Complete mitochondrial genome of the razor-backed musk turtle (Sternotherus carinatus, testudines: emydidae) in Korea

Abstract

Sternotherus carinatus has been considered as a potential invasive species in Korea. However, the mitochondrial genome information of S. carinatus which can be used to control its effect on ecosystem is lacking. In this study, the complete mitochondrial genome of S. carinatus in Korea was sequenced and characterized. The mitochondrial genome consists of 37 genes (13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes) and a noncoding region. Phylogenetic analysis based on the mitochondrial genome sequences showed that S. carinatus from Korea is separated from other turtles which are the invasive species in Korea. Sequence divergence calculations indicated near-zero divergence between S. carinatus populations in Korea, the USA, and China, suggesting limited genetic differentiation. In the context of the broader issue of invasive species disrupting ecosystems, this research contributes to the identification of mitochondrial genomes for various freshwater turtle species, emphasizing the need for extended data collection to discern genetic mixing trends between native and non-native species. This study is a significant step toward managing S. carinatus as a potential invasive species in Korea.

Keywords:

• Sternotherus carinatus
• emydidae
• mitochondrial genome

Introduction

The razor-backed musk turtle (Sternotherus carinatus, Gray 1856) is native to the USA (Lindeman 2008) (Figure 1). However, it has spread to other countries including Korea due to pet trade and intentional releases (Koo et al. 2020). This increase causes many problems in the native water ecosystem such as habitat and food competition, hybridization, and parasites (Cadi and Joly 2004; Silbernagel et al. 2013). Many researchers have tried to identify the mitochondrial genome of freshwater turtles and to decipher the relationships between native and non-native native species (Baek et al. 2022; Cheon et al. 2023; Chung et al. 2022; Kundu et al. 2022; Park et al. 2021, 2022; Ryu et al. 2021; Suzuki et al. 2014). Though not officially labeled as an invasive species in Korea, further discoveries of this species may lead to its designation as such. The comprehensive mitochondrial genome information of S. carinatus is crucial for effective species management and the protection of native species from potential impacts. Unfortunately, only limited information regarding the mitochondrial genome of S. carinatus is available. GenBank holds just one complete S. carinatus mitochondrial genome, alongside nine partial Cytb sequences, three partial 12S-rRNA sequences, two partial 16S-rRNA genes, one partial COX1 gene, and a complete tRNA-Val gene disclosed in GenBank.

Figure 1. The image of Sternotherus carinatus. This picture was taken by Seung-Min Park.

In this study, we sequenced the complete mitochondrial genome of S. carinatus, providing valuable data for phylogenetic studies and exotic species management.

Materials and methods

The S. carinatus specimen was captured from Gwangju (35°11′0.61″N, 126°53′26.64″E), Korea, and the tail tissue was acquired while the specimen was in a live state. This research received official sanction from Yeongsangang River Basin Environmental Office, a division of the Korea Ministry of Environment (permission number: 2021-8). Total genomic DNA was extracted from the tail tissue using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) and deposited at the Museum of Wildlife, located in Research Center of Ecomimetics, Chonnam National University, Korea (https://biology.jnu.ac.kr; Ha-Cheol Sung; [email protected]) under species voucher number 2023-RCE-SC001. The mitochondrial genome was analyzed using the Illumina NovaSeq X plus platform (Illumina, San Diego, CA), which was performed by Macrogen (Seoul, Korea). The raw sequence data were assessed using FastQC and filtered to remove adaptor sequences, low-quality reads, reads with more than 10% unknown bases, and ambiguous bases using Trimmomatic v0.36 (Andrews 2010; Bolger et al. 2014). From 46,393,912 raw reads, 39,846,366 filtered reads were obtained. De novo assembly was conducted with SPAdes 3.15.0, and the filtered reads were aligned using BLAST (Altschul et al. 1990; Bankevich et al. 2012). The coverage depth map is shown in Supplementary Figure 1, and the average read depth was 411.44. The complete sequence was annotated using the MITOS2 web server (Bernt et al. 2013).

To elucidate the relationship between S. carinatus and both native species and non-native species in Korea and investigate the phylogenetic position of S. carinatus, we selected Chrysemys picta, Pseudemys peninsularis, P. concinna, Trachemys scripta, Mauremys sinensis, and S. carinatus to represent the non-native species, while M. reevesii and Pelodiscus sinensis were chosen to represent the native species. We extracted their complete mitochondrial genome sequences from GenBank. All protein-coding genes (PCGs), rRNA genes, and tRNA genes were concatenated for analysis. The phylogenetic tree was constructed using MEGA X software (Kumar et al. 2018). Specifically, the sequences were aligned using the MUSCLE algorithm, and the phylogenetic tree was made using the maximum likelihood method and GTR + G model with 1000 bootstrap replicates (Edgar 2004; Waddell and Steel 1997). The GTR + G substitution model was selected as the best-fit model by MEGA X.

Results

The complete mitochondrial genome of S. carinatus spans 16,552 bp in length and deposited in GenBank (Accession number: OR253895). It comprises 13 PCGs, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a putative long non-coding control region. Notably, 12 PCGs, 14 tRNA genes, and 2 rRNA genes are encoded on the heavy strand, whereas 1 PCG (NADH dehydrogenase subunit 6) and 8 tRNA genes are encoded on the light strand (Figure 2). Most PCGs use canonical mitochondria start codons (AUG for ND2, COX1, COX2, ATP8, ATP6, COX3, ND3, ND5, ND6, and Cytb, and AUA for ND4L), except for ND1 and ND4, which have alternative initiation codon (AUU and GUG, respectively). Additionally, ND2, ND4, and COX1 have incomplete stop codons ending with U, and Cytb with UA.

Figure 2. Mitochondrial genome map of Sternotherus carinatus. Genes encoded on the heavy strand are written outside the circle and genes on the light strand inside the circle. The dark and light parts in the grey circle represent GC and at contents, respectively.

The nucleotide composition of the S. carinatus mitochondrial genome is A = 35.8%, T = 27.3%, G = 12.0%, and C = 24.9%., which is identical to that of S. carinatus from China (HQ114563; A = 35.8%, T = 27.3%, G = 12.0%, and C = 24.9%) and similar to that of Pelodiscus sinensis from Korea (AY962573; A = 35.4%, T = 27.3%, G = 11.8%, and C = 25.6%). While S. carinatus from Korea shares a 99% sequence identity with the Chinese counterpart, it exhibits lower similarity with Mauremys reevesii from Korea (FJ469674; 78%), Pseudemys concinna from Korea (OM935747; 78%), or Pelodiscus sinensis from Korea (AY962573; 75%).

A phylogenetic tree, constructed using the complete mitochondrial genome sequence of S. carinatus from Korea along with sequences from 16 other species, corroborates the genetic similarity. S. carinatus from Korea clusters closely with S. carinatus from China while distinctly separating from other turtles, including P. concinna, M. reevesii, and P. sinensis (Figure 3).

Figure 3. Phylogenetic tree of S. carinatus and other related species based on mitochondrial genome sequences. Phylogenetic analysis was performed using MEGA X software. The names of country referred behind each species indicate the locations where the sample was collected, as referenced on GenBank. GenBank accession numbers of each mt genome sequence are given in the bracket after the species name, and the following sequences were used: AF069423 (Mindell et al. 1999), KF874616 (Jiang et al. 2016), OM935748 (Chung et al. 2022), OM935747 (Park et al. 2022), KM216748 (Yu et al. 2016), MW019443 (Park et al. 2021), FJ392294 (Russell and Beckenbach 2008), KC333650 (Fang et al. 2013), FJ871126 (reference unavailable), AP019398 (Asami et al. 2019), FJ469674 (Shin et al. 2015), KJ700438 (reference unavailable), HQ114563 (Li et al. 2017), AY962573 (Jung et al. 2006), MG431983 (Zhang et al. 2019), AY687385 (reference unavailable), and AF039066 (Zardoya and Meyer 1998). the bootstrap value based on 1000 replicates is represented on each node. Pelomedusa subrufa was used as an outgroup to root the tree.

We also determined the divergence between S. carinatus from Korea and China, yielding a divergence value of 0.067%. Furthermore, comparing Cytb sequences of S. carinatus available in GenBank indicates negligible or minimal divergence when comparing the Korean population with those from the USA (Table S1).

Discussion and conclusion

We identified the complete mitochondrial genome of S. carinatus from Korea and studied its phylogenetic relationship with other turtles. In the phylogenetic tree, S. carinatus from Korea is placed in the distinct cluster apart from M. reevesii and P. sinensis which are the native species in Korea and P. concinna and T. s. elegans which are the invasive species in Korea. Furthermore, S. carinatus in Korea is separated from S. carinatus in China, despite being in the same cluster.

We calculated the sequence divergence between S. carinatus populations in Korea and those in the USA or China, revealing near-zero divergence. According to Morgan-Richards et al. (2017), a divergence exceeding 3% typically suggests significant population divergence over numerous generations. Considering the long lifespan of S. carinatus (20–50 years) and the relatively recent occurrence of its trade, it appears that S. carinatus from non-native habitats like Korea and China may not exhibit substantial genetic differentiation from their native habitat counterparts. Nonetheless, accumulating mitochondrial genome data over an extended period is essential for a more comprehensive investigation into the impact of S. carinatus on native species.

The disturbance of ecosystems by invasive species is a severe issue in many countries including Korea. We and other scientific groups have culminated in the identification of mitochondrial genomes for various freshwater turtle species to understand their mitogenomic relationships. Typically, when comparing species from different genera, non-native species have been distinctly separated from the native species, indicating minimal genetic mixing from invasive species. Nonetheless, it is evident that more extensive and prolonged data collection is necessary. This study serves as a valuable contribution to the management of S. carinatus, a potential invasive species in Korea.

Authors contributions

According to the International Committee of Medical Journal Editors (ICMJE) criteria, all authors including Jaehong Park, Seung-Ju Cheon, Jae-Hyuk Choi, Seung-Min Park, Ha-Cheol Sung, and Dong-Hyun Lee participated in the conception and design of the work, acquisition, and analysis of data, drafting and revising the work, and final approval of the version to be published. Also, all authors agreed to be accountable for all aspects of the work.

Ethical statement

This study was approved by the Yeongsangang River Basin Environmental Office of the Korea Ministry of Environment (permission number: 2021–8). The experiments were conducted in accordance with the International Union for Conservation of Nature (IUCN) policies.

Disclosure statement

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

Data availability statement

GenBank accession number from the complete mitochondrial genome of Sternotherus carinatus (OR253895) has been registered with the NCBI database (https://www.ncbi.nlm.nih.gov/OR253895). The associated BioProject, BioSample, and SRA accession numbers are PRJNA993827, SAMN36409325, and SRR25243167, respectively.

Additional information

Funding

This work was supported by the Korea Environment Industry & Technology Institute (KEITI) through the Project for the Development of Biological Diversity Threats Outbreak Management Technology (2018002270004), funded by the Korea Ministry of Environment (MOE).