Abstract
We analyzed Clonorchis sinensis ancient DNA (aDNA) acquired from the specimens of the Joseon mummies. The target regions were cytochrome C oxidase subunit 1 (CO1), internal transcribed spacer 1 (ITS1), nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase subunits 2 (NAD2) and 5 (NAD5). The sequences of C. sinensis aDNA was completely or almost identical to modern C. sinensis sequences in GenBank. We also found that ITS1, NAD2 and NAD5 could be good markers for molecular diagnosis between C. sinensis and the other trematode parasite species. The current result could improve our knowledge about genetic history of C. sinensis.
Keywords: Clonorchis sinensis, Ancient DNA, Phylogenetic Analysis, Mummies, Republic of Korea
Graphical Abstract
Clonorchis sinensis infects approximately 35 million people worldwide, causing various subclinical or clinical signs known as clonorchiasis.1,2,3,4,5 People are infected by ingestion of undercooked or raw freshwater fish harboring metacercariae of C. sinensis.6,7,8 In a historical context, C. sinensis infection was one of the most common trematode infections in Korea, especially due to the cuisine based on raw fish, which was enjoyed by the inhabitants of the country.9,10,11
To reveal the genetic characteristics of C. sinensis, researchers have attempted DNA analysis. Recently, parasitologists diagnosed C. sinensis through DNA analysis on internal transcribed spacer (ITS),12,13,14,15 cytochrome C oxidase subunit (CO),5,10 and nicotinamide adenine dinucleotide hydrogen dehydrogenase (NAD) subunits.5,12 The molecular analyses claimed that C. sinensis is genetically distinct from other trematodes.1,12,13,14,15,16
Meanwhile, paleoparasitologists have also tried to reveal the genetic characteristics of ancient C. sinensis through research on the samples collected at archeological sites. One of such studies was carried out in Korea. Shin et al.17 successfully analyzed ancient DNA (aDNA) sequences of C. sinensis eggs collected from the 17th century Korean mummy feces. They showed that amplified sequences of C. sinensis ITS1, ITS2 and CO1 were completely identical to modern C. sinensis sequences in GenBank.17
Although this pioneering work was to reveal genetic traits of ancient C. sinensis, the number of aDNA cases reported so far was too insufficient to get detailed information of ancient C. sinensis genetics. Fortunately, by paleoparasitological studies in Korea over the past several years, we collected a number of pre-modern Korean mummy feces or tissue specimens in which the presence of ancient Clonorchis eggs was microscopically confirmed.18,19 Utilizing the ancient specimens, in this study, we analyzed CO1, ITS1, NAD2 and NAD5 of C. sinensis aDNA. The current report could expand the spatiotemporal scope of parasitological research about the genetic history of C. sinensis.
The samples used in this study were obtained from the 16th to 17th century Joseon mummies (n = 5; Andong, Cheongdo, Dalsung, Hadong1 and Mungyeong) (Table 1, Supplementary Figs. 1 and 2). The specimens were coprolites retrieved from mummy intestines (Andong, Dalsung, and Hadong1) or mummified livers (Cheongdo and Mungyeong). We followed the Criteria of Authentication for authentic aDNA analysis.20
Table 1. The archaeological information of Korean mummies in this study.
| Cases | Estimated date, century | Sample condition | Sample type | Gender |
|---|---|---|---|---|
| Andong | 16 | Mummy | Coprolites | M |
| Cheongdo | 17 | Mummy | Mummified liver | M |
| Dalsung | 16–17 | Mummy | Coprolites | W |
| Hadong1 | 17 | Half mummified | Coprolites | W |
| Mungyeong | 17 | Mummy | Mummified liver | W |
M = men, W = women.
For aDNA extraction, we followed the method in our previous report.21 The specimens (0.3 g) were treated in a lysis buffer (1 mL) for 24 hours at 56°C. DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and then chloroform/isoamyl alcohol (24:1). DNA isolation/purification was performed by a QIAmp PCR purification kit (Qiagen, Hilden, Germany). Extract DNA (10 μL) was treated with 1 unit of uracil-DNA-glycosylase (New England Biolabs, Ipswich, MA, USA) for 30 minutes at 37°C. It (40 ng) was then mixed with a reagent premix containing 10 pmol of each primer (Table 2) and 1X AmpliTaq Gold® 360 Master Mix (Life Technologies, Camarillo, CA, USA). PCR conditions were as follows: pre-denaturation at 95°C for 10 minutes; 45 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C–63°C for 30 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 10 minutes. The amplified PCR products separated on 2.5% agarose gel (Invitrogen, Waltham, MA, USA) were stained by ethidium bromide. Electrophoresis also included negative (extraction) controls.
Table 2. List of primers used for the amplification of C. sinensis DNA in this study.
| DNA region | Set | Primers | Sequence, 5′ to 3′ | Annealing Temp., °C | Length, bp |
|---|---|---|---|---|---|
| CO1 | CO1 | CO1 F | GTG TTA ATA TTG CCG GGG TTT GG | 54 | 207 |
| CO1 R | ACC TAT AAT CAT AGT AAC CG | ||||
| ITS1 | ITS1-2 | ITS1 F2 | CTG GCA CGT GTA CCC AAT A | 56 | 122 |
| ITS1 R2 | TCA CCC CCA ATA TGG ACT | ||||
| ITS1-3 | ITS1 F3 | TCG GTA TGC TCG CTT CCG TTG | 62 | 151 | |
| ITS1 R3 | CGG TTT GAA ATG AAC AAC AA | ||||
| ITS1-4 | ITS1 F4 | GAG TGG GCA TGA TGT GTC TC | 63 | 215 | |
| ITS1 R4 | GGC GTT ATC AGT CGT ACC CGG | ||||
| NAD2 | NAD2-1 | NAD2 F1 | GCT ATG TTG TTG TTT CTG GTG | 56 | 194 |
| NAD2 R1 | ACG ACC TCT TCA AAA TGG TT | ||||
| NAD2-2 | NAD2 F2 | TGA AGT TTG GTC TTT TTC CA | 54 | 260 | |
| NAD2 R2 | TGA TGC ACT GGA ACT AAT CA | ||||
| NAD2-3 | NAD2 F3 | TGG GGG TTT AAC GTT TAT TT | 56 | 195 | |
| NAD2 R3 | CTC AGC AAC ATA ACC ACC AT | ||||
| NAD2-4 | NAD2 F4 | GAG CTT TCT CCT GAT TTG CT | 56 | 164 | |
| NAD2 R4 | ATG GAT AAA GAC CCT GGA AA | ||||
| NAD2-5 | NAD2 F5 | CCG CAG TTG GGA TAT ATT TT | 54 | 159 | |
| NAD2 R5 | ATA AAA CTG CTC CGA AAT GC | ||||
| NAD5 | NAD5-1 | NAD5 F1 | GAT GCC GTC CTT GAT ATT TT | 54 | 164 |
| NAD5 R1 | CCC AAT TCT GAA AAT GAC CT | ||||
| NAD5-2 | NAD5 F2 | TGC TAA ACC TCG GAG TAT GC | 58 | 191 | |
| NAD5 R2 | CCA CCA ACC AGG AAA TAA AT | ||||
| NAD5-3 | NAD5 F3 | CAG AAT TGG GTT GGT ATG TG | 56 | 200 | |
| NAD5 R3 | CCC CTG ATA GCA GAA TAA CG | ||||
| NAD5-4 | NAD5 F4 | CCC CAG TTA GTT GTT TGG TT | 56 | 211 | |
| NAD5 R4 | GCA ACA TTT TTG CAG GTA GA |
CO = cytochrome C oxidase subunit, ITS = internal transcribed spacer, NAD = nicotinamide adenine dinucleotide hydrogen dehydrogenase subunits.
The PCR amplicon was isolated by a QIAquick Gel Extraction Kit (Qiagen). Bacterial transformation was done using a pGEM-T Easy Vector system (Promega Corporation, Madison, WI, USA). Transformed bacteria were then grown on agar plate containing X-GAL (40 μg/μL), ampicillin (50 μg/mL) and 0.5 mM IPTG for 14 hours. After colonies were grown in LB media for 12 hours, the cultured bacteria were purified by a QIAprep® Spin Miniprep kit (Qiagen). Each amplified DNA strand was sequenced by an ABI Prism BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Waltham, MA, USA) and 3730xl Automatic Sequencer (Applied Biosystems).
To obtain consensus sequence, multiple sequence alignment was performed for each aDNA region by Clustal W implemented in MEGA7.22 We compared the consensus sequences of ancient C. sinensis to GenBank taxa by NCBI/BLAST tools.23 The evolutionary relationship of ancient C. sinensis and other parasites of NCBI GenBank was inferred by the Phylogeny Reconstruction analysis implemented in MEGA7. We used Maximum Likelihood method. Selected parameters are Tamura 3-parameter24 (CO1), Kimura 2-parameter25 (ITS1), Hasegawa-Kishino-Yano model26 (NAD2 and 5) for Model/Method. We performed bootstrap test to estimate the reliability of the tree. The number of bootstrap replicates was 500.27
To select the specimens used for aDNA analysis, we screened all the mummy coprolite samples using PCR with C. sinensis primers for CO1 (206 bp), ITS1-2 (122 bp), NAD2-1 (194 bp) and NAD5-1 (164 bp). In agarose gel electrophoresis, negative (extraction) controls exhibited no amplified bands. In Andong feces, the PCR products were detected for C. sinensis CO1, ITS1-2, NAD2-1 and NAD5-1. Mungyeong specimen also showed positive results for C. sinensis CO1 and ITS1-2 (Supplementary Fig. 3). We thus used the Andong and Mungyeong specimens for subsequent aDNA analysis.
To get the consensus aDNA sequences of C. sinensis CO1, ITS1, NAD2 and NAD5, we tried to do cloning and sequencing of each specific amplicon. By these trials, 9–10 clone sequences were successfully acquired for CO1, ITS1, NAD2 and NAD5 amplicons (Supplementary Fig. 4). The total sizes of consensus sequences obtained by multiple sequence alignment were 162 bp (CO1), 431 bp (ITS1), 588 bp (NAD2) and 443 bp (NAD5), respectively. The C. sinensis consensus sequences of Andong and Mungyeong specimens were almost the same to each other, except for a little difference at a nucleotide position (transversions occurred in the positions CO1: 100 and ITS1: 167) (Fig. 1). Considering these results, we conjecture that genetic characteristics of ancient C. sinensis might not have been uniform during the Joseon period.
Fig. 1. BLAST analyses of the consensus DNA sequences from ancient C. sinensis and other sequences in GenBank. (A) CO1, (B) ITS1, (C) NAD2, and (D) NAD5 DNA regions.
CO = cytochrome C oxidase subunit, ITS = internal transcribed spacer, NAD = nicotinamide adenine dinucleotide hydrogen dehydrogenase subunits.
In BLAST searching, the C. sinensis consensus sequences of Andong and Mungyeong specimens were completely or almost identical to C. sinensis CO1, ITS1, NAD2 and NAD 5 sequences reported in GenBank (Table 3 and Fig. 1). Briefly, the current ancient C. sinensis CO1 sequences were 100% identical to GenBank sequences of C. sinensis reported from Korea (KY564177.1), China (FJ965391.1; FJ965383.1; AF188122.2; AF184619.2), Russia (MF406205.1; MF406204.1), and Vietnam (MF287785.1; KJ204609.1). C. sinensis ITS1 sequences of Korean mummies also exhibited very high similarities (99%) to the GenBank ITS1 sequences reported from Korea (JN638318.1; JN638320.1), China (KJ137226.1; KF740425.1; HQ186255), Russia (JQ048578.1; KC987517.1) and Vietnam (MF319655.1; MF319650.1; MF319653.1). The aDNA sequences of Andong and Mungyeong specimens were also completely or almost (99%) identical to GenBank C. sinensis NAD2 and NAD sequences from Korea (NAD2, JF729304.1; NAD5, FJ729304.1), China (NAD2, KC170192.1; NAD5, KY564177.1), Russia (NAD2, FJ381664.2; NAD5, FJ381664.1) and Vietnam (NAD2, AY264851.1) (Table 3 and Fig. 1).
Table 3. BLAST searching results of ancient C. sinensis CO1, ITS1, NAD2, and NAD5 consensus sequences obtained from Andong mummy.
| DNA region | Species | Coverage, % | Percent identity, % | Accession No. | Geographical region |
|---|---|---|---|---|---|
| CO1 | C. sinensis | 100 | 100 | KY564177.1 | Korea |
| 100 | 100 | MF287785.1 | Vietnam | ||
| 100 | 100 | MF406205.1 | Russia | ||
| 100 | 100 | FJ965391.1 | China | ||
| 100 | 100 | EF688130.1 | Japan | ||
| 100 | 99 | FJ654383.1 | China | ||
| 100 | 99 | KJ204609.1 | Vietnam | ||
| 100 | 99 | JX040566.1 | Russia | ||
| 100 | 99 | JF729304.1 | Korea | ||
| 100 | 98 | MF406206.1 | Russia | ||
| 100 | 97 | AF188122.2 | China | ||
| 100 | 96 | AF184619.2 | China | ||
| P. summa | 100 | 100 | AF181884.3 | Korea | |
| T. spiralis | 98 | 100 | AF182302.1 | Unknown | |
| O. viverrini | 100 | 95 | AY055380.1 | Laos | |
| M. xanthosomus | 96 | 93 | FJ423740.1 | Unknown | |
| ITS1 | C. sinensis | 100 | 100 | JN638318.1 | Korea |
| 100 | 100 | MF319655.1 | Vietnam | ||
| 100 | 100 | KJ137226.1 | China | ||
| 100 | 100 | JQ048578.1 | Russia | ||
| 100 | 99 | JN638320.1 | Korea | ||
| 100 | 99 | KF740425.1 | China | ||
| 100 | 99 | MF319650.1 | Vietnam | ||
| 100 | 99 | KC987517.1 | Russia | ||
| 100 | 98 | MF319653.1 | Vietnam | ||
| 100 | 98 | HQ186255.1 | China | ||
| M. bilis | 95 | 93 | KY356536.1 | Russia | |
| O. felineus | 95 | 93 | DQ456831.1 | Russia | |
| O. viverrini | 94 | 91 | KX378012.1 | Vietnam | |
| NAD2 | C. sinensis | 100 | 99 | JK729304.1 | Korea |
| 100 | 99 | KC170213.1 | China | ||
| 100 | 99 | FJ381664.2 | Russia | ||
| 100 | 98 | AY264851.1 | Vietnam | ||
| O. sudarikovi | 100 | 77 | MK033132.1 | Pakistan | |
| M. orientalis | 100 | 76 | KT239342.1 | China | |
| O. felineus | 100 | 76 | EU921260.2 | Russia | |
| NAD5 | C. sinensis | 100 | 100 | JF729304.1 | Korea |
| 100 | 99 | KY564177.1 | China | ||
| 100 | 99 | FJ381664.2 | Russia | ||
| O. sudarikovi | 99 | 81 | MK033132.1 | Pakistan | |
| M. orientalis | 99 | 81 | KT239342.1 | China | |
| O. felineus | 98 | 80 | EU921260.2 | Russia |
CO = cytochrome C oxidase subunit, ITS = internal transcribed spacer, NAD = nicotinamide adenine dinucleotide hydrogen dehydrogenase subunits.
In the analyses, we found that CO1 region could not be an effective marker for differential diagnosis between C. sinensis and other trematode species because the CO1 sequences of Pygidiopsis summa (AF184884.3) and Trichinella spiralis (AF182302.1) were not distinguishable from C. sinensis CO1 sequences. Meanwhile, C. sinensis ITS1, NAD2 and NAD5 sequences were clearly distinct from those of other trematode species (Fig. 1). We identified similar patterns in phylogenetic analyses (Fig. 2). In case of CO1, ancient Andong and Mungyeong sequences belonged to the clade not only with C. sinensis, but also with P. summa and M. xanthosomus. On the other hand, ITS1, NAD2 and NAD5 of C. sinensis and other trematode species were separately clustered into different clades (Fig. 2). Actually, previous studies proposed that the interspecific sequence variations within zoonotic trematodes were observed for ITS1, NAD2 and NAD5.13 In this study, we re-confirmed the usefulness of ITS1, NAD2 and NAD5 as molecular markers for differential diagnosis of C. sinensis from other trematode species.
Fig. 2. Phylogenetic analyses (Maximum Likelihood method) of the current ancient C. sinensis (red dots) and the other trematodes in GenBank. (A) CO1, (B) ITS1, (C) NAD2, and (D) NAD5 DNA regions. Blue dots, ancient C. sinensis sequence previously reported by Shin et al.17.
CO = cytochrome C oxidase subunit, ITS = internal transcribed spacer, NAD = nicotinamide adenine dinucleotide hydrogen dehydrogenase subunits.
In summary, our present study about C. sinensis aDNA retrieved from Korean mummies is designed to uncover invaluable genetic information of C. sinensis prevalent among pre-20th century Korean people. Although detailed understanding of C. sinensis genetics require a future retrieval of ancient or modern DNA sequences in wider geo-historical scope, our current report represent a significant step to improve our knowledge about genetic history of C. sinensis.
Footnotes
Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No. NRF-2016R1A2B4015669).
Disclosure: The authors have no potential conflicts of interest to disclose.
- Conceptualization: Shin DH, Seo M.
- Data curation: Hong JH, Seo M, Shin DH.
- Investigation: Hong JH, Oh CS, Seo M.
- Writing - original draft: Hong JH, Seo M, Shin DH.
- Writing - review & editing: Hong JH, Chai JY, Seo M, Shin DH.
SUPPLEMENTARY MATERIALS
The map of Korea. Red dots represent the sites where the mummies of the current studies were found. 1 = Andong, 2 = Cheongdo, 3 = Dalsung, 4 = Hadong1, 5 = Mungyeong.
The archaeological information of Korean mummy specimens used for this study. (A) The tomb of Joseon period. (B) A mummy (Andong) used in this study.
Agarose gel electrophoresis of the PCR products amplified from ancient C. sinensis samples. Specific bands were indicated by arrows.
Aligned clone sequences of CO1, ITS1, NAD2 and 5 DNA fragments from Joseon Dynasty mummies. (A) Andong mummy, (B) Mungyeong mummy.
References
- 1.Park GM, Yong TS. Geographical variation of the liver fluke, Clonorchis sinensis, from Korea and China based on the karyotypes, zymodeme and DNA sequences. Southeast Asian J Trop Med Public Health. 2001;32(Suppl 2):12–16. [PubMed] [Google Scholar]
- 2.Kaewkes S. Taxonomy and biology of liver flukes. Acta Trop. 2003;88(3):177–186. doi: 10.1016/j.actatropica.2003.05.001. [DOI] [PubMed] [Google Scholar]
- 3.Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, et al. Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect Dis. 2005;5(1):31–41. doi: 10.1016/S1473-3099(04)01252-6. [DOI] [PubMed] [Google Scholar]
- 4.Keiser J, Utzinger J. Emerging foodborne trematodiasis. Emerg Infect Dis. 2005;11(10):1507–1514. doi: 10.3201/eid1110.050614. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Sun J, Huang Y, Huang H, Liang P, Wang X, Mao Q, et al. Low divergence of Clonorchis sinensis in China based on multilocus analysis. PLoS One. 2013;8(6):e67006. doi: 10.1371/journal.pone.0067006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kang SY, Ahn IY, Park CY, Chung YB, Hong ST, Kong Y, et al. Clonorchis sinensis: molecular cloning and characterization of 28-kDa glutathione S-transferase. Exp Parasitol. 2001;97(4):186–195. doi: 10.1006/expr.2001.4606. [DOI] [PubMed] [Google Scholar]
- 7.Yu SH, Masanori K, Li XM, Xu LQ, Lan CG, Lin R. Epidemiological investigation on Clonorchis sinensis in human population in an area of South China. Jpn J Infect Dis. 2003;56(4):168–171. [PubMed] [Google Scholar]
- 8.Lim JU, Joo KR, Shin HP, Cha JM, Lee JI, Lim SJ. Obstructive jaundice caused by Clonorchiasis-associated duodenal papillitis: a case report. J Korean Med Sci. 2011;26(1):135–137. doi: 10.3346/jkms.2011.26.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Seo M, Shin DH, Guk SM, Oh CS, Lee EJ, Shin MH, et al. Gymnophalloides seoi eggs from the stool of a 17th century female mummy found in Hadong, Republic of Korea. J Parasitol. 2008;94(2):467–472. doi: 10.1645/GE-1365.1. [DOI] [PubMed] [Google Scholar]
- 10.Ki HC, Shin DH, Seo M, Chai JY. Infection patterns of trematode parasites among Joseon people. J Korean Med Assoc. 2014;57(10):866–875. [Google Scholar]
- 11.Jeong YI, Shin HE, Lee SE, Cheun HI, Ju JW, Kim JY, et al. Prevalence of Clonorchis sinensis infection among residents along 5 major rivers in the Republic of Korea. Korean J Parasitol. 2016;54(2):215–219. doi: 10.3347/kjp.2016.54.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Lee SU, Huh S. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J Parasitol. 2004;42(3):145–148. doi: 10.3347/kjp.2004.42.3.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Xiao JY, Gao JF, Cai LS, Dai Y, Yang CJ, Luo L, et al. Genetic variation among Clonorchis sinensis isolates from different hosts and geographical locations revealed by sequence analysis of mitochondrial and ribosomal DNA regions. Mitochondrial DNA. 2013;24(5):559–564. doi: 10.3109/19401736.2013.770490. [DOI] [PubMed] [Google Scholar]
- 14.Tatonova YV, Chelomina GN, Nguyen HM. Inter-individual and intragenomic variations in the ITS region of Clonorchis sinensis (Trematoda: Opisthorchiidae) from Russia and Vietnam. Infect Genet Evol. 2017;55:350–357. doi: 10.1016/j.meegid.2017.10.008. [DOI] [PubMed] [Google Scholar]
- 15.Liu WQ, Liu J, Zhang JH, Long XC, Lei JH, Li YL. Comparison of ancient and modern Clonorchis sinensis based on ITS1 and ITS2 sequences. Acta Trop. 2007;101(2):91–94. doi: 10.1016/j.actatropica.2006.08.010. [DOI] [PubMed] [Google Scholar]
- 16.Park GM. Genetic comparison of liver flukes, Clonorchis sinensis and Opisthorchis viverrini, based on rDNA and mtDNA gene sequences. Parasitol Res. 2007;100(2):351–357. doi: 10.1007/s00436-006-0269-x. [DOI] [PubMed] [Google Scholar]
- 17.Shin DH, Oh CS, Lee HJ, Chai JY, Lee SJ, Hong DW, et al. Ancient DNA analysis on Clonorchis sinensis eggs remained in samples from medieval Korean mummy. J Archaeol Sci. 2013;40(1):211–216. [Google Scholar]
- 18.Seo M, Oh CS, Chai JY, Jeong MS, Hong SW, Seo YM, et al. The changing pattern of parasitic infection among Korean populations by paleoparasitological study of Joseon Dynasty mummies. J Parasitol. 2014;100(1):147–150. doi: 10.1645/12-60.1. [DOI] [PubMed] [Google Scholar]
- 19.Seo M, Oh CS, Hong JH, Chai JY, Cha SC, Bang Y, et al. Estimation of parasite infection prevalence of Joseon people by paleoparasitological data updates from the ancient feces of pre-modern Korean mummies. Anthropol Sci. 2017;125(1):9–14. [Google Scholar]
- 20.Hofreiter M, Serre D, Poinar HN, Kuch M, Pääbo S. Ancient DNA. Nat Rev Genet. 2001;2(5):353–359. doi: 10.1038/35072071. [DOI] [PubMed] [Google Scholar]
- 21.Kim YS, Oh CS, Lee SJ, Park JB, Kim MJ, Shin DH. Sex determination of Joseon people skeletons based on anatomical, cultural and molecular biological clues. Ann Anat. 2011;193(6):539–543. doi: 10.1016/j.aanat.2011.07.002. [DOI] [PubMed] [Google Scholar]
- 22.Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–1874. doi: 10.1093/molbev/msw054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Tamura K. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol Biol Evol. 1992;9(4):678–687. doi: 10.1093/oxfordjournals.molbev.a040752. [DOI] [PubMed] [Google Scholar]
- 25.Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980;16(2):111–120. doi: 10.1007/BF01731581. [DOI] [PubMed] [Google Scholar]
- 26.Hasegawa M, Kishino H, Yano T. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol. 1985;22(2):160–174. doi: 10.1007/BF02101694. [DOI] [PubMed] [Google Scholar]
- 27.Hall BG. Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol. 2013;30(5):1229–1235. doi: 10.1093/molbev/mst012. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
The map of Korea. Red dots represent the sites where the mummies of the current studies were found. 1 = Andong, 2 = Cheongdo, 3 = Dalsung, 4 = Hadong1, 5 = Mungyeong.
The archaeological information of Korean mummy specimens used for this study. (A) The tomb of Joseon period. (B) A mummy (Andong) used in this study.
Agarose gel electrophoresis of the PCR products amplified from ancient C. sinensis samples. Specific bands were indicated by arrows.
Aligned clone sequences of CO1, ITS1, NAD2 and 5 DNA fragments from Joseon Dynasty mummies. (A) Andong mummy, (B) Mungyeong mummy.