The investigation of intraspecific characteristics and comparative analyses of the complete mitochondrial genome of Stegobium paniceum (Linnaeus,1758) (Coleoptera: Ptinidae) assembled from public NGS raw reads of the black truffle,Tuber melanosporum

Abstract

Due to the rapid development of NGS technologies, a huge amount of NGS raw reads have been accumulated in public repositories, such as the Short Read Archive of NCBI. We successfully rescued the complete mitochondrial genome of Stegobium paniceum, a drug store beetle, from public NGS raw reads of truffle generated from the whole genome project. The circular mitogenome of S. paniceum is 15,474 bp long including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs, and a single large non-coding region of 803 bp. Intraspecific transfer RNAs structure and sequence variations were investigated and simple sequence repeats identified from three S. paniceum mitochondrial genomes were compared showing their diversities as fundamental data to utilize them in various aspects including developing efficient molecular markers in the family, Ptinidae. Phylogenetic analysis of 23 Bostrichoidea mitochondrial genomes presented better species identification based on phylogenetic analyses and the optimal options for constructing phylogenetic trees based on Bostrichoidea mitochondrial genomes. Our results present not only utilization of public NGS raw read sequences but also intraspecific features of S. paniceum mitochondrial genomes and comparative analysis of Bostrichoidea mitochondrial genomes in various aspects.

Keywords

mitochondrial genome truffle NGS reads intraspecific variations simple sequence repeats phylogenetic analysis Coleoptera Bostrichoidea Ptinidae

Introduction

Recent rapid development and utilization of next-generation sequencing (NGS) technologies have driven us to generate a large amount of NGS raw read sequences for various purposes,^1–3 such as the sequencing of the whole genome,^4–6 organelle genomes,^7–9 and phylogenetic and phylogenomic studies.^10,11 NGS raw read sequences can provide additional research opportunities because usually they cover almost all genomic DNA. For example, NGS raw reads from whole genomic DNA contains their organelle genome sequences, such as Rosa rugosa chloroplast¹² and mitochondrial¹³ genomes, Vibunum amplificatum, V. dilatatum,¹⁴ and Plantago ovata (Park et al. under review) chloroplast genomes and Monomorium pharaonis (Park et al. in preparation) and fourteen Pseudomyrmecinae mitochondrial genomes¹⁵ were rescued from public SRA datasets generated for various purposes.^11,16 In addition, organelle genomes of unexpected organisms were also retrieved, such as Rhopalosiphym mitochondrial genome¹⁷ from the whole genome of Nymphaea colorata¹⁸ and fungal mitochondrial genome of insect endosymbiont of Ricania speculum¹⁹ and Sogatella furcifera.²⁰ These examples also imply that these datasets may have additional DNA sequence reads originated from endophytic or pathogenic species unintendedly once these organisms were included in DNA samples.

Stegobium paniceum, known as the drugstore beetle or the biscuit beetle is a worldwide pest of stored products. They have an extremely polyphagous diet including all sorts of organic matter starting from food ingredients such as flour, dry mixes, mushrooms, and spices to non-food materials such as paper, wool, leather, museum specimens, and even prescription drugs, where its common name was originated. Adult beetles can pierce through light packages and then can lay up to 75 eggs on the substrate ²¹. The larval stage can vary from 4 to 20 weeks and is considered the most destructive stage as they bore tunnels in the food source. S. paniceum carries symbiotic yeasts, which produces nutrients such as vitamin B, compensating the poor nutrition uptake of the beetles.²²

Whole genome sequencing project of truffle, Tuber melanosporum, was published in 2010 with generating genome sequences from five individuals of truffle.²³ During assembling complete mitochondrial genome sequence of truffle, one insect mitochondrial genome was unexpectedly obtained from NGS raw reads of one individual of truffle (SRA accession: SRR5005499) which presents high similarity to that of S. paniceum. Considering with characteristics of S. paniceum, this insect might live inside the truffle and it was included unintendedly when DNA of this individual sample was prepared.

Based on obtained complete mitochondrial genome of S. paniceum together with already published mitochondrial genomes of S. paniceum,²⁴ identifications of intraspecific variations and SSRs among three mitochondrial genomes, nucleotide diversity analysis, configuration of 14 mitochondrial genomes of Anobiidae and Ptinidae, and phylogenetic analysis were conducted. Completion of our mitochondrial genome as a weak as diverse comparative analyses provides insights of characteristics of mitochondrial genomes in Anobiidae as well as a possibility of unexpected mitochondrial genomes which can be obtained from NGS raw reads publicly available.

Materials and methods

De novo assembly of the mitochondrial genome of S. paniceum from NGS raw reads of truffle

NGS raw reads of truffle (SRA accession: SRR5005499) were downloaded from NCBI and processed under the Genome Information System (GeIS, http://geis.infoboss.co.kr) which has been utilized in various genomics studies.^{7–9,13,14,17,19,20,25–83} SRR5005499 dataset used in this study contains 29,010,538 100-bp-reads (2,901,053,800 bp). In the pipeline, de novo assembly and confirmation were done by Velvet v1.2.10⁸⁴ with the options, minimum coverage is 80x and expected coverage is 100x, after filtering raw reads using Trimmomatic v0.33 with default option.⁸⁵ Gap-filling was conducted by GapCloser v1.12⁸⁶ with changing overlap parameters from 17 to 31 and assembled sequences were confirmed with BWA v0.7.17 with default alignment options⁸⁷ and the tview module of SAMtools v1.9.⁸⁸

Mitochondrial genome annotation

Geneious R11 v11.1.5 (Biomatters LSPANtd., Auckland, New Zealand) was used to annotate the mitochondrial genome of S. paniceum based on pairwise alignment with published S. paniceum mitochondrial genome.²⁴ ARWEN⁸⁹ was used for annotating tRNAs on the mitochondrial genome.

Drawing the circular map of the mitochondrial genome

The annotated GenBank format file of S. paniceum mitochondrial genome was used to draw the circular map using OGDRAW v1.3.1 with default options.⁹⁰

Prediction of tRNA structure

The annotated GenBank format file of S. paniceum mitochondrial genome was subjected to the MITOS web server with genetic code “05-invertebrate”,⁸⁷ and ARWEN web server with default option⁸⁹ for predicting tRNA structure. The prediction results were reviewed manually and draw final tRNA structure.

Calculation of codon usage

Codon usage was calculated based on gene annotations provided from NCBI database together with the pipelines of the revised insect mitochondrial genome database (Park et al. in preparation). Amino acids corresponding to codon sequence were investigated with a counting frequency of codons. After calculating all PCGs on the mitochondrial genome, statistics were generated in the environment of the revised insect mitochondrial genome database. The graph was drawn using Excel with adding codon labels.

Identification of intraspecific variations

Pair-wise sequence alignments among complete mitochondrial genomes of S. paniceum including the mitochondrial genome sequenced in this study were conducted by MAFFT v7.450⁹¹ with the default option. Identifying intraspecific variations, SNPs and INDELs, on the mitochondrial genomes of S. paniceum was conducted using the function, ‘Find variations/SNPs,’ implemented in the Geneious R11 v11.1.5 (Biomatters Ltd, Auckland, New Zealand), which has been utilized in the previous insect mitochondrial genome studies.^{8,25,26,33,34,71–79,83} When the number of INDELs was calculated, continuous INDEL bases were considered one INDEL region.

Identification of simple sequence repeats on S. paniceum mitochondrial genomes

Simple sequence repeats (SSRs) were identified on the mitochondrial genome sequence using the pipeline of the SSRDB (http://ssrdb.infoboss.co.kr). Based on the conventional definition of SSR on organelle genomes: monoSSR (1 bp) to hexaSSR (6 bp) and total length of SSRs is above 10 bp. Because of different criteria of SSRs on organelle genomes,^92–98 we adopted the criteria used in organelle genomes of Figulus binodulus,⁶² Sogatella furcifera (Park et al. ⁹⁹), endosymbiont of Sogatella furcifera,²⁰ Dysphania ambrosioides,⁵⁷ Arabidopsis thaliana,⁶⁸ Chenopodium album,⁶⁹ Rosa rugosa,¹³ Plnatago (Park et al. under review), and Agrimonia species (Park et al. in submission): monoSSR (unit sequence length is 1 bp) to hexaSSR (6 bp) are used as normal SSRs and heptaSSR (7 bp) to decaSSR (10 bp) are defined as extended SSRs. Among normal SSRs, pentaSSRs and hexaSSRs of which unit number is 2 are classified as potential SSRs.

Comparison of simple sequence repeats among three S. paniceum mitochondrial genome

The SSRDB provides a comparison pipeline based on flanking sequences of identified SSRs named as ‘SSR Comparison History,’ which has been utilized in the previous studies.^20,62,68,69 Because of the different coordination of mitochondrial genomes, nucleotide position is not an absolute criterion to identify SSRs found in the same region. This pipeline finds similar SSRs by selecting SSRs of which 500-bp flanking sequences of both sides of SSR are similar (sequence coverage is above 90% and sequence identity is above 90%). After selecting the candidates of similar SSRs, unit sequence and repeat number of SSRs are investigated to know whether sets of similar SSRs, called group SSRs, contain similar SSR or not.

Nucleotide diversity analysis including S. paniceum mitochondrial genomes

Nucleotide diversity was calculated based on the method proposed by Nei and Li¹⁰⁰ using the perl script based on two sets of multiple sequence alignments, which has been utilized in the previous studies.^{14,59,62,68–70,83} One is from four Ptinidae mitochondrial genomes including S. paniceum (MK947052), Gastrallus laevigatus (KX087292), Lasioderma redtenbacheri (KX087303), and Lasioderma serricorne (NC_038197) and the other is from eighteen mitochondrial genomes of superfamily Bostrichoidea (S. paniceum (MK947052), Gastrallus laevigatus (KX087292), Lasioderma redtenbacheri (KX087303), Lasioderma serricorne (NC_038197), Anthrenus verbasci (KX087239), Anthrenus verbasci (MG017450), Dermestes tessellatocollis (NC_044849), Dermestes maculatus (NC_037200), Dermestes frischii (NC_044850), Dermestes coarctatus (NC_044851), Nosodendron fasciculare (KX087322), Nosodendron sp. (KX035152), Anobiidae sp. (KT696225), Thorictus sp. (MK692678), Rhyzopertha dominica (NC_042820), Dinoderus minutus (KX087284), Bostrichoidea sp. (KX035220), and Apatides fortis (NC_013582)). Window size is 500 bp and step size is 200 bp for sliding-window method. Genomic coordination of each window was compared with gene annotation of mitochondrial genome.

Construction of phylogenetic trees

Twenty-three complete or partial mitochondrial genomes containing all 13 Protein-coding genes (PCGs) and 2 rRNA genes from superfamily Bostrichoidea were selected and retrieved from the NCBI. Together with the newly completed S. paniceum mitochondrial genome and an outgroup, Chaetosoma scaritides, four datasets were created: i) 13 PCGs with the third codon sites removed (called as dataset I), ii) 13 PCGs with all codon sites included (called as dataset II), iii) 13 PCGs with the third codon sites removed and 2 rRNA genes (called as dataset III), and iv) 13 PCGs with all codon sites and 2 rRNA genes (called as dataset IV). Codon-based alignments were individually made for each PCG using MAFFT provided on the Translator X web server with the default settings.¹⁰¹ rRNA genes were also aligned using MAFFT 7.450.⁹¹ Exclusion of the third codon sites and the concatenating of the aligned sequences were conducted under the environment of Geneious R11 v11.1.5. The maximum-likelihood (ML) trees were reconstructed in MEGA X.¹⁰² During the ML analysis, a heuristic search was used with nearest-neighbor interchange (NNI) branch swapping, the Tamura-Nei model, and uniform rates among sites. All other options were set to their default values. Bootstrap analyses with 1000 pseudoreplicates were conducted with the same options. The posterior probability of each node was estimated using the Bayesian inference (BI) using MrBayes v3.2.6.¹⁰³ The GTR model with gamma rates was used as a molecular model. A Markov-chain Monte Carlo algorithm was employed for 1,000,000 generations, sampling trees every 200 generations, with four chains running simultaneously. Trees from the first 100,000 generations were discarded as burn-in.

Results and discussions

The complete mitochondrial genome of S. paniceum

From the public NGS raw reads of truffle, we rescued insect mitochondrial genome sequence when we tried to obtain truffle mitochondrial genome. After trimming 30 kb assembled sequences because the sequence was doubled due to circular form of the insect mitochondrial genome, we confirmed that it is from S. paniceum based on the similarity of COI sequence. 39,635 out of 29,010,538 100-bp-reads (0.13%) were used for S. paniceum mitochondrial genome sequences displaying 265x coverage.

Here, we present the complete mitochondrial genome of S. paniceum (GenBank accession: MK947052) of which length is 15,474 bp, containing 13 PGCs, 22 transfer RNAs (tRNAs), and 2 ribosomal RNAs (rRNAs; Figure 1; Table 1), which is the typical configuration of insect mitochondrial genomes. Its GC ratio is 21.9%, which is similar to that of S. paniceum complete mitochondrial genome (21.7%; NC_036678).²⁴ It is slightly higher than the three complete mitochondrial genomes of the Xyletininae family (Table 2). The order of PGCs on S. paniceum mitochondrial genome is the same as those of the Anobiidae family; while the tRNA order of mitochondrial genomes between Anobiidae and Xyletininae families are different: e.g., strand of tRNA-Gln (UUG) on S. paniceum mitochondrial genome is reversed in comparison to that of Lasioderma serricorne mitochondrial genome.

Fig. 1.

Complete mitochondrial genome sequence of S. paniceum. Grey graph inside circular diagram presents GC ratio of mitochondrial genome. Colourful bars on outer circular form indicate PCGs (yellow, pink, green, and purple), tRNAs (dark blue), and rRNAs.

Table 1.

Summary of S. paniceum mitochondrial genome.

Gene	Strand	Start	End	Size (bp)	Anticodon	Start codon	Stop codon	Intergenic nucleotide (bp)*
COX1	Y	15439	15474	1531		AAA	T	N/A
		1	1495
tRNA-Leu	Y	1496	1559	64	UAG	TTT	A	0
COX2	Y	1560	2232	673		ATA	T	0
tRNA-Lys	Y	2233	2302	70	CUU	CAT	A	0
tRNA-Asp	Y	2302	2364	63	GUC	AAA	TTA	−1
ATP8	Y	2365	2520	156		ATT	TAA	0
ATP6	Y	2514	3182	669		ATG	TAA	−7
COX3	Y	3182	3968	787		ATG	T	−1
tRNA-Gly	Y	3969	4031	63	UCC	ATT	ATA	0
ND3	Y	4032	4385	354		ATA	TAG	0
tRNA-Ala	Y	4384	4447	64	UGC	AGG	A	−2
tRNA-Arg	Y	4448	4510	63	UCG	TAA	TTT	0
tRNA-Asn	Y	4509	4572	64	GUU	TTA	A	−2
tRNA-Ser	Y	4572	4638	67	UCU	AGA	T	−1
tRNA-Glu	Y	4639	4701	63	UUC	ATT	ATA	0
tRNA-Phe	N	4700	4762	63	GAA	TAC	AGT	−2
ND5	N	4763	6476	1714		AAA	T	0
tRNA-His	N	6477	6539	63	GUG	AAC	AGT	0
ND4	N	6540	7869	1330		ATA	T	0
ND4L	N	7866	8141	276		TTA	CAT	−4
tRNA-Thr	Y	8144	8206	63	UGU	GTT	CCA	2
tRNA-Pro	N	8207	8269	63	UGG	TCA	CTG	0
ND6	Y	8271	8747	477		ATA	TAA	1
CYTB	Y	8747	9883	1137		ATG	TAG	−1
tRNA-Ser	Y	9882	9947	66	UGA	AGT	CTT	−2
ND1	N	9968	10912	945		CTA	CAA	20
tRNA-Leu	N	10914	10975	62	UAA	TAT	AT	1
16S rRNA	N	10976	12229	1254		AGT	AAA	0
tRNA-Val	N	12230	12297	68	UAC	TCA	TG	0
12S rRNA	N	12298	13248	951		TCT	TTT	0
D-Loop	Y	13249	14051	803				0
tRNA-Ile	Y	14052	14113	62	GAU	AAT	TA	0
tRNA-Gln	N	14111	14179	69	UUG	TTA	ATA	−3
tRNA-Met	Y	14179	14248	70	CAU	AAA	A	−1
ND2	Y	14252	15259	1008		ATA	TAA	3
tRNA-Trp	Y	15258	15321	64	UCA	AAG	A	−2
tRNA-Cys	N	15314	15376	63	GCA	AAG	GCT	−8
tRNA-Tyr	N	15376	15437	62	GUA	TAA	TT	−1

*This column indicates the distance between two adjunct genes.

Table 2.

List of 28 available mitochondrial genomes from superfamily Bostrichoidea including S. paniceum completed in this study.

Family	Subfamily	Species name	Complete	GenBank accession	Length (bp)	Gap length (bp)	# of PCGs	# of tRNAs	# of rRNAs	GC ratio (%)	AT ratio (%)	GC-skew	AT-skew	Reference
Ptinidae	–	Anobiidae sp. BMNH 1274383	Y	KT696225	18,471	0	13	22	2	20.42	79.58	0.1838	−0.0288	Unpub.
	Ernobiinae	Ernobius pini	N	KX087290	6656	0	7	16	0	34.31	65.69	−0.1751	0.0252	Unpub
	Anobiinae	Anobiinae sp. GENSP01	N	JX412730	12,786	0	13	20	0	23.75	76.25	−0.1689	0.0772	Unpub
		Gastrallus laevigatus	N	KX087292	15,361	0	13	22	2	23.58	76.42	−0.2176	0.1137	Unpub
		Stegobium paniceum	Y	MK947052	15,474	0	13	22	2	21.89	78.11	−0.1692	0.0626	This study
		Stegobium paniceum	Y	NC_036678	15,271	0	13	22	2	21.68	78.32	−0.1640	0.0629	²⁴
		Stegobium paniceum	N	MH817140	14,101	0	13	19	2	22.49	77.51	−0.1595	0.0711	¹⁰⁴
		Xyletinus sp. XYL01	N	JX313671	9719	0	11	15	0	22.73	77.27	0.0575	−0.0115	Unpub
	Xyletininae	Lasioderma redtenbacheri	N	KX087303	15,042	0	13	21	2	22.55	77.45	−0.0501	−0.0357	Unpub
		Lasioderma serricorne	Y	MF417629	15,958	0	13	22	2	21.41	78.59	−0.0357	−0.0292	¹⁰⁵
		Lasioderma serricorne	Y	MH817138	14,550	0	13	22	2	21.71	78.29	−0.0408	−0.0289	¹⁰⁴
		Lasioderma serricorne	Y	NC_038197	14,476	0	13	22	2	21.59	78.41	−0.0377	−0.0322	¹⁰³
Ptinidae	–	Niptus hololeucus	N	KX087321	7828	0	8	14	0	26.90	73.10	−0.2070	0.0587	Unpub
Ptinidae	–	Ptinus rufipes	N	HQ232819	10,453	182	12	13	0	27.85	72.15	−0.1664	0.1610	¹⁰⁶
Bostrichidae	Bostrichinae	Apatides fortis	Y	NC_013582	16,171	0	13	22	2	32.81	67.19	−0.2946	0.1704	¹⁰⁷
	Dinoderinae	Dinoderus minutus	Y	KX087284	15,230	0	13	22	2	26.48	73.52	−0.2368	0.1773	Unpub
	Dinoderinae	Rhyzopertha dominica	Y	NC_042820	15,859	0	13	22	2	25.66	74.34	−0.2077	0.1905	Unpub
Dermestidae	Megatominae	Anthrenus verbasci	N	KX087239	15,818	0	13	22	2	26.68	73.32	−0.2741	0.1606	Unpub
	Megatominae	Anthrenus verbasci	N	MG017450	14,793	0	13	22	2	27.66	72.34	−0.2444	0.1898	Unpub
	Dermestinae	Dermestes maculatus	Y	MN900863	16,368	0	13	22	2	23.37	76.63	−0.1909	0.0713	Unpub
		Dermestes maculatus	Y	NC_037200	16,390	0	13	22	2	23.03	76.97	−0.1989	0.0602	¹⁰⁸
		Dermestes tessellatocollis	Y	NC_044849	16,218	0	13	22	2	23.13	76.87	−0.1903	0.0725	Unpub
		Dermestes frischii	Y	NC_044850	15,873	0	13	22	2	23.21	76.79	−0.183	0.0573	Unpub
		Dermestes coarctatus	Y	NC_044851	15,873	0	13	22	2	23.42	76.58	−0.2074	0.076	Unpub
	Thorictinae	Thorictus sp.	N	MK692678	16,473	0	13	N/A	2	28.18	71.82	−0.2611	0.1188	¹⁰⁹
Nosodendridae		Nosodendron fasciculare	N	KX087322	15,896	0	13	22	2	19.3	80.7	−0.2106	0.0355	Unpub
Nosodendridae		Nosodendron sp.	Y	KX035152	16,704	0	13	22	2	23.02	76.98	−0.2865	0.0394	Unpub
		Bostrichoidea sp.	Y	KX035220	15,462	0	13	22	2	31.73	68.27	−0.2964	0.1341	Unpub

The length of tRNAs ranges from 62 bp to 70 bp (Table 1). From the predicted tRNA secondary structure, the length of the nucleotide-amino acid acceptor arm (AA arm) is 7 bp in 22 tRNAs and anticodon arm (AC arm) length is from 4 bp to 5 bp (Figure 2). Compared to AA and AC arms, the length of the dihydrouridine arm (D arm) and TΨC arm (T arm) are more variable, ranging from 2 bp to 5 bp.¹⁰² All 22 tRNAs were shaped into a typical cloverleaf structure except tRNA-Ser (UCU) which lacks a D arm structure (Figure 2). Lack of D arm in tRNA-Ser on the mitochondrial genome is already a known phenomenon in many metazoan species.¹¹¹ On the other hand, tRNA-Val (UAC) on S. paniceum mitochondrial genome has a typical D arm structure, which is different from thrips mitochondrial genomes which miss D arm structure in tRNA-Val (UAC).¹⁰⁴ Twenty-four mismatches in 22 tRNAs were identified based on MITOS web server: eighteen out of twenty-four (75.00%) were G-U mismatch which can be explainable as G-U mismatch could make two hydrogen bonds and it has distinctive features and functions.¹¹² Four of twenty-four mismatches (16.67%) are U-U mismatches, one (4.17%) is U-C mismatch, and one (4.17%) is A-G mismatch. Predicted tRNA-Ser (UCU) structures from MITOS and ARWEN webserver display incongruent manner: there is C between U-A, and A-U bonds in AC arm in MITOS prediction (Figure 2); however, prediction structure from ARWEN showed two mismatches, A-A, and U-C in the same position of U-A and A-U bonds.

Fig. 2.

22 tRNA structure originated from S. paniceum mitochondrial genome. Structure of twenty-two tRNAs are presented with base pair of tRNAs. Names of tRNAs and anticodons are displayed on the right side under their structure. G-U mismatches (Wobble pairs) are coloured blue, and other mismatches were coloured red. Also, distinctive structure in Ser (UCU) is coloured red.

Codon usage of mitochondrial genomes of S. paniceum was analysed (Figure 3B). Based on the codon table of the invertebrate mitochondrial genome, there are two codons for methionine; however, two more codons, ATT (2 times) and TTG (1 time), were identified (see red colour codons in Figure 3B), caused by RNA editing events. It is different from that of Lasioderma serricorne (Coleoptera: Anobiidae) displaying the three exceptional codons: ATT, ATC, and TTG.¹⁰⁴ In addition, one of four codons for proline (CCG) and one of four codons for arginine (CGC) were not used in this mitochondrial genome (Figure 3B).

Fig. 3.

Codon usage of S. paniceum mitochondrial genome. (A) X-axis presents amino acids and Y-axis presents the number of amino acids along with the direction of genes ( + strand is blue colour and – strand is red colour) (B) Frequency of codons along with amino acids. Codons are displayed on the bars or around the bars with arrows. Red coloured codons indicate exceptional cases caused by an RNA editing event.

Identification and analysis of intraspecific variations on S. paniceum mitochondrial genomes

Intraspecific variations of insect mitochondrial genomes can be used for molecular markers to distinguish their origins in the level of populations^113–118 or to identify cryptic species;^119–121 however, these variations were not investigated intensively even though there are several complete or near-complete mitochondrial genomes of the specific species till now. To understand intraspecific characteristics of the mitochondrial genome of S. paniceum, one complete and one partial mitochondrial genome of S. paniceum (Table 2) were used for multiple sequence alignments to identify intraspecific variations. The genetic similarity among three S. panecium mitochondrial genomes is above 0.98 (Figure 4A), indicating three mitochondrial genomes are similar to each other.

Fig. 4.

Genetic distance and identified SNPs and INDELs among three S. paniceum mitochondrial genomes. (A) displays the table of genetic distances (left-down area) and number of SNPs and INDELs (right-up area) identified among three S. paniceum mitochondrial genomes. (B) Circular diagrams indicate three mitochondrial genomes of S. paniceum and boxes with an orange colour border mean genes containing synonymous SNPs and boxes with blue colour border indicate genes containing non-synonymous genes. Numbers of SNPs and INDELs are displayed around black arrows.

Eleven SNPs and 205 INDELs were identified between the mitochondrial genome in this study (GenBank accession: MK947052) and NC_036678²⁴ (Figure 4B). Three non-synonymous SNPs (nsSNPs) were found in ND4, ND1, and ND2 genes, respectively, and two synonymous SNPs (sSNPs) were found in ND2 and ND3 genes, respectively (Figure 4B). Interestingly, one INDEL was found in tRNA-Phe, which presents that three continuous cytosines on this mitochondrial genome; while four cytosines in NC_036678. The remaining SNPs and INDELs were in the 3′ of rRNA and putative control region. Twenty-two SNPs and 2 INDELs were identified between NC_036678²⁴ and the partial mitochondrial genome (GenBank accession: MH817140) which missed a part of 12S rRNA and control region (Figure 4B). Three nsSNPs were found in COX3, CYTB, and ND1, respectively, and ND4 and ND2 genes cover two nsSNPs each. Five sSNPs were found in COX2, ND3, ND5, ND1, and ND2 genes, respectively (Figure 4). One INDEL was also found in tRNA-Phe which is the same tRNA containing one INDEL in the previous comparison and one SNP was found in tRNA-Leu. The remaining SNPs and INDELs were in 16S and 5′ of 12S rRNAs. 14 SNPs without INDEL were found between MK947052 and MH817140 (Figure 4): four nsSNPs were in COX3, ND4, CYTB, and ND2 genes, respectively, three sSNPs were in COX2, ND5, and ND1 genes, respectively, and one SNP was found in tRNA-Leu (Figure 4B). Based on the small amount of sequence variations between two complete mitochondrial genomes of S. paniceum and partial mitochondrial genome (GenBank accession: MH817140), especially for INDELs, the control region of S. paniceum mitochondrial genomes may contain most sequence variations.

In tRNA, most of the tRNA sequences were consensus except tRNA-Leu (UAG), and tRNA-Phe (GAA).⁶ Intraspecific variations identified on tRNAs did not affect tRNAs secondary structure such as stems or anticodon (Figure 5A and 5B). In detail, substitution occurs in D-loop (U into G; Figure 5A) of tRNA-Leu (UAG) and insertion occurs in the variable loop (G was inserted; Figure 5B) of tRNA-Phe.⁶ These intraspecific variations of tRNAs indicate that sequence variations can occur on tRNAs but they will not affect their secondary structure because of their essential function in the mitochondrion.

Fig. 5.

Intraspecific variations of tRNAs in S. paniceum mitochondrial genomes. Variation of tRNAs are indicated as red circles. Also wobble pair is coloured blue and other mismatches coloured red. (A) Substitution of tRNA-Leu (UAG) in D-loop. (B) Insertion of tRNA-Phe in variable loop.

The number of sequence variations (11 SNPs and 205 INDELs) identified between S. paniceum complete mitochondrial genomes is similar or less level to those of Spodoptera frugiperda (275 SNPs and 43 INDELs between MN385596 and NC_027836, 351 SNPs and 49 INDELs between MN385596 and KU877172, 247 SNPs and 74 INDELs between MN385596 and KJ778892)⁸, Laodelphax striatellus (141 SNPs and 166 INDELs between MK862265 and NC_013706, 41 SNPs and 118 INDELs between MK862265 and JX880068),³⁷ and Chilo suppressalis (85 SNPs and 199 INDELs between MK207057 and NC_015612, 360 SNPs and 124 INDELs between MK207057 and HQ860290, and 836 SNPs and 243 INDELs between HQ860290 and NC_015612).³⁵ It is higher than those of Nilaparvata lugens (90 SNPs and 10 INDELs between MK606371 and MK590088²⁵ and 112 SNPs and 59 INDELs between MK590088 and NC_021748),³³ Aphis gossypii (61 SNPs and 3 INDELs between MN102349 and NC_024581,³⁴ 66 SNPs and 176 INDELs between MT430940 and NC_024581, and 11 SNPs and 173 INDELs between MT430940 and MN943499⁷¹), Hipparchia autonoe (19 SNPs),²⁶ Monomorium pharaonis (12 SNPs; Park et al. in preparation), and Laodelphax striatellus between MK838101 and MK862265 which were captured in Korea (only one SNP).³⁷ Considering with different origins of the samples and species-specific characteristics of each insect species of which intraspecific variations were investigated, the amount of intraspecific variations of S. paniceum mitochondrial genomes is enough to be utilized for distinguishing their origin; however, more mitochondrial genomes will be needed to establish stable molecular markers. Because of the utilization of the control region on mitochondrial genome,¹²² many intraspecific variations identified on S. paniceum mitochondrial genomes can be also utilized as molecular markers.

Comparative analyses of 28 mitochondrial genomes of superfamily bostrichoidea

GC ratio, GC-skew, AT ratio, and AT-skew values of 28 Bostrichoidea mitochondrial genomes were calculated based on their mitochondrial genomes (Table 2) and plotted in the two graphs (Figure 6A and 6B). Three mitochondrial genomes of S. paniceum display similar characteristics as we expected because of the small number of intraspecific variations in comparison to those of inter-specific variations (Figure 6). In addition, one partial mitochondrial genome (GenBank accession: MH817140) is placed away from the other two complete mitochondrial genomes (See star marks in circles labelled as S. paniceum) because part of control region sequences was missed (Figure 6).

Fig. 6.

At ratio vs AT skew and GC ratio vs GC skew in 14 Anobiidae and Ptinidae mitochondrial genomes. (A) X-axis present GC ratio and Y-axis show GC-skew. (B) X-axis presents AT ratio and Y-axis shows AT-skew. Coloured circles indicate complete or partial mitochondrial genomes. Red circle indicates three S. paniceum including this study. Stars on coloured circles mean partial mitochondrial genome.

Subfamilies Xyletininae, and Dermestinae were well clustered from the remaining subfamilies (Figure 6A and 6B) which can be caused by their narrow taxonomical range: mitochondrial genomes from both families are in one genus. Interestingly, three S. paniceum (family Anobiinae) mitochondrial genomes were clustered, while Gastrallus laevigatus (family Anobiinae) is away from them (Figure 6), indicating that taxonomic distance is reflected in these plots. Anobiidae sp. (GenBank accession: KT696225) was far from those of superfamily Bostrichoidea in GC Skew plot (Figure 6B), which is congruent to the constructed phylogenetic tree that Anobiidae sp. is basal species in comparison to the other species. Taken together, GC ratio/GC skew and AT ratio/AT skew plots can be used as another barometer to measure their taxonomical distances beside phylogenetic trees.

Configuration of 28 mitochondrial genomes of Bostrichoidea

Based on multiple sequence alignment of available 28 mitochondrial genomes including three S. paniceum mitochondrial genomes assembled in this study, all 28 mitochondrial genomes present the same gene order (Figure 7A). Three S. paniceum mitochondrial genomes display the same gene order. In addition, all 28 mitochondrial genomes have the same gene order including PCGs, tRNAs, and rRNAs (Figure 7A). In addition, control regions of 16 complete mitochondrial genomes display low conserveness in comparison to the rest regions (Figure 7A), which is a similar trend to the other insect mitochondrial genomes,^7,29–32,36 so that further analysis is required.

Fig. 7.

Genome configuration of 22 Bostrichoidea mitochondrial genomes. (A) displays multiple sequence alignment of 22 Bostrichoidea complete or partial mitochondrial genomes. NCBI accessions of mitochondrial genome are displayed on the left side. Bolded accession indicates mitochondrial genome of S. paniceum assembled in this study. Black dotted box indicates the control region of mitochondrial genomes used in the multiple sequence alignment. (B) presents multiple sequence alignment of control region sequences extracted from sixteen mitochondrial genomes of which the control regions are available.

Control region, which is the most variable region in the mitochondrial genome¹²³ and covers displacement loop (D-loop) region comprising a third strand of DNA creating a semi-stable structure,^122,124 of the 16 complete mitochondrial genomes were collected and aligned to measure its conserveness (Figure 7B). As we mentioned above, most of the intraspecific variations of S. paniceum mitochondrial genomes were found in the control region, reflecting that the control region is the most variable region on the mitochondrial genome. Nucleotide diversity of control region including D-loop region was calculated, resulting 0.2131 which is higher than nucleotide diversity of conserved regions covering 13 protein-coding genes, 2 ribosomal RNAs, and 13 transfer RNAs (0.08368 in family Ptinidae and 0.1287 in superfamily Bostrichoidea; see the section, Nucleotide diversity of Bostrichoidea mitochondrial genomes). Nucleotide diversity of the control region is also higher than those of other insect species, such as tribe Figulini (0.1331)⁶² and family Thripidae (0.1329; Seo et al. in preparation).

Simple sequence repeat analysis of S. paniceum mitochondrial genomes

Simple sequence repeat (SSR) was investigated on S. paniceum mitochondrial genomes using the pipeline of the SSR database (See Material and methods section). In total, 90 SSRs were identified (Table 3): Number of pentaSSRs is the largest (35 SSRs; 38.89%) and that of hexaSSR is the second largest (28 SSRs; 31.11%); while there are only one triSSR (1.11%), one octaSSR (1.11%), three heptaSSRs (3.33%) and four monoSSRs (4.44%; Figure 8A). This trend is similar to that of F. binodulus⁶² even though the total number of SSRs of S. paniceum is almost two times than that of F. binodulus (50 SSRs); while it is different from that of Monomorium pharaonis (99 SSRs and 101 SSRs from that of USA and Korean isolates; Park et al. in preparation).

Fig. 8.

Distribution of simple sequence repeats of S. paniceum mitochondrial genome. (A) X axis presents SSR types and Y axis indicates number of SSRs for each type. Three colours, blue, orange, and grey, indicate mitogenomes of this study (GenBank accession: MK947052), NC_036678, and MH817140 (partial genome), respectively. The numbers on the bars indicates the number of SSRs. (B) The pie graph displays the number of SSR groups and the transparent blue area indicates that singleton SSRs consist of three origins of SSRs displayed in (C).

Table 3.

List of SSRs identified from S. paniceum mitochondrial genomes.

No	Name	SSR type	Type	Cooridnation		Unit sequence	Repeat number	Position	Genic
1	cP0000001	PotentialSSR	PentaSSR	10	19	TATTT	2	Genic	COX1
2	cH0000001	PotentialSSR	HexaSSR	1644	1655	TTAATT	2	Genic	COX2
3	cP0000002	PotentialSSR	PentaSSR	2486	2495	TTAAA	2	Genic	ATP8
4	cH0000003	PotentialSSR	HexaSSR	2589	2600	TTAATT	2	Genic	ATP6
5	cP0000003	PotentialSSR	PentaSSR	2636	2645	TAATA	2	Genic	ATP6
6	cH0000004	PotentialSSR	HexaSSR	2787	2798	ACAAGA	2	Genic	ATP6
7	cH0000005	PotentialSSR	HexaSSR	2812	2823	CTTTAT	2	Genic	ATP6
8	cH0000006	PotentialSSR	HexaSSR	3058	3069	TAATTA	2	Genic	ATP6
9	cP0000004	PotentialSSR	PentaSSR	3204	3213	CATTT	2	Genic	COX3
10	cH0000007	PotentialSSR	HexaSSR	3255	3266	TTTTAA	2	Genic	COX3
11	cH0000008	PotentialSSR	HexaSSR	3335	3346	ATTATA	2	Genic	COX3
12	cP0000005	PotentialSSR	PentaSSR	3696	3705	TTTAC	2	Genic	COX3
13	cH0000009	PotentialSSR	HexaSSR	4317	4328	TTTATT	2	Genic	ND3
14	cP0000006	PotentialSSR	PentaSSR	5133	5142	AATAA	2	Genic	ND5
15	cP0000007	PotentialSSR	PentaSSR	5251	5260	AAAGA	2	Genic	ND5
16	cM0000001	SSR	MonoSSR	5329	5338	A	10	Genic	ND5
17	cD0000001	SSR	DiSSR	5341	5356	AT	8	Genic	ND5
18	cP0000008	PotentialSSR	PentaSSR	5615	5624	AAAAT	2	Genic	ND5
19	cP0000009	PotentialSSR	PentaSSR	5675	5684	TCAAA	2	Genic	ND5
20	cP0000010	PotentialSSR	PentaSSR	5974	5983	AAAAT	2	Genic	ND5
21	cP0000011	PotentialSSR	PentaSSR	6210	6219	TAAAA	2	Genic	ND5
22	cH0000010	PotentialSSR	HexaSSR	6321	6332	AAATAA	2	Genic	ND5
23	cH0000011	PotentialSSR	HexaSSR	6392	6403	TTAAAA	2	Genic	ND5
24	cP0000012	PotentialSSR	PentaSSR	6422	6431	AAAAT	2	Genic	ND5
25	cH0000012	PotentialSSR	HexaSSR	6439	6450	AAAAAG	2	Genic	ND5
26	cM0000002	SSR	MonoSSR	6451	6460	A	10	Genic	ND5
27	cP0000013	PotentialSSR	PentaSSR	6570	6579	AAAAT	2	Genic	ND4
28	cH0000013	PotentialSSR	HexaSSR	6682	6693	AAATAA	2	Genic	ND4
29	cD0000002	SSR	DiSSR	7156	7165	TA	5	Genic	ND4
30	cP0000014	PotentialSSR	PentaSSR	7326	7335	AAAAT	2	Genic	ND4
31	c70000003	ExtenedSSR	HeptaSSR	7393	7406	AAAATAG	2	Genic	ND4
32	cTe0000001	SSR	TetraSSR	7542	7553	AAAT	3	Genic	ND4
33	cH0000014	PotentialSSR	HexaSSR	7741	7752	ATAATT	2	Genic	ND4
34	cP0000015	PotentialSSR	PentaSSR	8157	8166	AAATA	2	Genic	tRNA-Thr
35	cH0000015	PotentialSSR	HexaSSR	8285	8296	ATTAAT	2	Genic	ND6
36	cH0000016	PotentialSSR	HexaSSR	8455	8466	TTATTT	2	Genic	ND6
37	cD0000003	SSR	DiSSR	8574	8585	TA	6	Genic	ND6
38	cT0000001	SSR	TriSSR	8612	8623	AAT	4	Genic	ND6
39	cH0000017	PotentialSSR	HexaSSR	9111	9122	TTATAG	2	Genic	CYTB
40	cP0000016	PotentialSSR	PentaSSR	9917	9926	ATAAA	2	Genic	tRNA-Ser
41	cH0000018	PotentialSSR	HexaSSR	10067	10078	ATAACG	2	Genic	ND1
42	cM0000003	SSR	MonoSSR	10362	10372	A	11	Genic	ND1
43	cH0000019	PotentialSSR	HexaSSR	10388	10399	ATAAAA	2	Genic	ND1
44	cP0000017	PotentialSSR	PentaSSR	10442	10451	TAAAA	2	Genic	ND1
45	cH0000020	PotentialSSR	HexaSSR	10592	10603	AAAATT	2	Genic	ND1
46	cP0000018	PotentialSSR	PentaSSR	10624	10633	GAAAA	2	Genic	ND1
47	cH0000021	PotentialSSR	HexaSSR	10682	10693	AAAATT	2	Genic	ND1
48	cP0000019	PotentialSSR	PentaSSR	10986	10995	AAAAT	2	Genic	16S rRNA
49	cTe0000002	SSR	TetraSSR	11272	11283	TAAA	3	Genic	16S rRNA
50	cP0000020	PotentialSSR	PentaSSR	11528	11537	ATTAA	2	Genic	16S rRNA
51	cP0000021	PotentialSSR	PentaSSR	11557	11566	TTATA	2	Genic	16S rRNA
52	cTe0000003	SSR	TetraSSR	11722	11733	TAAA	3	Genic	16S rRNA
53	cP0000022	PotentialSSR	PentaSSR	11887	11896	TAAAT	2	Genic	16S rRNA
54	cTe0000004	SSR	TetraSSR	11992	12003	AAAT	3	Genic	16S rRNA
55	cP0000023	PotentialSSR	PentaSSR	12080	12089	TAAAT	2	Genic	16S rRNA
56	cP0000024	PotentialSSR	PentaSSR	12097	12106	TTAAA	2	Genic	16S rRNA
57	cP0000025	PotentialSSR	PentaSSR	12165	12174	AATAA	2	Genic	16S rRNA
58	cH0000022	PotentialSSR	HexaSSR	12438	12449	ATTAAT	2	Genic	12S rRNA
59	cH0000023	PotentialSSR	HexaSSR	12699	12710	AAATTT	2	Genic	12S rRNA
60	cTe0000005	SSR	TetraSSR	12772	12783	AATA	3	Genic	12S rRNA
61	cD0000004	SSR	DiSSR	12991	13000	TA	5	Genic	12S rRNA
62	cH0000024	PotentialSSR	HexaSSR	13291	13302	ATTTAT	2	Intergenic
63	cM0000004	SSR	MonoSSR	13307	13316	T	10	Intergenic
64	cP0000026	PotentialSSR	PentaSSR	13360	13369	TTTAA	2	Intergenic
65	cD0000005	SSR	DiSSR	13475	13484	TA	5	Intergenic
66	cD0000006	SSR	DiSSR	13515	13530	TA	8	Intergenic
67	cD0000007	SSR	DiSSR	13545	13560	AT	8	Intergenic
68	cP0000027	PotentialSSR	PentaSSR	13563	13572	ATATT	2	Intergenic
69	cD0000008	SSR	DiSSR	13586	13599	AT	7	Intergenic
70	cP0000029	PotentialSSR	PentaSSR	13617	13626	ATAAT	2	Intergenic
71	cP0000030	PotentialSSR	PentaSSR	13638	13647	TATTT	2	Intergenic
72	cP0000031	PotentialSSR	PentaSSR	13659	13668	TAATA	2	Intergenic
73	cP0000032	PotentialSSR	PentaSSR	13682	13691	ATATA	2	Intergenic
74	cD0000009	SSR	DiSSR	13704	13713	TA	5	Intergenic
75	cTe0000006	SSR	TetraSSR	13727	13738	ATTA	3	Intergenic
76	cH0000028	PotentialSSR	HexaSSR	13740	13751	TTTATA	2	Intergenic
77	cD0000010	SSR	DiSSR	13797	13806	AT	5	Intergenic
78	cD0000011	SSR	DiSSR	13817	13832	TA	8	Intergenic
79	cD0000012	SSR	DiSSR	13835	13848	TA	7	Intergenic
80	cP0000033	PotentialSSR	PentaSSR	13942	13951	CTTTA	2	Intergenic
81	cP0000034	PotentialSSR	PentaSSR	14045	14054	TTAAT	2	Intergenic, Genic	tRNA-Ile
82	c70000005	ExtenedSSR	HeptaSSR	14358	14371	TATTATC	2	Genic	ND2
83	cP0000035	PotentialSSR	PentaSSR	14565	14574	CATTT	2	Genic	ND2
84	cH0000029	PotentialSSR	HexaSSR	14904	14915	TTAATC	2	Genic	ND2
85	cH0000030	PotentialSSR	HexaSSR	14928	14939	AATTAA	2	Genic	ND2
86	cH0000031	PotentialSSR	HexaSSR	14959	14970	TATTAA	2	Genic	ND2
87	c80000003	ExtenedSSR	OctaSSR	15047	15062	AATGATTT	2	Genic	ND2
88	cP0000036	PotentialSSR	PentaSSR	15158	15167	TTTTA	2	Genic	ND2
89	cH0000032	PotentialSSR	HexaSSR	15227	15238	TTAATC	2	Genic	ND2
90	c70000006	ExtenedSSR	HeptaSSR	15402	15415	AATTTAC	2	Genic	tRNA-Tyr

With the same pipeline, SSRs of two mitochondrial genomes were identified: 89 and 70 SSRs were found from NC_036678 and MH817140, respectively (Figure 8A). The number of SSRs identified from MH817140 is less than those of the rest two mitochondrial genomes because MH817140 mitogenome is shorter than the rest two mitogenomes missing part of 12S rRNA and control region. The overall trend of the number of SSRs along with ten types in three mitochondrial genomes is similar to each other (Figure 8A).

Comparison of simple sequence repeats among three S. paniceum mitochondrial genome

To understand the diversity of SSRs identified from the three mitochondrial genomes of S. paniceum, we grouped all 249 SSRs using the pipeline of the SSRDB (See Materials and Methods). In total, 65 SSR groups originating from three mitochondrial genomes (195 SSRs; 78.31%) were identified; while 11 partial SSR groups (22 SSRs; 8.84%) containing SSRs from the two mitochondrial genomes and 32 singleton SSRs (32 SSRs; 12.85%) were found (Figure 8B). Because of MH817140 partial mitochondrial genome, all 11 partial SSR groups contain SSRs only from NC_036678 and MK947052. Taken together, 32 singletons SSRs indicate SSR variations among three mitochondrial genomes. They cover five SSRs originating from MH817140 partial mitochondrial genome, 13 SSRs from NC_036678 and 14 SSRs from MK947052 (this study; Figure 8C). This diversity of SSRs can also be utilized to distinct its origin or populations like SNPs and INDELs on the mitochondrial genomes.

Nucleotide diversity analysis of Bostrichoidea mitochondrial genomes

To understand nucleotide diversity along with mitochondrial genome sequences, we calculated nucleotide diversity based on the two datasets: one consists of four mitochondrial genomes of Ptinidae species and the other covers eighteen species including the four Ptinidae species without species redundancy. The average nucleotide diversity of the first dataset is 0.08368 (Figure 9A), which is lower than those of tribe Figulini (0.1331)⁶² and family Thripidae (0.1329; Seo et al. in preparation) but is higher than that of Aphididae (0.0432; Lee et al. in preparation). Considering with low taxonomic coverage of the four species, it can be larger than now once additional mitochondrial genomes in this family are available. Nucleotide diversity of the second dataset displays a similar trend to that of the first dataset (Figure 9B); however, average nucleotide diversity is 0.1287, larger than that of the first dataset because its taxonomical coverage is increased in comparison to the first dataset.

Fig. 9.

Nucleotide diversity plots of Ptinidae family and Bostrichoidea superfamily mitochondrial genomes. (A) displays nucleotide diversity plot using four mitochondrial genomes of Ptinidae family and (B) shows nucleotide diversity plot using eighteen mitochondrial genomes of Bostrichoidea superfamily. In both panels, X-axis indicates start position of sliding window (500 bp) used for calculating nucleotide diversity. Y-axis shows nucleotide diversity value (Pi value). Blue line means nucleotide diversity of each window and yellow dotted line is average nucleotide diversity value. Grey dotted lines show boundary of 13 PCGs displayed on the bottom of graph.

Most of PCGs display similar trend in both nucleotide plots except NAD1 which shows variations of nucleotide diversity inside NAD1 gene in superfamily Bostrichoidea is larger than that of Ptinidae family (Figure 9). It reflects that some of the four families including Ptinidae may have a different trend of nucleotide diversity in the NAD1 gene. In addition, CYTB and NAD1 genes present lowest nucleotide diversity among all 13 PCGs in both plots; while ATP8 and NAD5 genes show the highest in both plots (Figure 9), which is different from those of Tribe Figulini presenting NAD6 and ATP8 genes are the highest⁶² and 88 Laodelphax striatellus mitochondrial genomes showing that ATP8 and CYTB genes are the highest.²⁸ From these results, ATP8 gene presents the highest nucleotide diversity as same as previous studies.^105,106

Nucleotide diversity of COX1 gene display gradually increase from N-terminal to C-terminal in both plots (Figure 9); while NAD4, NAD5, and COX3 genes present decrease from N-terminal and then increase toward C-terminal (Figure 9), indicating that this trend of nucleotide diversity inside the gene can also be considered for developing efficient molecular markers for diverse purposes, e.g. N-terminal of COX1 gene will show better resolution of species identification in superfamily Bostrichoidea.

Phylogenetic interpretation of superfamily Bostrichoidea species based on available 23 mitochondrial genomes

Bootstrapped phylogenetic trees of 23 mitochondrial genomes of superfamily Bostrichoidea were constructed with various options (See Materials and Methods). Overall, the topology of each tree was congruent to each other (Figure 10 and Supplementary Figures S1 to S8), especially in the BI trees (Supplementary Figures S1 to S4) where only a single topology difference was found in the tree of the dataset I in the placement of genus Stegobium. ML trees showed a bit more disagreements in the topologies, particularly in the tree of the dataset I.

Fig. 10.

Phylogenetic trees of 23 mitochondrial genomes of superfamily Bostrichoidea. Bayesian inference (1,000,000 generations) and the maximum likelihood (bootstrap repeat: 10,000) phylogenetic trees of 23 mitochondrial genomes from superfamily Bostrichoidea and an outgroup. Subfamily and family names are displayed on the vertical bars on the right side. Support values are shown in the following order: BI-i/BI-ii/BI-iii/BI-iv/ML-i/ML-ii/ML-iii/ML-iv. The figured tree was based on BI-iv tree and branches not supported in other trees were shown by ‘-’. High values of 1 in BI trees and 95 or over in ML trees are depicted with an asterisk (*).

All four species of family Ptinidae including S. paiceum were clustered into a single clade (Figure 10). Subfamily Anobiinae of Ptinidae however was only supported in trees generated with dataset I and was paraphyletic in other six trees with Stegobium grouping with Lasioderma of subfamily Xyletininae. The partial mitogenome, KT696225, was identified as “Anobiidae sp.”, a junior synonym of family Ptinidae, however, did not cluster with the other Ptinid species. Instead, it clustered with genus Nosodendron of family Nosodendridae and the branch was found in seven of the eight trees (Figure 10).

Three species each representing a separate genus of Bostrichidae were well clustered in all eight trees. KX087284 identified as “Bostrichoidea sp.” and Apatides of subfamily Bostrichinae were clustered with highly supported in all eight trees (Figure 10) implying that KX087284 is likely from a member of the subfamily, based on the branch length compared to the other subfamily Dinoderinae. The whole clade of Bostrichidae was sister to Ptinidae except for ML trees using datasets II and IV which are the sets including untrimmed PCG sequences.

Two sequences of Dermestes maculatus were used in the phylogenetic analysis however, the two did not cluster into a single branch as in the three sequences of S. paniceum or L. serricorne. Instead, MN900863 formed a single clade together with D. tessellatocollis. We have compared the sequence with multiple COX1 sequences of the two species retrieved from NCBI and confirmed MN900863 is likely a misidentification of D. tessellatocollis. Anthrenus verbasci also had two mitochondrial genome sequences, however, the two genomes showed separate branches as in the former examples. Further analysis on the COX1 region, we found MG017450 mitochondrial genome was identical to that of A. unicolor japonicus. Two sequences of Nosodendron of Nosodendridae, a family sometimes considered a member of the neighbouring superfamily Derodontoidea, was nested within Dermestidae in ML tree of dataset I but was not supported elsewhere.

Taken together, our phylogenetic analyses including S. paniceum present that this approach can provide better species identification of unidentified mitochondrial genomes. Moreover, maximum likelihood phylogenetic trees with dataset I displays incongruent or low supportive values of phylogenetic tree (Figure 10), indicating that topology of phylogenetic tree with this option can be considered as relatively low confident phylogenetic tree. Further in-depth analysis for understanding this phenomenon should be required in near future using more up-coming mitochondrial genomes in this superfamily.

Supplemental Material

sj-pptx-1-sci-10.1177_00368504211072355 - Supplemental material for The investigation of intraspecific characteristics and comparative analyses of the complete mitochondrial genome of Stegobium paniceum (Linnaeus, 1758) (Coleoptera: Ptinidae) assembled from public NGS raw reads of the black truffle, Tuber melanosporum

Supplemental material, sj-pptx-1-sci-10.1177_00368504211072355 for The investigation of intraspecific characteristics and comparative analyses of the complete mitochondrial genome of Stegobium paniceum (Linnaeus, 1758) (Coleoptera: Ptinidae) assembled from public NGS raw reads of the black truffle, Tuber melanosporum by Jongsun Park, Jungmo Lee and Jonghyun Park in Science Progress

Footnotes

Acknowledgements

This study was carried out with the support of the two grants: InfoBoss Research Grant (IBI-0004) and “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ013389052019)”, Rural Development Administration, Republic of Korea.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Supplemental material

Supplemental material for this article is available online.

ORCID iD

Jongsun Park

Author biographies

Jongsun Park PhD is the co-CEO and CTO in Infoboss Inc. His research interest is bioinformatics, genomics, phylogenetics, plant systematics, entomology, biodiversity, and machine learning/deep learning for analyzing heterogenous Bio-BigData.

Jungmo Lee is a researcher who is interested in entomology.

Jonghyun Park is a researcher who is interested in ants.

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