Complete Mitochondrial Genome and Phylogenetic Analysis of Tarsiger indicus (Aves: Passeriformes: Muscicapidae)

Tarsiger indicus (Vieillot, 1817), the White-browed Bush Robin, is a small passerine bird widely distributed in Asian countries. Here, we successfully sequenced its mitogenome using the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA) for PE 2 × 150 bp sequencing. Combined with other published mitogenomes, we conducted the first comprehensive comparative mitogenome analysis of Muscicapidae birds and reconstructed the phylogenetic relationships between Muscicapidae and related groups. The T. indicus mitogenome was 16,723 bp in size, and it possessed the typical avian mitogenome structure and organization. Most PCGs of T. indicus were initiated strictly with the typical start codon ATG, while COX1 and ND2 were started with GTG. RSCU statistics showed that CUA, CGA, and GCC were relatively high frequency in the T. indicus mitogenome. T. cyanurus and T. indicus shared very similar mitogenomic features. All 13 PCGs of Muscicapidae mitogenomes had experienced purifying selection. Specifically, ATP8 had the highest rate of evolution (0.13296), whereas COX1 had the lowest (0.01373). The monophylies of Muscicapidae, Turdidae, and Paradoxornithidae were strongly supported. The clade of ((Muscicapidae + Turdidae) + Sturnidae) in Passeriformes was supported by both Bayesian Inference and Maximum likelihood analyses. The latest taxonomic status of many passerine birds with complex taxonomic histories were also supported. For example, Monticola gularis, T. indicus, and T. cyanurus were allocated to Turdidae in other literature; our phylogenetic topologies clearly supported their membership in Muscicapidae; Paradoxornis heudei, Suthora webbiana, S. nipalensis, and S. fulvifrons were formerly classified into Muscicapidae; we supported their membership in Paradoxornithidae; Culicicapa ceylonensis was originally classified as a member of Muscicapidae; our results are consistent with a position in Stenostiridae. Our study enriches the genetic data of T. indicus and provides new insights into the molecular phylogeny and evolution of passerine birds.


Introduction
Passerines (Aves: Passeriformes) include a large number of species and are adapted to various ecological environments.The latest data show that the group has 145 families and 6695 species, accounting for 60% of all bird species; moreover, Muscicapidae is the third-largest family after Tyrannidae and Thraupidae, with 351 species from 53 genera [1].Tarsiger indicus (Vieillot, 1817) (Figure 1), the White-browed Bush Robin, is a small Muscicapidae bird widely distributed in Asian countries, including India, Nepal, Bhutan, Myanmar, Vietnam, and China [2].In China, T. indicus is found in Sichuan, Gansu, Shanxi, Hubei, Yunnan, Tibet, and Taiwan [3][4][5].It generally inhabits the coniferous forests and the mixed broadleaf-conifer forests between alpine rock valleys at altitudes of 2440-4270 m above sea level in western China; in addition, it also inhabits the bottom shrubland of dense forests at altitudes of 2300-3200 m above sea level in Taiwan Island of China.In the past, the Whitebrowed Bush Robin has been divided into three subspecies, including T. indicus indicus, T. i. yunnanensis, and T. i. formosanus [3].Recently, an integrative taxonomic investigation found the Taiwan endemic T. i. formosanus to be distinctive in genetics, song, and morphology from T. i. indicus and T. i. yunnanensis of the Sino-Himalayan mountains [6].In view of this, the T. i. formosanus subspecies has been suggested to be upgraded to the species T. formosanus, named the Taiwan Bush Robin [6,7].In addition, T. indicus has been included in the updated List of Terrestrial Wild Animals of Important Ecological, Scientific, and Social Value in China [8].Due to its wide geographical distribution and large population size, the conservation status of T. indicus is Least Concern in both the IUCN Red List of Threatened Species [2] and the Red List of China's Vertebrates [9].
Tarsiger indicus (Vieillot, 1817) (Figure 1), the White-browed Bush Robin, is a small Muscicapidae bird widely distributed in Asian countries, including India, Nepal, Bhutan, Myanmar, Vietnam, and China [2].In China, T. indicus is found in Sichuan, Gansu, Shanxi, Hubei, Yunnan, Tibet, and Taiwan [3][4][5].It generally inhabits the coniferous forests and the mixed broadleaf-conifer forests between alpine rock valleys at altitudes of 2440-4270 m above sea level in western China; in addition, it also inhabits the bottom shrubland of dense forests at altitudes of 2300-3200 m above sea level in Taiwan Island of China.In the past, the White-browed Bush Robin has been divided into three subspecies, including T. indicus indicus, T. i. yunnanensis, and T. i. formosanus [3].Recently, an integrative taxonomic investigation found the Taiwan endemic T. i. formosanus to be distinctive in genetics, song, and morphology from T. i. indicus and T. i. yunnanensis of the Sino-Himalayan mountains [6].In view of this, the T. i. formosanus subspecies has been suggested to be upgraded to the species T. formosanus, named the Taiwan Bush Robin [6,7].In addition, T. indicus has been included in the updated List of Terrestrial Wild Animals of Important Ecological, Scientific, and Social Value in China [8].Due to its wide geographical distribution and large population size, the conservation status of T. indicus is Least Concern in both the IUCN Red List of Threatened Species [2] and the Red List of China's Vertebrates [9].Vertebrate mitochondrial genomes (mitogenomes) are circular, typically 14,000-20,000 bp, and contain 13 protein-coding genes (PCGs), two ribosomal RNA (rRNAs), 22 transfer RNA genes (tRNAs), and one large non-coding D-loop region [10,11].The mitogenome has been extensively used in population genetics, population dynamics, and adaptive evolution studies of various animal groups [12][13][14][15][16], particularly in phylogenetic reconstruction among animal species [14,[16][17][18][19].It is worth emphasizing that mitochondrial genomes are more reliable in phylogenetic reconstruction than a single mitochondrial gene [20][21][22].However, the mitogenomes of the Muscicapidae family, a complex lineage of passerines, has been studied very little.So far, complete mitochondrial genomes of only 24 species (ca.7% of the overall clade) from 15 genera (ca.28%) within Muscicapidae family have published in the GenBank database (Table 1), mainly focusing on simple mitogenomic descriptions [23][24][25][26][27][28][29].Vertebrate mitochondrial genomes (mitogenomes) are circular, typically 14,000-20,000 bp, and contain 13 protein-coding genes (PCGs), two ribosomal RNA (rRNAs), 22 transfer RNA genes (tRNAs), and one large non-coding D-loop region [10,11].The mitogenome has been extensively used in population genetics, population dynamics, and adaptive evolution studies of various animal groups [12][13][14][15][16], particularly in phylogenetic reconstruction among animal species [14,[16][17][18][19].It is worth emphasizing that mitochondrial genomes are more reliable in phylogenetic reconstruction than a single mitochondrial gene [20][21][22].However, the mitogenomes of the Muscicapidae family, a complex lineage of passerines, has been studied very little.So far, complete mitochondrial genomes of only 24 species (ca.7% of the overall clade) from 15 genera (ca.28%) within Muscicapidae family have published in the GenBank database (Table 1), mainly focusing on simple mitogenomic descriptions [23][24][25][26][27][28][29].Genetic data on T. indicus are currently rare.In the GenBank database, only 39 nucleotide sequences have been uploaded as of August 2023, including 16 sequences of mitochondrial Cytb and ND2 genes.An accurate understanding of phylogeny is an important prerequisite for many studies of ecology and evolution [6,48].However, in terms of phylogenetic status, T. indicus was previously placed into the genus Luscinia [49] and is now still placed into the Turdidae family in some publications [50].
In order to better understand the mitogenome characteristics and the phylogenetic relationship of T. indicus, we sequenced its mitochondrial genome through high-throughput sequencing technology here.Combined with other published data, we conduct the first comprehensive comparative mitogenome analysis of Muscicapidae birds and reconstruct the phylogenetic relationships between Muscicapidae and related groups using a mitogenomic approach.

Materials
A subadult window victim, which was found dead, was collected from Yingjing Area of the Giant Panda National Park, Scihuan Province, China (29 • 33 ′ 39.50 ′′ N, 102 • 51 ′ 4.10 ′′ E, 2428 m above sea level) on 30 July 2022, and it was identified as T. indicus by morphological characters and mitochondrial Cytb blast.The extraction of genomic DNA from a pectoral muscle was carried out using the Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech Co., Ltd., Shanghai, China), according to the manufacturer's protocol.The specimen and its DNA were deposited at the Chengdu Research Base of Giant Panda Breeding (Dr.Jiabin Liu, jiabin_liu2013@126.com)with the voucher number PB2022027.

Mitogenomic Phylogenetic Analyses
Two rRNAs and 13 PCGs of T. indicus and 40 other Passeriformes birds belonging to 26 genera and seven families were used for mitogenomic phylogenetic analyses (Table 1).The taxonomy of all birds is based on the IOC World Bird List v13.2 [1].Pitta sordida (Passeriformes: Pittidae) was used as an outgroup based on its well-documented distant phylogenetic position from the ingroup [60][61][62].Two rRNA sequences were aligned in batches with MAFFT v7.505 [56] using '-auto' strategy and normal alignment mode, and 13 PCGs sequences were aligned in batches using the codon-aware program MACSE v2.06 [63], which preserves reading frame and allows incorporation of sequencing errors or sequences with frameshifts.Ambiguously aligned fragments of these 15 alignments were removed in batches using Gblocks v0.91b [64] with the following parameter settings: minimum number of sequences for a conserved/flank position (22/22), maximum number of contiguous non-conserved positions (8), minimum length of a block (10), allowed gap positions (with half).The 15 alignments were eventually concatenated into one multi-gene dataset consisting of a 13,893 bp sequence using PhyloSuite v1.2.3 [65].The concatenated multi-gene dataset was used to clarify the phylogeny using Bayesian Inference (BI) and Maximum Likelihood (ML) methods.A best-fit partition model (edge-linked) was selected by ModelFinder v2.2.0 [66] using a BIC criterion, and the results are shown in Table S1.BI phylogenies were inferred using MrBayes v3.2.6 [67] under a partition model (2 parallel runs, ten million generations, sampling every one thousand generations), in which the initial 25% of sampled data were discarded as burn-in.ML phylogenies were inferred using IQ-TREE v2.2.0 [68] under an edge-linked partition model for one hundred thousand ultrafast [69] bootstraps.

Structure and Organization of the T. indicus Mitogenome
Herein, the complete mitogenome of T. indicus (GenBank accession number: OR459825) was successfully sequenced and annotated.It was a circular and double-stranded DNA molecule, consisting of a typical structure with 13 PCGs, 2 rRNAs, 22 tRNAs, and a major non-coding D-loop region (Table 2; Figure 2).Among these 37 genes, 28 were located on the heavy strand, while the remaining nine genes, including eight tRNAs (trnQ, trnA, trnN, trnC, trnY, trnS2, trnE and trnP) and one PCG (ND6), were located on the light strand (Table 2; Figure 2).T. indicus showed the typical avian mitogenome order [21,70], which was also the ancestral avian arrangement found in many lineages of Passeriformes [21].The mitogenome structure and organization of T. indicus was consistent with those of T. cyanurus, but the T. indicus mitogenome (16,723 bp) was smaller in size than the T. cyanurus mitogenome (16,803 bp), and the interspecific difference was mainly caused by the size difference in the D-loop region located between trnE and trnF (Table 2).

Codon Usage
Among the 13 PCGs, the smallest one was ATP8, and the largest one was ND5, ranging from 168 bp to 1818 bp (Table 2).Most PCGs of T. indicus were initiated with the typical start codon ATG, while COX1 and ND2 were started with GTG (Table 2).The unusual start codon GTG was also observed in COX1 from other bird groups, such as Sittidae [71,72], Accipitridae [73,74], Phasianidae [75], Columbidae [76], and other Passeriformes species [24,25,30,36,45].The stop codons of 13 PCGs were quite varied in T. indicus.ATP6, ATP8, COX2, Cytb, ND3, ND4L, and ND6 were terminated with the representative stop codon TAA or TAG, COX1, ND1, and ND5 ended with AGA or AGG, while COX3, ND2, and ND4 were occasionally terminated with the truncated stop codon TA or T (Table 2).The incomplete stop codons TA and T are common in metazoan mitogenomes [19,20,51,72], and they can be converted to TAA by post-transcriptional modifications during the mRNA maturation process [77].The start and stop codons of the 13 PCGs were very similar in the mitogenomes of T. indicus and T. cyanurus, and the only difference was the stop codon of the ND6 gene: the former was TAG, while the latter was AGG (Table 2).
In addition, RSCU is a reference value to evaluate the frequency of codons encoding the same amino acid [80].When the RSCU ratio was greater than 1, it indicated that the codon occurred many times [80].Statistics on the RSCU showed that CUA (3.14), CGA (2.34), and GCC (2.18) were relatively high-frequency in T. indicus mitogenome (Figure 3; Table S2).RSCU values of T. cyanurus mitogenome was also summarized and compared with T. indicus, and these two mitogenomes had very similar characteristics of utilization rate of synonymous codon of single amino acids (Figure 3; Table S2).

Codon Usage
Among the 13 PCGs, the smallest one was ATP8, and the largest one was ND5, ranging from 168 bp to 1818 bp (Table 2).Most PCGs of T. indicus were initiated with the typical start codon ATG, while COX1 and ND2 were started with GTG (Table 2).The unusual start codon GTG was also observed in COX1 from other bird groups, such as Sittidae [71,72], Accipitridae [73,74], Phasianidae [75], Columbidae [76], and other Passeriformes species [24,25,30,36,45].The stop codons of 13 PCGs were quite varied in T. indicus.ATP6, ATP8, COX2, Cytb, ND3, ND4L, and ND6 were terminated with the representative stop codon TAA or TAG, COX1, ND1, and ND5 ended with AGA or AGG, while COX3, ND2, and ND4 were occasionally terminated with the truncated stop codon TA or T (Table 2).The incomplete stop codons TA and T are common in metazoan mitogenomes [19,20,51,72], and they can be converted to TAA by post-transcriptional modifications during the mRNA maturation process [77].The start and stop codons of the 13 PCGs were very similar in the mitogenomes of T. indicus and T. cyanurus, and the only difference was the stop codon of the ND6 gene: the former was TAG, while the latter was AGG (Table 2).
In addition, RSCU is a reference value to evaluate the frequency of codons encoding the same amino acid [80].When the RSCU ratio was greater than 1, it indicated that the codon occurred many times [80].Statistics on the RSCU showed that CUA (3.14), CGA (2.34), and GCC (2.18) were relatively high-frequency in T. indicus mitogenome (Figure 3; Table S2).RSCU values of T. cyanurus mitogenome was also summarized and compared with T. indicus, and these two mitogenomes had very similar characteristics of utilization rate of synonymous codon of single amino acids (Figure 3; Table S2).

Nucleotide Composition, Diversity, and Evolution
The overall nucleotide composition of the T. indicus mitogenome was 32.88% C, 29.63% A, 22.75% T, and 14.73% G, indicating that the mitogenomes were biased towards C and A bases, which had also been the case in previous studies of avian mitochondrial genomes [18,81].Its overall G + C content was 47.62%, which was similar to the 47.03% of the T. cyanurus mitogenome (Figure 4).Similar to most other birds [18,37,72], overall G + C content of the whole mitogenomes of all 25 Muscicapidae birds was slightly lower than their overall A + T content (Table S3).In terms of a single mitochondrial gene of Muscicapidae species including T. indicus, the individual G + C contents were very close to 50% (Table S3; Figures 4 and 5).Although T. indicus and T. cyanurus were closely related species, their individual G + C content had an inconsistent trend among all genes (Figure 4).
We also calculated the nucleotide skew of mitochondrial gene in 25 Muscicapidae species.The AT-skew values of the entire genome, concatenated rRNAs, concatenated PCGs, and single rRNA and PCG (except ND6) were positive, while the GC-skew values were negative (Figure 5), as was common in mitogenomes of Strigiformes [18] and Accipitriformes [74], indicating that Cs were more abundant than Gs, and As were more abundant than Ts.AT-skew and GC-skew were due to the different distribution of nucleotides between the two DNA strands, which further led to an asymmetry in the DNA strands [51,80].We also analyzed the correlation between nucleotide content and corresponding skew of all mitogenomes of Muscicapidae (Figure 5), but the correlation was weak and further confirmation was needed with more data.
To further understand the role of selective pressure on the mitochondrial PCGs among the Muscicapidae species, we calculated and compared the average Ka/Ks ratio for each PCG (Figure 6B).Ka/Ks ratio = 1 denotes neutral mutations, Ka/Ks ratio < 1 denotes negative selection, and Ka/Ks ratio > 1 denotes positive selection [82,83].Here, the average Ka/Ks ratio for all PCGs were consistently far lower than 1, indicating that all PCGs of Muscicapidae mitogenomes had experienced purifying selection.Among the 13 PCGs, ATP8 had the highest rate of evolution (0.13296), whereas COX1 had the lowest (0.01373) (Figure 6B), which was congruent with the previous studies in Passeriformes [51,71], Piciformes [79], Strigiformes [18], and penguins [84], as well as frogs [85].Therefore, our findings confirmed that COX1 experienced the strongest purifying selection and COX1 might play important roles in the evolution of avian mitogenomes.

Nucleotide Composition, Diversity, and Evolution
The overall nucleotide composition of the T. indicus mitogenome was 32.88% C, 29.63% A, 22.75% T, and 14.73% G, indicating that the mitogenomes were biased towards C and A bases, which had also been the case in previous studies of avian mitochondrial genomes [18,81].Its overall G + C content was 47.62%, which was similar to the 47.03% of the T. cyanurus mitogenome (Figure 4).Similar to most other birds [18,37,72], overall G + C content of the whole mitogenomes of all 25 Muscicapidae birds was slightly lower than their overall A + T content (Table S3).In terms of a single mitochondrial gene of Muscicapidae species including T. indicus, the individual G + C contents were very close to 50% (Table S3; Figures 4 and 5).Although T. indicus and T. cyanurus were closely related species, their individual G + C content had an inconsistent trend among all genes (Figure 4).Muscicapidae mitogenomes had experienced purifying selection.Among the 13 PCGs, ATP8 had the highest rate of evolution (0.13296), whereas COX1 had the lowest (0.01373) (Figure 6B), which was congruent with the previous studies in Passeriformes [51,71], Piciformes [79], Strigiformes [18], and penguins [84], as well as frogs [85].Therefore, our findings confirmed that COX1 experienced the strongest purifying selection and COX1 might play important roles in the evolution of avian mitogenomes.

Mitochondrial Phylogenomics
The ML and BI trees of the 13PCGs + 2rRNAs dataset had similar topologies, and most nodes were supported by high bootstrap percentages (BP) and Bayesian posterior probabilities (BPP) (Figures 7 and S1).

Mitochondrial phylogenomics
The ML and BI trees of the 13PCGs + 2rRNAs dataset had similar topologies, and most nodes were supported by high bootstrap percentages (BP) and Bayesian posterior probabilities (BPP) (Figures 7 and S1).Our results showed that Muscicapidae, Turdidae, and Paradoxornithidae were clustered into two monophyletic groups, and species of the same genus were clustered together with a high degree of confidence.Muscicapidae and Turdidae were sister groups (BP = 85, BPP = 1.00), and they clustered together with Sturnidae (BP = 100, BPP = 1.00),Our results showed that Muscicapidae, Turdidae, and Paradoxornithidae were clustered into two monophyletic groups, and species of the same genus were clustered together with a high degree of confidence.Muscicapidae and Turdidae were sister groups (BP = 85, BPP = 1.00), and they clustered together with Sturnidae (BP = 100, BPP = 1.00), which was consistent with a previous study [38].T. indicus and T. cyanurus were clustered together with high confidence (BP = 100, BPP = 1.00).These two Tarsiger birds were previously placed in the genus Luscinia [49].Although many species of Muscicapidae, such as M. gularis, T. indicus, and T. cyanurus were allocated to Turdidae in some older works [50,86] and the up-to-date NCBI taxonomy database; our phylogenetic topologies clearly supported their membership in the Muscicapidae family.It is important to note that the phylogenetic relationships between some genera within Muscicapidae are problematic between our study and a previous study [23].The position of C. semirufa in our ML and BI trees was not consistent, and different from the ML tree based on a 13 PCGs dataset in a Yang et al. study [23], and the degree of confidence of related branches was not high (Figures 7 and S1).Our ML and BI trees showed consistent topology (Calliope + Larvivora) + Ficedula (Figures 7  and S1); however, the ML tree of the Yang et al. study showed the diametrical topology Calliope + (Ficedula + Larvivora) with low bootstrap percentages [23].Complete mitogenomes may provide more accurate signals than gene fragments for phylogenetic reconstruction.Overall, the current 25 species represent only 7% of the old-world flycatchers group, so, in order to better resolve the phylogenetic relationships within Muscicapidae, it is still necessary to obtain more mitochondrial genome sequences of old-world flycatchers.
In addition, P. heudei, S. webbiana, S. nipalensis, and S. fulvifrons were classified into Muscicapidae in previous studies [57,71] and the NCBI taxonomy database, but our results showed that these species clustered into the Paradoxornithidae family [87].The taxonomic history of C. ceylonensis was also complex [72].C. ceylonensis was originally classified into the Muscicapidae family based on external morphology, reproductive habits, and nesting characteristics [86].Subsequently, it was classified into the family Rhipiduridae [88].Lately, the phylogenetic analyses based on multilocus sequence data revealed that C. ceylonensis was in fact a member of the Stenostiridae family [62].Here, we also clarified its taxonomic validity based on mitochondrial genome approach.

Conclusions
In this study, we successfully sequenced the mitogenome of T. indicus using the Illumina Novaseq 6000 platform with a paired-end read length of 150 bp.We also annotated and summarized its mitogenomic characteristics in detail.Importantly, we conducted the first comprehensive mitogenome analysis of Muscicapidae.The mitogenome of T. indicus mitogenome contained the typical avian mitochondrial gene arrangement.T. cyanurus and T. indicus shared very similar mitogenomic features.All 13 PCGs of the mitogenomes of Muscicapidae had experienced purifying selection.The monophylies of Muscicapidae, Turdidae, and Paradoxornithidae were strongly supported.The clade of ((Muscicapidae + Turdidae) + Sturnidae) in Passeriformes was supported by both BI and ML analyses.The current taxonomic status of many passerine birds with complex taxonomic histories were also supported.Our study provides the first complete mitochondrial genome of T. indicus to enrich its genetic data.A large number of studies on the mitochondrial genome of Muscicapidae are still needed in the future to further solve some phylogenetic problems.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes15010090/s1,Table S1: The partition and best-fit partition models used in this study; Table S2: The codon usage in the mitogenomes of T. indicus and T. cyanurus; Table S3: The nucleotide composition and skew in the mitogenomes of 25 species of Muscicapidae;

Figure 1 .
Figure 1.Reference image of adult T. indicus.The photo was taken by Taihu Hu on 20 February 2022 in Yingjing County, Ya'an City, Sichuan Province, China.

Figure 1 .
Figure 1.Reference image of adult T. indicus.The photo was taken by Taihu Hu on 20 February 2022 in Yingjing County, Ya'an City, Sichuan Province, China.

Figure 2 .
Figure 2. Graphical representation of Tarsiger indicus mitogenome.Genes outside the outer multicolored circle are located on the light strand counterclockwise, and those inside the outer circle

Figure 2 .
Figure 2. Graphical representation of Tarsiger indicus mitogenome.Genes outside the outer multicolored circle are located on the light strand counterclockwise, and those inside the outer circle are located on the heavy strand clockwise.Different colors indicate different types of genes and regions.The inner blue circle represents the local GC content.

Figure 5 .
Figure 5. Correlation between nucleotide content and corresponding skew in the mitogenomes of 26 species of Muscicapidae.(A) A + T content (%AT) vs. AT-skew; (B) G + C content (%GC) vs. GC-skew.Each dot represents a mitogenome.

Figure 6 .
Figure 6.Evolutionary rates of mitochondrial genes of 25 species of Muscicapidae.(A) Nucleotide diversity and percentage of variable sites; (B)The ratio of non-synonymous substitution rate and synonymous substitution rate.

Figure 6 .
Figure 6.Evolutionary rates of mitochondrial genes of 25 species of Muscicapidae.(A) Nucleotide diversity and percentage of variable sites; (B)The ratio of non-synonymous substitution rate and synonymous substitution rate.

Figure 7 .
Figure 7.The phylogenetic relationships of Passeriformes inferred by ML method based on the 13PCGs + 2rRNAs dataset.Numbers on nodes are the bootstrap percentages.

Figure 7 .
Figure 7.The phylogenetic relationships of Passeriformes inferred by ML method based on the 13PCGs + 2rRNAs dataset.Numbers on nodes are the bootstrap percentages.

Table 1 .
List of 41 species used for the comparative mitogenomic analyses and the mitogenomic phylogenetic analyses in this study.

Table 2 .
The mitochondrial genome comparison between T. indicus and T. cyanurus.
+ represents heavy strand, and -represents light strand.+represents heavy strand, and -represents light strand.

24 ,
15, x FOR PEER REVIEW 7 of 17 are located on the heavy strand clockwise.Different colors indicate different types of genes and regions.The inner blue circle represents the local GC content.