater in clade A than in clades BE (table 1
), strongly suggesting that clade A contains functional mitochondrial sequences, whereas clades BE consist of nuclear pseudogenes derived from mitochondrial DNA. Support for this assumption comes from the following: (1) All sequences in clade A are translatable, whereas all members of clades D and E and one of the clade C sequences (Chumeralifer 3.21.1) have mitochondrial stop codons. (2) Using conserved primers L14724 and CCRH2, a 2.2-kb segment of mitochondrial DNA containing cytochrome b and control region was amplified from samples Cjacchus 292 and Cebuella 592 and directly sequenced. The clean sequences thus obtained matched the clade A sequences obtained from these samples using primers CBAF and CBAR.
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The failure to obtain sequences from all clades from all individuals is more likely to be for technical reasons (e.g., mismatches in primer sites, sequencing of a small number of clones) than the actual absence of the sequences in those individuals. We have no evidence that nuclear paralogs of mtDNA are more likely to be obtained from hair samples than from blood samples, as has been described for elephants (Greenwood and Pååbo 1999
), since members of all four nuclear clades BE were obtained from blood samples as well as hair samples.
Comparisons of Substitution Pattern Among Clades
As expected, the mitochondrial sequences evolve more rapidly and more unevenly with respect to codon position than do the nuclear clades: mean pairwise intraclade distances were almost five times as high for the mitochondrial clade as for the nuclear clades, and, in addition to the high codon position 3:2 ratio, the mean number of substitutions for codon position 3 to codon position 1 was higher for the mitochondrial than for the nuclear clades. The transition/transversion (Ts/Tv) ratio for the mitochondrial clade (4.25) was lower than expected, probably because of saturationpairwise comparisons among closely related species gave much higher ratios.
Our data provide an unusual opportunity to compare evolution among multiple nuclear mtDNA paralogs. The overall rates of evolution of the clades are very similar (mean intraclade distances are presented in table 1 ), but there is an almost twofold variation in the Ts/Tv ratio. Additionally, there is some variation in rates of evolution by codon position away from the expected value of 1:1:1. As nuclear paralogs of mtDNA are unlikely to be functional, the most likely explanation for these variations is the small number of substitutions involved. For example, the relatively low Ts/Tv ratio and relatively high ratios of change by codon positions 3:1 and 3:2 for clade C is largely explained by the contribution from the four variable sites (at nucleotide positions 34, 113, 325, and 334) which split the jacchus and argentata species groups: of these four sites, three are at codon position 3 (two transversions and one transition) and one is at codon position 1 (transition). Also, the largest ratios of change by codon positions 3:1 and 3:2 occur among nuclear clade D, which has the smallest number of sequences (three).
Age of Insertion Events
In order to determine when the nuclear insertion events occurred, TA cloning from three primer pairs (L15375/H5, CBAF/CBAR, and CBEF/CBER) was performed for the remaining callitrichine genera (Callimico, Saguinus, and Leontopithecus) and members of two outgroups to the callitrichines (Saimiri and the more distant Alouatta). In all callitrichine genera and Saimiri, multiple cytochrome blike sequences were found, whereas a single sequence was found for Alouatta. The following were identified as mitochondrial sequences: Saguinus 296.52.3, Soerstedtii 128.63.2, and Alouatta 584.mt (all confirmed by direct sequencing of L14724/CCRH2 product) and Leontopithecus 22.64.2 and Callimico 1.66.2 (these were obtained with primers CBAF/CBAR and do not contain stop codons, and their ratio of change by codon position is consistent with mtDNA).
All of these sequences, combined with published cytochrome b sequences of Saimiri sciureus and several Old World monkeys and apes (Collura and Stewart 1995
), were used in phylogenetic reconstructions. In maximum-parsimony and neighbor-joining analyses, the five clades of marmoset sequences were conserved, and certain of the new sequences consistently grouped with one of the five clades. However, relationships of many of the new sequences and relationships among many of the clades were unstable.
Figure 2 shows a 50% consensus tree of 500 bootstrap replicates using maximum parsimony that is consistent with other trees generated. The five clades of marmoset sequences from the tree in figure 1 are still present and well supported. Sequences from the three other callitrichine genera (Saguinus, Leontopithecus, and Callimico) are present in an expanded nuclear clade E, and one sequence from Saguinus is present in nuclear clade D. Interestingly, none of the other callitrichine D or E sequences contain deletions like those in the marmoset sequences, showing that the deletions occurred in the common ancestor of marmosets (clade E) or since the split with Saguinus (clade D). None of the sequences from the other taxa were grouped with marmoset mitochondrial clade A or nuclear clade B or C.
Three further independent nuclear paralogs were identified: Callimico 2.66.2, which contains a stop codon and has a sister relationship with Callimico 2.66.1, the putative mitochondrial sequence from Callimico, and Soerstedtii 128.55.5 and 128.55.3.
The failure to resolve relationships among the mitochondrial sequences (clade A and the sequences mentioned above) may at first appear surprising. But this relatively short segment of cytochrome b also fails to resolve relationships among the catarrhines (fig. 2
), which is probably at least partly due to the well-known rate variation in mtDNA evolution in hominoids (Irwin, Kocher, and Wilson 1991
). The failure is not due to the number of sequences included, as the species phylogeny was not recovered for either catarrhines or platyrrhines when all nuclear sequences were excluded from the phylogenetic analysis.
The tree in figure 2 shows weak support for a clade containing clades A, B, and C to the exclusion of clades D and E, and all other sequences. Otherwise, the relationship between clades A to E is unresolved, and, in particular, it is not clear whether clades B and C or clades D and E represent separate insertion events or duplications following insertion. Returning to the question of the age of the insertion events, there is weak evidence that the insertions leading to clades B and C occurred after marmosets split from the other callitrichines. In contrast, the presence of sequences from Saguinus in clades D and E, along with the distant relationship between Callithrix and Saguinus within callitrichines, shows that the insertions leading to clades D and E occurred at least before the split between the callitrichine genera.
Nuclear transpositions of the same segment of cytochrome b have been described in Old World monkeys and apes (Collura and Stewart 1995
). When these additional nuclear pseudogenes were included in phylogenetic reconstructions (not shown), they grouped with the other catarrhine (Old World monkey and ape) sequences and thus represent insertion events independent of those reported here in New World monkeys.
Do Clades BE Represent Independent Nuclear Insertion Events or Duplication Following Insertion?
As standard methods of phylogenetic reconstruction had failed to resolve this issue, an alternative approach was taken based on the different substitution rates by codon position expected in functional mtDNA and nonfunctional nuclear inserts. The rationale for this approach is shown in figure 3
, which shows the three possible phylogenies for mitochondrial clade A and nuclear clades B and C. In the two cases where nuclear insertion occurred independently (trees 1 and 2 in fig. 3
), the interclade difference between clades B and C includes a period of mitochondrial evolution corresponding to the time between the two insertion events. In contrast, if clades B and C represent duplication from a single insertion event (tree 3 in fig. 3
), the comparison between them does not include a period of mitochondrial evolution. The greatly elevated rate of third-position substitution in mitochondrial DNA and the overall slow rate of evolution of nuclear DNA mean that the signature of a period of mitochondrial DNA evolution may be detectable in interclade comparisons.
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Thus, knowledge of mode of sequence evolution can be a useful tool in phylogenetic analysis and can give better resolution for certain specific questions than traditional methods of phylogenetic reconstruction. The use of this method is not restricted to nuclear pseudogenes; it can be applied in any case in which different modes of sequence evolution are involved. The power of the method will increase when several sequences from different clades are available and when transposition events occurred at well-spaced time intervals.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Keywords: mitochondrial DNA
cytochrome b,
nuclear paralog
Numt
Callithrix.
2 Address for correspondence: Nicholas I. Mundy, Institute of Biological Anthropology, University of Oxford, 58 Banbury Road, Oxford OX2 6QS, United Kingdom. E-mail: nick.mundy{at}bioanth.ox.ac.uk
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