Characterization of the complete mitochondrial genome of Spirobolus grahami (Diplopoda: Spirobolidae) with phylogenetic analysis

Diplopoda is one of the most diverse and important groups of soil arthropods, but little research has been done on their phylogenetic relationship and evolution. Here, we sequenced and annotated the complete mitochondrial genomes of Spirobolus grahami. The total mitogenome of S. grahami was typical circular, double-stranded molecules, with 14,875 bp in length, including 13 protein-coding genes, 22 tRNAs, two rRNAs, and one control region. Base composition analysis suggested that the mitochondrial sequences were biased toward A and T, with A + T content of 58.68%. The mitogenomes of S. grahami exhibited negative AT and positive GC skews. Most of the 13 PCGs had ATN as the start codon, except COX1 start with CGA, and most PCGs ended with the T stop codon. The dN/dS values for most PCGs were lower than 1, suggesting that purifying selection was likely the main driver of mitochondrial PCG evolution. Phylogenetic analyses based on 13 PCGs using BI and ML methods support the classification of genus Spirobolus and Tropostreptus. Glomeridesmus spelaeus is distantly related to the other Diplopoda species.

Invertebrate mitochondrial genome (mitogenome) is typically double-stranded and closed circular molecules, approximately 15-18 kb in length 1,2 .Invertebrate mitogenome consists of 13 protein-coding genes (PCGs), 22  transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and one non-coding control region (CR) 2,3 .The mitogenome has characteristics such as small size, simple structure and fast evolution, it has been extensively studied and widely used for species identification and molecular phylogeny researches 4,5 .
Diplopoda (millipedes) is one of the most diverse groups of arthropods, with more than 7000 species described 6 .Millipedes Spirobolus grahami belongs to the Spirobolidae family of the Diplopoda class 7 .Millipedes are an important part of modern terrestrial ecosystems and play an important role in the decomposition of organic matter 6,8,9 .However, few studies have documented the phylogeny, evolution, behavior, physiology, and ecology of Millipedes 8,10,11 .Therefore, the use of mitogenome might be expected to provide valuable data on their phylogenetic relationship.
In order to further investigate the relationship between the Diplopoda, in this study, we firstly sequenced and characterized the mitogenome of S. grahami.The structural organization, nucleotide composition, codon usage, and AT/GC-skew were analyzed.Additionally, we conducted phylogenetic analyses based on 13 PCGs available elsewhere for the purpose of investigating the phylogenetic position of S. grahami within Diplopoda, which we believe might be helpful for further evolutionary and phylogenetic studies on millipedes within the Diplopoda.

Mitochondrial genome organization
The total mitogenome of S. grahami was typical circular, double-stranded molecules, with 14,875 bp in length (Fig. 1).The mitogenome has been submitted to GenBank (Table 1).Mitogenomes of S. grahami encoded all 37 classical mitochondrial genes (13 PCGs, 22 tRNAs, and 2 rRNAs) and one CR.In this mitogenome, 15 genes (four PCGs, two rRNAs, and nine tRNAs) were transcribed from the majority strand (J strand), and the remaining 22 genes were transcribed from the minority strand (N strand) (Table 2), which is identical to S. bungii of the same genus 11 .The gene order of S. grahami was also consistent with that of S. bungii and Spirobolus walkeri in the same genus 11 .The gene order of millipede mitogenome is diverse 24 , but the gene order of this genus is relatively stable.
Base composition analysis suggested that this mitogenome was biased toward A and T, the content ratio of A + T is 58.68% (Table 3), which is consistent with a previous study 11 .Besides, the PCGs, tRNAs, rRNAs, and CR were all biased in nucleotide composition (A + T > G + C), which is consistent with other invertebrate researches 25,26 .The AT-skew of S. grahami was negative, while the GC-skew was positive.The low GC-skew values of the analyzed mitogenome indicated the occurrence of more Cs than Gs.However, the AT-skew of tRNAs and CR were slight positive.
Multiple overlaps between contiguous genes were calculated.There were five gene overlaps in this mitogenome, ranging from 3 to 7 bp.The longest overlap region of the mitogenome was found between Cytb and ND6, as well tRNA-Cys and tRNA-Trp, with 7 bp in length.

Protein-coding genes and codon usage
The total length of the PCGs was 10,988 bp, accounting for 73.87%.Four PCGs, ND1, ND4L, ND4, and ND5 were transcribed from the J-stand, and the other PCGs from the N-strand.The sizes of 13 PCGs ranged from 156 (ATP8) to 1702 bp (ND5) in the mitogenome.The start codon of all PCGs is ATN (ATG, ATT, and ATA), except COX1 starts with the CTA codon.This unusual start codon, CTA, have previously been reported in Spirobolus 11 .In addition, three stop codons were found in the PCGs of S. grahami, namely TAA, TAG, and T. In the mitogenome, the occurrence frequency of the stop codon T was higher than those of the other two stop codons, while the stop codon TAG occurred the least.www.nature.com/scientificreports/ The RSCUs of the PCGs in the mitogenome were calculated, as shown in Fig. 2. The RSCUs analysis of S. grahami showed that codons tended to use more A or T at the third codon, which is consistent with some previous studies 27,28 .The dN/dS of the PCGs in the mitogenome of Spirobolus (S bungii, S. grahami, and S. walkeri) were calculated, too (Table 4).In evolutionary analysis, it is necessary to understand the rate at which dN and dS mutations occur, analyzing their ratios to detect selective pressures, if any, among PCGs.In this study, ND4L having the lowest evolutionary rate, and COX1 having the highest sequence variability.The faster evolution of COX1 of the genus Spirobolus might result in greater amino acid diversity, indicating its potential as an effective marker for classification.The dN/dS values for most PCGs were lower than 1, suggesting that purifying selection was likely the main driver of mitochondrial PCG evolution 29 .
Transfer RNA, ribosomal RNA genes and control regions 22 tRNAs and two rRNAs were discontinuously distributed throughout the whole mitogenome.The tRNA genes of the mitogenome were 1376 bp, which account for 9.3% of the entire mitogenome.There were nine tRNAs from the J-strand and 14 transcribed from the N-strand.Among all secondary structures of the 22 tRNA genes from the S. grahami mitogenome, except for tRNA-Ser1, all had a typical cloverleaf structure (Fig. 3), as observed in other Diplopoda mitogenomes 8,11,30 .The 16S rRNA (1033 bp) was encoded between tRNA-Val and tRNA-Leu1, and the 12S rRNA was 757 bp long.The total size of the two rRNAs was 1790 bp, accounting for 12.03%.
One CR was found between the genes tRNA-Ile and 12S rRNA in the mitogenome, with 450 bp in length, accounting for 3.03%.The content of A + T is 71.78%, consistent with research that mitochondrial CR is typically characterized by high A + T content in most invertebrates 25,31,32 .

Phylogenetic analyses
We included 23 species of Diplopoda in the phylogenetic analyses and selected L. forficatus in Chilopoda as an outgroup to root the phylogenetic trees, using BI and ML methods.Phylogenetic trees were constructed based on sequences of 13 PCGs (Fig. 4).The topologies of the BI and ML trees were similar to each other.S. grahami is clustered together with S. bungii and S. walkeri, which belong to the same genus Spirobolus.Narceus annularus is closely related to genus Spirobolus, which is consistent with the result of previous study 11 .Glomeridesmus spelaeus is distantly related to the other Diplopoda species, similar to the previous study 33 .In addition, phylogenetic trees also support the classification of genus Tropostreptus.Previous study on millipede mitochondria have shown www.nature.com/scientificreports/ that genus Tropostreptus is phylogenetically more closely related to Archispirostreptus gigas and Macrolenostreptus orestes 34 .The results of the our study on the phylogenetic analysis of mitochondria also support this.We demonstrate that the mitogenome might be an effective tool for millipede classification.Our study shows that mitogenome sequences are effective molecular markers for studying the phylogenetic relationships and evolution within Diplopoda, but the data that covered only 22 species, meaning it's still limited.

Figure 2 .
Figure 2. Relative synonymous codon usage of S. grahami, the stop codon is not included.

Figure 4 .
Figure 4. Phylogenetic trees of 23 Diplopoda species and an outgroup (Lithobius forficatus) based on 13 PCGs using the BI (a) and ML (b) method.

Table 1 .
The mitogenomes used in phylogenetic analyses.

Table 2 .
Annotation and organization of the complete mitogenome of S. grahami.

Table 4 .
The dN/dS values among Spirobolus species.