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The complete mitochondrial genome of Macaca thibetana and a novel nuclear mitochondrial pseudogene
Gene 429 (2009) 31–36
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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e
The complete mitochondrial genome of Macaca thibetana and a novel nuclear mitochondrial pseudogene Deming Li a,1, Longqing Fan a,1, Bo Zeng a, Hailin Yin b, Fangdong Zou a, Hongxing Wang c, Yang Meng a, Emily King a, Bisong Yue a,⁎ a b c
Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, China Laboratory Animal Center, Sichuan University, Chengdu, Sichuan 610041, China Institute of Laboratory Animal Science, Academy of Medical Sciences, Chengdu, Sichuan 610065, China
a r t i c l e
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Article history: Received 5 April 2008 Received in revised form 3 October 2008 Accepted 4 October 2008 Available online 25 October 2008 Received by M. Di Giulio Keywords: Macaca thibetana Mitochondrial genome Nuclear mitochondrial pseudogene Phylogenetic analysis
a b s t r a c t The complete mitochondrial genome of Macaca thibetana was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequencing methods. It is 16,540 bp and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region. Most codon usage followed the typical pattern of vertebrates. Two rare start codons were found, in which GTG initiated the NADH dehydrogenase subunit 1 (ND1) gene and the ATP8 gene, and ATT initiated the ND2 gene. A new mitochondrial DNA-like sequence (2003 bp) in the nuclear genome of M. thibetana was found. It matched with the 3′ end of the ND1 gene, the tRNAIle-tRNAGln-tRNAMet genes, the ND2 gene, and the 5′ end of the tRNATrp gene. Sequence divergence between the nuclear pseudogene and the mitochondrial homologue suggested that the translocation of this mtDNA fragment into the nuclear genome occurred 3.16 ∼ 3.48 million years ago (MYA). Molecular phylogenetic analysis of 16 Cercopithecidae species was performed using sequences from 12 concatenated heavy-strand encoded protein coding genes. The results provided more evidence to support previous morphological and chromosomal studies on Cercopithecidae. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Tibetan macaques (Macaca thibetana), large primates with short, stump-like tails, are mainly found in Fujian, Anhui, Guangdong, Guangxi, Guizhou, Shanxi, Sichuan, Tibet, and Yunnan provinces, China. It was commonly considered an endemic monkey species to China and occupies subtropical, deciduous and broadleaf evergreen forests, between 800 and 2500 m above sea level (Jiang et al., 1996; Wang, 1998). It has also been reported in the extreme north of India (Kumar et al., 2005). The most serious threat to the Tibetan macaques come from humans and include habitat destruction and illegal poaching; as such this species is protected by Chinese law and listed in Appendix II of CITES (Wang, 1998). Extant Old World monkeys, family Cercopithecidae, are the largest and most species-rich group of nonhuman primates alive today. Cercopithecidae includes at least 21 genera, with about 11 in
Abbreviations: bp, base pairs; LA-PCR, long and accurate polymerase chain reaction; ATP6 and 8, ATPase subunit 6 and 8; COI–III, cytochrome oxidase subunits I–III; Cytb, cytochrome b; NADH1–6 and 4L, NADH dehydrogenase subunit 1–6 and 4L; tRNA, transfer RNA; CSB, conserved sequence block; ETAS, extended termination associated sequence; numt, pseudogene; MYA, million years ago; mtDNA, mitochondrial DNA. ⁎ Corresponding author. E-mail address: [email protected] (B. Yue). 1 These two authors contributed equally to this work. 0378-1119/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.10.010
subfamily Cercopithecinae and 10 in Colobinae (Groves, 2001; Disotell, 2003). The genus Macaca, a very successful genus in Cercopithecidae including more than 20 well characterized species, is distributed in different geographical areas in Asia, as well as limited regions in North Africa (Fooden, 1976). Many molecular studies on the phylogenetic relationships within Cercopithecidae have been done, but still many questions remain (Harris and Disotell, 1998; Page et al., 1999; Tosi et al., 2004; Hayasaka et al., 1996). This is in part due to the limited amount of sequence information. Many studies have shown that several concatenated genes have great potential in phylogenetic inferences of deeper branches (Flynn and Nedbal, 1998; Flynn et al., 2000; Miya et al., 2003; Yu et al., 2007). In recent years, more and more complete mitochondrial genome sequences have become available and they are widely applied in phylogenetic analysis (Jondeung et al., 2007; Peng et al., 2006; Peng et al., 2007; Zhang et al., 2008). The complete mtDNA sequence of Macaca thibetana will be valuable in elucidating relationships within Cercopithecidae and primates in the future. Over the last 30 years, nuclear insertions of mitochondrial DNA (mtDNA) or ‘numts’ have been extensively reported in many eukaryotic organisms, including more than 60 animal and plants species (Bensasson et al., 2001). They are segments of mitochondrial DNA that have translocated into the nuclear genome (Lopez et al., 1994; Ricchetti et al., 2004). In this study, we describe a novel pseudogene in Macaca thibetana and analyze its evolutionary history.
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D. Li et al. / Gene 429 (2009) 31–36
M. mulatta. The tRNA genes were identified with tRNAscan-SE v.1.21 (Lowe and Eddy, 1997). Two tRNA genes, which were not found with tRNAscan-SE, were identified by comparison with homologues from M. mulatta.
2. Materials and methods 2.1. DNA sample A blood sample from a female M. thibetana (M. t. thibetana) was obtained from Jianyang County, Sichuan Province, China, during a medical exam. Total genomic DNA was extracted from the blood following the method of Sambrook and Russell (2001). 2.2. PCR amplification and sequencing The M. thibetana mitochondrial genome was amplified in ten overlapping fragments using the long and accurate polymerase chain reaction (LA-PCR) technique according to the manufacturer's instruction (TaKaRa, China). Primers were designed based on the conserved sequences of five species in Cercopithecinae: M. mulatta (NC_005943), M. sylvanus (NC_002764), Chlorocebus aethiops (NC_007009), C. sabaeus (NC_008066), and Papio hamadryas (Y18001). All primers used in this study are listed in Table 1. PCR cycles were 5 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 50 s at 55–62 °C, and 1.5–4 min at 72 °C, with a final extension at 72 °C for 10 min. The ten PCR products were purified from a 1% agarose gel with the DNA Agarose Gel Extraction Kit (Omega, USA) and then sequenced directly in the ABI 3730 DNA sequencer. The primer walking method was applied to sequence LA-PCR products. Each segment overlapped the next fragment by about 150 bp. Another primer pair (Xm2.3) was designed based on neighboring sequences in the mitochondrial genome because the amplified product from primer pair X2.3 was not in conjunction with neighboring fragments. PCR products of primer pair X2.3 were ligated with the pMD18-T vector (TaKaRa, China) and transformed into E. coli JM109 competent cells. Colonies were picked and DNA sequencing was conducted in both directions. Internal sequencing primers W1Fn5′-ATACCCATCGCAATCTCC-3′ and W1Fm 5′-TTCTGATTCGCCCTCTATG-3′ were applied for the products of primer X2.3 and Xm2.3, respectively. 2.3. Sequence analysis DNA sequences were analyzed using the MEGA3.1 program (Kumar et al., 2004). The locations of protein-coding and rRNA genes were determined by comparison with known sequences from
Table 1 Primer locations and sequences Primer locations
Primer sequence (5′–3′)
X0
F:5′-GCATCCATAATATACTTCGCCAC-3′ R:5′-CGTCTTATTGTTTTATGTCCGTCTC-3′ F: 5′-AAAAGTAAAAGGAACTCGGC-3′ R: 5′-CGATTAGGGCGTAGTTTGAGT-3′ F: 5′-ATTGCCCTCCTCCTATGAAC-3′ R: 5′-GGCTTTGAAGGCTCTTGGTC-3′ F:5′-ACTTCTACATACGCCTAATCTACAC-3′ R: 5′-TAGGTGTTGATATAGGATAGGGTC-3′ F: 5′-TTAGGAGCC ATCAACTTCATTACC-3′ R: 5′-GGTTAGTTTTGTTGTGAGTGTTGAG-3′ F: 5′-CCAGTTCAACTAAGCCTACAAGA-3′ R: 5′-CCTTGGTATG TGCTTTCTCG-3′ F: 5′-AACAACACCTAATGACCCACCA-3′ R: 5′-GGTTGTTAGTTTTTGGTCTGTGAGTG-3′ F: 5′-TAAATGGGGCAACCAAGCA-3′ R: 5′-GCTTGAGGTGGAGAAGGCTAC-3′ F: 5′-TGGGGGCTATTACTACCCTATTCAT-3′ R: 5′-GTGGGGCTATCAATGGCGTAT-3′ F: 5′-CTCATACCTCCTCCTAGAAACCTG-3′ R: 5′-GAATGCCAGCTTTGGGTGTT-3′ Fm5′-CACCCTTCTCCTATGAACCCCT-3′ Rm5′-GGCTTTGAAGGCTCTTGGTC-3′
X1 X2.3 X4 X5 X6 X7 X8 X9 X10 Xm2.3
2.4. Phylogenetic analysis To obtain a more complete understanding of the evolutionary history of M. thibetana, a total of 10,798 bp from the 12 concatenated H-strand protein-coding genes in the mitochondrial genome were used for phylogenetic analysis. Multiple alignments of these fragments from M. thibetana and 16 other species were performed using MEGA3.1 (Kumar et al., 2004) with the default settings. The complete mitochondrial genome sequences of 15 other Cercopithecidae species are available from GenBank and the sequence accession numbers of these species are as follows: M. mulatta (NC_005943), M. sylvanus (NC_002764), Chlorocebus aethiops (NC_007009), C. sabaeus (NC_0 08066), Papio hamadryas (Y180 01), C. pygerythrus (NC_009747), C. tantalus (NC_009748), Colobus guereza (NC_006901), Nasalis larvatus (NC_008216), Presbytis melalophos (NC_008217), Procolobus badius (NC_008219), Pygathrix nemaeus (NC_008220), P. roxellana (NC_008218), Semnopithecus entellus (NC_008215), Trachypithecus obscurus (NC_006900), and Cebus albifrons (NC_002763). The C. albifrons sequence was used as an outgroup. Maximum parsimony (MP) analyses were performed using PAUP⁎4.0b10 (Swofford, 2003). A consensus tree was made for the equally-weighted MP analysis using the majority rule. We assessed the reliability of clades in phylogenetic trees by bootstrap probabilities (BSP) computed using 1000 replicates with 20 random additional sequencing replicates for each bootstrap replicate. Bayesian analysis was conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Bayesian posterior probabilities (BPP) used models estimated with Modeltest 3.7 (Posada and Crandall, 1998) under the Akaike Information Criterion (AIC), performing two separate runs with four Markov chains. Each run was conducted with 200 million generations and sampled every 100 generations. We discarded the first 20% of the trees as part of a burn-in procedure, and used the remaining 16,001 sampling trees to construct a 50% majority rule consensus tree. Molecular dating for the numt origin was estimated from the overall divergence (δ) between the numt and M. thibetana mitochondrial homologue, applying the equation δ = (λN + λC)T (Li et al., 1981), where λC = 2.5 × 10− 8 substitutions/sites/year for mitochondrial DNA (Hasegawa et al., 1985) and λN = 2.0 × 10− 9 substitutions/sites/year for the rate of neutral substitution of human genomes (Kumar and Subramanian, 2002) or λN = 4.7 × 10− 9 substitutions/sites/year for human pseudogenes (Li et al., 1981). 3. Results and discussion 3.1. Structure of the mitochondrial genome The complete mitochondrial genome of M. thibetana was sequenced and it is 16,540 bp including 13 protein-coding genes, two rRNA genes (12S rRNA, 16S rRNA), 22 tRNA genes, and a control region. It was deposited in GenBank under accession number EU294187. The gene organization in line with M. mulatta, represented as the H-strand with the first nucleotide in the middle of the D-loop region, is shown in Table 2. It is obvious that the gene distributions are the same as those of other mammalian mitochondrial genomes (Anderson et al., 1981, 1982; Hiendleder et al., 1998; Lin et al., 1999) and that most genes are encoded on the H strand, except for the ND6 gene and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Pro, tRNA-Glu), which are encoded on the L strand. The overall base composition of the H strand is as follows: 31.70% A, 25.10% T, 30.23% C, and 12.97% G.
D. Li et al. / Gene 429 (2009) 31–36 Table 2 Characteristics of the M. thibetana mitochondrial genome Gene
Control region tRNA-Phe 12s-rRNA tRNA-Val 16s-rRNA tRNA-Leu(UUR) ND1 tRNA-Ile tRNA-Gln tRNA-Met ND2 tRNA-Trp tRNA-Ala tRNA-Asn OL tRNA-Cys tRNA-Tyr COI tRNA-Ser(UCN) tRNA-Asp COII tRNA-Lys ATP8 ATP6 COIII tRNA-Gly ND3 tRNA-Arg ND4L ND4 tRNA-His tRNA-Ser(AGY) tRNA-Leu(CUN) ND5 ND6 tRNA-Glu CYTB tRNA-Thr tRNA-Pro
Position
Size (bp)
From
To
15992,1 522 595 1542 1611 3171 3248 4203 4269 4342 4410 5452 5526 5596 5670 5701 5769 5839 7380 7452 7521 8270 8334 8495 9175 9959 10,027 10,373 10,438 10,728 12,106 12,175 12,234 12,305 14,117 14,645 14,718 15,859 15,924
16540, 542 594 1541 1610 3170 3245 4204 4271 4340 4409 5453 5518 5594 5668 5702 5763 5834 7377 7448 7519 8204 8334 8540 9175 9959 10,026 10,372 10,437 10,734 12,105 12,174 12,233 12,304 14,116 14,644 14,713 15,858 15,922 15,991
1091 73 947 69 1560 75 957 69 72 68 1044 67 69 73 63 71 1540 69 68 684 65 207 681 785 68 346 65 297 1378 69 59 71 1812 528 49 1141 64 68
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Table 3 Codon usage in the M. thibetana mitochondrial genome Strand
H H H H H H H H L H H H L L L L L H L H H H H H H H H H H H H H H H L L H H L
Codon Start
Stop
GTG
TAG
ATT
TAG
ATG
?
ATG
TAA
GTG ATG ATG
TAA TAA TA
ATC
T
ATG ATG
TAA T
ATA ATG
TAA AGG
ATG
T
Codon
Count
Codon
Count
Codon
Count
Codon
Count
UUU UUC Phe UUA UUG CUU CUC CUA Leu CUG AUU AUC Ile AUA Met AUG GUU GUC GUA Val GUG
66 136 65 9 72 140 286 41 119 188 181 24 30 32 59 4
UCU UCC UCA UCG CCU CCC CCA Pro CCG ACU ACC ACA Thr ACG GCU GCC GCA Ala GCG
48 70 73 10 48 100 64 8 61 136 163 11 48 118 73 5
UAU UAC Tyr UAA UAG CAU CAC His CAA Gln CAG AAU AAC Asn AAA Lys AAG GAU GAC Asp GAA Glu GAG
44 80 0 0 28 68 90 8 34 131 87 3 18 43 63 11
UGU UGC Cys UGA Trp UGG CGU CGC CGA Arg CGG AGU AGC Ser AGA AGG GGU GGC GGA Gly GGG
3 22 84 13 7 26 27 2 8 45 0 0 18 81 52 26
3.3. Sequence features of the ribosomal and transfer RNA genes The 12S and 16S rRNA genes of M. thibetana are 947 and 1560 bp, respectively. They are located between the tRNA-Phe and tRNA-Leu (UUR) genes, and are separated by the tRNA-Val gene with the same situation found in other vertebrates (Inoue et al., 2000). The complete mitochondrial sequence contains 22 tRNA genes, which are interspersed in the genome and range in size from 49–75 bp. Only one long 67 bp spacer between COII and tRNA-Lys was found. Twenty tRNA genes have the typical cloverleaf secondary structure. tRNA-Ser (AGY) and tRNA-Cys which were not found by the tRNAscan-SE, were identified by comparison with M. mulatta counterparts. The structure
3.2. Sequence features of the protein-coding genes In the 13 protein-coding genes, eight use ATG as the start codon while ND1 and ATP8 use GTG (Table 2). ND2, ND3 and ND5 start with ATT, ATC, and ATA, respectively. In fact, ATG and ATA are commonly used as initiation codons in mammals (Xu and Arnason, 1994). With regards to stop codons (TAA, TAG, AGG), 5 genes (COII, ATP8, ATP6, ND4L, ND5) use the complete stop codon TAA, whereas 4 genes have incomplete stop codons, TA_ (COIII) and T_ (ND3, ND4, CYTB), which were presumably completed as TAA by posttranscriptional polyadenylation (Anderson et al., 1981). The COI gene stop codon was not determined, as was the case in 7 sequenced Cercopithecinae species: M. mulatta (NC_005943), M. sylvanus (NC_002764), Chlorocebus aethiops (NC_007009), C. sabaeus (NC_008066), Papio hamadryas (Y18001), C. pygerythrus (NC_009747), and C. tantalus (NC_009748). Codon usage in the M. thibetana mitochondrial genome indicated that CTA (Leu), ATC (Ile) and ATA (Met) occur at a higher frequency than others (Table 3). G is found in the lowest proportion (4.9%) at the third codon position in the 12 H-strand protein-coding genes, as compared to 37.9% A, 39.3% C and 17.9% T, which is similar to other findings in vertebrate mitochondrial genomes (Lopez et al., 1996; Janke and Arnason, 1997; Janke et al., 2001; Zhang et al., 2003). Two pairs of protein-coding genes (ATP8 and ATP6, ND4L and ND4), both located on the H-strand, overlapped in readingframes for 46 and 7 bp. Only 1 bp overlap was found in the ATP6 and COIII genes.
Fig. 1. Phylogenetic tree of the numt and its homologues as inferred from the nucleotide sequences of the concatenated ND2 gene and the 3′ end of the ND1 gene using Bayesian analysis. The numbers beside the nodes are Bayesian posterior probabilities.
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D. Li et al. / Gene 429 (2009) 31–36
of the tRNA-Ser (AGY) gene was analyzed by RNAstructure, version 4.5 (http://rna.urmc.rochester.edu/rnastructure.html) and it was found that the complete dihydrouridine arm (D-arm) was lacking. Aberrant tRNA can also fit the ribosome by adjusting its structural conformation and function in a similar way to that of usual tRNAs in the ribosome (Ohtsuki et al., 2002). 3.4. Sequence features of the non-coding regions The origin of L-strand replication (OL) in M. thibetana is 33 bp and located in a cluster of five tRNA genes (WANCY region: tRNA-Trp, tRNA-Ala, tRNA-Asn, tRNA-Cys, and tRNA-Tyr). We found that it can fold into a stable stem–loop secondary structure with 10 bp in the stem and 13 bp in the loop. This region overlaps the tRNA-Cys gene by 2 bp. The M. thibetana mtDNA control region spans 1091 bp and is located between the tRNA-Pro and tRNA-Phe genes. Within this sequence, we identified conserved sequence blocks (CSB) and extended termination associated sequences (ETAS) based on the description by Sbisa et al. (1997). CSB-1, CSB-2 and CSB-3 were located in positions 212–236, 297–312, and 343–360, respectively. ETAS1 and ETAS2 were close to the 5′ end of the control region, at positions 16046–16102 and 16264–16323, respectively. 3.5. Characterization of the numt sequence and the time of nuclear integration A roughly 2.0 kb fragment was amplified using primer pair X2.3 from the total genomic DNA of M. thibetana and double peaks were observed while sequencing. Regular peaks were obtained when the
fragment was then cloned into the vector and sequenced again. It is 2003 bp in length; however the sequences along both sides didn't overlap completely with neighboring mitochondrial sequences while the sequence amplified using primer Xm2.3 was in good conjunction with the neighboring sequences on both sides. The protein coding regions of these two sequences could be correctly translated into amino acids based on mammalian mitochondrial codes. Therefore, we concluded that a nuclear mitochondrial pseudogene (numt) was obtained and the sequence was deposited in GenBank under accession number EU340344. BLAST search for the numt sequence in GenBank showed that this sequence matched the mitochondrial genomes of primates. There are 3 homologues in the M. mulatta nuclear genome and they share 83.2%, 82.5% and 81.4% similarity. It matches with the 3′ end (717 bp) of the ND1 gene, the tRNAIle-tRNAGln-tRNAMet genes, the ND2 gene, and the 5′ end (37 bp) of the tRNATrp gene. It covers 12.11% of the complete M. thibetana mitochondrial genome and shares 90.6% sequence identity with its mitochondrial homologue. The ratio of transitions to transversions in these two sequences was 12.5. The base composition of the mitochondrial fragment is 24.3% T, 33.0% C, 32.3% A, and 10.4% G, as compared to 25.2% T, 32.1% C, 32.4% A, and 10.4% G in the numt. In order to estimate when the mtDNA fragment translocated into the nuclear DNA, sequences from the protein-coding region of the numt and its homologues from 16 Cercopithecidae species (Old World monkeys) and a Cebidae species, C. albifrons (a New World monkey, which is regarded as an outgroup), were used to reconstruct their phylogenetic relationship using the Bayesian analysis (Fig. 1). Using an overall divergence (δ) of 0.094 between the numt and the M. thibetana
Fig. 2. Phylogenetic tree of M. thibetana and 15 other Cercopithecidae species as inferred from the nucleotide sequences of 12 concatenated protein-coding genes. (A) Bayesian analysis. The numbers beside the nodes are Bayesian posterior probabilities. (B) Maximum parsimony (MP) method in PAUP⁎4.0b10 analysis. Percentage bootstrap values are shown on interior branches using 1000 replicates with 20 random additional sequencing replicates.
D. Li et al. / Gene 429 (2009) 31–36
mitochondrial homologue, we estimated that the numt translocated into the nuclear genome around 3.16 ∼ 3.48 million years ago (MYA), just before the divergence of the macaques about 3 MYA or later (Hayasaka et al., 1996). 3.6. Phylogenetic analyses Similar phylogenetic trees were obtained when Bayesian and MP methods were applied based on the 12 protein-coding genes of the 16 Cercopithecidae species (Fig. 2). The results support the traditional classification scheme, i.e., extant Old World monkeys can be divided into two ecologically and morphologically distinct subfamilies: Cercopithecinae (cheek-pouched monkeys) and Colobinae (leaf-eating monkeys) (Delson, 1992; Disotell, 2003; Groves, 1993, 2001). There are two clades in Cercopithecinae and one clade consists of three macaques and P. hamadryas. In this clade, M. thibetana and M. mulatta formed a sister group to M. sylvanus, then grouped with P. hamadryas, which suggests that M. thibetana emerged later than P. hamadryas and M. sylvanus in Cercopithecinae. Another clade consists of 4 chlorocebus species. In Colobinae, the phylogenetic tree supports reciprocal monophyly of African (C. guereza, P. badius) and Asian (P. nemaeus, P. roxellana, N. larvatus, S. entellus, P. melalophos, T. obscurus) colobines, which lends more support to previous morphological (Napier and Napier, 1970; Szalay and Delson, 1979; Groves, 2001), chromosomal (Bigoni et al., 2003, 2004), and molecular studies (Messier and Stewart, 1997; Zhang and Ryder, 1998; Page et al., 1999; Xing et al., 2005). Asian colobines were divided into three clades, an odd-nosed clade (N. larvatus, P. nemaeus and P. roxellana), a langur clade (P. melalophos and T. obscurus) and the S. entellus clade. Except for the S. entellus clade, this result is congruent with previous phylogenetic studies (Groves, 1970; Jablonski and Peng, 1993; Jablonski, 1998). Acknowledgments We thank Mr. Yao Fang for collecting the M. thibetana blood sample during the health exams. This research was funded by the National Basic Research Program of China (973 Project: 2007CB411605) and the “985 project” of the Central University of Nationalities. References Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H.L., Coulson, A.R., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J.H., Staden, R., Young, I.G., 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457–464. Anderson, S., de Bruijn, M.H., Coulson, A.R., Eperon, I.C., Sanger, F., Young, I.G., 1982. Complete sequence of bovine mitochondrial DNA: conserved features of the mammalian mitochondrial genome. J. Mol. Biol. 156, 683–717. Bensasson, D., Zhang, D.X., Hartl, D.L., Hewitt, G.M., 2001. Mitochondrial pseudogenes: evolution's misplaced witnesses. Trends Ecol. Evol. 16, 314–321. Bigoni, F., Houck, M.L., Ryder, O.A., Wienberg, J., Stanyon, R., 2004. Chromosome painting shows that Pygathrix nemaeus has the most basal karyotype among Asian colobines. Int. J. Primatol. 25, 679–688. Bigoni, F., Stanyon, R., Wimmer, R., Schempp, W., 2003. Chromosome painting shows that the proboscis monkey (Nasalis larvatus) has a derived karyotype and is phylogenetically nested within Asian colobines. Am. J. Primatol. 60, 85–93. Delson, E., 1992. Evolution of old word monkeys. In: Johns, J.S., Martin, R.D., Pilbeam, D., Bunney, S. (Eds.), The Cambridge Encyclopedia of Human Evolution. Cambridge University Press, Cambridge, pp. 217–222. Disotell, T.R., 2003. Primates: Phylogenetics. Encyclopedia of the Human Genome. Nature Publishing Group, London. Flynn, J.J., Nedbal, M.A., 1998. Phylogeny of the Carnivora (Mammalia): congruence vs incompatibility among multiple data sets. Mol. Phylogenet. Evol. 9, 414–426. Flynn, J.J., Nedbal, M.A., Dragoo, J.W., Honeycutt, R.L., 2000. Whence the red panda? Mol. Phylogenet. Evol. 17, 190–199. Fooden, J., 1976. Provisional classification and key to living species of macaques (primates: Macaca). Folia Primatol 25, 225–236. Groves, C., 1970. The forgotten leaf-eaters, and the phylogeny of the Colobinae. In: Napier, J.R., Napier, P.H. (Eds.), Old World Monkeys: Evolution, Systematics and Behavior. Academic Press, New York, pp. 555–587. Groves, C., 2001. Primate Taxonomy. Pages Smithsonian Press, Washington, DC.
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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e
The complete mitochondrial genome of Macaca thibetana and a novel nuclear mitochondrial pseudogene Deming Li a,1, Longqing Fan a,1, Bo Zeng a, Hailin Yin b, Fangdong Zou a, Hongxing Wang c, Yang Meng a, Emily King a, Bisong Yue a,⁎ a b c
Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, China Laboratory Animal Center, Sichuan University, Chengdu, Sichuan 610041, China Institute of Laboratory Animal Science, Academy of Medical Sciences, Chengdu, Sichuan 610065, China
a r t i c l e
i n f o
Article history: Received 5 April 2008 Received in revised form 3 October 2008 Accepted 4 October 2008 Available online 25 October 2008 Received by M. Di Giulio Keywords: Macaca thibetana Mitochondrial genome Nuclear mitochondrial pseudogene Phylogenetic analysis
a b s t r a c t The complete mitochondrial genome of Macaca thibetana was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequencing methods. It is 16,540 bp and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region. Most codon usage followed the typical pattern of vertebrates. Two rare start codons were found, in which GTG initiated the NADH dehydrogenase subunit 1 (ND1) gene and the ATP8 gene, and ATT initiated the ND2 gene. A new mitochondrial DNA-like sequence (2003 bp) in the nuclear genome of M. thibetana was found. It matched with the 3′ end of the ND1 gene, the tRNAIle-tRNAGln-tRNAMet genes, the ND2 gene, and the 5′ end of the tRNATrp gene. Sequence divergence between the nuclear pseudogene and the mitochondrial homologue suggested that the translocation of this mtDNA fragment into the nuclear genome occurred 3.16 ∼ 3.48 million years ago (MYA). Molecular phylogenetic analysis of 16 Cercopithecidae species was performed using sequences from 12 concatenated heavy-strand encoded protein coding genes. The results provided more evidence to support previous morphological and chromosomal studies on Cercopithecidae. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Tibetan macaques (Macaca thibetana), large primates with short, stump-like tails, are mainly found in Fujian, Anhui, Guangdong, Guangxi, Guizhou, Shanxi, Sichuan, Tibet, and Yunnan provinces, China. It was commonly considered an endemic monkey species to China and occupies subtropical, deciduous and broadleaf evergreen forests, between 800 and 2500 m above sea level (Jiang et al., 1996; Wang, 1998). It has also been reported in the extreme north of India (Kumar et al., 2005). The most serious threat to the Tibetan macaques come from humans and include habitat destruction and illegal poaching; as such this species is protected by Chinese law and listed in Appendix II of CITES (Wang, 1998). Extant Old World monkeys, family Cercopithecidae, are the largest and most species-rich group of nonhuman primates alive today. Cercopithecidae includes at least 21 genera, with about 11 in
Abbreviations: bp, base pairs; LA-PCR, long and accurate polymerase chain reaction; ATP6 and 8, ATPase subunit 6 and 8; COI–III, cytochrome oxidase subunits I–III; Cytb, cytochrome b; NADH1–6 and 4L, NADH dehydrogenase subunit 1–6 and 4L; tRNA, transfer RNA; CSB, conserved sequence block; ETAS, extended termination associated sequence; numt, pseudogene; MYA, million years ago; mtDNA, mitochondrial DNA. ⁎ Corresponding author. E-mail address: [email protected] (B. Yue). 1 These two authors contributed equally to this work. 0378-1119/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.10.010
subfamily Cercopithecinae and 10 in Colobinae (Groves, 2001; Disotell, 2003). The genus Macaca, a very successful genus in Cercopithecidae including more than 20 well characterized species, is distributed in different geographical areas in Asia, as well as limited regions in North Africa (Fooden, 1976). Many molecular studies on the phylogenetic relationships within Cercopithecidae have been done, but still many questions remain (Harris and Disotell, 1998; Page et al., 1999; Tosi et al., 2004; Hayasaka et al., 1996). This is in part due to the limited amount of sequence information. Many studies have shown that several concatenated genes have great potential in phylogenetic inferences of deeper branches (Flynn and Nedbal, 1998; Flynn et al., 2000; Miya et al., 2003; Yu et al., 2007). In recent years, more and more complete mitochondrial genome sequences have become available and they are widely applied in phylogenetic analysis (Jondeung et al., 2007; Peng et al., 2006; Peng et al., 2007; Zhang et al., 2008). The complete mtDNA sequence of Macaca thibetana will be valuable in elucidating relationships within Cercopithecidae and primates in the future. Over the last 30 years, nuclear insertions of mitochondrial DNA (mtDNA) or ‘numts’ have been extensively reported in many eukaryotic organisms, including more than 60 animal and plants species (Bensasson et al., 2001). They are segments of mitochondrial DNA that have translocated into the nuclear genome (Lopez et al., 1994; Ricchetti et al., 2004). In this study, we describe a novel pseudogene in Macaca thibetana and analyze its evolutionary history.
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M. mulatta. The tRNA genes were identified with tRNAscan-SE v.1.21 (Lowe and Eddy, 1997). Two tRNA genes, which were not found with tRNAscan-SE, were identified by comparison with homologues from M. mulatta.
2. Materials and methods 2.1. DNA sample A blood sample from a female M. thibetana (M. t. thibetana) was obtained from Jianyang County, Sichuan Province, China, during a medical exam. Total genomic DNA was extracted from the blood following the method of Sambrook and Russell (2001). 2.2. PCR amplification and sequencing The M. thibetana mitochondrial genome was amplified in ten overlapping fragments using the long and accurate polymerase chain reaction (LA-PCR) technique according to the manufacturer's instruction (TaKaRa, China). Primers were designed based on the conserved sequences of five species in Cercopithecinae: M. mulatta (NC_005943), M. sylvanus (NC_002764), Chlorocebus aethiops (NC_007009), C. sabaeus (NC_008066), and Papio hamadryas (Y18001). All primers used in this study are listed in Table 1. PCR cycles were 5 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 50 s at 55–62 °C, and 1.5–4 min at 72 °C, with a final extension at 72 °C for 10 min. The ten PCR products were purified from a 1% agarose gel with the DNA Agarose Gel Extraction Kit (Omega, USA) and then sequenced directly in the ABI 3730 DNA sequencer. The primer walking method was applied to sequence LA-PCR products. Each segment overlapped the next fragment by about 150 bp. Another primer pair (Xm2.3) was designed based on neighboring sequences in the mitochondrial genome because the amplified product from primer pair X2.3 was not in conjunction with neighboring fragments. PCR products of primer pair X2.3 were ligated with the pMD18-T vector (TaKaRa, China) and transformed into E. coli JM109 competent cells. Colonies were picked and DNA sequencing was conducted in both directions. Internal sequencing primers W1Fn5′-ATACCCATCGCAATCTCC-3′ and W1Fm 5′-TTCTGATTCGCCCTCTATG-3′ were applied for the products of primer X2.3 and Xm2.3, respectively. 2.3. Sequence analysis DNA sequences were analyzed using the MEGA3.1 program (Kumar et al., 2004). The locations of protein-coding and rRNA genes were determined by comparison with known sequences from
Table 1 Primer locations and sequences Primer locations
Primer sequence (5′–3′)
X0
F:5′-GCATCCATAATATACTTCGCCAC-3′ R:5′-CGTCTTATTGTTTTATGTCCGTCTC-3′ F: 5′-AAAAGTAAAAGGAACTCGGC-3′ R: 5′-CGATTAGGGCGTAGTTTGAGT-3′ F: 5′-ATTGCCCTCCTCCTATGAAC-3′ R: 5′-GGCTTTGAAGGCTCTTGGTC-3′ F:5′-ACTTCTACATACGCCTAATCTACAC-3′ R: 5′-TAGGTGTTGATATAGGATAGGGTC-3′ F: 5′-TTAGGAGCC ATCAACTTCATTACC-3′ R: 5′-GGTTAGTTTTGTTGTGAGTGTTGAG-3′ F: 5′-CCAGTTCAACTAAGCCTACAAGA-3′ R: 5′-CCTTGGTATG TGCTTTCTCG-3′ F: 5′-AACAACACCTAATGACCCACCA-3′ R: 5′-GGTTGTTAGTTTTTGGTCTGTGAGTG-3′ F: 5′-TAAATGGGGCAACCAAGCA-3′ R: 5′-GCTTGAGGTGGAGAAGGCTAC-3′ F: 5′-TGGGGGCTATTACTACCCTATTCAT-3′ R: 5′-GTGGGGCTATCAATGGCGTAT-3′ F: 5′-CTCATACCTCCTCCTAGAAACCTG-3′ R: 5′-GAATGCCAGCTTTGGGTGTT-3′ Fm5′-CACCCTTCTCCTATGAACCCCT-3′ Rm5′-GGCTTTGAAGGCTCTTGGTC-3′
X1 X2.3 X4 X5 X6 X7 X8 X9 X10 Xm2.3
2.4. Phylogenetic analysis To obtain a more complete understanding of the evolutionary history of M. thibetana, a total of 10,798 bp from the 12 concatenated H-strand protein-coding genes in the mitochondrial genome were used for phylogenetic analysis. Multiple alignments of these fragments from M. thibetana and 16 other species were performed using MEGA3.1 (Kumar et al., 2004) with the default settings. The complete mitochondrial genome sequences of 15 other Cercopithecidae species are available from GenBank and the sequence accession numbers of these species are as follows: M. mulatta (NC_005943), M. sylvanus (NC_002764), Chlorocebus aethiops (NC_007009), C. sabaeus (NC_0 08066), Papio hamadryas (Y180 01), C. pygerythrus (NC_009747), C. tantalus (NC_009748), Colobus guereza (NC_006901), Nasalis larvatus (NC_008216), Presbytis melalophos (NC_008217), Procolobus badius (NC_008219), Pygathrix nemaeus (NC_008220), P. roxellana (NC_008218), Semnopithecus entellus (NC_008215), Trachypithecus obscurus (NC_006900), and Cebus albifrons (NC_002763). The C. albifrons sequence was used as an outgroup. Maximum parsimony (MP) analyses were performed using PAUP⁎4.0b10 (Swofford, 2003). A consensus tree was made for the equally-weighted MP analysis using the majority rule. We assessed the reliability of clades in phylogenetic trees by bootstrap probabilities (BSP) computed using 1000 replicates with 20 random additional sequencing replicates for each bootstrap replicate. Bayesian analysis was conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Bayesian posterior probabilities (BPP) used models estimated with Modeltest 3.7 (Posada and Crandall, 1998) under the Akaike Information Criterion (AIC), performing two separate runs with four Markov chains. Each run was conducted with 200 million generations and sampled every 100 generations. We discarded the first 20% of the trees as part of a burn-in procedure, and used the remaining 16,001 sampling trees to construct a 50% majority rule consensus tree. Molecular dating for the numt origin was estimated from the overall divergence (δ) between the numt and M. thibetana mitochondrial homologue, applying the equation δ = (λN + λC)T (Li et al., 1981), where λC = 2.5 × 10− 8 substitutions/sites/year for mitochondrial DNA (Hasegawa et al., 1985) and λN = 2.0 × 10− 9 substitutions/sites/year for the rate of neutral substitution of human genomes (Kumar and Subramanian, 2002) or λN = 4.7 × 10− 9 substitutions/sites/year for human pseudogenes (Li et al., 1981). 3. Results and discussion 3.1. Structure of the mitochondrial genome The complete mitochondrial genome of M. thibetana was sequenced and it is 16,540 bp including 13 protein-coding genes, two rRNA genes (12S rRNA, 16S rRNA), 22 tRNA genes, and a control region. It was deposited in GenBank under accession number EU294187. The gene organization in line with M. mulatta, represented as the H-strand with the first nucleotide in the middle of the D-loop region, is shown in Table 2. It is obvious that the gene distributions are the same as those of other mammalian mitochondrial genomes (Anderson et al., 1981, 1982; Hiendleder et al., 1998; Lin et al., 1999) and that most genes are encoded on the H strand, except for the ND6 gene and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Pro, tRNA-Glu), which are encoded on the L strand. The overall base composition of the H strand is as follows: 31.70% A, 25.10% T, 30.23% C, and 12.97% G.
D. Li et al. / Gene 429 (2009) 31–36 Table 2 Characteristics of the M. thibetana mitochondrial genome Gene
Control region tRNA-Phe 12s-rRNA tRNA-Val 16s-rRNA tRNA-Leu(UUR) ND1 tRNA-Ile tRNA-Gln tRNA-Met ND2 tRNA-Trp tRNA-Ala tRNA-Asn OL tRNA-Cys tRNA-Tyr COI tRNA-Ser(UCN) tRNA-Asp COII tRNA-Lys ATP8 ATP6 COIII tRNA-Gly ND3 tRNA-Arg ND4L ND4 tRNA-His tRNA-Ser(AGY) tRNA-Leu(CUN) ND5 ND6 tRNA-Glu CYTB tRNA-Thr tRNA-Pro
Position
Size (bp)
From
To
15992,1 522 595 1542 1611 3171 3248 4203 4269 4342 4410 5452 5526 5596 5670 5701 5769 5839 7380 7452 7521 8270 8334 8495 9175 9959 10,027 10,373 10,438 10,728 12,106 12,175 12,234 12,305 14,117 14,645 14,718 15,859 15,924
16540, 542 594 1541 1610 3170 3245 4204 4271 4340 4409 5453 5518 5594 5668 5702 5763 5834 7377 7448 7519 8204 8334 8540 9175 9959 10,026 10,372 10,437 10,734 12,105 12,174 12,233 12,304 14,116 14,644 14,713 15,858 15,922 15,991
1091 73 947 69 1560 75 957 69 72 68 1044 67 69 73 63 71 1540 69 68 684 65 207 681 785 68 346 65 297 1378 69 59 71 1812 528 49 1141 64 68
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Table 3 Codon usage in the M. thibetana mitochondrial genome Strand
H H H H H H H H L H H H L L L L L H L H H H H H H H H H H H H H H H L L H H L
Codon Start
Stop
GTG
TAG
ATT
TAG
ATG
?
ATG
TAA
GTG ATG ATG
TAA TAA TA
ATC
T
ATG ATG
TAA T
ATA ATG
TAA AGG
ATG
T
Codon
Count
Codon
Count
Codon
Count
Codon
Count
UUU UUC Phe UUA UUG CUU CUC CUA Leu CUG AUU AUC Ile AUA Met AUG GUU GUC GUA Val GUG
66 136 65 9 72 140 286 41 119 188 181 24 30 32 59 4
UCU UCC UCA UCG CCU CCC CCA Pro CCG ACU ACC ACA Thr ACG GCU GCC GCA Ala GCG
48 70 73 10 48 100 64 8 61 136 163 11 48 118 73 5
UAU UAC Tyr UAA UAG CAU CAC His CAA Gln CAG AAU AAC Asn AAA Lys AAG GAU GAC Asp GAA Glu GAG
44 80 0 0 28 68 90 8 34 131 87 3 18 43 63 11
UGU UGC Cys UGA Trp UGG CGU CGC CGA Arg CGG AGU AGC Ser AGA AGG GGU GGC GGA Gly GGG
3 22 84 13 7 26 27 2 8 45 0 0 18 81 52 26
3.3. Sequence features of the ribosomal and transfer RNA genes The 12S and 16S rRNA genes of M. thibetana are 947 and 1560 bp, respectively. They are located between the tRNA-Phe and tRNA-Leu (UUR) genes, and are separated by the tRNA-Val gene with the same situation found in other vertebrates (Inoue et al., 2000). The complete mitochondrial sequence contains 22 tRNA genes, which are interspersed in the genome and range in size from 49–75 bp. Only one long 67 bp spacer between COII and tRNA-Lys was found. Twenty tRNA genes have the typical cloverleaf secondary structure. tRNA-Ser (AGY) and tRNA-Cys which were not found by the tRNAscan-SE, were identified by comparison with M. mulatta counterparts. The structure
3.2. Sequence features of the protein-coding genes In the 13 protein-coding genes, eight use ATG as the start codon while ND1 and ATP8 use GTG (Table 2). ND2, ND3 and ND5 start with ATT, ATC, and ATA, respectively. In fact, ATG and ATA are commonly used as initiation codons in mammals (Xu and Arnason, 1994). With regards to stop codons (TAA, TAG, AGG), 5 genes (COII, ATP8, ATP6, ND4L, ND5) use the complete stop codon TAA, whereas 4 genes have incomplete stop codons, TA_ (COIII) and T_ (ND3, ND4, CYTB), which were presumably completed as TAA by posttranscriptional polyadenylation (Anderson et al., 1981). The COI gene stop codon was not determined, as was the case in 7 sequenced Cercopithecinae species: M. mulatta (NC_005943), M. sylvanus (NC_002764), Chlorocebus aethiops (NC_007009), C. sabaeus (NC_008066), Papio hamadryas (Y18001), C. pygerythrus (NC_009747), and C. tantalus (NC_009748). Codon usage in the M. thibetana mitochondrial genome indicated that CTA (Leu), ATC (Ile) and ATA (Met) occur at a higher frequency than others (Table 3). G is found in the lowest proportion (4.9%) at the third codon position in the 12 H-strand protein-coding genes, as compared to 37.9% A, 39.3% C and 17.9% T, which is similar to other findings in vertebrate mitochondrial genomes (Lopez et al., 1996; Janke and Arnason, 1997; Janke et al., 2001; Zhang et al., 2003). Two pairs of protein-coding genes (ATP8 and ATP6, ND4L and ND4), both located on the H-strand, overlapped in readingframes for 46 and 7 bp. Only 1 bp overlap was found in the ATP6 and COIII genes.
Fig. 1. Phylogenetic tree of the numt and its homologues as inferred from the nucleotide sequences of the concatenated ND2 gene and the 3′ end of the ND1 gene using Bayesian analysis. The numbers beside the nodes are Bayesian posterior probabilities.
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D. Li et al. / Gene 429 (2009) 31–36
of the tRNA-Ser (AGY) gene was analyzed by RNAstructure, version 4.5 (http://rna.urmc.rochester.edu/rnastructure.html) and it was found that the complete dihydrouridine arm (D-arm) was lacking. Aberrant tRNA can also fit the ribosome by adjusting its structural conformation and function in a similar way to that of usual tRNAs in the ribosome (Ohtsuki et al., 2002). 3.4. Sequence features of the non-coding regions The origin of L-strand replication (OL) in M. thibetana is 33 bp and located in a cluster of five tRNA genes (WANCY region: tRNA-Trp, tRNA-Ala, tRNA-Asn, tRNA-Cys, and tRNA-Tyr). We found that it can fold into a stable stem–loop secondary structure with 10 bp in the stem and 13 bp in the loop. This region overlaps the tRNA-Cys gene by 2 bp. The M. thibetana mtDNA control region spans 1091 bp and is located between the tRNA-Pro and tRNA-Phe genes. Within this sequence, we identified conserved sequence blocks (CSB) and extended termination associated sequences (ETAS) based on the description by Sbisa et al. (1997). CSB-1, CSB-2 and CSB-3 were located in positions 212–236, 297–312, and 343–360, respectively. ETAS1 and ETAS2 were close to the 5′ end of the control region, at positions 16046–16102 and 16264–16323, respectively. 3.5. Characterization of the numt sequence and the time of nuclear integration A roughly 2.0 kb fragment was amplified using primer pair X2.3 from the total genomic DNA of M. thibetana and double peaks were observed while sequencing. Regular peaks were obtained when the
fragment was then cloned into the vector and sequenced again. It is 2003 bp in length; however the sequences along both sides didn't overlap completely with neighboring mitochondrial sequences while the sequence amplified using primer Xm2.3 was in good conjunction with the neighboring sequences on both sides. The protein coding regions of these two sequences could be correctly translated into amino acids based on mammalian mitochondrial codes. Therefore, we concluded that a nuclear mitochondrial pseudogene (numt) was obtained and the sequence was deposited in GenBank under accession number EU340344. BLAST search for the numt sequence in GenBank showed that this sequence matched the mitochondrial genomes of primates. There are 3 homologues in the M. mulatta nuclear genome and they share 83.2%, 82.5% and 81.4% similarity. It matches with the 3′ end (717 bp) of the ND1 gene, the tRNAIle-tRNAGln-tRNAMet genes, the ND2 gene, and the 5′ end (37 bp) of the tRNATrp gene. It covers 12.11% of the complete M. thibetana mitochondrial genome and shares 90.6% sequence identity with its mitochondrial homologue. The ratio of transitions to transversions in these two sequences was 12.5. The base composition of the mitochondrial fragment is 24.3% T, 33.0% C, 32.3% A, and 10.4% G, as compared to 25.2% T, 32.1% C, 32.4% A, and 10.4% G in the numt. In order to estimate when the mtDNA fragment translocated into the nuclear DNA, sequences from the protein-coding region of the numt and its homologues from 16 Cercopithecidae species (Old World monkeys) and a Cebidae species, C. albifrons (a New World monkey, which is regarded as an outgroup), were used to reconstruct their phylogenetic relationship using the Bayesian analysis (Fig. 1). Using an overall divergence (δ) of 0.094 between the numt and the M. thibetana
Fig. 2. Phylogenetic tree of M. thibetana and 15 other Cercopithecidae species as inferred from the nucleotide sequences of 12 concatenated protein-coding genes. (A) Bayesian analysis. The numbers beside the nodes are Bayesian posterior probabilities. (B) Maximum parsimony (MP) method in PAUP⁎4.0b10 analysis. Percentage bootstrap values are shown on interior branches using 1000 replicates with 20 random additional sequencing replicates.
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mitochondrial homologue, we estimated that the numt translocated into the nuclear genome around 3.16 ∼ 3.48 million years ago (MYA), just before the divergence of the macaques about 3 MYA or later (Hayasaka et al., 1996). 3.6. Phylogenetic analyses Similar phylogenetic trees were obtained when Bayesian and MP methods were applied based on the 12 protein-coding genes of the 16 Cercopithecidae species (Fig. 2). The results support the traditional classification scheme, i.e., extant Old World monkeys can be divided into two ecologically and morphologically distinct subfamilies: Cercopithecinae (cheek-pouched monkeys) and Colobinae (leaf-eating monkeys) (Delson, 1992; Disotell, 2003; Groves, 1993, 2001). There are two clades in Cercopithecinae and one clade consists of three macaques and P. hamadryas. In this clade, M. thibetana and M. mulatta formed a sister group to M. sylvanus, then grouped with P. hamadryas, which suggests that M. thibetana emerged later than P. hamadryas and M. sylvanus in Cercopithecinae. Another clade consists of 4 chlorocebus species. In Colobinae, the phylogenetic tree supports reciprocal monophyly of African (C. guereza, P. badius) and Asian (P. nemaeus, P. roxellana, N. larvatus, S. entellus, P. melalophos, T. obscurus) colobines, which lends more support to previous morphological (Napier and Napier, 1970; Szalay and Delson, 1979; Groves, 2001), chromosomal (Bigoni et al., 2003, 2004), and molecular studies (Messier and Stewart, 1997; Zhang and Ryder, 1998; Page et al., 1999; Xing et al., 2005). Asian colobines were divided into three clades, an odd-nosed clade (N. larvatus, P. nemaeus and P. roxellana), a langur clade (P. melalophos and T. obscurus) and the S. entellus clade. Except for the S. entellus clade, this result is congruent with previous phylogenetic studies (Groves, 1970; Jablonski and Peng, 1993; Jablonski, 1998). Acknowledgments We thank Mr. Yao Fang for collecting the M. thibetana blood sample during the health exams. This research was funded by the National Basic Research Program of China (973 Project: 2007CB411605) and the “985 project” of the Central University of Nationalities. References Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H.L., Coulson, A.R., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J.H., Staden, R., Young, I.G., 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457–464. Anderson, S., de Bruijn, M.H., Coulson, A.R., Eperon, I.C., Sanger, F., Young, I.G., 1982. Complete sequence of bovine mitochondrial DNA: conserved features of the mammalian mitochondrial genome. J. Mol. Biol. 156, 683–717. Bensasson, D., Zhang, D.X., Hartl, D.L., Hewitt, G.M., 2001. Mitochondrial pseudogenes: evolution's misplaced witnesses. Trends Ecol. Evol. 16, 314–321. Bigoni, F., Houck, M.L., Ryder, O.A., Wienberg, J., Stanyon, R., 2004. Chromosome painting shows that Pygathrix nemaeus has the most basal karyotype among Asian colobines. Int. J. Primatol. 25, 679–688. Bigoni, F., Stanyon, R., Wimmer, R., Schempp, W., 2003. Chromosome painting shows that the proboscis monkey (Nasalis larvatus) has a derived karyotype and is phylogenetically nested within Asian colobines. Am. J. Primatol. 60, 85–93. Delson, E., 1992. Evolution of old word monkeys. In: Johns, J.S., Martin, R.D., Pilbeam, D., Bunney, S. (Eds.), The Cambridge Encyclopedia of Human Evolution. Cambridge University Press, Cambridge, pp. 217–222. Disotell, T.R., 2003. Primates: Phylogenetics. Encyclopedia of the Human Genome. Nature Publishing Group, London. Flynn, J.J., Nedbal, M.A., 1998. Phylogeny of the Carnivora (Mammalia): congruence vs incompatibility among multiple data sets. Mol. Phylogenet. Evol. 9, 414–426. Flynn, J.J., Nedbal, M.A., Dragoo, J.W., Honeycutt, R.L., 2000. Whence the red panda? Mol. Phylogenet. Evol. 17, 190–199. Fooden, J., 1976. Provisional classification and key to living species of macaques (primates: Macaca). Folia Primatol 25, 225–236. Groves, C., 1970. The forgotten leaf-eaters, and the phylogeny of the Colobinae. In: Napier, J.R., Napier, P.H. (Eds.), Old World Monkeys: Evolution, Systematics and Behavior. Academic Press, New York, pp. 555–587. Groves, C., 2001. Primate Taxonomy. Pages Smithsonian Press, Washington, DC.
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