The Complete Mitochondrial Genome of the Longhorn Beetle Dorysthenes paradoxus (Coleoptera: Cerambycidae: Prionini) and the Implication for the Phylogenetic Relationships of the Cerambycidae Species.

The longhorn beetle Dorysthenes paradoxus (Faldermann, 1833) (Coleoptera: Cerambycidae) is not only a serious agricultural pest but also a traditionally edible insect in China. However, no genetic information on this species has been acquired. In the present study, we report the mitochondrial genome (mitogenome) of Do. paradoxus, as the first complete mitogenome of Prioninae. The circular mitogenome of 15,922 bp encodes 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs), and it contains an A+T-rich region. This mitogenome exhibits the lowest A+T content (71.13%) but harbors the largest AT skew (0.116) among the completely sequenced Cerambycidae species. Eleven of the 13 PCGs have a typical ATN start codon, whereas COI and ND1 are tentatively designated by AAT and TTG, respectively. Only 4 of the 13 PCGs harbor a complete termination codon, and the remaining 9 possess incomplete termination codons (T or TA). Apart from tRNASer(AGN), the other 21 tRNAs can fold into a typical clover-leaf secondary structures. The Do. paradoxus A+T-rich region contains two poly-T stretches and a tandem repeat that comprises two 47-bp-long copies. Both Bayesian inference and Maximum likelihood analyses confirmed the subfamily ranks of Cerambycidae ([Prioninae + Cerambycinae] + Lamiinae) and the close relationship between Philinae and Prioninae/Cerambycinae. However, the data did not support the monophyly of Prioninae and Cerambycinae. The mitogenome presented here provides basic genetic information for this economically important species.

The beetles are the most diverse group of insects with over 360,000 described species worldwide (Sheffield et al. 2008), representing approximately 40% of all insect species described to date and approximately 25% of all animals (Hunt et al. 2007). Beetles are prominent in human culture: some are serious agricultural, forestry, and horticultural pests, and over 300 species are used as human food. The ecological and morphological diversity in Coleoptera have attracted the attention of biologists (Hunt et al. 2007), as has the phylogenetic relationship within this order (Zhang et al. 2016). esearchers have attempted to sequence the mitochondrial genome (mitogenome) as the reference sequence for target species, which is widely considered an effective molecular marker for research on phylogenetic analysis and phylogeography (Simon et al. 2006). In recent years, the number of coleopteran mitogenome sequences has increased dramatically in the GenBank database. Cerambycidae is a large family in the Coleoptera order, and more than 25,000 species of Cerambycidae have been described worldwide (Sama et al. 2010). Currently, 12 complete mitogenomes of Cerambycidae species are available in GenBank. Among these, seven are in the subfamily Lamiinae, three are in the subfamily Cerambycinae, one is in the subfamily Lepturinae, and one is in subfamily Prioninae (Table 1). Information about the subfamily Prioninae mitogenome is still limited. One partial mitogenome in Prioninae is available, but the taxonomic status of the specimen sequenced remains unidentified.

Table 1.

List of mitogenomes of Cerambycoidea. *Partial genome

Family/subfamily/species	Accessionno.	Size(bp)	A%	T%	C%	G%	A+T %	ATskew	GCskew	References
Cerambycidae
Prioninae
Dorysthenes paradoxus	MG460483	15,922	39.67	31.45	17.87	11	71.13	0.116	–0.238	This study
Prioninae sp.*	JX220991	10,679	39.53	31.44	17.71	11.29	70.97	0.114	–0.222
Cerambycinae
Aeolesthes oenochrous	NC_025243	15,747	39.44	31.75	18.3	10.5	71.19	0.108	–0.271	Chiu et al. (2016)
Massicus raddei	NC_023937	15,858	38.61	33.18	17.06	11.16	71.78	0.076	–0.209	Wang et al. (2016)
Xylotrechus grayii	NC_030782	15,540	41.35	33.94	15.03	9.68	75.29	0.098	–0.216
Lamiinae
Anoplophora chinensis	NC_029230	15,805	39.48	38.17	13.57	8.78	77.65	0.017	–0.214	Li et al. (2016b)
Anoplophora glabripennis	NC_008221	15,774	39.61	38.7	13.07	8.59	78.31	0.012	–0.207	Fang et al. (2016)
Apriona swainsoni	NC_033872	15,412	38.9	35.81	15.99	9.29	74.71	0.041	–0.265
Batocera lineolata	NC_022671	15,418	38.75	35.73	16.21	9.31	74.48	0.040	–0.271	Wang et al. (2012)
Monochamus alternatus	NC_024652	15,874	39.76	39.26	12.3	8.68	79.02	0.006	–0.173	Li et al. (2016a)
Psacothea hilaris	NC_013070	15,856	38.76	37.88	14.17	9.2	76.63	0.011	–0.213	Kim et al. (2009)
Thyestilla gebleri	NC_034752	15,505	38.5	35.56	16.64	9.28	74.07	0.040	–0.284
Lepturinae
Rhagium mordax*	JX412743	10,676	41.14	36.76	12.84	9.25	77.9	0.056	–0.194
Chrysomelidae
Galeruca daurica	NC_027114	16,615	40.51	37.63	13.11	8.73	78.15	0.048	–0.251	Zhou et al. (2016)
Diabrotica barberi	KF669870	16,366	41.26	37.91	12.29	8.54	79.17	0.042	–0.220	Coates (2014)
Diabrotica virgifera	KF658070	16,650	41.56	37.97	12.09	8.38	79.53	0.045	–0.221	Coates (2014)
Crioceris duodecimpunctata	AF467886	15,880	40.14	36.74	13.44	9.67	76.89	0.044	–0.195	Stewart and Beckenbach (2003)
Vesperidae
Philinae
Spiniphilus spinicornis	NC_029515	15,707	38.58	30.94	19.35	11.13	69.52	0.11	–0.369	Nie et al. (2017)

The longhorn beetle Dorysthenes paradoxus (Faldermann, 1833) (Coleoptera: Cerambycidae) belongs to the insect subfamily Prioninae, within the family Cerambycidae. This species is widely distributed in China. In general, this species is considered a serious agricultural pest that attacks the underground stems of gramineous crops, such as maize, sorghum, rice, sugarcane, and bluegrass (Yan et al. 1997). In the Shanxi and Liaoning Provinces of China, the adult beetle has been traditionally accepted and used as an edible insect with a long history of use. In the sample collection site used in this study, each year on the day that autumn begins, many people like to capture the adults of the beetle after a rain. In recent years, the price of this beetle has soared. However, little attention has been paid to biological studies of this beetle, although its life cycle, habits, and morphological characteristics of four developmental stages and the digestive tract have been investigated (Yan et al. 1997, Hao et al. 1999). In the GenBank database, only four nucleotide sequences for three mitochondrial DNA fragments encoding partial COI (GU130426 and KJ159158), lrRNA (GU130418), and srRNA (GU130410) genes are available. Even though the genetic background is in high demand, no genetic information has been acquired.

With the aim to provide more useful information for studies of the population structure and genetic diversity of Do. paradoxus, in the present study, the complete mitogenome sequence of this economically important species was determined. This genome is the first mitogenome in the subfamily Prioninae, so we performed a phylogenetic analysis based on the available complete mitogenome sequences to provide insight into the phylogenetic relationships within the family Cerambycidae.

Materials and Methods

Specimen and DNA Extraction

Adult beetles were captured at Beiqiuzhang village (N35°32′42.05″; E111°27′39.42″), Gujiang town, Jiangxian county, Shanxi Province, China on 8 August 2015. The freshly collected specimens were preserved immediately in 95% ethanol. The specimens were identified as Do. paradoxus based on the morphology by Prof. Hong Fang (Department of Entomology, College of Plant Protection, Shenyang Agricultural University, China). Total genomic DNA was extracted from the leg muscle tissue of an adult individual using the Trace DNA Extraction Method (Wang et al. 1997). The partial leg was immersed in the extraction buffer (10 mM/liter Tris–HCl, pH 8.0, 10 mM/liter EDTA, pH 8.0, 100 mM/liter NaCl, 2% SDS, 0.039 M/liter DTT, and 100 mg/liter Proteinase K) and then treated with the traditional phenol/chloroform method. In our laboratory, this method has been confirmed to be suitable to extract total genomic DNA from animal, insect, plant, and microorganism samples.

PCR Amplification and Sequencing

The entire mitogenome of Do. paradoxus was amplified in 14 overlapping fragments (Table 2). In the first step, four fragments were successfully amplified with universal insect PCR primers (Simon et al. 2006). Then, seven species specific primer pairs (Dp1-14) were designed, based on the initial sequences of the four obtained fragments and three mitochondrial DNA fragments available in the GenBank database (COI, lrRNA, srRNA). Finally, we designed three primer pairs (Dp15-20) to fill the remaining gaps corresponding to the sequences from GU130426, GU130418, and GU130410. PCR amplifications were done using TaKaRa LA Taq or EX Taq polymerase (Takara Biotechnology Co. Ltd., Dalian, China), under the following procedure: 94°C for 2 min, followed by 35 cycles of 1 min at 94°C and 1 min at 50–55°C, with a subsequent 10 min final extension at 72°C. All amplified products were sequenced firstly using upstream and downstream primers from both directions and later by primer walking. Sanger sequencing was performed at SinoGenoMax Co., Ltd., Beijing, China.

Table 2.

Primers used for PCR amplification

Fragment	Primer	Sequence（5ʹ–3ʹ）	Target	Position
Universal primers (Simon et al. 2006)
F1	TK-J3790	CATTAgATgACTgAAAgCAAgTA	tRNA Lys	3,703–3,725
F1	A6-N4552	ATgTCCWgCAATYATATTWgC	ATP6	4,481–4,501
F2	C3-J4792	gTTgATTATAgACCWTgRCC	COIII	4,695–4,715
F2	C3-N5460	TCAACAAAATgTCARTAYCA	COIII	5,389–5,408
F3	N5-J7572	AAAgggAATTTgAgCTCTTTTWgT	ND5	7,395–7,418
F3	N4-N8727	AAATCTTTRATTgCTTATTCWTC	ND4	8,570–8,592
F4	CB-J10933	gTTCTACCTTgAggNCAAATRTC	Cytb	10,728–10,750
F4	CB-N11526	TTCTACTggTCgRgCTCCAATYCA	Cytb	11,343–11,363
Species specific primers (This study)
F5	Dp2-F	gAATgACTACAAAAACTTCCACCTgC	COI	2,887–2,912
F5	Dp3-R	TTAAggggTgATATTTgTgggATT	ATP8	3,845–3,868
F6	Dp4-F	AACCCTTgCTTTACCCTTATgATT	ATP6	4,303–4,326
F6	Dp5-R	ATTgAAAAACCTAATAGTAATAAA	COIII	4,794–4,817
F7	Dp6-F	CCTCgTTCTTTgTggCAACTggAT	COIII	5,255–5,278
F7	Dp7-R	TTAgggTTggTTTCTTATTgTTTggT	ND5	7,618–7,643
F8	Dp8-F	TAAACCAgAATAATAAgAACCAT	ND5	8,170–8,192
F8	Dp9-R	gCATTATCAACggCgAATCCTCC	Cytb	10,830–10,852
F9	Dp10-F	AATCCTATTTTgATCgTTggTAg	Cytb	11,300–11,322
F9	Dp11-R	AACTTTTTTTTCATTTTTACACTT	16SrRNA	12,875–12,898
F10	Dp12-F	TTCTCATCCAACCATTCATTCCAg	16SrRNA	13,071–13,094
F10	Dp13-R	AAAAggggAgATgggTTACAAT	12SrRNA	14,133–14,158
F11	Dp14-F	TTCAgAATAATAgggTATCTAATCC	12SrRNA	14,380–14,404
F11	Dp1-R	TCCTCTTTCTTgTCTgATAATgTggg	COI	2,174–2,199
F12	Dp15-F	ggAggTggAgATCCTATTCTAT	COI	2,080–2,101
F12	Dp16-R	ggACgCTCTATCTTggAgTAAT	COII	3,039–3,060
F13	Dp17-F	AATCCTATTTTgATCgTTggTAg	Cytb	11,300–11,322
F13	Dp18-R	TATATATTTTTgTTTATgggggTA	16SrRNA	13,264–1,3287
F14	Dp19-F	TgTTACgACTTATTTCACCTTggg	12SrRNA	13,986–14,009
F14	Dp20-R	gTTTgTgCCAgCAGTTgCggTTA	12SrRNA	145,49–14,571
For amplification and sequencing of the A+T-rich region
	Dp21-F	TAAAAAGAAGATCAAAATCCCAG	AT-rich	14,977–14,999
	Dp23-R	CTATAAgTTATTAAAAACTAAT	AT-rich	15,566–15,587
	Dp24-F	ATTAgTTTTTAAAAACTTATAg	AT-rich	15,566–15,587
	Dp22-R	TTgATACTTTAggAggTggTTT	tRNA Gln	85–106
	N = [A, C, G, T]; R = [A, G]; Y = [C, T]; W = [A, T]

When we attempted to sequence the AT-rich region, the occurrence of a polyT(12) at positions 15,132–15,143 and a polyA(21) at positions 15,841–15,861 made their internal sequencing results unreadable. To address this issue, several primers based on the possible sequence deduced by the indefinite sequencing results of primers Dp21-F and Dp22-R were used. Fortunately, primer Dp23-F and its reverse complement primer Dp24-R, paired with Dp21-F and Dp22-R, respectively, were suitable for amplification and sequencing which allowed us to cover the remaining gaps.

Sequence Annotation and Genomic Analysis

The partial mitogenome of Prioninae sp. (Coleoptera: Cerambycidae) is available in GenBank, so we used it as the reference sequence for mapping the amplified fragments of Do. paradoxus. The overlapping sequences were manually checked and assembled into a complete mitogenome via the alignment of neighboring fragments using the Clustal X program (Thompson et al. 1997). Protein-coding genes (PCGs) were identified by comparison with sequences from other Cerambycidae species. The 5ʹ ends of PCGs were assumed to be at the first legitimate in-frame start codon ATN in an open reading frame (ORF) that was not located within an upstream gene encoded on the same strand. A truncated stop codon (T or TA) adjacent to the beginning of the downstream gene was designated the termination codon (Wolstenholme 1992). The transfer RNA (tRNA) genes and their secondary structures were identified using the MITOS web server (Bernt et al. 2013). The large rRNA (lrRNA) gene was annotated to extend to the boundaries of the flanking tRNA(CUN) and tRNA. The 3ʹ end of the small rRNA (srRNA) gene was annotated to be adjacent to the start of tRNA, whereas the 5ʹ end was determined by comparing orthologous sequences of other known mitogenomes. The entire A+T-rich region was subjected to a search for the tandem repeats using Tandem Repeats Finder program (Benson 1999). Composition skew was used to describe the base composition of nucleotide sequences, with the relative number of As to Ts (AT skew = [A – T]/[A + T]) and Gs to Cs (GC skew = [G – C]/[G + C]) (Perna and Kocher 1995). Overlapping regions and intergenic spacers between genes were counted manually. Circular genome maps were generated by OrganellarGenomeDRAW (Lohse et al. 2013) and revised by hand. The sequence data were deposited in GenBank under accession MG460483.

Phylogenetic Analysis

To reconstruct the phylogenetic relationships among Chrysomeloidae species, a total of 17 available Cerambycidea mitogenomes, including 13 Cerambycinae and 4 Chrysomelidae, were used (Table 1). The mitogenome of Spiniphilus spinicornis (Lin & Bi, 2011) (Coleoptera: Vesperidae), which belongs to Philinae of Vesperidae, was also included due to its association with Prioninae of Chrysomeloidae in their adult morphology (Nie et al. 2017). One neuropteran species, Polytoechotes punctatus (Fabricius) (Neuroptera: Polystoechotidae) (Beckenbach and Stewart 2009), served as an outgroup. The nucleotide sequences of each of the 13 PCGs were translated into amino acid sequences then aligned with default settings, and these resultant alignments were retranslated into nucleotide alignments by MEGA 6.06 (Tamura et al. 2013). The alignment was done with Muscle using default settings. The sequences of 13 PCGs were then concatenated to allow a more complete analysis (Hassanin 2006). In the present study, the nucleotide sequence of ND5 in Massicus raddei (Blessig) (Coleoptera: Cerambycidae) was excluded due to probable sequencing misread. Bayesian Inference (BI) and Maximum likelihood (ML) methods were used for phylogenetic analysis. The GTR + G + I model was selected by the Akaike information criterion in MEGA 6.06. BI analysis was performed using MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001), with posterior distributions estimated using Markov chain Monte Carlo (MCMC) sampling. The MCMC search was conducted for 100,000 generations, and sampling was done every 100 generations. The average standard deviation of split frequencies was below 0.01. Then, the first 25% of the trees were discarded as ‘burn-in’, and posterior probabilities were estimated for each node. For ML analysis, PhyML v3.0 (Guindon and Gascuel 2003) was used with the following conditions: equilibrium frequencies as ‘empirical’, proportion of invariable sites as ‘estimated’, number of substitution rates categorized in fours, gamma shape parameter as ‘estimated’, and starting tree as ’BIONJ’. The confidence values of the ML tree were evaluated via a bootstrap test with 100 replications. The obtained phylogenetic trees were visualized with Treeview 1.6.6 (Page 1996).

Results and Discussion

General Features of the Do. paradoxus Mitogenome

In the present study, we sequenced the entire mitogenome of Do. paradoxus by PCR amplification in 14 overlapping fragments with universal insect primers and species specific primers (Table 2). The complete mitogenome sequence of Do. paradoxus is 15,922 bp in size, which is the largest among the completely sequenced Cerambycidae insects. This mitogenome shares typical metazoan genes (Fig. 1, Table 3), including 13 PCGs, 22 tRNA genes, 2 rRNAs, and a noncoding A+T-rich region (Wolstenholme 1992). The gene arrangement and orientation of this mitogenome are identical to those of the completely sequenced coleopteran insects, with the ancestor gene order tRNA-tRNA-tRNA located between the A+T-rich region and ND2.

Fig. 1.

The gene map of the mitogenome of Do. paradoxus. The circle inside the GC content graph marks the 50% threshold. Genes are shown as blocks facing out if they are transcribed in the clockwise direction or facing in if they are transcribed in the counter-clockwise direction. COI, COII, and COIII refer to the cytochrome oxidase subunits, Cytb refers to cytochrome b, and ND1-6 refers to NADH dehydrogenase components. tRNA genes are denoted as one-letter symbols according to the IUPAC-IUB single-letter amino acid codes. The one-letter symbols L1, L2, S1, and S2 denote tRNA(CUN), tRNA(UUR), tRNA(AGN), and tRNA(UCN), respectively. lrRNA and srRNA refer to large and small rRNA, respectively.

Table 3.

Annotation of the mitogenome of Do. paradoxus

Gene	Direction	Nucleotide no.	Size (bp)	Anticodon	IGS	OL	Start codon	Stop codon
tRNA Ile	F	1–64	64	GAT	3
tRNA Gln	R	68–136	69	TTG		1
tRNA Met	F	136–204	69	CAT
ND2	F	205–1,213	1009				ATG	T
tRNA Trp	F	1,214–1,280	67	TCA	1
tRNA Cys	R	1,282–1,344	63	GCA	9
tRNA Tyr	R	1,354–1,418	65	GTA	1
COI	F	1,420–2,953	1534				AAT	T
tRNA Leu(UUR)	F	2,954–3,018	65	TAA
COII	F	3,019–3,699	681		3		ATC	TAG
tRNA Lys	F	3,703–3,772	70	TTT
tRNA Asp	F	3,773–3,845	73	GTC
ATP8	F	3,846–4,001	156			7	ATC	TAA
ATP6	F	3,995–4,668	674				ATG	TA
COIII	F	4,669–5,457	789		6		ATG	TAA
tRNA Gly	F	5,464–5,527	64	TCC
ND3	F	5,528–5,880	353				ATT	TA
tRNA Ala	F	5,881–5,946	66	TGC		1
tRNA Arg	F	5,946–6,011	66	TCG		1
tRNA Asn	F	6,011–6,075	65	GTT
tRNA Ser(AGN)	F	6,076–6,142	67	TCT
tRNA Glu	F	6,143–6,206	64	TTC	2
tRNA Phe	R	6,209–6,273	65	GAA
ND5	R	6,274–7,994	1,721				ATT	TA
tRNA His	R	7,995–8,063	69	GTG
ND4	R	8,064–9,396	1,333		1		ATG	T
ND4L	R	9,398–9,677	280				ATG	T
tRNA Thr	F	9,690–9,754	65	TGT
tRNA Pro	R	9,755–9,820	66	TGG	2
ND6	F	9,823–10,331	509				ATT	TA
Cytb	F	10,332–11,473	1,142				ATG	TA
tRNA Ser(UCN)	F	11,474–11,541	68	TGA	21
ND1	R	11,563–12,513	951				TTG	TAA
tRNA Leu(CUN)	R	12,514–12,579	66	TAG
lrRNA	R	12,580–13,868	1,289
tRNA Val	R	13,869–13,938	70	TAC
srRNA	R	13,939–14,742	804
A+T-rich region		14,743–15,922	1,180

The direction of the genes is presented as F for forward and R for reverse direction. OL denotes inside genes overlap. IGS represents the intergenic spacer sequences.

The nucleotide composition of the mitogenome of Do. paradoxus is biased toward As and Ts, which account for 71.13% of the base pairs (A 39.67%, G 11.00%, T 31.45%, and C 17.87%). The bias value is the lowest among the completely sequenced Cerambycidae species (Table 1).

The AT skew of the forward strand of the mitogenome of Do. paradoxus is slightly positive (0.116), indicating the occurrence of more As than Ts. This case is the largest among previously sequenced Cerambycidae mitogenomes. The GC skew of Do. paradoxus mitogenome is -0.238, which is well within the range of all analyzed Cerambycidae mitogenomes.

Coding Regions of the Do. paradoxus Mitogenome

Among 13 PCGs, 11 have a typical ATN start codon: 6 have an ATG (ND2, ATP6, COIII, ND4, ND4L, Cytb), 2 have an ATC (COII, ATP8), and 3 have an ATT (ND3, ND5, ND6). The remaining two PCGs begin with atypical codons: COI with AAT and ND1 with TTG, as found in Aeolesthes oenochrous (Fairmaire) (Coleoptera: Cerambycidae) (Chiu et al. 2016). AAT (Asparagine) is highly conserved in coleopteran Polyphaga (Sheffield et al. 2008), and TTG (Leucine) has been proposed as the start codon of ND1 in Anopheles quadrimaculatus Say (Diptera: Culicidae) and Tricholepidion gertschi Wygodzinsky (Insecta, Zygentoma) (Mitchell et al. 1993, Nardi et al. 2003). Only 4 of the 13 PCGs harbor a complete termination codon (three TAA and one TAG), the remaining 9 possess incomplete termination codons (4 T and 5 TA).

The Do. paradoxus mitogenome bears a typical set of 22 tRNA genes (1 specific for each amino acid and 2 each for Leucine and Serine). These tRNAs are scattered throughout the mitogenome, between rRNAs and PCGs, with sizes ranging from 63 bp for tRNA to 73 bp for tRNA. The anticodons are identical to those observed in other Cerambycidae insects. With the exception of tRNA(AGN), which lacks a stable stem-loop structure in the DHU arm, the other 21 tRNAs can fold into a typical clover-leaf secondary structures. This abnormal structure of tRNA(AGN) has been observed in many metazoan species, including insects (Wolstenholme 1992).

Two rRNA genes (lrRNA and srRNA), located between tRNA (CUN), and tRNA, and between tRNA and the A+T-rich region, respectively, were identified in the mitogenome of Do. paradoxus. The length of lrRNA and srRNA were 1,289 and 804 bp, respectively, which were well within the range observed in the completely sequenced Cerambycidae insects.

Noncoding Regions of the Do. paradoxus Mitogenome

The Do. paradoxus mitochondrial genes harbor a total of 49-bp intergenic spacer sequences, which are spread over 10 regions and range in size from 1 to 21 bp. The longest one is located between tRNA (UCN) and ND1 contains the 5-bp-long motif (TACTA), which is conserved across Cerambycidae species (Fig. 2A). This region is present in most insect mitochondrial DNA, even though the nucleotide sequence can be quite divergent (Cameron and Whiting 2008, Kim et al. 2009). This motif has been suggested to be a possible recognition site for mtTERM protein, the transcription termination peptide (Taanman 1999).

Fig. 2.

Sequence alignment results. (A) The intergenic spacer region between tRNA(UCN) and ND1 of Cerambycidae species. The bold nucleotides indicate the conserved 5 bp-long motif (TACTA) found in all sequenced Cerambycidae insects. (B) Tandem repeat units detected in the Do. paradoxus A+T-rich region. The nucleotide position is provided at each end of the sequence with regard to the mitogenome of Do. paradoxus.

The Do. paradoxus mitogenome has a total of 10-bp overlapping sequences that vary in length from 1 to 7 bp in four regions. The longest overlap of 7-bp overlap was found with the reading frames of ATP8/ATP6, which is common in insect mitogenomes (Boore 1999). The remaining three overlaps are involved in tRNAs.

The 1,180-bp A+T-rich region, located between srRNA and tRNA, exhibits the highest A+T content of 82.71% (A 44.07%, G 6.95%, T 38.64%, C 10.34%) in the mitogenome. In coleopteran insects, the presence of tandem repeats in the mitochondrial A+T-rich region has been observed, including 21 copies of a 58-bp repeat element in Trachypachus holmbergi Mannerheim (Coleoptera: Trachypachidae), 6 tandem repeats of a 312-bp sequence in Priasilpha obscura (Herbst) (Coleoptera: Phloeostichidae), 3 ~96-bp tandem repeat copies in Chaetosoma scaritides Westwood (Coleoptera: Melyridae), and 7 copies of 57-bp tandem repeats in Psacothea hilaris Pascoe (Coleoptera: Cerambycidae) (Sheffield et al. 2008, Kim et al. 2009). In the Do. paradoxus A+T-rich region, a tandem repeat comprising two 47-bp-long copies was identified, beginning from nucleotide number 15,315 with regard to the Do. paradoxus mitogenome. The nucleotide sequence of repeat 1 is almost identical to repeat 2 (Fig. 2B). The repeat sequence comprises >91.67% A+T, which is higher than that of the whole A+T-rich region. Moreover, the Do. paradoxus A+T-rich region includes two poly-T stretches: one is 12 bp long (position: 15,132–15,143) in the majority strand, and the other is 21-bp long (position: 15,841–15,861) in the minority strand near the tRNA. It has been verified that the latter poly-T stretch is a structural signal for the recognition of proteins that are involved in replication initiation, at least among holometabolous insects (Zuker et al. 1999).

Phylogenetic Relationships

In the present study, we intended to use the 18 complete or near complete mitogenomes available to date, including 13 Cerambycidae, four Chrysomelidae and one Vesperidae, to evaluate the phylogenetic relationship within the superfamily Chrysomeloidea. According to the morphological characters, the 13 Cerambycidae mitogenomes used in this study are derived from four subfamilies, i.e., Prioninae (Do. paradoxus, Prioninae sp.), Cerambycinae (Ae. oenochrous, Ma. raddei, Xy. grayii), Lamiinae (An. chinensis, An. glabripennis, Ap. swainsoni, Ba. lineolata, Mo. alternatus, Ps. hilaris, Th. gebleri), and Lepturinae (Rh. mordax). The mitogenome of Sp. spinicornis, belonging to Philinae of Vesperidae was also included due to its problematic status (Nie et al. 2017). Their phylogenetic relationships were reconstructed based on the nucleotide sequences of 13 PCGs with BI and ML methods (Fig. 3A and B). The phylogenetic analyses yielded almost identical topological relationships, indicating the presence of two distinct groups: Cerambycidae and Chrysomelidae, which is consistent with the morphological analysis and the previous findings (Nie et al. 2017). In our phylogenetic analyses, Lepturinae (Rh. mordax) is closely related to Chrysomelidae, rather than Cerambycidae, indicating that the taxonomy status of Rh. mordax needs to be reconsidered.

Fig. 3.

Phylogeny of Chrysomeloidea species. Phylogenetic tree inferred from the nucleotide sequences of 13 PCGs by Bayesian inference (A) and maximum likelihood (B). The numbers close to the nodes specify posterior probability values and bootstrap percentages, and values higher than 50% are shown.

Both BI and ML analyses confirmed the subfamily rank of Cerambycidae ([Prioninae + Cerambycinae] + Lamiinae) with high support values (100% for BI and 63% for ML), the monophyly of Lamiinae, with the nearest relationship being between Philinae (Sp. spinicornis) and Prioninae/Cerambycinae. BI analysis supported the monophyly of Prioninae and Cerambycinae, and the nearest relationship between Prioninae and Philinae with 100% posterior probabilities. However, the monophyly of Prioninae and Cerambycinae, and the nearest relationship between Prioninae and Philinae were not recovered in ML analysis. These results suggested that the monophyly of Prioninae and Cerambycinae and their relationship with Prioninae should be further studied in the future.