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.


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. 2021Park et al. , 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.
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 0.61 00 N, 126 � 53 0 26.64 00 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; shcol2002@jnu.ac.kr) 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 þ G model with 1000 bootstrap replicates (Edgar 2004;Waddell and   Steel 1997).The GTR þ G substitution model was selected as the best-fit model by MEGA X.
Additionally, ND2, ND4, and COX1 have incomplete stop codons ending with U, and Cytb with UA.
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.

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

Figure 2 .
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.