The complete mitochondrial genome of Eremias yarkandensis (Reptilia, Squamata, Lacertidae) from Kyrgyzstan

Abstract

The Yarkand racerunner, Eremias yarkandensis Blandford, 1875, is only distributed in China and Kyrgyzstan. Its complete mitogenome was determined by next-generation sequencing for the first time. The mitogenome was 18,743 bp in length, including 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and 1 control region. Its gene arrangement was similar to the typical mtDNA of vertebrates. The 13 concatenated PCGs were used to perform Bayesian phylogenetic analyses together with several congeners as well as two representative species of Lacerta with mitogenome data in GenBank. The resulting phylogenetic tree recovered the monophyly of both Eremias and its viviparous group, with E. yarkandensis being more closely related to E. przewalskii than to E. dzungarica. The mitogenome of E. yarkandensis will provide fundamental data for the exploration of the mitogenome evolution in racerunners.

Keywords:

• Kyrgyzstan
• mitochondrial genome
• next-generation sequencing
• phylogenetic tree
• Eremias
• viviparity

The viviparous Yarkand racerunner, Eremias yarkandensis Blandford, 1875, is endemic to East Alai of Kyrgyzstan and Xinjiang Uyghur Autonomous Region of China, with terra typica in the Yarkand County, Kashgaria, China. Historically, it was assigned to subspecies status of Eremias multiocellata yarkandensis (Boulenger 1921; Schmidt 1926; Szczerbak 1974; Eremchenko et al. 1992). However, its status was confirmed as distinct biological species through long-term research including hybridization experiments (Eremchenko and Panfilov 1999). To date, little is known about its genetic affinities with other viviparous species in genus Eremias albeit with limited understanding of viviparity evolution in racerunner lizards (Guo et al. 2011; Orlova et al. 2017; Liu et al. 2021).

In this study, we determined for the first time the complete sequence of the mitogenome of E. yarkandensis by next-generation sequencing through the Illumina NovaSeq platform. A female adult (voucher number Guo4719) was collected from Kyrgyzstan (N39.64809°, E73.86512°; 2963 meters above sea level) in August 2014. The collection of lizard used for this study obeyed the Law ‘On the Animal World’ No. 59 of Kyrgyzstan, and followed the guidelines in the Institute of Biology and Soil, National Academy of Science of the Kyrgyz Republic. Its liver tissue was fixed with 95% ethanol, and stored at −20 °C in the Chengdu Institute of Biology (CIB), Chinese Academy of Sciences (Contact person: Xianguang Guo, E-mail: [email protected]). A small amount of liver tissue was shipped to Genepioneer Biotechnologies (Nanjing, China) for genomic extraction and 150-base-pair paired-end library construction as well as sequencing. The CIB Animal Care and Use Committee approved all experimental procedures (No. 20200767).

The raw data were processed with fastp v.0.20.0 (Chen et al. 2018), by trimming adapters and primers, filtering reads with phred quality < Q5, and filtering reads with N base number > 5. De novo assembly of clean data (6,996,357,900 pair-end reads) was performed using SPAdes v.3.10.1 (Bankevich et al. 2012). The average coverage of reads aligned on the mitogenome was 625.4147. We used a published sequence (GenBank accession number MW250881; Wang et al. 2021) as the reference for queries to assemble the mitogenome. Subsequently, the mitogenome was annotated with the MITOS WebServer (Bernt et al. 2013). Specifically, 22 tRNA genes were identified by using the web server of tRNA scan-SE (Lowe and Chan 2016). The base composition was calculated in MEGA v.7.0 (Kumar et al. 2016).

The gene content, arrangement, and composition exhibited a typical vertebrate mitogenome feature. All of 37 genes were completely recovered including two ribosomal RNA genes, 13 protein-coding genes (PCGs), 22 transfer RNA (tRNAs), and a control region (CR or D-loop). The mitogenome of E. yarkandensis was 18,743 bp in length, which was composed of 28.4%(T), 27.2%(C), 31.1%(A), 13.3%(G). In 13 PCGs, the shortest was ATP8 gene (162 bp) and the longest was ND5 (1824 bp). Eleven PCGs used ATG as start codons, the remaining two (COX1, ND1) used GTG as start codons. As for stop codons, six PCGs (ND1, ND4L, ND5, ATP6, ATP8, Cytb) used TAA; five PCGs (ND2, ND3, ND4, COX2, COX3) used T as an incomplete stop codon; two PCGs (COX1, ND6) used AGG. In addition, 12S rRNA, 16S rRNA, and D-loop were 951 bp, 1559 bp, and 3339 bp, respectively. The majority of the genes in the mtDNA of E. yarkandensis were distributed on H-strand, except for ND6 gene and eight tRNAs (tRNA-Glu, Ala, Asn, Cys, Tyr, Ser^[UGA], Gln, and Pro).

The Bayesian phylogenetic tree was used to assess mitochondrial sequence authenticity of E. yarkandensis and its phylogenetic placement by using the concatenated 13 PCGs of E. yarkandensis together with two other representative Lacerta lizards in GenBank. Gene partitioning, model selection, and tree reconstruction were made in the plug-in programs of PhyloSuite v.1.2.1 (Zhang et al. 2020). PartitionFinder v.2.1.1 was used to select the best-fitting substitution models and partitioning schemes with the Bayesian information criterion (Lanfear et al. 2017). Bayesian inference was conducted using MrBayes v.3.2.6 (Ronquist et al. 2012). Two independent runs were carried out with four Monte Carlo Markov chains (MCMCs) for 10 million generations with parameters and topologies sampled every 1000 generations. The convergence of the runs was assessed by the standard deviation of split frequencies (<0.01). A 50% majority-rule consensus tree and posterior probability (PP) of clades were assessed by combining the sampled trees from the two independent runs after a 25% burn-in phase.

As shown in Figure 1, the monophyly of both genus Eremias and its viviparous group was recovered with strong support (Guo et al. 2011; Orlova et al. 2017). Eremias yarkandensis was more closely related to E. przewalskii than to E. dzungarica albeit with moderate support (PP = 0.81). The mitogenome of E. yarkandensis will provide fundamental data for the exploration of the mitogenome evolution in racerunners.

Figure 1. A majority-rule consensus tree inferred from Bayesian inference using MrBayes with the best model for each partition, based on the PCGs of Eremias spp. and two other representative Lacerta lizards retrieved from GenBank. The phylogenetic placement of E. yarkandensis is highlighted. GenBank accession numbers are given with species names. Node numbers show Bayesian posterior probabilities. Branch lengths represent means of the posterior distribution.

Author contributions

Conceptualization, X.G. and B.Z.; Data curation, M.C., J.L., X.G.; Analysis and interpretation of the data, S.W. and J.L.; Funding acquisition, J.L. and X.G.; Writing – original draft, S.W.,. J.L., X.G.; Writing – review & editing, M.C., B.Z., X.G. All authors have read and agreed to the published version of the manuscript. All authors agree to be accountable for all aspects of the work.

Acknowledgments

We thank Dr. Anar Alymkulova (the Institute of Biology and Soil, National Academy of Science of the Kyrgyz Republic) and Dr. Roman A. Nazarov (Zoological Museum, Department of Herpetology, Moscow State University) for assistance in collecting the sample during the fieldwork.

Disclosure statement

No potential conflict of interest was reported by the authors. The authors alone are responsible for the content and writing of this article.

Data availability statement

The data that support the findings of this study are openly available in NCBI at https://www.ncbi.nlm.nih.gov/nuccore/OK585048, reference number OK585048 . The associated BioProject, SRA, and Bio-Sample numbers are PRJNA773190, SRR16508134, and SAMN22445465, respectively.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [32000288 and 32070433], the Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region [NJYT-19-A13].