The Complete Mitochondrial Genome of Sphaeniscus atilius (Walker, 1849) (Diptera: Tephritidae) and Implication for the Phylogeny of Tephritidae

Shibao Guo1, Junhua Chen1, Nan Song2, Fangmei Zhang1*

1Xinyang Agriculture and Forestry University, Xinyang 464000, China

2Henan Agriculture University, Zhengzhou 450002, China

ABSTRACT

The complete mitochondrial genome of Sphaeniscus atilius was characterized and annotated in this study. The mitogenome was 16,854 bp in length and encoded 37 typical mitochondrial genes, including 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 1 control regions. The total length of the 13 PCGs was 11,140 bp, and the AT content was 79.8%. There were five types of start codons, ATT (nad2, nad3, nad5, and nad6), ATG (cox2, cox3, atp6, nad4, nad4l, and cob), CGA (cox1), as well as ATC (atp8) and ATA (nad1). Most of the PCGs had typical TAA stop codons, except nad5 which terminated with incomplete forms T-. Ile, Phe, Leu and Asn were the most frequently used amino acids in mitochondrial PCGs. Most tRNA genes could be folded into the typical cloverleaf structure, except trnS1 and trnT which lacked the dihydrouridine (DHU) and TΨC arms, respectively. Phylogenetic analyses based on 13 protein-coding genes among the available sequenced species of family Tephritidae by maximum likelihood and bayesian inference methods suggested the genus relationship of Tephritidae: ((Bactrocera, Dacus, Zeugodacus), Felderimyia, Anastrepha), (Acrotaeniostola, (Neoceratitis, Ceratitis), Euleia, Rivellia), (Procecidochares, (Tephritis, Sphaenisscus))))). Our results presented the first mitogenome from Sphaeniscus and provide insights into the species identification, taxonomy and phylogeny of S. atilius.

Article Information

Received 12 September 2023

Revised 09 December 2023

Accepted 26 December 2023

Available online 04 April 2024

(early access)

Published 08 November 2024

Authors’ Contribution

Data curation: SG, NS. Formal analysis: FZ, NS. Investigation: JC. Methodology: FZ, NS. Funding acquisition: SG. Supervision: JC. Writing-original draft: SG, FZ. Writing-review and editing: SG, FZ, NS.

Key words

Tephritidae, Sphaeniscus atilius, Mitochondrial genome, Phylogeny

DOI: https://dx.doi.org/10.17582/journal.pjz/20230912053433

* Corresponding author: zhangfm@xyafu.edu.cn

0030-9923/2024/0006-2929 $ 9.00/00

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Tephritidae, one of the largest families of Diptera, consists of more than 500 genera and almost 5,000 named species and predominantly distributes throughout the temperate and tropical areas of the world (Pape et al., 2009; Aluja and Norrbom, 2000; Mazzon et al., 2021). This family is also referred to as true fruit flies, with hundreds of fruit-eating species accounting for about 40% of the species. It has been reported to attack a great variety of fruit plants, bamboo culms, vegetables, flowers, and seeds (Korneyev, 1999; Dohm et al., 2014). In practice, some of the fruit-eating species in the Anastrepha, Bactrocera, Ceratitis, Dacus, and Rhagoletis genera have been considered serious agricultural pests due to their significant economic impact on the production of fruit crops and stored fruit (White and Elson-Harris, 1992; Aluja and Mangan, 2008). Melon fly, Zeugodacus cucurbitae (Siderhurst and Jang, 2010), medfly, Ceratitis fasciventris (Drosopoulou et al., 2017), together with Bactrocera latifrons (Yong et al., 2016), are well-known examples.

The insect mitochondrial genome has been regarded as a useful molecular marker in studies of phylogenetic and evolutionary analysis, genetic diversity, and species delimitation at the genus or species level, due to its small size, high copy numbers, maternal inheritance, unambiguous orthologous genes, conserved gene composition, and high evolutionary rate (Cameron, 2014; Song et al., 2016; Wilson and Xu, 2012). Generally, the typical insect mitochondrial genome is a highly conserved circular molecule ranging in size from approximately 14 to 40 kbp, encoding a fixed set of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region (CR) or the A+T-rich region (Du et al., 2021; Wolstenholme, 1992), with only a few exceptions. For example, long gene intergenic spacers, gene rearrangements, and gene loss have also been reported in different orders of insects (Du et al., 2017; Gong et al., 2018; Yan et al., 2022).

Partial mitochondrial gene sequences have become a preferred approach for inferring phylogenetic and molecular systematic studies in several insect groups, such as Arma custos and Picromerus lewisi (Hemiptera) (Mu et al., 2022), Episymploce splendens (Blattodea) (Yan et al., 2022), and Haematopinus tuberculatus (Psocodea) (Fu et al., 2022), Coomaniella copipes, Coomaniella dentata, and Dicerca corrugate (Coleoptera) (Huang et al., 2022), including the Tephritidae family (Ceratitis fasciventris (Drosopoulou et al., 2017), Bactrocera carambolae (Drosopoulou et al., 2019), Bactrocera biguttula (Teixeira et al., 2019), Zeugodacus cucurbitae (Zhou et al., 2020), and Lepidotrigona flavibasis (Wang et al., 2021). The aforementioned studies of mitochondrial gene sequences explored the origin and evolution of insects, explained the species and evolution of the system, and revealed the geographical distribution of intraspecific polymorphism.

Sphaeniscus atilius (Diptera: Tephritidae: Sphaeniscus), which is distributed from India to Russia, Korea, Japan, Australasian and Oceanian regions, can be morphologically distinguished from any other tephritid species based on clear diagnostic morphological features, including an almost entirely dark brown body, the number of orbital frontal setae, and an almost perpendicular base of the discal band of the wing (Han et al., 2010). To date, the complete mitochondrial genomes (mitogenomes) of 46 species, belonging to 14 genera of Tephritidae, are available in GenBank (https://www.ncbi.nlm.nih.gov/nucleotide/). All of these species belong to 5 subfamilies, Sciomyzidae, Sepsidae, Lauxaniidae, Celyphidae, Platystomatidae, and Tephritidae, respectively. However, there are no reports on the molecular phylogeny studies of the mitochondrial genome information in S. atilius, which limits our comprehensive understanding of the evolutionary and phylogenetic relationships of S. atilius.

In the current study, we sequenced, annotated, and described the complete mitogenome of S. atilius using next-generation sequencing, which is the first complete mitogenome sequence reported in the genus Sphaeniscus. We predicted and analyzed the gene organization, base composition, PCGs, codon usage, and the structure of the tRNAs and rRNAs of its mitochondrial genome. Additionally, we carried out phylogenetic analyses based on maximum likelihood (ML) and Bayesian Inference (BI) methods to assess the phylogenetic position of S. atilius. These results will be greatly helpful for clarifying the phylogenetic status and relationships between different species of Tephritidae.

MATERIALS AND METHODS

Taxon sampling and DNA extraction

Specimens of S. atilius were collected in Mount Jigong, Xinyang, Henan Province, China (31°48′43″N, 114°05′43″E), in June 2020. Specimens were preserved in 100% ethanol, and stored at -20°C. After morphological identification, total genomic DNA was extracted from muscle tissue of pre-thoraxes using Tissue DNA kit (TIANGEN Biotech, Beijing, China) according to the manufacture’s protocol. The impurities and concentration were detected by agarose (1%) electrophoresis and Nanodrop spectrophotometer (ThermoFisher Scientific, Waltham, MA), respectively.

Sequencing and assembling of mitochondrial genome

Genome sequencing was performed on an Illumina HiSeq 2500 platform (150 bp paired-end reads). The TruSeq library was prepared with an insert size of 400 bp. Quantity of sequencing data for each sample was at least 20 Gb. Sequencing was performed at Beijing Novogene Bioinformatics Technology Co., Ltd, China. A total of 2 Gb raw paired reads were generated. Data filtering was conducted using NGS QC-Toolkit v2.5 (Patel and Jain, 2012). After removing the connector and the unmatched, short, and poor-quality reads, the high-quality reads (Q20 > 90% and Q30 > 80%) were used for genome assembly.

De novo assembly was performed with IDBA-UD v. 1.1.1 (Peng et al., 2012). The parameter settings were as follows: 200 minimum sizes of contig, 41 as the minimum k-mer size, 10 as an iteration size, and 91 as the maximum k-mer size. The pre sequenced mitochondrial cox1 gene was used to bait the mitogenome from the assembled contigs.

Mitochondrial genome annotation and analysis

Preliminary annotation of mitogenome was conducted using MITOS (http://mitos.bioinf.uni-leipzig.de/index.py) (Bernt et al., 2013). The gene boundaries were refined by blasting against closely related species. The secondary structures of tRNA genes were predicted in MITOS. The structure map of the mitogenome was drawn using OGDRAW v1.3.1 (Greiner et al., 2019). The annotated mitogenome sequence of S. atilius was deposited at GenBank (accession number OQ909100).

The mitogenome nucleotide composition and relative synonymous codon usage (RSCU) were computed using MEGA 7.0 (Kumar et al., 2016). AT and GC skews were calculated following the formula: AT Skew = (A − T)/(A + T), GC Skew = (G − C)/(G + C) (Perna and Kocher, 1995). The nucleotide diversity (Pi) and nonsynonymous (Ka)/synonymous (Ks) mutation rate ratios were calculated by DnaSP v5.10.01 (Librado and Rozas, 2009).

Phylogenetic analysis

The new mitogenome sequence of S. atilius was merged with the existing dipteran mitogenome sequences. The amino acid sequences of 13 protein coding genes of 38 dipteran species were aligned individually using MAFFT with default parameters (Katoh and Standley, 2013). We chose Pachycerina (Pachycerina decemlineata) and Sciomyza (Sciomyza simplex) as outgroups. The alignments were trimmed by trimAl (Capella-Gutierrez et al., 2009). The resulting alignments were concatenated using SequenceMatrix 1.8 (Vaidya et al., 2011).

The phylogenetic analysis was conducted using ML and BI methods. ML tree was reconstructed using IQ-TREE v2.2.3 (Minh et al., 2020). The best-fitting model for the alignment was estimated by ModelFinder (Kalyaanamoorthy et al., 2017). Branch support was assessed by 10,000 ultrafast bootstrap replicates (Hoang et al., 2018). MrBayes 3.1 was used to reconstruct the BI tree, and four independent Markov chain runs were performed for 1,000,000 metropolis–coupled (MCMC) generations, sampling a tree every 100 generations.

RESULTS

Genome organization

The length of the S. atilius mitogenome was 16,854 bp in length, which consisted of the typical 37 genes including PCGs, 22 tRNA genes, and two rRNA genes (Fig. 1, Table I). In addition, a major non-coding region known as the control region (CR) or A+T-rich region was found between rrnS and trnI. The heavy chain (H-strand) encoded 23 genes (nine PCGs and 14 tRNAs). The remaining 4 PCGs, 8 tRNAs and 2 rRNAs were transcribed in the light chain (L-strand).

 

Table I. Summary of the mitogenome of S. atilius.

Gene	Strand	Location	Size (bp)	Anti codon	Start codon	Stop codon	Intergenic nucleotides
trnI	H	1-67	67	GAT			7
trnQ	L	67-135	69	TTG			-1
trnM	H	157-228	72	CAT			21
nad2	H	243-1250	1008		ATT	TAA	14
trnW	H	1242-1309	68	TCA			-8
trnC	L	1311-1376	66	GCA			1
trnY	L	1377-1443	67	GTA			47
cox1	H	1491-3026	1536		CGA	TAA	-5
trnL2	H	3021-3086	66	TAA			10
cox2	H	3110-3793	684		ATG	TAA	23
trnK	H	3810-3880	71	CTT			16
trnD	H	3881-3954	74	GTC			0
atp8	H	3947-4111	165		ATC	TAA	-7
atp6	H	4240-4917	678		ATG	TAA	28
cox3	H	4940-5725	786		ATG	TAA	22
trnG	H	5726-5791	66	TCC			0
nad3	H	5864-6217	354		ATT	TAA	72
trnA	H	6245-6313	69	TGC			27
trnR	H	6342-6408	68	TCG			28
trnN	H	6409-6471	65	GTT			0
trnS1	H	6474-6540	67	GCT			2
trnE	H	6532-6997	66	TTC			-8
trnF	L	7058-7125	68	GAA			60
nad5	L	7141-8854	1714		ATT	T(AA)	15
trnH	L	8855-8920	66	GTG			0
nad4	L	8919-10259	1341		ATG	TAA	-1
nad4L	L	10262-10552	291		ATG	TAA	2
trnT	H	10553-10616	64	TGT			0
trnP	L	10637-10702	66	TGG			20
nad6	H	10700-11206	507		ATT	TAA	-2
cob	H	11265-11401	1137		ATG	TAA	58
trnS2	H	11419-11485	67	TGA			17
nad1	L	11496-12423	939		ATA	TAA	10
trnL1	L	12424-12505	66	TAG			16
16sRNA	L	12547-13855	1309				41
trnV	L	13854-13925	72	TAC			-1
12sRNA	L	13913-14711	799				-12
CR		14712-16854	2143				0

 

Note: Strand of the genes is presented as L for majority and H for minority strand. In the column for intergenic length, a positive sign indicates the interval in base pairs between genes, while the negative sign indicates overlapping base pairs between genes.

 

Table II. Composition and skewness of the S. atilius mitogenome.

S. atilius	Size (bp)	A (%)	T (%)	G (%)	C (%)	A+T (%)	G+C (%)	AT-skew	GC-skew
Genome	16,854	41.80	39.92	7.67	10.61	81.72	18.28	0.02	-0.16
Protein-coding genes	11,140	34.15	45.67	10.30	9.88	79.82	20.18	-0.14	0.02
tRNA genes	1,490	41.41	39.06	10.94	8.59	79.47	19.53	0.03	0.12
rRNA genes	2108	39.28	42.88	11.48	6.36	82.16	17.84	-0.04	0.28
Detected CR	1674	45.65	42.60	1.61	7.46	88.25	9.07	0.03	0.64

 

CR, control region

There were five intergenic overlapping regions totaling 29 bp, with varying lengths of 1-8 bp, which ware mainly present in the tRNA genes. The two longest overlapping regions, both with a length of 8 bp, occured between nad2 and trnW and between trnS1 and trnE. Sixteen intergenic spacer regions were identified, totaling 555 bp in length, with the longest spacer sequence (72 bp) located between trnG and nad3, followed by a 60 bp spacer between trnE and trnF. There were also three regions without gene overlaps or intergenic spacers.

The mitogenome nucleotide composition was 41.80% for A, 39.92% for T, 7.67% for G, and 10.61% for C, respectively. The mitogenome was significantly biased to A+T (81.72%). The whole mitogenome of S. atilius exhibited a positive AT skew (0.02) and a negative GC skew (-0.16) (Table II).

Protein-coding genes

The total length of the 13 PGGs of in the S. atilius mitogenome was 11,140 bp, accounting for 66.10% of the whole mitogenome sequence, and encoding a total of 3,612 codons. Among them, nad5 (1,714 bp) was found to be the longest sequence, and nad4L (291 bp) was the shortest (Table I). Nine PCGs (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, and cob) were coded on the H-strand, while the remaining four PCGs (nad5, nad4, nad4L, and nad1) were located on the L-strand. The content of AT and GC was 79.82% and 20.18% in the 13 PCGs, exhibiting a highly AT bias (Table II). The AT skew was negative (-0.14), while the GC skew was positive (0.02) in the PCGs. Additionally, six PCGs (cox2, cox3, atp6, nad4, nad4L, and cob) initiated with an ATG start codons, four PCGs (nad2, nad3, nad5 and nad6) used ATT as the start codon, nad1 used ATA, atp8 used ATC, and cox1 used CGA. The termination codons of 12 PCGs were TAA. Only the nad5 used an incomplete stop codon T.

The amino acid usage of the 13 PCGs and the relative synonymous codon usage (RSCU) frequency are shown in Figure 2 and Table III. The most frequently used amino acids in mitochondrial PCGs were Ile, Phe, Leu, and Asn, accounting for 16.68%, 12.51%, 10.86%, and 8.13% of the total amino acids, respectively (Fig. 2A). The most frequently used codons were UUU (436), AUU (436), UUA (342), and AAU (277). The relatively scarce amino acids were Met (0.62%), Trp (0.67%), Arg1 (0.93%), and Asp (1.23%). The most frequent synonymous codons were UUA, AGA, and GUU, with UUA having the highest frequency of relative synonymous codons (RSCU = 4.86) (Fig. 2B).

.

The nucleotide diversity (Pi) of the PCGs among 46 species was calculated, ranging from 0.14 to 0.22 (Fig. 3). Among them, nad6 (Pi=0.22) showed the most diverse nucleotide variability among all PCGs, followed by nad2 (Pi=0.21), nad3 (Pi=0.19), and atp8 (Pi=0.18). The nad5 (Pi=0.14), nad1 (Pi=0.15), and cox3 (0.15) genes exhibited relatively low values of nucleotide variability. The ratio of Ka/Ks was calculated for each gene of the 13 PCGs (Fig. 3). The value of the cox3 gene (Ka/Ks=1.26) was higher than others. Meanwhile, the ratio of Ka/Ks of other 12 PCGs were all significantly less than 1, with the value of cox1 gene being the lowest (Ka/Ks=0.06).

 

Table III. Codon number and RSCU in S. atilius mitochondrial PCGs.

Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	431	1.77	UCU(S)	82	1.65	UAU(Y)	183	1.8	UGU(C)	36	1.18
UUC(F)	55	0.23	UCC(S)	27	0.54	UAC(Y)	20	0.20	UGC(C)	25	0.82
UUA(L)	342	4.86	UCA(S)	109	2.19	UAA(*)	60	1.05	UGA(*)	93	1.62
UUG(L)	42	0.60	UCG(S)	13	0.26	UAG(*)	19	0.33	UGG(W)	26	1.00
CUU(L)	23	0.33	CCU(P)	32	1.58	CAU(H)	56	1.93	CGU(R)	8	0.35
CUC(L)	0	0	CCC(P)	8	0.40	CAC(H)	2	0.07	CGC(R)	0	0
CUA(L)	15	0.21	CCA(P)	40	1.98	CAA(Q)	47	1.96	CGA(R)	27	1.17
CUG(L)	0	0	CCG(P)	1	0.05	CAG(Q)	1	0.04	CGG(R)	1	0.04
AUU(I)	436	2.01	ACU(T)	68	1.59	AAU(N)	277	1.75	AGU(S)	44	0.88
AUC(I)	33	0.15	ACC(T)	22	0.51	AAC(N)	39	0.25	AGC(S)	24	0.48
AUA(I)	183	0.84	ACA(T)	67	1.57	AAA(K)	123	1.73	AGA(R)	59	2.57
AUG(M)	24	1.00	ACG(T)	14	0.33	AAG(K)	19	0.27	AGG(R)	43	1.87
GUU(V)	52	2.31	GCU(A)	48	2.13	GAU(D)	43	1.79	GGU(G)	32	0.98
GUC(V)	2	0.09	GCC(A)	4	0.18	GAC(D)	5	0.21	GGC(G)	1	0.03
GUA(V)	33	1.47	GCA(A)	36	1.60	GAA(E)	54	1.96	GGA(G)	91	2.78
GUG(V)	3	0.13	GCG(A)	2	0.09	GAG(E)	1	0.04	GGG(G)	7	0.21

 

tRNAs and rRNAs

The 22 tRNA genes of the mitogenome of S. atilius had a total of 1,490 bp in length, 9.51% of the entire mitogenome, ranging from 65 bp (trnN) to 74 bp (trnD) (Table I). Among these, 14 genes (trn1, trnM, trnW, trnL2, trnK, trnD, trnG, trnA, trnR, trnN, trnS1, trnE, trnT, and trnS2) were located on the H-strand and the remaining eight genes (trnQ, trnC, trnY, trnF, trnH, trnP, trnL1, and trnV) were located on the L-strand. Through the analysis of the secondary structure of the tRNAs (Fig. 4), most tRNA genes could be folded into the typical cloverleaf secondary structure, while trnS1 and trnT lacked the dihydrouridine (DHU) and TΨC arms, respectively. In the secondary structures of tRNAs of S. atilius (Fig. 3), three or four base pairs in the DHU arms, and four or five base pairs in the TΨC arms. Except the classic base pairs (A-U and C-G), fourteen wobble base pairs (G-U) were detected in nine genes (trnA, trnC, trnF, trnG, trnH, trnP, trnQ, trnT, and trnV), which occured in the amino acid-accepting arms, anticodon arms, TψC arms or DHU arms. Of them, trnH had the highest rate (three pairs each). Besides, five pairs of U-U base mismatches in trnA, trnG, trnL1, trnR, trnV, and trnW and one mismatches base in trnN were found in the TψC arms. The AT and GC content were 79.47% and 19.53% in the 22 tRNA genes, respectively, with a positive AT skew (0.03) and GC skew (0.12) (Table II).

There were two rRNAs in the mitogenome of S. atilius: a 1,309 bp 16S rRNA (rrnL) and a 799 bp 12S rRNA (rrnS) (Table I). The 16S rRNA gene was located between trnL1 and trnV, while the 12S rRNA gene was located between trnV and the control region. Both 16S rRNA and 12S rRNA were embedded in the L-strand. The AT content was 82.16%, with a negative AT skew (-0.04) and positive GC skew (0.28) (Table II).

 

Control region

The mitogenome of S. atilius contained one large putative control region (CR), and the length of the CR was 2143 bp. It was located between the rrnS and trnI, trnE and trnF, nad6 and cob, and rrnS and trnI, respectively. The AT content of non-coding regions (88.25%) was obviously higher than other regions. Additionally, the positive AT skew (0.03) and GC skew (0.64) were detected in the control region (Table II).

 

 

Phylogenetic analysis

The same phylogenetic relationships of S. atilius and other Tephritidae species were produced by ML and BI methods. The result revealed that the thirteen genera of Tephritidae species followed the following monophyletic relationships: ((Bactrocera, Dacus, Zeugodacus), Felderimyia, Anastrepha), (Acrotaeniostola, (Neoceratitis, Ceratitis), Euleia, Rivellia), (Procecidochares, (Tephritis, Sphaenisscus))))). Of them, Bactrocera (12 exemplars) formed a separate clade at the top of phylogenetic tree, and formed the sister group of a clade including Dacus (4 exemplars), Zeugodacus (8 exemplars), Felderimyia and Anastrepha. Ceratitis (4 exemplars) formed a monophyletic group and was sister to a clade comprising Acrotaeniostola and Neoceratit. Euleia and Rivellia formed the separate clades, respectively. Sphaenisscus and Tephritis clustered together showing a high statistical support value (PP=1, BS=100), and formed a sister group to Procecidochares (1 exemplar). S. atilius and T. femoralis were closely related and formed a sister group to Procecidochares utilis.

DISCUSSION

The complete mitogenome of S. atilius was a circular, double-stranded DNA molecule with a total length of 16, 854 bp, which was similar to that of other Tephritoidea insects analyzed, ranging from 15,117 bp (Tephritis femoralis) to 16,739 bp (Anastrepha fraterculus) (Table IV). It has the typical organization and composition of an insect mitochondrion, including 13 PCGs, 22 tRNA genes, and 2 rRNA genes, which was consistent with the existing mitogenomes of Tephritoidea, such as Bactrocera carambolae (Drosopoulou et al., 2019), Zeugodacus cucurbitae (Zhou et al., 2020), Dacus haikouensis (Wang et al., 2022). The nucleotide composition of all regions showed a strong AT bias (Nguyen et al., 2020), as seen in other insects (Yan et al., 2022; Mu et al., 2022; Lv et al., 2021). The AT-skew of the entire mitogenome was positive (0.02), while the GC skew is negative (-0.16), indicating that the content of bases C was higher than that of G, and A was higher than T in the whole genome.

The mitogenome commonly exhibited compact arrangement, such as small intergenic spacers or overlapping genes (Ojala et al., 1981). In the present study, intergenic overlapping regions ranging from 1 to 8 bp, with a total length of 29 bp. Overlapping regions of similar size were common among Tephritoidea insects (Drosopoulou et al., 2017, 2019; Yong et al., 2016; Teixeira et al., 2019), while their positions varied across species. For example, the longest overlaps were found between atp8 and atp6 in B. carambolae (Drosopoulou et al., 2019), and between nad2 and trnW, and between trnS1 and trnE in S. atilius. Most of the gene overlaps occurred in tRNA genes due to the lower evolutionary constraints of these genes (Yuan et al., 2021).

The non-coding intergenic spacers, which were composed of less than 10 non-coding nucleotides in the mitochondria of most animals, which contributed to species identification and the evolution of insect mitochondrial genomes (Yan et al., 2022). Sixteen intergenic spacer regions were examined, with a total length of 555 bp, and the longest spacer sequence (72 bp) was located between trnG and nad3. Larger intergenic spacers in mitogenomes had been reported in Tephritoidea insects, such as 94 bp in Bactrocera latifrons, 82 bp in Bactrocera melastomatos and 79 bp in Bactrocera umbrosa (Yong et al., 2016). Generally, the duplication/random loss model and slipped-strand mispairing can be used to explain the origin of mitogenome intergenic spacers (Du et al., 2017; Cheng et al., 2016). Whether these long spacer regions being functional was controversial (Yan et al., 2022).

The 13 PCGs of the S. atilius mitogenome were found to be 11,140 bp in length and used a variety of start codons, including ATG for cox2, cox3, atp6, nad4, nad4L, and cob; ATT for nad2, nad3, nad5, and nad6; ATA for nad1; CGA for cox1; and ATC for atp8, as reported for Bactrocera biguttula (Teixeira et al., 2019) and Bactrocera carambolae PCGs (Drosopoulou et al., 2019). Alternative start codons had also been found in other insects, such as TTG in Arma custos and Picromerus lewisi (Mu et al., 2022) and GTG in Nisia fuliginosa (Lv et al., 2021). The cox1 gene in the S. atilius mitogenome used CGA as the starting codon, consistent with other known insects (Yang et al., 2019). However, the starting codon of cox1 was not always uniform, for example, TTG in Episymploce splendens (Yan et al., 2022) and ATA in Anastatus fulloi (Yi et al., 2022). The typical termination codons TAA was employed in 12 PCGs, which was common among metazoans (Yan et al., 2022; Yi et al., 2022), with one exception of an incomplete stop codon T for nad5. This exception was commonly observed in arthropod mitogenomes (Huang et al., 2022; Yi et al., 2022), and might be attributed to post-transcriptional modification during the mRNA maturation process (Boore, 1999; Lv et al., 2021).

The four most frequently used codons, UUU (Phe), AUU (Ile), UUA (Leu), and AAU (Asn) were observed in the S. atilius mitogenome, which was similar to other insect mitogenomes, such as those of Ephemeroptera (Li et al., 2021), Coleoptera (Zeng et al., 2021) and Hemiptera (Nguyen et al., 2020). Meanwhile, the RSCU analysis of the PCGs also indicated that A and U were the components that contributed to the high A+T bias of the full mitogenome. The nucleotide diversity (Pi) and the ratio of Ka/Ks of the PCGs among 43 Tephritoidea species

 

Table IV. List of taxa used for phylogenetic analysis.

Superfamily	Family	Genus	Species	GenBank accession	Length (bp)
Tephritoidea	Tephritidae	Acrotaeniostola	Acrotaeniostola dissimilis	MH900079	15,384
		Anastrepha	Anastrepha fraterculus	KX926433	16,739
			Bactrocera biguttula	MK293875	15,829
			Bactrocera carambolae	EF014414	15,915
			Bactrocera correcta	JX456552	15,936
			Bactrocera dorsalis	DQ845759	15,915
			Bactrocera limbifera	MG566056	15,860
			Bactrocera melastomatos	KT881557	15,945
			Bactrocera oleae	GU108463	15,821
			Bactrocera ritsemai	MF668132	15,927
			Bactrocera rubigina	MN714223	15,285
			Bactrocera ruiliensis	MN477221	15,870
			Bactrocera tuberculata	MT196006	15,273
			Bactrocera zonata	KP296150	15,935
		Ceratitis	Ceratitis capitata	AJ242872	15,980
			Ceratitis fasciventris	KY436396	16,017
			Ceratitis quilicii	MT998948	16,035
			Ceratitis rosa	MT997010	16,047
		Dacus	Dacus bivittatus	MG962404	15,833
			Dacus conopsoides	MH351199	15,852
			Dacus longicornis	NC_032690	16,253
			Dacus trimacula	MK940811	15,851
		Euleia	Euleia heraclei	MT410819	15,514
		Felderimyia	Felderimyia fuscipennis	MT702879	16.536
		Neoceratitis	Neoceratitis asiatica	MF434829	15,481
		Procecidochares	Procecidochares utilis	KC355248	15,922
		Sphaeniscus	Sphaeniscus atilius	OQ909100	16,285
		Zeugodacus	Zeugodacus caudatus	KT625491	15,866
			Zeugodacus cilifer	MT702880	15,843
			Zeugodacus cucurbitae	JN635562	15,825
			Zeugodacus depressus	KY131831	15,832
			Zeugodacus diaphorus	KT159730	15,890
			Zeugodacus proprediaphora	MN688227	15,829
			Zeugodacus scutellatus	KP722192	15,915
			Zeugodacus tau	KP711431	15,687
	Platystomatidae	Rivellia	Rivellia syngenesiae	MT410799	17,835
Lauxanioidea	Lauxaniidae	Pachycerina	Pachycerina decemlineata	NC_034923	16,286
Sciomyzoidea	Sciomyzidae	Sciomyza	Sciomyza simplex	MT410781	16,553

 

were calculated. The results showed that nad6 exhibited the most diverse nucleotide variability among all PCGs, while nad5, nad1, and cox3 exhibited a relatively low variation rate and were the most conserved genes. The overall ratios of Ka/Ks for most PCG genes were significantly less than 1, which suggests that these PCGs were under purifying selection. The cox1 gene had the lowest Ka/Ks ratio (Ka/Ks=0.06), indicating that this gene had a relatively slow evolutionary rate (Hurst, 2002). This phenomenon occurred in almost all animals (Xiao et al., 2019), and had been subjected to species identification and evolutionary analysis in various arthropod species in Tephritoidea, as well as like in other insects (Demari-Silva et al., 2015; Zhou et al., 2020).

As in other insects, most tRNA genes in S. atilius could be folded into the typical clover-leaf secondary structure, while trnS1 lacked the dihydrouridine (DHU) arm and trnT lacked the TΨC arm. This feature of trnS1 had been observed in many other insect mitogenomes (Huang et al., 2022; Lv et al., 2021; Yuan et al., 2021; Yi et al., 2022; Soumia et al., 2022). However, the feature of trnT was less found in other insects. Other tRNAs, trnA also lacked the TΨC arm as described in Chrysodeixis acuta (Soumia et al., 2022). So, we speculated that this phenomenon could cause by the loss of gene in the process of evolution. We have added reason in the discussion. In addition, 14 wobble base pairs (G-U) and 5 pairs of U-U base mismatches in the tRNA genes of the S. atilius mitogenome were observed. Previous reports had suggested that wobble and mismatched pairs, which commonly occurred in insect tRNAs, were usually corrected through the editing process and sustain the transport function (Varani and McClain, 2000; Lavrov et al., 2000). The length, location, and base composition of the two rRNA genes were similar to those of other Tephritoidea insects, such as Bactrocera arecae (Yong et al., 2015) and Bactrocera biguttula (Teixeira et al., 2019).

In addition, the S. atilius mitogenome contained one large non-coding regions of which, the one located between rrnS and trnI was supposed to act as the origin of genome replication and gene transcription (Wolstenholme, 1992; Boore, 1999). The control region of insect mitogenomes ranged in size from tens of to several thousands of base pairs (Zhang et al., 1995; Lewis et al., 1995; Inohira et al., 1997). The control region was a source of length variation in the mitogenomes (Zhang et al., 1995; Lewis et al., 1995; Inohira et al., 1997). In the S. atilius mitogenome, 2143 bp in length was sequenced, which was more than the longest control region of 1,141 bp in Tephritidae insects (Mu et al., 2022). The control region was located between rrnS and trnI genes and also had a higher AT content (88.25%), compared to other Tephritidae insects, such as Bactrocera arecae (86.0%) (Yong et al., 2015), Bactrocera melastomatos (89.0%) (Yong et al., 2016), and Ceratitis fasciventris (90.24%) (Drosopoulou et al., 2017).

Phylogenetic analyses were based on the nucleotide sequences of the 13 PCGs used ML and BI methods to construct phylogenetic trees from the mitogenomes of 38 species of Diptera to elaborate phylogenetic relationship. The same phylogenetic relationships of S. atilius and other Tephritidae species were produced by two methods. In the present study, the phylogenetic relationships within Tephritidae could be presented as follows: ((Bactrocera, Dacus, Zeugodacus), Felderimyia, Anastrepha), (Acrotaeniostola, (Neoceratitis, Ceratitis), Euleia, Rivellia), (Procecidochares, (Tephritis, Sphaenisscus))))), in concordance with the findings of previous phylogenetic studies (Drosopoulou et al., 2019; Teixeira et al., 2019; Jia et al., 2019; Yang et al., 2020). The results showed that four genera (Felderimyia, Zeugodacus, Dacus and Bactrocera) were closely related and formed a sister group. Dacus and Zeugodacus constituted a sister to Bactrocera. This pattern had been demonstrated in the previous studies (Krosch et al., 2012; Virgilio et al., 2015; Jiang et al., 2016; San et al., 2018), which further be verified by our results. Besides, S. atilius and T. femoralis were closely related, and both of them formed the sister group of P. utilis. In this study, the phylogenetic placement of S. atilius was firstly investigated. Although the phylogenetic analyses on S. atilius were still limited, this result contributed to a molecular basis for the classification and phylogeny of S. atilius within Tephritidae.

CONCLUSION

In conclusion, the mitogenome of S. atilius, was the first one from the genus Sphaenisscus. This mitogenome showed high conservation in terms of gene size, organization, AT bias and secondary structures of tRNAs. The phylogenetic placement S. atilius was investigated and clarified, revealing that S. atilius and T. femoralis were closely related, and both of them formed the sister group of P. utilis. These results provided a framework for further studies of the phylogenetics and evolution of S. atilius.

Declarations

Funding

This study was supported by Special funds for Henan Provinces Scientific and Technological Development Guided by the Central Government (Z20221341063), Natural Science Foundation of Henan Province (No. 212300410229), Key Project for University Excellent Young Talents of Henan Province (No. 2020GGJS260), the Project of Science and Technology Innovation Team (No. XNKJTD-007 and KJCXTD-202001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Statement of conflict of interest

The authors declare that there is no conflict of interest.

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