Mitochondrial Genomes of Two Bombycoidea Insects and Implications for Their Phylogeny

Abstract

The mitochondrial genome (mt genome) provides important information for understanding molecular evolution and phylogenetics. As such, the two complete mt genomes of Ampelophaga rubiginosa and Rondotia menciana were sequenced and annotated. The two circular genomes of A. rubiginosa and R. menciana are 15,282 and 15,636 bp long, respectively, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A + T-rich region. The nucleotide composition of the A. rubiginosa mt genome is A + T rich (81.5%) but is lower than that of R. menciana (82.2%). The AT skew is slightly positive and the GC skew is negative in these two mt genomes. Except for cox1, which started with CGA, all other 12PCGs started with ATN codons. The A + T-rich regions of A. rubiginosa and R. menciana were 399 bp and 604 bp long and consist of several features common to Bombycoidea insects. The order and orientation of A. rubiginosa and R. menciana mitogenomes with the order trnM-trnI-trnQ-nad2 is different from the ancestral insects in which trnM is located between trnQ and nad2 (trnI-trnQ-trnM-nad2). Phylogenetic analyses indicate that A. rubiginosa belongs in the Sphingidae family, and R. menciana belongs in the Bombycidae family.

Introduction

Insect mitochondrial DNA (mtDNA) is a double-stranded, circular molecule that is 14–19 kb in length and contains 13 PCGs: subunits 6 and 8 of the ATPase (atp6 and atp8), cytochrome c oxidase subunits 1–3 (cox1–cox3), cytochrome B (cob), NADH dehydrogenase subunits 1–6 and 4 L (nad1–6 and nad4L). It also contains two rRNA genes, small and large subunit rRNAs (rrnL and rrnS), 22 tRNA genes and a non-coding element termed the A + T-rich region¹. The A + T-rich region has a higher level of sequence and length variability than other regions of the genome^2–5 and regulates the transcription and replication of mt genomes⁶. As an informative molecular marker, mtDNA can provide important information for rearrangement patterns and phylogenetic analysis due to its rapid evolutionary rate and lack of genetic recombination⁷. Therefore, mtDNA has been widely used for diverse evolutionary studies among species⁸.

Recent advances in sequencing technologies have led to the rapid increase in mt genome data in GenBank, including Bombycoidea mt genomes. Bombycoidea is a superfamily of moths that contains the silk moths, emperor moths, sphinx moth, and relatives⁹. Some complete mt genomes of Bombycoidea insects are currently available in GenBank (Table 1). Several representative families were studied in this paper. Two families, Bombycidae and Saturniidae, are silk-producing insects with economic values in Bombycoidea¹⁰. The Sphingidae are a family of Bombycoidea, commonly known as hawk moths, sphinx moths, and hornworms; this family includes approximately 1,450 species^{11, 12}. Brahmaeidae are a family of Bombycoidea^{11, 12}. The Lasiocampidae are also a family of Bombycoidea, known as eggars, snout moths, or lappet moths. Over 2,000 species occur worldwide, and it is likely that not all have been named or studied¹³.

Table 1.

List of Bombycoidea species analysed in this paper with their respective GenBank accession numbers.

Superfamily	Family	Species	Size (bp)	GBAN*
Bombycoidea	Sphingidae	Ampelophaga rubiginosa	15,282	KT153024
Bombycoidea	Bombycidae	Rondotia menciana	15,636	KT258908
Bombycoidea	Bombycidae	Rondotia menciana	15,301	KC881286
Bombycoidea	Bombycidae	Rondotia menciana	15,364	KJ647172
Bombycoidea	Bombycidae	Andraca theae	15,737	KX365419
Bombycoidea	Bombycidae	Bombyx mandarina	15,928	AB070263
Bombycoidea	Bombycidae	Bombyx mori	15,643	AF149768
Bombycoidea	Bombycidae	Bombyx huttoni	15,638	KP216766
Bombycoidea	Saturniidae	Samia cynthia ricini	15,384	JN215366
Bombycoidea	Saturniidae	Actias selene	15,236	JX186589
Bombycoidea	Saturniidae	Antheraea pernyi	15,566	AY242996
Bombycoidea	Saturniidae	Antheraea yamamai	15,338	EU726630
Bombycoidea	Saturniidae	Eriogyna pyretorum	15,327	FJ685653
Bombycoidea	Saturniidae	Saturnia boisduvalii	15,360	EF622227
Bombycoidea	Saturniidae	Antheraea assama	15,312	KU301792
Bombycoidea	Saturniidae	Samia cynthia cynthia	15,345	KC812618
Bombycoidea	Saturniidae	Antheraea frithi	15,338	KJ740437
Bombycoidea	Saturniidae	Attacus atlas	15,282	KF006326
Bombycoidea	Saturniidae	Actias artemis aliena	15,243	KF927042
Bombycoidea	Saturniidae	Samia canningi	15,384	KJ159909
Bombycoidea	Lasiocampidae	Dendrolimus spectabilis	15,411	KM244678
Bombycoidea	Lasiocampidae	Dendrolimus tabulaeformis	15,411	KJ913817
Bombycoidea	Lasiocampidae	Dendrolimus punctatus	15,411	KJ913813
Bombycoidea	Lasiocampidae	Apatelopteryx phenax	15,552	KJ508055
Bombycoidea	Lasiocampidae	Trabala vishnou guttata	15,281	KU884483
Bombycoidea	Lasiocampidae	Euthrix laeta	15,368	KU870700
Bombycoidea	Sphingidae	Daphnis nerii	15,247
Bombycoidea	Sphingidae	Agrius convolvuli	15,349
Bombycoidea	Sphingidae	Manduca sexta	15,516	EU286785
Bombycoidea	Sphingidae	Sphinx morio	15,299	KC470083
Bombycoidea	Sphingidae	Notonagemia analis scribae	15,303	KU934302
Bombycoidea	Brahmaeidae	Brahmaea hearseyi	15,442	KU884326

*GenBank accession number.

Here, we sequenced the complete mt genomes of two species, A. rubiginosa and R. menciana. We aimed to analyse the mt genomes of these two species and to investigate the phylogeny of Bombycoidea insects. We were particularly interested in the phylogenetic position of Sphingidae and Bombycidae based on the 32 Bombycoidea complete mt genomes available to date.

Materials and Methods

Specimen collection

The moths of A. rubiginosa and R. menciana were collected in Xuancheng, Anhui Province. Total DNA was isolated using the Genomic DNA Extraction Kit (SangonBiotech, China) according to manufacturer instructions. Extracted DNA was used to amplify the complete mt genomes by PCR.

PCR amplification and sequencing

For amplification of the entire mt genomes of A. rubiginosa and R. menciana, specific primers were designed based on mt genomes sequences obtained from other Lepidopteran insects^{14, 15} (Table 2). The complete mt genomes were obtained using a combination of conventional PCR and long PCR to amplify overlapping fragments spanning the complete mt genomes. All amplifications were performed on an Eppendorf Mastercycler and Mastercycler gradient in 50 µl reaction volumes with 5 µl of 10 × Taq Buffer (Mg²⁺) (Aidlab), 4 µl of dNTPs (2.5 mM, Aidlab), 2 µl of each primer (10 µM), 2 µl of DNA (~100 ng), 34.5 µl of ddH₂O, and 0.5 µl of Red Taq DNA polymerase (5U, Aidlab). PCR was performed under the following conditions: 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 1–3 min at 54–60 °C (depending on primer combination), elongation at 72 °C for 30 s to 4 min (depending on the fragment length) and final extension at 72 °C for 10 min. The PCR products were separated by agarose gel electrophoresis (1% w/v) and purified using a DNA gel extraction kit (Transgene, China). The purified PCR products were ligated into the T-vector (SangonBiotech, China) and sequenced.

Table 2.

Primers used in this study.

Primer	Sequence (5′–3′)	Annealing temperature	Region
F1	GCTTTTGGGCTCATACCTCA	56 °C	trnM-cox1
R1	GATGAAATACCTGCAAGATGAAG
F2	TGGAGCAGGAACAGGATGAAC	55 °C	cox1-trnK
R2	GAGACCADTACTTGCTTTCAG
F3	ATTTGTGGAGCTAATCATAG	56 °C	cox2- cox3
R3	GGTCAGGGACTATAATCTAC
F4	TCGACCTGGAACTTTAGC	55 °C	atp6- nad5
R4	GCAGCTATAGCCGCTCCTACT
F5	TAAAGCAGAAACAGGAGTAG	54 °C	nad5
R5	ATTGCGATATTATTTCTTTTG
F6	CCCCAGCAGTAACTAAAGTAGAAG	54 °C	nad5-cob
R6	GTTAAAGTGGCATTATCT
F7	GGAGCTTCTACATGAGCTTTTGG	56 °C	nad4-rrnL
R7	GTTTGCGACCTCGATGTTG
F8	GGTCCCTTACGAATTTGAATATATCCT	60 °C	nad1-rrnS
R8	AAACTAGGATTAGATACCCTATTAT
F9	CTCTACTTTGTTACGACTTATT	55 °C	rrnS-nad2
R9	TCTAGGCCAATTCAACAACC

Sequence analysis

Annotation of sequences were performed using the blast tools in NCBI web site (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were edited and assembled using EditSeq and SeqMan (DNAStar package, DNAStar Inc. Madison, WI, USA). The graphical maps of A. rubiginosa and R. menciana complete mt genomes were drawn using the online mitochondrial visualization tool mtviz (http://pacosy.informatik.uni-leipzig.de/mtviz). The nucleotide sequences of PCGs were translated with the invertebrate mt genome genetic code. Alignments of A. rubiginosa and R. menciana PCGs with various Bombycoidea mt genomes were performed using MAFFT¹⁶. Composition skewness was calculated according to the following formulas:

$$\mathrm{AT}\mathrm{skew}=[\mathrm{A}-\mathrm{T}]/[\mathrm{A}+\mathrm{T}\left];\mathrm{GC}\mathrm{skew}=\right[\mathrm{G}-\mathrm{C}]/[\mathrm{G}+\mathrm{C}].$$

Nucleotide composition statistics and codon usage were computed using MEGA 5.0¹⁷.

Phylogenetic analysis

Thirty complete Bombycoidea mt genomes were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). In addition, mt genomes of Biston panterinaria and Phthonandria atrilineata were downloaded from GenBank and used as outgroup taxa. GenBank sequence information is shown in Table 1.

We estimated the taxonomic status of A. rubiginosa and R. menciana within Bombycoidea by constructing phylogenetic trees. Sequences from the PCGs of 34 mt genomes were combined. Two inference methods were used for analysis: Bayesian inference (BI) and Maximum likelihood (ML). BI was performed with MrBayes v 3.2.1¹⁸. While ML was performed with raxmlGUI¹⁹. Nucleotide substitution model selection was done using the Akaike information criterion implemented in MrModeltest v 2.3²⁰. ProtTest version 1.4²¹ was used to select the amino acid substitution model. The GTR + I + G model was the best for nucleotide data, and the MtREV + I + G + F model was the best for amino acids. ML analysis was performed on 1000 bootstrapped datasets. The Bayesian analysis ran as 4 simultaneous MCMC chains for 10,000,000 generations, sampled every 100 generations, with a burn-in of 5000 generations. Convergence was tested for the Bayesian analysis by ensuring that the average standard deviation of split frequencies was less than 0.01. Additionally, we tested for sufficient parameter sampling by ensuring an ESS of more than 200 using the software Tracer v1.6²². The resulting phylogenetic trees were visualized in FigTree v1.4.2²³.

Results and Discussion

Genome structure, organization and composition

The complete sequences of A. rubiginosa and R. menciana, 15,282 bp and 15,636 bp in size, respectively, were determined and submitted to GenBank (Accession No. KT153024 and KT258908). These two mt genomes both contain 13 PCGs, two rRNA genes, 22 tRNA genes, and an A + T-rich region. Four of the 13 PCGs (ND5, ND4, ND4L, and ND1), 8 tRNAs (trnQ, trnC, trnY, trnF, trnH, trnP, trnL (CUN), and trnV) and two rRNAs (rrnL and rrnS) are coded with the minority-strand, while the remaining 23 genes are encoded by the majority-strand in A. rubiginosa and R. menciana (Fig. 1, Table 3). The length of the R. menciana mt genome (15,636 bp) is larger than A. rubiginosa (15,282 bp) and smaller than that of Bombyx mandarina (15,928 bp), B. mori (15,643 bp) and B. huttoni (15,638 bp), but it falls within the range (15,236–15,928 bp) of other known Bombycoidea mt genomes in our study (Table 1). The nucleotide composition of the A. rubiginosa mt genome is as follows (Table 4): A = 6,334 (41.4%), T = 6,126 (40.1%), G = 1,144 (7.5%), and C = 1,678 (11.0%). The nucleotide composition of the A. rubiginosa mt genome is A + T rich (81.5%) but is lower than that of R. menciana (82.2%). The AT skew²⁴ is slightly positive and the GC skew is negative in these two mt genomes (Table 4), indicating an obvious bias towards the use of As and Cs. The order and orientation of genes in the A. rubiginosa and R. menciana mt genomes are identical to other bombicoid insects sequenced to date²⁵, but differ from ancestral insects²⁶. The placement of the trnM gene in the A. rubiginosa and R. menciana mt genome is trnM-trnI-trnQ, while in ancestral insects, it is trnI-trnQ-trnM (Fig. 2). Ghost moths exhibited the ancestral insect placement of the trnM gene cluster²⁷. The hypothesis that the ancestral arrangement of the trnM gene cluster underwent rearrangement after Hepialoidea diverged from other Lepidopteran lineages was supported by our results in A. rubiginosa and R. menciana. The tRNA rearrangements are generally presumed to be a consequence of tandem duplication of partial mt genomes^28–31, followed by random or non-random loss of the duplicated copies^{28, 32, 33}.

Figure 1.

Circular map of the mt genomes of A. rubiginosa (A) and R. Menciana (B). tRNA-Ser1, tRNA-Ser2, tRNA-Leu1 and tRNA-Leu2 denote codons tRNA-Ser1 (AGN), tRNA-Ser2 (UCN), tRNA-Leu1 (CUN), and tRNA-Leu2 (UUR), respectively.

Table 3.

Summary of the mt genomes of A. rubiginosa and R. menciana.

Gene	Direction	Location	Size	Anticodon	Start codon	Stop codon	Intergenic nucleotides
trnM	F	1–68	68	CAT	—	—	0
trnI	F	69–132	64	GAT	—	—	−3
trnQ	R	130–198	69	TTG	—	—	56
nad2	F	255–1266	1012	—	ATT	T	0
trnW	F	1267–1334	68	TCA	—	—	−8
trnC	R	1327–1391	65	GCA	—	—	0
trnY	R	1392–1456	65	GTA	—	—	6
cox1	F	1463–2990	1528	—	CGA	T	0
trnL2(UUR)	F	2991–3058	68	TAA	—	—	0
cox2	F	3059–3740	682	—	ATG	T	0
trnK	F	3741–3811	71	CTT	—	—	2
trnD	F	3814–3881	68	GTC	—	—	0
atp8	F	3882–4043	162	—	ATT	TAA	−7
atp6	F	4037–4714	678	—	ATG	TAA	0
cox3	F	4715–5506	792	—	ATG	TAA	2
trnG	F	5509–5574	66	TCC	—	—	0
nad3	F	5575–5926	352	—	ATT	T	0
trnA	F	5927–5993	67	TGC	—	—	1
trnR	F	5995–6058	64	TCG	—	—	0
trnN	F	6059–6124	66	GTT	—	—	0
trnS1(AGN)	F	6125–6186	62	GCT	—	—	9
trnE	F	6196–6263	68	TTC	—	—	−2
trnF	R	6262–6327	66	GAA	—	—	27
nad5	R	6355–8076	1722	—	ATT	A	15
trnH	R	8092–8155	64	GTG	—	—	0
nad4	R	8156–9490	1335	—	ATG	TAA	0
nad4L	R	9491–9781	291	—	ATG	TAA	4
trnT	F	9786–9851	66	TGT	—	—	−1
trnP	R	9851–9916	66	TGG	—	—	6
nad6	F	9923–10,453	531	—	ATG	TAA	6
cob	F	10,460–11,608	1149	—	ATG	TAA	−1
trnS2(UCN)	F	11,608–11,672	65	TGA	—	—	21
nad1	R	11,694–12,629	936	—	ATG	TAA	0
trnL1(CUN)	R	12,630–12,696	67	TAG	—	—	0
rrnL	R	12,697–14,040	1344	—	—	—	0
trnV	R	14,041–14,108	68	TAC	—	—	0
rrnS	R	14,109–14,883	775	—	—	—	0
A + T-rich region		14,884–15,282	399	—	—	—
trnM	F	1–68	68	CAT	—	—	0
trnI	F	69–132	64	GAT	—	—	−3
trnQ	R	130–198	69	TTG	—	—	52
nad2	F	251–1264	1014	—	ATT	TAA	7
trnW	F	1272–1338	67	TCA	—	—	−8
trnC	R	1331–1394	64	GCA	—	—	0
trnY	R	1395–1459	65	GTA	—	—	9
cox1	F	1469–2999	1531	—	CGA	T	0
trnL2(UUR)	F	3000–3066	67	TAA	—	—	0
cox2	F	3067–3748	682	—	ATG	T	0
trnK	F	3749–3819	71	CTT	—	—	−1
trnD	F	3819–3884	66	GTC	—	—	0
atp8	F	3885–4046	162	—	ATC	TAA	−7
atp6	F	4040–4717	678	—	ATG	TAA	3
cox3	F	4721–5509	789	—	ATG	TAA	2
trnG	F	5512–5577	66	TCC	—	—	0
nad3	F	5575–5931	357	—	ATA	TAA	27
trnA	F	5959–6032	74	TGC	—	—	10
trnR	F	6043–6105	63	TCG	—	—	0
trnN	F	6106–6173	68	GTT	—	—	6
trnS1(AGN)	F	6180–6248	69	GCT	—	—	1
trnE	F	6250–6314	65	TTC	—	—	3
trnF	R	6318–6385	68	GAA	—	—	0
nad5	R	6386–8124	1739	—	ATT	TA	0
trnH	R	8125–8190	66	GTG	—	—	10
nad4	R	8201–9541	1341	—	ATG	TAA	5
nad4L	R	9547–9837	291	—	ATG	TAA	2
trnT	F	9840–9904	65	TGT	—	—	0
trnP	R	9905–9970	66	TGG	—	—	2
nad6	F	9973–10,503	531	—	ATG	TAA	7
cob	F	10,511–11,665	1155	—	ATG	TAA	10
trnS2(UCN)	F	11,676–11,727	52	TGA	—	—	33
nad1	R	11,761–12,699	939	—	ATG	TAA	1
trnL1(CUN)	R	12,701–12,770	70	TAG	—	—	0
rrnL	R	12,771–14,186	1416	—	—	—	0
trnV	R	14,187–14,252	66	TAC	—	—	0
rrnS	R	14,253–15,032	780	—	—	—	0
A + T-rich region		15,033–15,636	604	—	—	—

Table 4.

Composition and skewness in the A. rubiginosa and R. menciana mt genomes.

A. rubiginosa	Size (bp)	A (bp)	tCT (bp)	G (bp)	C (bp)	A %	T %	G %	C %	AT %	AT skew	GC skew
Whole genome	15,282	6334	616126	1144	1678	41.4	40.1	7.5	11.0	81.5	0.017	−0.189
Protein-coding genes	11,175	3894	5090	1135	1056	34.8	45.5	10.2	9.5	80.3	−0.133	0.038
tRNA genes	1461	602	589	116	154	41.2	40.3	7.9	10.6	81.5	0.011	−0.141
rRNA genes	2119	906	887	104	222	42.8	41.9	4.9	10.4	84.7	0.011	−0.362
A + T-rich region	399	174	194	14	17	43.6	48.6	3.5	4.3	92.2	−0.054	−0.097
Whole genome	15,636	6561	6290	1122	1663	42.0	40.2	7.2	10.6	82.2	0.021	−0.194
Protein-coding genes	11,205	3934	5107	1114	1050	35.1	45.6	9.9	9.4	80.7	−0.130	0.030
tRNA genes	1460	606	588	115	151	41.5	40.3	7.9	10.3	81.8	0.015	−0.135
rRNA genes	2196	959	927	100	210	43.7	42.2	4.5	9.6	85.9	0.017	−0.355
A + T-rich region	604	281	287	18	18	46.5	47.5	3.0	3.0	94.0	−0.011	0

Figure 2.

The mitochondrial gene order of ancestral insects and A. rubiginosa and R. menciana.

Protein-coding genes

Summaries of the genes that make up the mt genomes of A. rubiginosa and R. menciana are given in Table 3. Twelve of the thirteen PCGs use standard ATN start codons in A. rubiginosa and R. menciana, except for cox1, which is initiated by the CGA codon (arginine). The CGA codon is highly conserved across most insect groups^{14, 34}. In A. rubiginosa, eight PCGs (atp8, atp6, cox3, nad4, nad4L, nad6, cob, and nad1) have the complete stop codon TAA, while the remaining five terminate with either T (nad2, cox1, cox2, and nad3) or A (nad5). In R. menciana, ten PCGs (nad2, atp8, atp6, cox3, nad3, nad4, nad4L, nad6, cob, and nad1) have the complete stop codon TAA, while the remaining three terminate with either T (cox1 and cox2) or TA (nad5). For A. rubiginosa, the average AT content of the 13 PCGs is 80.3%, and the overall AT and GC skews are –0.133 and 0.038, showing that T and G are more abundant than A and C. Similarly, the A + T composition of the 13 PCGs in the mt genome of R. menciana is 80.7%, while the AT and GC skews are –0.130 and 0.030, showing that T and G are more abundant than A and C (Table 4). Relative synonymous codon usage (RSCU) values for the A. rubiginosa and R. menciana mt genomes are summarized in Table 5 and Fig. 3, which show that NNT and NNA are more frequent than NNG and NNC, indicating a strong A or T bias in the third codon position. The most common amino acids for A. rubiginosa and R. menciana mitochondrial proteins are Leu (UUR), Ile, and Phe (Fig. 4).

Table 5.

Codon number and RSCU in the A. rubiginosa and R. menciana mitochondrial PCGs.

Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	347	1.88	UCU(S)	91	2.35	UAU(Y)	184	1.86	UGU(C)	31	1.82
UUC(F)	23	0.12	UCC(S)	1	0.03	UAC(Y)	14	0.14	UGC(C)	3	0.18
UUA(L)	482	5.32	UCA(S)	103	2.66	UAA(*)	10	2	UGA(W)	91	1.94
UUG(L)	14	0.15	UCG(S)	0	0	UAG(*)	0	0	UGG(W)	3	0.06
CUU(L)	26	0.29	CCU(P)	63	1.98	CAU(H)	57	1.73	CGU(R)	13	1
CUC(L)	2	0.02	CCC(P)	12	0.38	CAC(H)	9	0.27	CGC(R)	0	0
CUA(L)	20	0.22	CCA(P)	52	1.64	CAA(Q)	63	2	CGA(R)	37	2.85
CUG(L)	0	0	CCG(P)	0	0	CAG(Q)	0	0	CGG(R)	2	0.15
AUU(I)	452	1.91	ACU(T)	83	2.26	AAU(N)	239	1.85	AGU(S)	22	0.57
AUC(I)	22	0.09	ACC(T)	6	0.16	AAC(N)	19	0.15	AGC(S)	0	0
AUA(M)	276	1.86	ACA(T)	56	1.52	AAA(K)	102	1.92	AGA(S)	92	2.37
AUG(M)	21	0.14	ACG(T)	2	0.05	AAG(K)	4	0.08	AGG(S)	1	0.03
GUU(V)	74	2.26	GCU(A)	75	2.59	GAU(D)	58	1.9	GGU(G)	62	1.29
GUC(V)	1	0.03	GCC(A)	0	0	GAC(D)	3	0.1	GGC(G)	0	0
GUA(V)	55	1.68	GCA(A)	40	1.38	GAA(E)	70	1.87	GGA(G)	111	2.31
GUG(V)	1	0.03	GCG(A)	1	0.03	GAG(E)	5	0.13	GGG(G)	19	0.4
UUU(F)	369	1.9	UCU(S)	94	2.39	UAU(Y)	178	1.87	UGU(C)	29	1.81
UUC(F)	20	0.1	UCC(S)	10	0.25	UAC(Y)	12	0.13	UGC(C)	3	0.19
UUA(L)	478	5.32	UCA(S)	97	2.47	UAA(*)	11	2	UGA(W)	90	1.94
UUG(L)	14	0.16	UCG(S)	0	0	UAG(*)	0	0	UGG(W)	3	0.06
CUU(L)	24	0.27	CCU(P)	60	1.98	CAU(H)	57	1.73	CGU(R)	14	1.06
CUC(L)	3	0.03	CCC(P)	10	0.33	CAC(H)	9	0.27	CGC(R)	0	0
CUA(L)	19	0.21	CCA(P)	48	1.59	CAA(Q)	60	2	CGA(R)	38	2.87
CUG(L)	1	0.01	CCG(P)	3	0.1	CAG(Q)	0	0	CGG(R)	1	0.08
AUU(I)	452	1.9	ACU(T)	67	1.91	AAU(N)	246	1.82	AGU(S)	30	0.76
AUC(I)	25	0.1	ACC(T)	6	0.17	AAC(N)	24	0.18	AGC(S)	1	0.03
AUA(M)	285	1.89	ACA(T)	67	1.91	AAA(K)	107	1.88	AGA(S)	82	2.09
AUG(M)	17	0.11	ACG(T)	0	0	AAG(K)	7	0.12	AGG(S)	0	0
GUU(V)	66	2.08	GCU(A)	65	2.39	GAU(D)	62	1.88	GGU(G)	52	1.09
GUC(V)	1	0.03	GCC(A)	3	0.11	GAC(D)	4	0.12	GGC(G)	1	0.02
GUA(V)	56	1.76	GCA(A)	39	1.43	GAA(E)	66	1.83	GGA(G)	126	2.65
GUG(V)	4	0.13	GCG(A)	2	0.07	GAG(E)	6	0.17	GGG(G)	11	0.23

Figure 3.

The relative synonymous codon usage (RSCU) in the mt genomes of A. rubiginosa (A) and R. menciana (B).

Figure 4.

Amino acid composition in the mt genomes of A. rubiginosa (A) and R. menciana (B).

Transfer RNA and ribosomal RNA genes

A. rubiginosa and R. menciana both contain 22 tRNAs. Eight of these tRNAs (trnQ, trnC, trnY, trnF, trnH, trnP, trnL(CUN), and trnV) are coded with the minority-strand, while the remaining 14 tRNA genes are encoded by the majority-strand in A. rubiginosa and R. menciana (Table 3). The total length of the 22 tRNAs in the mt genome of A. rubiginosa is 1461 bp, and their A + T content is 81.5%. Similarly, the total length of the 22 tRNAs in the mt genome of R. menciana is 1460 bp and their A + T content is 81.8%. The AT skew is slightly positive and the GC skew is negative in the 22 tRNAs of A. rubiginosa and R. menciana (Table 4). The rrnL and rrnS genes of A. rubiginosa and R. menciana are located between trnL1(CUN) and trnV and between trnV and the A + T-rich region, respectively. The A + T content of the two rRNA genes is 84.7% in A. rubiginosa, which is lower than that of R. menciana (85.9%) (Table 4).

A + T-rich region

The A + T-rich regions of A. rubiginosa and R. menciana are located between rrnS and trnM and were 399 bp and 604 bp long, respectively. The A + T-rich regions contain 92.2% and 94.0% A + T contents in A. rubiginosa and R. menciana, respectively, which were the highest across the studied mt genomes (Table 4). The AT skew and GC skew of A. rubiginosa are −0.054 and −0.097, indicating an obvious bias towards the use of T and C. However, in the R. menciana A + T-rich region, AT skew is −0.011 and the number of G and C is the same, meaning that T is more abundant than A and that the usage of G and C is equal. Several conserved structures found in other bombicoid species mt genomes are also observed in the A + T-rich regions of A. rubiginosa and R. menciana. The conserved “ATAGA + poly T” motif is located downstream of the rrnS gene in the A + T-rich region of A. rubiginosa and R. menciana, which may represent the origin of minority or light strand replication³¹, and is conserved in lepidopteran mt genomes. Multiple tandem repeat elements are typically present in the A + T-rich region of most insects. Only one tandem repeat was found in the A. rubiginosa mt genome (Fig. S1). We identified two tandem repeats elements in the A + T-rich region of R. menciana (Fig. S2).

The mt genome of R. menciana has been previously sequenced, and two complete mt genomes of the species are available^{35, 36}. However, in the present study, there was a difference of approximately 300 nt in the length of the mt genome of R. menciana compared to the two published sequences^{35, 36}. The excess 300 nt of R. menciana in the present study mainly arose from the upper area of the A + T-rich region (Fig. S2). The A + T-rich regions of the R. menciana (Ankang Shaanxi) and R. menciana (Korea) mt genomes were identical. The length of tandem repeats of the A + T-rich region of R. menciana in this study was greater than the two published sequences.

Phylogenetic analysis

Phylogenetic analyses were based on sequences of 13 PCGs of 34 mt genomes using two methods (BI and ML) and alignments performed by MAFFT. B. panterinaria and P. atrilineata were used as outgroups. Thirty bombycoid species mt genomes that were downloaded from GenBank (plus A. rubiginosa and R. menciana) represent five families belonging to the Bombycoidea: Bombycidae, Lasiocampidae, Saturniidae, Brahmaeidae and Sphingidae. It is obvious that A. rubiginosa and Daphnis nerii ³⁷ are clustered on one branch in the phylogenetic tree with high nodal support values. The analyses show that A. rubiginosa belongs in the Sphingidae family. The three phylogenetic trees consistently showed that R. menciana from Ankang was remarkably different from those of Korea and Xuancheng. The bombycid species were Andraca theae + ((R. menciana (Ankang)³⁵ + (R. menciana (Xuancheng) + R. menciana (Korea)³⁶)) + (B. huttoni + (B. mandarina ³⁸ + B. mori))), indicating that R. menciana belongs in the Bombycidae family (Figs 5, 6 and 7).

Figure 5.

Phylogenetic tree derived for Bombycoidea using BI and ML analyses based on amino acid sequences and using MAFFT for alignment. Bayesian posterior probability (BPP) and bootstrap values (BP) of each node are shown as BPP/BP, with maxima of 1.00/100.

Figure 6.

Phylogenetic tree derived for Bombycoidea using BI analysis based on nucleotide sequences using MAFFT for alignment.

Figure 7.

Phylogenetic tree derived for Bombycoidea using BI and ML analyses based on 16S ribosomal RNA and 12S ribosomal RNA sequences of 33 species (there are no 16S ribosomal RNA and 12S ribosomal RNA sequences in the Apatelopteryx phenax (KJ508055)). Bayesian posterior probability (BPP) and bootstrap value (BP) of each node are shown as BPP/BP, with maxima of 1.00/100.

A problem remains with the phylogenetic relationships of families among the Bombycoidea in our study. The phylogenetic trees based on ML and BI analyses of amino acid sequences showed that the phylogenetic relationships were (Lasiocampidae + Brahmaeidae) + (Bombycidae + (Sphingidae + Saturniidae)) (Fig. 5), which is similar to some past studies^{10, 39}. However, the phylogenetic tree based on BI analysis of nucleotide sequences showed that the phylogenetic relationships were (Lasiocampidae + Brahmaeidae) + (Sphingidae + (Bombycidae + Saturniidae)) (Fig. 6). The phylogenetic relationships of families in our study (Figs 5, 6 and 7) differ from the findings of other previous studies, where the families group as Lasiocampidae + (Saturniidae + (Bombycidae + Sphingidae))⁴⁰. The reason for these differences may be the incorporation of complete mt genomes³⁹. The relationships in the Bombycoidea remain unsettled. More mt genomes from Bombycoidea insects are required to resolve the positions of Bombycoidea in the future.

Author Contributions

Q.N.L. and B.P.T. conceived and designed the experiments. Q.N.L., Z.Z.X., Y.L., X.Y.Z., and Y.W. performed the experiments. Q.N.L. and Z.Z.X. analysed the data. H.B.Z., D.Z.Z., C.L.Z. and B.P.T. contributed reagents and materials. Q.N.L. and Z.Z.X. wrote the paper. Z.Z.X., Q.N.L. and B.P.T. revised the paper.

Competing Interests

The authors declare that they have no competing interests.