The Journal of Heredity 2002:93(1)
© 2002 The American Genetic Association 93:9-18
Mitochondrial Sequence Diversity Within a Subspecies of Savanna Monkeys (Cercopithecus aethiops) Is Similar to That Between Subspecies
From the Primate Research Institute, Kyoto University (Shimada and Shotake), and Tsukuba Primate Center for Medical Science (Terao).
Address correspondence to Makoto K. Shimada, Division of Population Genetics, National Institute of Genetics, Yata 1777, Mishima 411-8540, Japan, or e-mail: mshimada{at}lab.nig.ac.jp.
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Abstract |
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Cercopithecus aethiops can be classified into four subspecies by morphology and by geographic distribution. However, the phylogenetic relationship between these subspecies is unclear. We previously found five distinct haplogroups of mitochondrial DNA (mtDNA) in the subspecies C. aethiops aethiops at the restriction fragment length polymorphism (RFLP) level, and found that those haplogroups are parapatrically distributed in their habitat. To determine the relationship between subspeciation and haplogroup formation in a subspecies, we compared mtDNA control region and 12S rRNA gene sequences (approximately 700 bp) in C. a. aethiops, two other subspecies of C. aethiops, and two species of Cercopithecus. The diversity between haplogroups in C. a. aethiops was almost the same as that between subspecies. This similar level of diversification between and within haplogroups may explain why a previously obtained mtDNA tree did not show monophyletic branching according to subspecies.
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Introduction |
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Savanna monkeys, or African green monkeys (Cercopithecus aethiops or Chlorocebus aethiops), are the most widely distributed nonhuman primates in Africa. These monkeys had been previously classified as several species on the basis of morphology, but as members of a single species group (e.g., Hill 1966). Napier (1981), however, proposed that they should be regarded as one species, Cercopithecus aethiops, with 22 subspecies gathered into four subspecies groups. Napier's 22 subspecies are often united into three to five subspecies for the purpose of simplicity in some molecular genetic studies (e.g., Ruvolo 1983; van der Kuyl et al. 1995a). Because of the complex diversity of savanna monkeys, many different classification schemes exist. For our study, we classified subspecies found in Ethiopia as grivet monkeys (Cercopithecus aethiops aethiops abbreviated Caa), those from Kenya as vervet monkeys (C. a. pygerythrus, abbreviated Cap), and those from Cameroon as tantalus monkeys (C. a. tantalus, abbreviated Cat) (Figure 1). Shimada (2000b) studied the geographic distribution of mitochondrial DNA (mtDNA) variations within grivet monkeys (Caa) in central Ethiopia with restriction fragment length polymorphism (RFLP), and classified the haplotypes into five haplogroups (or phylogroups) according to similarity. The divergence within each haplogroup was small (sequence divergence 0.17–0.38%), whereas that between haplogroups was rather large (sequence divergence 1.0–2.5%). Although these haplogroups may correspond to Napier's "subspecies'' (i.e., 22 subspecies classification) reported as djamdjamensis, hilgerti, and matschiei, we could not divide Caa into subcategories according to morphology at the field study site in Ethiopia, but we could easily distinguish Caa from other subspecies (Cap and Cat) according to morphology. This situation is consistent with the morphologists' view where both Caa and Cap are easily distinguished but differences within Caa are unclear (Dandelot and Prévost 1972; Lernould 1988); however, hybridization between these subspecies may make classification difficult (Kingdon 1997). An investigation and comparison of the degree of sequence divergence between these haplogroups and subspecies will advance our understanding of the relationship between these subspeciations. To clarify these issues, we sequenced the mtDNA control region (approximately 284 nt) from five different haplogroups of Caa, three subspecies of savanna monkeys, and two other species belonging to the Cercopithecus genus (i.e., guenon monkeys). Using a control region sequence, we compared the extent of divergence within subspecies with that between subspecies.
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The phylogenetic relation and divergence between these subspecies remains unclear (van der Kuyl et al. 1995a; Fomsgaard et al. 1997). Van der Kuyl et al. (1995a) studied the molecular phylogeny of African monkeys, including four subspecies of savanna monkeys, on the basis of the mitochondrial 12S rRNA gene. The clustering pattern of the obtained phylogenetic tree was not monophyletic in each subspecies. For example, three haplotypes found in four Caps appeared in different clusters on the tree. They explained this incongruence by arguing that the mitochondrial 12S rRNA genes were not sufficiently divergent to be informative for phylogenetic analysis.
Because speciation and subspeciation of African Old World monkeys is likely to be closely influenced by paleoclimate in Africa, calculating the divergence time within savanna monkeys and comparing fossil data may yield concrete answers regarding subspeciation and haplogroup formation. Unfortunately the number of molecular evolutionary studies on divergence time is much smaller for African Old World monkeys than for hominoids. Common historical events that cause speciation in both cercopithcoids and hominoids will be estimated by comparing the divergence times of both lineages. The evolutionary rate of the control region, which is broadly used for phylogenetic study, is too fast and is therefore unsuitable for comparing these distantly related lineages. To circumvent this problem we determined the divergence time between African Old World monkeys by comparing of 12S rRNA gene sequences (approximately 392 nt) with published sequences having well-studied divergence times. We discuss the phylogenetic relations among the grivet monkey haplogroups and between subspecies of savanna monkeys and the geographic event that caused the divergence within the savanna monkeys.
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Methods |
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Sampling
Table 1 lists the samples from which sequence data were obtained for this study. All mtDNA RFLP haplogroups (1–5) of the Caa were identified by Shimada (2000b). Caa and Cat samples were collected from the field (sampling sites and methods were described in Shimada and Shotake 1997), but other samples were collected from monkeys in captivity. To extract their DNA, we used the phenol/chloroform method followed by dialysis (Takenaka et al. 1989), or we used the DNA extraction kits QIAamp (Qiagen, Hilden, Germany) or Easy-DNA (Invitrogen, Carlsbad, CA).
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Sequencing
To amplify the region containing the mtDNA control region and 12S rRNA gene, we used polymerase chain reaction (PCR) with primers that we designed: L16507 (5'-CTGTATCCGGCATCTGGTTC-3') and H1073 (5'-AGCATAGTGGGGTATCTAATCC-3') (Figure 2). Each 25 µl PCR mixture contained 2.5–100 ng DNA, 1x PCR buffer, 0.625 units of TaKaRa Taq DNA polymerase (Takara, Otsu, Japan), 0.6 µM primer (each), and 200 µM dNTPs. The PCR mixtures were initially denatured at 94°C for 5 min, then amplified for 30 cycles of 94°C for 10 s, 55°C for 10 s, and 72°C for 10 s, and finally extended at 72°C for 3 min. After amplification the PCR products were purified by one of the following three methods: (1) MicroSpin column S400 HR (Amersham Pharmacia, Buckinghamshire, UK); (2) 0.9% agarose gel electrophoresis; or (3) 2.2% low melting temperature agarose (NuSieve GTG agarose, BMA, Rockland, ME) gel electrophoresis. The purified PCR products were labeled and sequenced directly from both ends with an ABI PRISM 310 automated sequencer and the ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems Division, Perkin-Elmer, Foster, CA). In addition to the L16507 and H1073 primers, we designed three other internal primers, Lc611 (5'-CCCAAAGCAAGACACTGAAA-3'), Hc272 (5'-GGGGGGTTTGATTAAAGGTT-3'), and Hc296 (5'-TTGTAGTGTTGTGGG-3'), for nested PCR amplification and for labeling reactions.
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The obtained sequences of the control region and 12S rRNA gene were separately aligned, and we constructed a neighbor-joining tree (Saitou and Nei 1987) with CLUSTAL W (Thompson et al. 1994) modified by K. Kryukov (personal distribution of a prereleased version, available by claire-williams{at}tamu.edu).
For control region sequences, we compared the range of sequence divergences in four categories: (1) within haplogroups, (2) between haplogroups within subspecies, (3) between subspecies within species, and (4) between species. We then evaluated the differences in the average of sequence divergence between these categories by t test.
We extracted only the informative site from our control region sequence data and then transformed the data from Fasta format to binary format by the Esper program (personal distribution of prereleased version by K. Krykov, available by kkryukov{at}lab.nig.ac.jp). A phylogenetic network was constructed by the Reduce Median network method with Network 3.0.1.2 software (Bandelt et al. 1995; Fluxus-Technology Ltd., <http://www.fluxus-engineering.com>).
The 12S rRNA gene sequences were aligned with published sequences (Y18001 for Papio, X99256 for Hylobates, X97707 for Pongo, X93347 for Gorilla, X93335 for Pan troglodytes, D38116 for Pan paniscus, X93334 for Homo) to construct the gene tree and calculate the divergence time with the use of a calibration point in the hominoid lineage. The root of the 12S rRNA gene tree was chosen with the use of the Cebus albifrons (AJ309866) sequence as an outgroup. To evaluate the evolutionary rate change between African Old World monkeys and hominoid lineages, relative rate tests (Nei and Kumar 2000; Rzhetsky and Nei 1992; Wu and Li 1985) were conducted with representative pairs of the two lineages. The longest branched operational taxonomic unit (OTU) in the Old World monkey lineage was compared to the shortest branched OTU in the hominoid lineage, and vice versa. We assumed that the divergence time between humans and chimpanzees is 5 million years and used this value to estimate the divergence time in Old World monkeys of our study. The transition/transversion ratio of nucleotide differences and its variance (Nei and Kumar 2000) were calculated with MEGA version 2 (Kumar et al. 2001) to check for saturation in substitution in the 12S rRNA gene region. The obtained phylogenetic trees and phylogenetic network were arranged manually for ease of interpretation.
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Results |
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Sequencing
Using the primer pair L16507 and H1073, we obtained a single PCR product that contains both control region and 12S rRNA gene sequences from Caa samples (Figure 2). Direct sequencing with these primers was successful for all but one of the Caa samples. However, these primers produced multiple PCR products and poor sequencing results with 3 samples from Cat and 10 samples from Cap. Since the obtained sequence data contained a region between the control region and 12S rRNA gene that was difficult to sequence, we performed nested PCR to eliminate the region (Figure 2). The total length of these two regions was 676–706 bp. Consequently we obtained 19 Caa, 5 Cat, 4 Cap, 1 C. a. spp., 1 C. neglectus, and 1 C. mitis. Only the control region was obtained from three Cap and one Cat sample (Figure 3).
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Comparison of Sequence Divergence Between Categories
The ranges of sequence divergence of the control region are as follows: within haplogroups (0–0.026; average 0.013), between haplogroups (0.040–0.120; average 0.087), between subspecies (0.058–0.162; average 0.098), and between species (0.093–0.220; average 0.167) (Table 2). Figure 4 shows the range of sequence divergence of the control region in each category. The distribution of sequence divergence between haplogroups and between subspecies was not significantly different. However, the distributions of sequence diversity were significantly different (P < .0001) between the within-haplogroup category and the between-haplogroup category and between the between-subspecies category and the between-species category.
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Phylogenetic Tree and Phylogenetic Network
Figure 5A shows the neighbor-joining (NJ) tree for the Cercopithecus control region sequences. Each sequence clusters according to its haplogroup and subspecies, showing high bootstrap values. Since most of the nucleotide substitutions within C. aethiops occurred in the control region (Figure 3), the phylogenetic tree for only this region was almost the same as that for both sequenced regions. Figure 6 shows the phylogenetic network of the Cercopithecus control region sequences.
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Figure 5B shows the NJ tree for the 12S rRNA gene sequences of the present study and of sequences obtained from published DNA databases. This NJ tree indicated that the longest branched OTU in the Old World monkey lineage was Caa1a1624 and the shortest branched OTU in the hominoid lineage was Hylobates. In contrast, the shortest branched OTU in the Old World monkey lineage was C.neg7446 and the longest branched OTU in the hominoid lineage was Pongo. Relative rate tests of both of these pairs did not show evolutionary rate change (P < .8 for Caa1a1624 versus Hylobates; .2 < P < .5 for C.neg7446 versus Pongo).
Assuming that the divergence time between human and chimpanzee (sequence divergence 0.056) is 5 million years, the following divergence times were obtained: 0.4–1.6 million years between haplogroups and subspecies of C. aethiops, 4.7–6.3 million years between Cercopithecus species, 8.9–11.2 million years between Papio and Cercopithecus, and 14.5–18.6 million years between cercopithecoids and hominoids. Table 3 shows transition/transversion ratios and their variances calculated from the number of different nucleotides in each pair.
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Discussion |
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Subspecies Phylogeny of Savanna Monkeys
The purpose of the present study was to explore the issue raised by van der Kuyl et al. (1995a) concerning the mtDNA 12S rRNA gene tree. Since parts of the mitochondrial genome have been known to be duplicated and transferred to the nuclear genome in primates (van der Kuyl 1995b), the results of mitochondrial phylogenetic studies should be carefully interpreted (Disotell 1996). Our results are congruent with our earlier RFLP study on the whole mitochondrial genome detected by Southern blotting (Shimada 2000b). This congruence means that our sequence data are from the mitochondrial genome and not the nuclear genome. Our results indicate that the divergence within a single subspecies is broadly comparable to that between subspecies (Figure 5A). Moreover, our results bring the paraphyly subspecies into Cap (Figures 5A and 6). However, the apparent cluster structure within subspecies Caa suggests substantial diversity ~within each subspecies. The clustering pattern of the NJ tree and phylogenetic network were congruent with each other except one OTU (Ca. spp.). Since the phylogenetic network method did not consider the substitution rate difference between transition and transversion, the long branches in the NJ tree were not expressed properly in the phylogenetic network.
Although the gene for CD4, which is the main receptor for SIV/HIV, may not be a neutral phylogenetic marker, Fomsgaard et al. (1997) found that the number of nucleotide substitutions between CD4 sequences from two individuals of C. a. sabaeus is similar to those between different subspecies of C. aethiops. This fact also may be attributed to the distinct subpopulational structure within a subspecies.
Most previous phylogenetic studies of savanna monkeys have been focused on viral evolution and have taken samples from only a few natural habitats in each subspecies (reviewed in Kurth and Norley 1994; Sharp et al. 1996). Using a neutral marker, we show here that subspecies of savanna monkeys diverged within a short period. Furthermore, by investigating multiple individuals within subspecies, we found that a haplogroup structure in the maternal lineage occurred at the time of subspecies divergence.
Divergence Times
The estimated divergence time of subspecies/haplogroups of C. aethiops occurred during the Pleistocene, and is shorter than that between Pan troglodytes and Pan paniscus (2.3 million years by this calculation). During the Pleistocene, a relatively rapid and dramatic climatic fluctuation is proposed to have affected the composition of fauna (Delay and Happold 1979). A climatic change during the Pleistocene may have triggered the formation of subspecies and haplogroups of C. aethiops. Fossil records suggest that many modern genera of Old World monkeys appear in the late Pliocene and Pleistocene (Gundling and Hill 2000). Although there are no fossils of the last common ancestor just prior to diversification between Cercopithecini and Papionini, Delson (1992) estimated from the available fossil record that the divergence time between Cercopithecini and Papionini was about 10 million years. This estimation is congruent with our calculations. We observed smaller transition/transversion ratios in pairs of distantly related taxa (Table 3). This suggests that there is saturation in nucleotide substitution, which produces an underestimation of the divergence time before the split of Pongo from the others.
Mitochondrial Haplogroups
From a survey of 77 wild monkeys, Shimada (2000a) showed that the mitochondrial haplogroups within Caa geographically distribute in clumps. Although mitochondrial haplogroups in the present study were studied only in Caa for sample correction difficulties, these results showed that the ranges of sequence divergence between haplogroups and between subspecies are greatly overlapping and are almost indistinguishable.
Among African primates, the subspecies diversity of mitochondrial DNA is probably most studied in chimpanzees (Gagneux et al. 1999; Morin et al. 1994). The divergence time estimate between chimpanzee subspecies is 1.6 million years, which is slightly older but closely overlaps our present estimates of this subspecies/haplogroup of C. aethiops. However, haplogroup structure as clearly diverged as subspecies difference and geographically clumped distribution of haplotypes have not been observed in chimpanzee mitochondrial diversity.
The rhesus macaques, which are the most widely distributed subspecies of Asian macaques, have two mitochondrial lineages, western and eastern, and the divergence between these two lineages was earlier than the separation of the eastern lineage from Japanese macaques (Melnick et al. 1993; Tosi et al. 2002). Although Melnick et al. (1993) did not describe the divergence time between these western and eastern lineages, the divergence time estimated from the sequence divergence is almost the same as that for the subspecies/haplogroup mitochondrial divergence in C. aethiops. Melnick et al. (1993) attributed this mitochondrial paraphyly to the high female philopatric nature and character of mitochondrial markers (i.e., no recombination and maternal inheritance). Intergroup gene flow of C. aethiops also depends on male migration as in Asian macaques (Shimada 2000b; Shimada and Shotake 1997), but not as in chimpanzees in which females migrate. Female philopatry is considered to affect the mitochondrial geographic differentiation of almost all Old World monkeys whose males migrate between groups in their social system (Melnick and Hoelzer 1992, 1996). The sex-dependent migration system may produce this similarity in mitochondrial diversity between haplogroup difference and subspecies difference. Shimada and Shotake (1997) attributed the lower genetic differentiation between local populations in Caa compared to other Old World monkeys to frequent migration of males. Since genes corresponding to morphology are encoded by the nuclear genome, such genes are exchanged over geographically wider ranges than mitochondrial genome exchange ranges. In the process of subspeciation, when geographic barriers divided gene flow range by male migration, the exchange range of the mitochondrial genome may have been divided within each nuclear genome exchange range. If this gene exchange range division of the mitochondrial genome occurred by the same geographic event in all subspecies, it would produce mitochondrial clusters with similar diversity in all subspecies. This type of phenomenon may be responsible for the haplogroup/subspecies difference observed in this study.
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Acknowledgments |
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We thank Dr. Y. Kawamoto and Dr. O. Hishida of the Primate Research Institute, Kyoto University for technical advice; Dr. K. Hayasaka and Mr. Kuraishi of the Japan Monkey Centre for supplying samples of C. aethiops tantalus and Cercopithecus mitis; the Department of Biology at Addis Ababa University, Wildlife Authority of Ethiopia, and the project team of the Ethiopian primate research project from Japan for assistance with the field study; and Drs. N. Saitou, A. J. Tosi, and A. Wyndham for reading and commenting on an earlier version of this manuscript. This study was supported by a Grant-in-Aid for Scientific Research (no. 06041065) from the Ministry of Education, Science, Sports, and Culture, Japan (to T.S.), the Nissan Science Foundation, and the Inamori Foundation (to M.K.S.).
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Footnotes |
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Corresponding Editor: Stephen J. O'Brien
Received January 23, 2001
Accepted November 26, 2001
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