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Mitochondrial DNA B Resour
Mitochondrial DNA B Resour
Mitochondrial DNA. Part B, Resources
2380-2359
Taylor & Francis

10.1080/23802359.2024.2403407
2403407
Version of Record
Data Note
Mitogenome Report
The ancient mitochondrial genome of a Siberian roe deer (Capreolus pygargus) from Jartai Pass Site in Xinjiang, China, and its phylogenetic relationships
G. Song et al.
Song Guangjie a
Wang Yongqiang b
Ruan Qiurong b
https://orcid.org/0000-0001-6650-0217
Cai Daw ei a
a Bioarchaeology Laboratory, Jilin University, Changchun, China
b Xinjiang Institute of Culture Relics and Archaeology, Urumqi, China
Supplemental data for this article can be accessed online at https://doi.org/10.1080/23802359.2024.2403407.

CONTACT Dawei Cai caidw@jlu.edu.cn Bioarchaeology Laboratory, Jilin University, Changchun, China.
16 9 2024
2024
16 9 2024
9 9 12321236
10 4 2024
7 9 2024
KnowledgeWorks Global Ltd.16 9 2024
published online in a building issue16 9 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

The ancient mitochondrial genome of a Siberian roe deer (Capreolus pygargus) coded as NJ26S from Jartai Pass Site was obtained by high throughput sequencing. The damage pattern demonstrated the authenticity and reliability of the ancient DNA data. The length of the mitogenome was 16,357 bp, which contained 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one control region. The total base composition of the mitochondrial genome is 28.17% A, 25.01% T, 11.89% G, 19.72% C, and 15.21% missing data with an AT composition of 53.18%. A maximum-likelihood phylogenetic tree was recovered including other roe deer sequences under the TIM2 + I + G4 model. This study presents molecular evidence indicating the presence of Capreolus pygargus in the Xinjiang Uygur Autonomous Region in China more than 3,000 years ago.

Keywords

Siberian roe deer (Capreolus pygargus)
China
Jartai Pass Site
phylogenetic analysis
high throughput sequencing
No funding was received.
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pmcIntroduction

The genus Capreolus (Cervidae:Capreolinae) comprises two species, the Siberian roe deer (C. pygargus) and the western roe deer (C. capreolus). (Sokolov and Gromov 1990; Hewison and Danilkin 2001). The Siberian roe deer is typically found in broad-leaved forests or mixed coniferous and broad-leaved forests, and it often prefers habitats with sparse and mostly deciduous trees in rugged mountainous regions (Wang et al. 2017). Its distribution range is extensive, with the species mainly inhabiting areas in China, Kazakhstan, South Korea, North Korea, Mongolia, and Russia (Wilson and Reeder 1993; Randi et al. 1998). In China, the Siberian roe deer is found in Inner Mongolia, Heilongjiang, Jilin, Liaoning, Hebei, Shandong, Henan, Shanxi, Shaanxi, Xinjiang, Qinghai, Sichuan, and Gansu, and it is classified as a second-class protected animal nationwide (An et al. 2020). Additionally, the Siberian roe deer is listed as Least Concern (LC) in the Red List of Threatened Species by the International Union for the Conservation of Nature (IUCN) (Lovari et al. 2016).

Jartai Pass Site (43°84’N, 82°77’E) is an Andronovo culture settlement site dating back to about 3,600 years ago, located in the east Nilek County, Ili Kazak Autonomous Prefecture, Xinjiang Uygur Autonomous Region (Wang et al. 2017) (see Figure 1A). This site has provided important data for studying the Bronze Age culture and settlement form, including evidence of hunting habits, like numerous roe deer skeletal remains.

Figure 1. (A) Location of the Jartai Pass Site; (B) Ancient skeletal remains analyzed in this study. Photographed by Guangjie Song.

In this study, we collected remains of an ancient Siberian roe deer skeleton from the Jartai Pass Site (see Figure 1B) and conducted mitochondrial genome sequencing. Additionally, we compared the structural composition of this ancient genome with modern sequences and constructed a phylogenetic tree.

Materials and methods

The metatarsal bone from ancient Siberian roe deer was deposited at Jilin University (www.jlu.edu.cn, contact person: Dawei Cai, Email: caidw@jlu.edu.cn) and was labeled with archaeological ID M48(T4) and lab code NJ26S. The samples were prepared by using an electric grinding tool and sterilized disposable drills to remove the 2 mm external surface of the bone. After cleaning, the samples were treated with a 10% chloramine solution, followed by decontamination with DEPC water and a 5-minute immersion in 70% ethanol. Subsequently, the samples were dried under UV light. The next day, the bones were ground into powder using an electric grinding tool, and 200 mg of each sample was collected. For DNA extraction, the method described by Yang et al. (1998) was followed using the QIAquick® PCR Purification Kit and ultrafiltration tubes (Millipore) to obtain the extraction solution. This solution was then used to generate a genomic library (Max Planck Institute, https://www.protocols.io/view/a-z-of-ancient-dna-protocols-for-shotgun-illumina-36wgq529xgk5/v2/guidelines). The library preparation was carried out as follows: We added 3 μL of T4 PNK and 1.65 μL of T4 polymerase into the DNA extract, and then incubated it for 20 min at 25 °C followed by 10 min at 12 °C. Next, we purified the sample using the MinElute® PCR purification kit. Then, we combined 20 μL of quick ligase buffer, 1 μL of adapter mix, and 1 μL of quick ligase with the DNA and incubated it for 20 min at 22 °C. After that, we repeated the previous step. Following this, we added 0.2 μL of dNTPs, 2 μL of Bst polymerase, and 4 μL of buffer into the DNA and incubated it for 30 min at 37 °C followed by 10 min at 80 °C. Finally, we added 25 μL of Q5 and 1 μL of double-ended index primer into the DNA, and the mix was amplified in the following cycles: 14 times at 98 °C for 30 s, 98 °C for 10 s, and 60 °C for 75 s, and then a final step at 60 °C for 10 min.

After purifying the DNA library, an Illumina Hiseq X 150 bp paired-end Ten platform was used for paired-end reads sequencing at Novogene Inc, Beijing, China. The raw data were processed through the Paleomix pipeline (Schubert et al. 2014), with the Capreolus pygargus mitochondrial genome (GenBank accession number NC_025271.1) as reference sequence. The data underwent analysis using MapDamage v2.2.1 (Jónsson et al. 2013) to assess terminal damage. MapDamage identifies potential damage effects by analyzing the frequency of each base at different positions within the reads. In ancient DNA, certain base frequencies may deviate from expected patterns due to postmortem damage. Statistical methods tests are employed to assess whether observed base frequencies at each position significantly deviate from the frequencies expected by the model. MapDamage generates charts that illustrate patterns of damage and damage frequencies at various positions in the DNA sequence. A consensus FASTA-format sequence was generated using ANGSD (Korneliussen et al. 2014). The mitochondrial genome sequence was annotated using the Proksee online web server (https://proksee.ca/) (Grant et al. 2023). To obtain the phylogenetic relationship of our ancient sample with other roe deer, we analyzed a dataset that included the NJ26S sequence, 10 modern mitogenomes of C. capreolus, 14 modern mitogenomes and four ancient mitogenomes of C. pygargus, using the water deer (Hydropotes inermis) as an outgroup (Table S1). We obtained a maximum-likelihood (ML) phylogenetic tree using the RAxML-ng program (Kozlov et al. 2019) with 1,000 replications under an unpartitioned TIM2 + I + G4 model, which was determined using ModelTest-NG v0.1.6 (Darriba et al. 2020). Bayesian phylogenetic tree (BI) was constructed with BEAST v2.6.7 (Bouckaert et al. 2014) using a strict molecular clock model running 50,000,000 iterations of sampling every 5,000 iterations under an unpartitioned GTR + I + G model, which was determined using jModelTest 2.1.1 (Darriba et al. 2012) to carry out statistical selection of best-fit models of nucleotide substitution against the Bayesian Information Criterion (BIC). Tracer v1.7.2 (Rambaut et al. 2018) was adopted to test all effective sample sizes greater than 300. Neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 11 (Tamura et al. 2021) with 1,000 replications under an unpartitioned TN93 + I + G model. The phylogenetic trees were depicted using iTOL (Letunic and Bork 2021).

Results

The mitogenome sequence of sample NJ26S (GenBank accession number PP068269) displayed an average coverage per base of 18.6X (Figure S1). The sequencing data of ancient DNA samples typically show a higher frequency of GC nucleotides at the 3′ and 5′ ends (Briggs et al. 2007). Our analysis confirmed that the C. pygargus from the Jartai Pass Site exhibited the typical damage pattern found in ancient DNA (Figure S2), ensuring the authenticity and reliability of our results.

The total length of the ancient mitogenome was 16,357 bp, which contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes (12S rRNA and 16S rRNA), and one control region (D-loop region). The base composition of the mitochondrial genome is 28.17% A, 25.01% T, 11.89% G, 19.72% C, and 15.21% N with an AT proportion of 53.18%. All protein-coding genes, except for the NAD6 gene, are transcribed in the same direction as the H-strand. Additionally, 14 out of the 22 tRNA genes and the two ribosomal RNA genes are also encoded on the H-strand (Figure 2). ​The lengths of the control region, 12S rRNA, and 16S rRNA are 929 bp, 955 bp, and 1,567 bp, respectively. The 22 tRNA genes ranged from 60 to 75 bp in length, which were the same as in other Siberian roe deer. The PCG Nd2 and Nd5 start with ATA, and Nd4L begins with GTG. ATG is used as the start codon for Nd1, Nd6, Cox1-Cox3, Atp6, and Cytb. Incomplete stop codons are found in Nd1, Nd2, Nd3, Nd4, and Cox3, while complete stop codons are present in Atp8 (TAG), Cytb (AGA), Nd5, Nd6, Cox1, Cox2, and Atp6 (TAA).

Figure 2. Mitochondrial gene map of an ancient capreolus pygargus specimen from Jartai Pass Site, China (sample NJ26S; 16,357 bp).

The ML (Figure 3), BI (Figure S4), and NJ (Figure S5) trees showed identical topologies and support the monophyly of the two Capreolus species, and the presence of three subclades within C. pygargus. Consistent with previous research (Deng et al. 2022), the European and Siberian roe deer clades each further divide into three distinct lineages (C1, C2, and C3; P1, P2, and P3). Additionally, subclades P1 and P2 are sister to each other, with the NJ26S sequence clustered in P1.

Figure 3. Maximum-likehood (ML) phylogenetic tree based on the mitogenomes of capreolus capreolus and capreolus pygargus, with Hydropotes inermis functioning as an outgroup (see supplementary Table S1 and Figure S3). Numbers at the branches representing the bootstrap values with 1,000 replications. The following sequences were used: Hydropotes inermis EU315254.1 (unpublished), Capreolus capreolus KJ681483.1 (Matosiuk et al. 2014), Capreolus capreolus KJ681484.1 (Matosiuk et al. 2014), Capreolus capreolus KJ681481.1 (Matosiuk et al. 2014), Capreolus capreolus KJ681482.1 (Matosiuk et al. 2014), Capreolus capreolus KJ681480.1 (Matosiuk et al. 2014), Capreolus capreolus KJ681487.1 (Matosiuk et al. 2014), Capreolus capreolus KJ681486.1 (Matosiuk et al. 2014), Capreolus capreolus KJ681485.1 (Matosiuk et al. 2014), Capreolus capreolus NC_020684.1 (unpublished), Capreolus capreolus KJ681485.1 (Hassanin et al. 2012), Capreolus pygargus KJ681494.1 (Matosiuk et al. 2014), Capreolus pygargus OK041026.1 (Deng et al. 2022), Capreolus pygargus OK323229.1 (Deng et al. 2022), Capreolus pygargus KJ681491.1 (Matosiuk et al. 2014), Capreolus pygargus OK041027.1 (Deng et al. 2022), Capreolus pygargus MK795818.1 (Li et al. 2020), Capreolus pygargus MN485773.1 (unpublished), Capreolus pygargus KJ681495.1 (Matosiuk et al. 2014), Capreolus pygargus OK041025.1 (Deng et al. 2022), Capreolus pygargus MN813763.1 (Ao et al. 2020), Capreolus pygargus NC_039093.1 (unpublished), Capreolus pygargus MF497305.1 (Kim et al. 2017), Capreolus pygargus PP068269 (NJ26S) (this study), Capreolus pygargus KJ681493.1 (Matosiuk et al. 2014), Capreolus pygargus KJ681488.1 (Matosiuk et al. 2014), Capreolus pygargus KJ681489.1 (Matosiuk et al. 2014), Capreolus pygargus KJ681490.1 (Matosiuk et al. 2014), Capreolus pygargus NC_025271.1 (Matosiuk et al. 2014), Capreolus pygargus KJ681492.1 (Matosiuk et al. 2014).

Discussion and conclusion

In this study, we obtained and annotated the ancient mitochondrial genome of a C. pygargus specimen. We then recovered highly supported phylogenetic trees that revealed the relationship to Capreolus. As can be observed in the phylogenetic trees, NJ26S is placed closer to some modern Capreolus pygargus from Russia and Poland than other region with a high node support, suggesting that ancient Capreolus pygargus genetically contributed to the inheritance of modern Capreolus pygargus worldwide.

Ancient DNA plays a crucial role in anthropology and biology by exploring human origins and migration patterns across different periods, revealing histories of interaction and mixing between human populations, and studying the origins and evolutionary history of biological populations, as well as the population structure and changes of existing species (Frantz et al. 2020). The extraction, sequencing, and data processing of ancient DNA are crucial steps in research because they directly impact our ability to obtain reliable information from ancient genetic materials. The extraction process requires specialized techniques to retrieve DNA preserved in ancient remains, while sequencing allows us to decipher the specific sequences of this DNA for genetic study. Data processing involves analyzing and comparing these sequence data to reveal evolutionary histories, population relationships, and genetic changes in humans and species (Orlando et al. 2021). Many analyses of C. pygargus have focused on modern samples, while ancient samples have received less attention. Phylogenetic analysis indicates that ancient and modern C. pygargus cluster within the same subclade, suggesting they share similar genetic backgrounds and stable genetic structures.

The Xinjiang Uygur Autonomous Region of China is one of the distribution areas of modern Capreolus pygargus. This study identified a Capreolus pygargus sample that existed in this region 3,600 years ago, providing substantial molecular evidence for the presence of Capreolus pygargus in this area over the last 3,000 years.

Supplementary Material

Figure S3 R2.tif

Supplementary Table S1 R2.docx

Figure S5 R2.tif

Figure S1.tif

Supplementary Figures R2.docx

Figure S4.tif

Figure S2.tif

Authors’ contributions

Guangjie Song and Dawei Cai conceived and designed the experiments. The specimen was provided by Yongqiang Wang and Qiurong Ruan. The experiments were performed by Guangjie Song. Guangjie Song and Dawei Cai conducted data analysis. The manuscript was written by Guangjie Song. Yongqiang Wang and Qiurong Ruan revised the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

The material involved in the article is an ancient remains excavated from an archaeological site, not a modern biological sample, therefore does not involve ethical conflicts. The ancient sample was provided and received experimental permission by the Xinjiang Institute of Cultural Relics and Archaeology in 2016.

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov/) under accession number PP068269. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA1096930, SRR28574121, and SAMN40806009, respectively.
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