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Corresponding author: Hongyi Liu ( hongyi_liu@njfu.edu.cn ) Academic editor: Lorenzo Prendini
© 2025 Wei Xu, Gaoji Zhang, Tangjun Xu, Ke He, Jiachen Wang, Zeyang Liu, Hongyi Liu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Scorpions, a diverse group of arachnids consisting of over 2,000 valid species, have received limited research attention in terms of their complete mitochondrial genomes (mitogenomes). To increase the taxonomic sampling frequency of species available for study based on mitogenomes, we reconstructed the complete mitogenomes of five scorpions, Androctonus amoreuxi (Audouin, 1826), Hottentotta tamulus (Fabricius, 1798), Leiurus quinquestriatus (Ehrenberg, 1828), Lychas mucronatus (Fabricius, 1798), and Teruelius flavopiceus (Kraepelin, 1900) within the family Buthidae. These five mitogenomes had a typical circular structure, with total sizes ranging from 14,504 to 15,083 bp. Nucleotide composition analysis indicated that the sequences were biased toward A and T. The Ka/Ks ratios within 13 protein-coding genes (PCGs) were lower than 1, suggesting that they had been subject to purifying selection in Buthidae. Our analyses provide additional evidence on that, in scorpions, the majority of mitogenome rearrangements occurred in tRNAs. Moreover, the genes tRNA-Asp, tRNA-Gln and tRNA-Ile were the hotspots of rearrangement in this order. Phylogenetic analyses based on PCGs supported taxonomic relationships in this taxon. Our results might provide useful insights into the gene arrangement features of scorpion mitogenomes and lay the foundation for further studies on the family Buthidae.
Buthidae, mitochondrial genome, tRNA, phylogenetic analysis, rearrangement
In eumetazoans, the circular mitochondrial genome (mitogenome, usually 15 to 18 kb) typically contains 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and a long noncoding region (CR) (
In recent decades, research focus has shifted to gene rearrangements as a valuable phylogenetic characteristic (
In this study, we sequenced the complete mitogenomes of five scorpion species (Androctonus amoreuxi Audouin, 1826, Hottentotta tamulus Fabricius, 1798, Leiurus quinquestriatus Ehrenberg, 1828, Lychas mucronatus Fabricius, 1798, and Teruelius flavopiceus Kraepelin, 1900) for the first time and analyzed them in comparison with other Scorpiones complete mitogenomes published in the NCBI (as of February 2024). We determined the nucleotide composition, codon usage, gene order, and phylogenetic relationship of a total of 16 complete mitogenomes, with the aim of discovering the patterns of gene rearrangements within Scorpiones, particularly within the family Buthidae, as well as supplementing the mitochondrial data of this order.
Samples used in this study were collected from insect markets in Linyi (Shandong, China) and Jieyang (Guangdong, China). The collected specimens were firstly morphologically characterized based on the images and morphological features on GBIF (https://www.gbif.org/), then identified through molecular identification according to the COX1 published on NCBI. The collection of specimens was reviewed and approved by Nanjing Forestry University, which conformed to the relevant Chinese laws. The animals were raised at the Zoology Laboratory of Nanjing Forestry University. Total DNA was extracted from muscular tissue using a FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme™, Nanjing, China). DNA was stored at –20°C for follow-up.
Library construction and sequencing were conducted by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China) utilizing the NovaSeq X Plus platform (Illumina, USA) for 150 bp paired-end reads.
In order to produce high-quality data, sequences of low quality were eliminated. Quality filtering included a sliding window approach for quality assessment. The window size was set to 9 bp, with a step size of 1 bp. The window was moved forward by one base each time, and the average Q-value for the 9 bases within the window was calculated. If the average Q-value of the window was less than 2, only the second-to-last base and all preceding bases within that window were retained. The clean reads were taken for the assembly of the complete mitogenomes, using the Geneious Prime 2023 software and Buthus occitanus (Amoreux, 1789) (EU523755.1) as a reference template (
Conservative domains of the mitogenomes were identified using BLAST CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and the MITOS server (http://mitos.bioinf.uni-leipzig.de/index.py) (Marchler and Bryant 2004;
Sixteen species from five families of Scorpiones, as well as Asemonea sichuanensis (Song & Chai, 1992) from Araneae-Salticidae as an outgroup taxon, were used for the phylogenetic analyses (Table
The mitogenomes of Buthidae, Chactidae, Scorpionidae, Scorpiopidae, Vaejovidae, and Salticidae used in this study.
Order | Family | Species | Size (bp) | GenBank No. |
Scorpiones | Buthidae | Androctonus amoreuxi | 15,064 | PP778689 |
Buthus occitanus | 15,060 | EU523755.1 | ||
Centruroides limpidus | 14,519 | AY803353.1 | ||
Centruroides vittatus | 14,602 | MF975702.1 | ||
Hottentotta tamulus | 14,990 | PP755025 | ||
Leiurus quinquestriatus | 15,083 | PP818816 | ||
Lychas mucronatus | 14,534 | PP874230 | ||
Mesobuthus gibbosus | 15,983 | AJ716204.2 | ||
Mesobuthus martensii | 15,034 | DQ340065.1 | ||
Mesobuthus martensii | 14,840 | MN597087.1 | ||
Teruelius flavopiceus | 14,504 | PP874229 | ||
Tityus serrulatus | 14,460 | KR024030.1 | ||
Chactidae | Uroctonus mordax | 14,840 | EU523756.1 | |
Scorpionidae | Heterometrus longimanus | 14,655 | KR190462.1 | |
Scorpiopidae | Scorpiops tibetanus | 14,825 | MT903349.1 | |
Vaejovidae | Vaejovis smithi | 14,492 | KX520650.1 | |
Araneae | Salticidae | Asemonea sichuanensis | 15,419 | MN651970.1 |
The five scorpions’ complete mitogenomes were typically circular, double-stranded molecules, with sizes of 15,064 bp, 14,990 bp, 15,083 bp, 14,534 bp, and 14,504 bp (Fig.
The nucleotide composition analysis indicated that five mitogenomes displayed a preference for Adenine (A) and Thymine (T), as illustrated in Fig.
The studied mitochondrial genomes were compact, with intergenic spaces not exceeding 24 bp and gene overlaps not longer than 52 bp (Table S1). Multiple overlaps between adjacent genes were detected: nine gene overlaps were observed in A. amoreuxi, six in H. tamulus, ten in L. quinquestriatus and L. mucronatus, and seven in T. flavopiceus. The largest overlap was located between 12S rRNA and tRNA-Asp.
The total sizes of the PCGs were 10,854 bp, 10,854 bp, 10,867 bp, 10,818 bp, and 10,807 bp, respectively, accounting for 72.05% (L. quinquestriatus) to 74.51% (T. flavopiceus) of their whole mitogenomes. All PCGs were encoded on the major strand, except for ND5, ND4, ND4L, and ND1, which were encoded on the minor strand (Table S1). Among the 13 PCGs presented in these five mitogenomes, ATP8 exhibited the smallest size, while ND5 displayed the largest.
The majority of PCGs in the five mitogenomes started with the ATG codon, while the rest used alternative initiation codons such as those found in other animal mitochondrial genomes like ATA, ATT, TTG, and GTG. The termination codon varied across these PCGs, with codons of TAA, TAG, and T. The frequency of the termination codon TAA was consistently higher than that of the other two termination codons, whereas the occurrence of the termination codon TAG was the lowest. Next, an analysis of the initiation and termination codon usage in 16 mitogenomes was conducted (Fig.
Initiation codon (a) and termination codon (b) usage for the mitochondrial genome protein-coding genes of Scorpiones mitogenomes; Ka/Ks values for the 13 PCGs of five Buthidae mitogenomes in this study (c); Relative synonymous codon usage of five Buthidae mitogenomes in this study (d), the termination codon is not included.
This study examined the evolutionary trajectory of PCGs through the analysis of Ka/Ks ratios (Fig.
Twenty-two tRNAs of A. amoreuxi, L. mucronatus, L. quinquestriatus, and T. flavopiceus, and twenty-one tRNAs of H. tamulus mitogenomes were scattered discontinuously over the complete mitogenomes (Table S1). H. tamulus lacked tRNA-Asp. The tRNA regions of these five mitogenomes were 1,313, 1,285, 1,309, 1,298, and 1,305 bp, accounting for 8.72%, 8.57%, 8.68%, 8.93%, and 9.00%, respectively, of the whole mitogenomes. These five mitogenomes have 22 (or 21) typical tRNA genes, with eight transcribed from the minor strand. The secondary structures of these tRNAs are shown in Fig.
In the five mitogenomes, two rRNA genes (12S rRNA and 16S rRNA) were transcribed from the minor strand (Table S1). The larger rRNA (16S rRNA) was found between tRNA-Leu1 and tRNA-Val, while the smaller rRNA (12S rRNA) was located between tRNA-Val and tRNA-Gln. The sizes of 12S rRNA ranged from 718 to 728 bp, and those 16S rRNA from 1,151 to 1,160 bp in the mitogenomes.
In the five mitogenomes, CRs were found between the genes 12S rRNA and tRNA-Gln (Table S1). Their sizes ranged from 513 bp (T. flavopiceus) to 1,078 bp (L. quinquestriatus), accounting for 3.54% to 7.15% of the whole mitogenomes. This accounted for a significant variation, which in turn influenced the overall size of the complete mitogenome. The AT skew of CRs exhibited consistently higher values than PCGs and RNAs, indicating a higher occurrence of A than T (Fig.
The structures of the five mitogenomes sequenced in this study differed in tRNAs. As mentioned above, unlike the “tRNA-Val, tRNA-Asp, tRNA-Gln” genetic arrangement of the other four scorpions, H. tamulus lacked tRNA-Asp to form “tRNA-Val, tRNA-Gln” (Table S1).
We also summarized and organized the gene arrangement of the five mitogenomes along with other published Scorpiones mitogenomes (Fig.
A total of 16 mitogenomes from five families of the order Scorpiones were included in the phylogenetic analyses (Table
The mitogenomes of the five studied scorpions contained 13 PCGs, 22 tRNAs (or 21 tRNAs), two rRNAs, and one noncoding CR, consistent with findings for other species in the family Buthidae (
Among the studied Scorpiones species, there was a tendency towards a conservative approach in the selection of initiation codons, predominantly favoring ATG (Fig.
Certain tRNAs could not be accurately predicted in terms of their secondary structures. This phenomenon of tRNA structures has also been reported by a previous study (
By obtaining numerous mitogenome sequences, it has been established that while the gene arrangement remains consistent across many invertebrates, rearrangements in a small number of tRNAs are also frequently observed (
The results of our phylogenetic analyses based on the complete mitogenomes obtained in this study and published complete Scorpiones mitogenomes were consistent with the classification of the families Buthidae. Furthermore, there are still a large number of Scorpiones species whose complete mitogenomes have not yet been published, and our knowledge on the structure of Scorpiones mitogenomes, especially the pattern and underlying mechanisms of gene rearrangements, is far from comprehensive. Therefore, it is necessary to obtain mitogenome data on further species in this group. Due to the fact that the samples in this study did not originate from their native habitats, but were acquired from commercial sources, we must acknowledge that the presence of these limitations may introduce some bias or uncertainty into our research results. Consequently, in future studies, we need to continue exploring more reliable and accurate sources of samples and identification methods in order to further enhance the accuracy and reliability of related data.
This study presents the structures and sequencing results of five mitogenomes in the family Buthidae, which are typical circular DNA molecules with sizes ranging from 14,504 to 15,083 bp. Nucleotide composition analysis revealed a bias towards A and T in the sequences. The Ka/Ks ratios of 13 PCGs were found to be below 1, indicating the presence of purifying selection within this family. Therefore, our findings suggest that mitogenome rearrangements in scorpions primarily occur in tRNAs. Specifically, tRNA-Asp, tRNA-Gln and tRNA-Ile were identified as key sites for rearrangements in our sample representatives of the order Scorpiones (mainly Buthidae), and phylogenetic analyses based on PCGs supported the taxonomic relationships within this order. These findings offer valuable insights into the gene arrangement patterns of scorpion mitogenomes and establish a sound basis for future investigations concerning the Buthidae family.
Authors’ contributions. LHY conceived the study. XW acquired the fund. XW and LZY conducted the sampling. XW, ZGJ, XTJ and WJC conducted the experiments. XW carried out the bioinformatics analysis. XW drafted the manuscript. LHY and HK reviewed and revised the manuscript. All authors approved the final manuscript.
Funding. This study was supported by the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX24_1382).
Data availability statement. DNA sequences: GenBank accession number PP778689 for Androctonus amoreuxi, PP755025 for Hottentotta tamulus, PP818816 for Leiurus quinquestriatus, PP874230 for Lychas mucronatus, and PP874229 for Teruelius flavopiceus.
Conflicts of interest. The authors declare no conflict of interest.
We thank Xinyi Wang for her support of this study and the anonymous reviewers for their work.
Table S1
Data type: .xlsx
Explanation notes: Comprehensive overview of the general features of the mitogenomes of five Buthidae species.