The Complete Mitochondrial Genomes of Penthe kochi (Coleoptera: Tetratomidae) with Its Phylogenetic Implications

Abstract

To explore the mitogenome characteristics of Tetratomidae and the phylogenetic position of this family in Tenebrionoidea, the mitogenome of Penthe kochi Mařan, 1940 was sequenced, annotated, and analyzed. The P. kochi mitogenome is consistent with Tenebrionoidea species in gene length, genomic organization, codon usage, and secondary structures of transfer genes (tRNAs). Most protein-coding genes (PCGs) originate with a typical ATN start codon, except nad1 and nad3, which start with TTG. In total, 10 PCGs are terminated with complete stop codon TAA and TAG, while cox1, cox2, and nad 4 contain an incomplete stop codon T-. Among the 13 PCGs, nad2 (Pi = 0.282) has the most diverse nucleotide composition, and cox2 is the most conserved gene with the lowest value (Pi = 0.154). The Ka/Ks ratio of cox1 (0.076) and cox2 (0.124) has a lower value. All the tRNAs can be folded in a typical clover-leaf secondary structure, except trnS1, which lacked a dihydrouridine arm. And phylogenetic analyses were performed based on 13 PCGs using the Bayesian inference (BI) method. The results showed that the clade of Tenebrionoidea was well separated from the outgroups, and Tetratomidae and Mycetophagidae were not well resolved. Phylogenetic analyses with more mitogenome samplings are needed to resolve the phylogeny of Tenebrionoidea.

1. Introduction

The family Tetratomidae, including 13 genera over 150 species in five subfamilies [1], is a small group in the superfamily Tenebrionoidea. The tetratomid species are widely distributed in the Palearctic, Oriental, Sino-Japanese, Australian, Oceanian, Afrotropical, and Nearctic regions. To date, eight genera and twenty-two species in five subfamilies have been recorded from China, primarily distributed in the southwest and southeast [2,3,4,5,6,7,8,9,10]. Adults are frequently found on the surface of Polyporaceae or Tricholomataceae at night, and their larvae feed internally on hyphal tissue [1].

Although many taxonomists have made important contributions to the Tenebrionoidea classification during phylogenetic analyses based on morphological characteristics and molecular data, the problems of the overall classification remain unsettled. Based on the morphological characteristics, Tenebrionoidea (26 families) was divided into five lineages, one of which was composed of Tetratomidae, Melandryidae, Mordellidae, and Ripiphoridae [11]. Based on the molecular data, Hunt et al. performed a comprehensive phylogenetic analysis of beetles inferred from three genes (16S, 18S, cox1); the results suggested that the 23 sampled families of Tenebrionoidea formed a clade [12]. Mckenna and Farrell constructed a Timetree of life of Coleoptera based on morphological characteristics, fossils, and molecular data, and the results suggested that the 21 sampled families of Tenebrionoidea formed a clade with the origin of Tenebrionoidea being ca. 236 Ma [13]. Gunter et al. reconstructed the phylogenetic tree of 24 tenebrionoid families based on four genes (ssu, lsu, rrnL, cox1), and the results showed that topology included four lineages [14]. Then, McKenna et al. reconstructed the phylogenetic tree of Coleoptera based on eight nuclear genes; the results exhibited that Tenebrionoidea included the 28 sampled families and Lymexyloidea was recovered within Tenebrionoidea [15]. And then, McKenna et al. inferred the phylogeny of beetles using 4818 genes for 146 species [16]; the results suggested that the phylogenetic position of Lymexylidae in Tenebrionoidea is suitable. However, Cai et al. used 68 single-copy nuclear protein-coding genes of 129 extant families to explore beetle evolution, and the results suggested that Lymexyloidea is a sister group of Tenebrionoidea [17]. However, Tetratomidae is not included in these studies. In Tenebrionoidea, the phylogenetic position of Tetratomidae is rather unclear.

Recently, the mitogenome emerged as a valuable source for higher-level phylogenetic analysis [18,19,20,21]. In this study, the first complete mitochondrial genome for Tetratomidae, the mitogenome of Penthe kochi Mařan, 1940, was sequenced, annotated, and comparatively analyzed. Moreover, phylogenetic analyses based on the Bayesian inference (BI) method was carried out to assess the phylogenetic position of Tetratomidae in Tenebrionoidea and to contribute to further understanding the phylogenetic relationships of each group of Tenebrionoidea.

2. Materials and Methods

2.1. Sampling, Identification, and DNA Extraction

The specimens of Penthe kochi Mařan, 1940 were collected from Dayaoshan Mountains, Jinxiu County, Guangxi Zhuang Autonomous Region, China, on 11 February 2021. Specimens were immediately preserved in 95% ethanol in the field after they were collected and then stored at −24 °C in laboratory. The specimens were examined using Olympus SZX10 and were identified based on the morphological characteristics described by Mařan [22]. Genomic DNA was extracted from the legs and thoracic muscle tissue. Next-generation sequencing and the assembly of mitogenome were performed by Beijing Aoweisen Gene Technology Co., Ltd. (Beijing, China).

2.2. Mitogenome Assembly, Annotation and Analysis

A whole genome shotgun strategy was used based on the Illumina HiSeq platform when the total genome DNA was quantified. Then, sequencing was performed with a strategy of 150 bp paired-end reads. The assembler MITO-bim was used for mitogenome assembly [23]. To check the correctness of the assembly, clean data were manually mapped on the mitochondrial scaffold using Geneious 11.0.2 software [24]. The 13 protein-coding genes (PCGs) and two ribosomal RNA genes (rRNAs) were identified using Geneious 11.0.2. The 22 transfer RNA genes (tRNAs) were re-identified using a tRNAScan-SE server v 1.21 and MITOS WebSever [25,26]. The mitogenome map was illustrated using the online tool Organellar Genome DRAW (OGDRAW; https://chlorobox.mpimp-golm.mpg.de/OGDraw.html, 11 September 2024) [27]. In an A + T-rich region, the tandem repeat elements were identified using an online tool, Tandem Repeats Finder [28]. The relative synonymous codon usage (RSCU) was calculated by Mega X [29]. The nucleotide diversity (Pi) and the ratio of nonsynonymous/synonymous (Ka/Ks) PCGS were calculated with DnaSP v 5 [30].

2.3. Phylogenetic Analysis

The mitochondrial genome of 33 species from 15 families of Tenebrionoidea were selected as ingroups (

Table 1. The mitogenomes of Tenebrionoidea and outgroups were used for phylogenetic analyses.

Family	Sufamily	Species	GenBank No.	References
Tetratomidae	Penthinae	Penthe kochi	ON113044	This study
Tetratomidae	Tetratominae	Tetratoma fungorum	NC_036276	[35]
Mordellidae	Mordellinae	Mordella atrata	NC_013254	[36]
Mordellidae		Mordellidae sp.	JX412844	[31]
Mycteridae	Hemipeplinae	Hemipeplus sp.	JX412852	[31]
Mycteridae	Lacconotinae	Stilponotus mexicanus	JX412811	[31]
Mycetophagidae	Mycetophaginae	Mycetophagus quadripustulatus	HQ232824	[37]
Anthicidae	Anthicinae	Formicomus sp.	JX412857	[31]
Anthicidae	Anthicinae	Omonadus floralis	HQ232825	[37]
Boridae	Borinae	Boros schneideri	HQ232823	[37]
Meloidae	Meloinae	Mylabris sp.	JX412732	[31]
Oedemeridae	Oedemerinae	Oedemera virescens	HQ232826	[37]
Oedemeridae	Oedemerinae	Ischnomera cyanea	JX412790	[31]
Prostomidae	Prostominae	Prostomis sp.	JX412787	[31]
Pyrochroidae	Ischaliinae	Ischalia sp.	HQ232827	[37]
Scraptiidae	Anaspidinae	Anaspis sp.	HQ232806	[37]
Scraptiidae	Anaspidinae	Anaspis sp.	JX412856	[31]
Melandryidae	Melandryinae	Mikadonius gracilis	JX412823	[31]
Melandryidae	Melandryinae	Osphya bipunctata	JX313675	[31]
Ciidae		Ciidae sp.	JX412846	[31]
Tenebrionidae	Pimeliinae	Asbolus verrucosus	NC_027256	[38]
Tenebrionidae	Tenebrioninae	Eutrapela sp.	JX412754	Unpublished
Tenebrionidae	Lagriinae	Adelium sp.	FJ613422	[39]
Tenebrionidae	Lagriinae	Eutrapela ruficollis	HQ232805	[37]
Tenebrionidae	Tenebrioninae	Tenebrio molitor	NC_024633	[40]
Tenebrionidae	Tenebrioninae	Tribolium confusum	NC_026702	[41]
Tenebrionidae	Blaptinae	Gonocephalum outreyi	KU236386	[42]
Tenebrionidae	Alleculinae	Paramarygmus sp.	JX412775	[31]
Tenebrionidae	Diaperinae	Platydema sp.	JX412842	[31]
Tenebrionidae	Stenochiinae	Strongylium suspicax	JX412780	[31]
Tenebrionidae		Paramarygmus sp.	JX412808	[31]
Lymexylidae (outgroups)	Hylecoetinae	Lymexylon navale	KX087311	Unpublished
	Melittommatinae	Hyloecetus dermestoides	HQ232820	[37]

), and mitochondrial genomes of Lymexylidae species were chosen as the outgroups, as they are phylogenetically distant from Tenebrionoidea in the Coleoptera [17,31]. The nucleotide sequences (13 PCGs) of 33 mitogenomes were aligned using ClustalW and trimmed using trimAl v 1.2 [32,33]. The best-fit model was determined by ModelFinder based on Bayesian information criterion. The phylogenetic trees were constructed using PhyloSuite v 1.2.2 [34], based on the Bayesian inference (BI) method. Bayesian analyses were run with two independent chains spanning 2,000,000 generations, four Markov chains, with sampling at every 100 generations and a burn-in period of 0.25 for each chain. The phylogenetic trees were edited and visualized by Figtree v 1.4.3.

3. Results and Discussion

3.1. Genome Organization and Base Composition

The mitogenome of P. kochi is deposited in GenBank with accession number (ON113044). The mitogenome sequence is 16,719 bp in length and encodes 37 typical mitochondrial genes: 13 protein-coding genes (13 PCGs), two ribosomal RNA genes (2 rRNAs), 22 transfer RNA genes (22 tRNAs), and an A + T-rich region (control region, CR) (Figure 1). The gene arrangement of the P. kochi mitogenome is identical to the hypothetical ancestral insect pattern [43]: 14 genes (4 PCGs, 8 tRNAs, and 2 RNAs) are N-strand, and the other genes (9 PCGs and 14 tRNAs) are J-strand (

Table 2. Summary of the characteristics of the mitogenome of Penthe kochi.

Genes	Strand	Position		Size	Start Condon	Stop Codon	IGN	Anticodon
		from	to
trnI	J	1	65	65			−	GAT
trnQ	N	63	131	69			0	AAG
trnM	J	132	200	69			0	CAT
nad2	J	201	1208	1008	ATA	TAA	−2
trnW	J	1207	1273	67			34	ACA
trnC	N	1308	1368	61			0	GCA
trnY	N	1369	1431	63			<2	GAA
cox1	J	<1433	2966	>1534	?/?/?	T(AA)	0
trnL2	J	2967	3030	64			0	AAA
cox2	J	3031	3718	688	ATT	T(AA)	0
trnK	J	3719	3789	71			−1	CAA
trnD	J	3789	3853	65			0	GAC
atp8	J	3854	4009	156	ATT	TAA	−7
atp6	J	4003	4674	672	ATG	TAA	5
cox3	J	4680	5468	789	ATG	TAA	−1
trnG	J	5468	5530	63			0	ACC
nad3	J	5531	5884	354	TTG	TAG	−2
trnA	J	5883	5947	65			−1	AGC
trnR	J	5947	6009	63			−1	ACG
trnN	J	6009	6071	63			0	GAA
trnS1	J	6072	6129	58			0	ACA
trnE	J	6130	6193	64			−1	AAC
trnF	N	6193	6256	64			0	GAA
nad5	N	6257	7969	1713	ATT	TAA	0
trnH	N	7970	8032	63			0	GAG
nad4	N	8033	9365	1333	ATG	T(AA)	−7
nad4l	N	9359	9646	288	ATG	TAA	2
trnT	J	9649	9711	63			0	AGA
trnP	N	9712	9775	9775			2	AGG
nad6	J	9778	10,284	507	ATT	TAA	−1
cytb	J	10,284	11,420	1137	ATG	TAA	1
trnS2	J	11,422	11,486	65			17	AGA
nad1	N	11,503	12,453	951	TTG	TAG	0
trnL1	N	12,454	12,515	62			0	AAG
rrnL	N	12,516	13,800	1285			0
trnV	N	13,801	13,865	65			0	AAC
rrnS	N	13,866	14,600	735			0
A + T-rich region		14,601	16,719	2119			0

IGN = intergenic nucleotide; J = the majority strand; N = the minority strand; and ? = not determined.

). This mitogenome contains 11 overlapping genes, and the longest overlap is 7 bp between nad4 and nad4l. In tRNAs, the gene overlap probably represents the lower evolutionary constraints [39,44]. In the P. kochi mitogenome, intergenic nucleotides range from 1 to 34 bp, with the longest region located between trnW and trnC.

The base composition of the mitogenome of P. kochi is A (40.97%), C (11.69%), G (7.70%), and T (39.63%) (Table 2 and Table S2). The nucleotide composition has a high A/T bias (80.60%), the same as other beetles [42,45,46,47,48], and exhibits a positive AT-skew (0.02) and a negative GC-skew (−0.21), indicating that A and C are more abundant than T and G.

3.2. Protein-Coding Genes and Codon Usage

The total length of 13 protein-coding genes (PCGs) is 11,130 bp, approximately accounting for 66.57% of the entire mitochondrial genome. In total, 4 of the 13 PCGs (nad1, nad4, and4L, and nad5) are located on the minority strand (N-strand), and the other nine (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, and cytb) are located on the majority strand (J-strand) (Table 2). The A/T nucleotide composition of PCGs is 78.99%, exhibiting a high A/T bias (Table S1). The AT-skew of the entire PCGs is −0.14. Among the 13 PCGs, nad5 (1713 bp) and atp8 (156 bp) were found to be the largest and smallest genes, respectively. Most PCGs originated with a typical ATN (ATA, ATT, ATG) start codon, except nad1 and and3, which started with TTG. Moreover, with the exception of cox1, cox2, and nad 4 that contain an incomplete stop codon T-, the other 10 PCGs were terminated with complete stop codon TAA (nad6, cytb, nad4l, nad5, atp8, atp6, cox3, and nad2) and TAG (nad1, nad3). In insects, lots of mitochondrial genes have incomplete stop codons, which is consistent with previous research [49].

The relative synonymous codon usage (RSCU) of the mitogenomes was calculated and is exhibited (Figure 2). The 13 PCGs contain 3699 codons, excluding stop codons (Table S2), and the most frequently used codons are UUA (508), AUU (429), UUU (311), and AAU (214). Accordingly, L2, I, F, and N are the most frequently used amino acids, accounting for 13.73%, 11.60%, 8.41%, and 5.78% of the total amino acids, respectively.

To determine the differences between Penthinae (P. kochi) and Tetratominae (T. fungorum), the authors compared the length, acid composition, and RSCU of PCGs. The whole mitogenome of P. kochi (16,719) is longer than the T. fungorum (15,089). In the 13 PCGs, the size of nad2, cox2, nad5, nad4l, nad6, cytb, and nad1 is longer in P. kochi than in T. fungorum, but cox1 is the opposite (Table S3). And the length of the rrnL and rrnS of P. kochi (1285 bp and 735 bp) is shorter than that of T. fungorum (1364 bp and 768 bp). In the P. kochi mitogenome, the CUG codon used for L1, the CCG codon used for P, the CGC codon used for R, and the GCG codon used for A are absent (Figure 2); however, their stability requires further investigation. In Penthinae (P. kochi) and Tetratominae (T. fungorum), the amino acid composition and proportion are similar.

In Tetratomidae, the nucleotide diversity (Pi) of 13 PCGs ranged from 0.154 (cox2) to 0.282 (nad2) (Figure 3, Table S1). Among the 13 PCGs, the gene nad2 (Pi = 0.282) was the most diverse nucleotide, followed by nad6 (Pi = 0.277) and atp8 (Pi = 0.260). In contrast, the gene cox2 was the most conserved gene with the lowest value (Pi = 0.154).

The ratio of nonsynonymous/synonymous (Ka/Ks) of PCGs was also calculated, which is representative of the evolutionary rate [50]. The Ka/Ks ratio of nad5 (3.484) is distinctly larger than one, indicating that this gene is under positive selection [51], while the other 12 PCGs are under purifying selection (Figure 3; Table S4). Among them, cox1 (0.076) and cox2 (0.124) have a lower value. These data suggest that the genes cox1 and cox2 can be used as barcodes for deducing the phylogenetic relationships of Tetratomidae.

3.3. Transfer and Ribosomal RNA Genes

The transfer RNA genes (tRNAs) are 1416 bp in total length, with a high A/T bias (A + T content is 80.08%). The AT-skew of whole tRNAs is 0.03 (Table S1). The tRNAs range from 58 to 71 bp in length. All the tRNAs can be folded in the typical clover-leaf secondary structure, except trnS1, which lacked a dihydrouridine arm (Figure 4), which is a common phenomenon for insects [52,53,54,55,56]. In the whole mitogenome, the longest intergenic nucleotide (34 bp) is located between trnC and trnW.

The lengths of rrnL and rrnS are 1285 bp and 735 bp, respectively. The A + T content is 82.52%, with a negative AT-skew (−0.04) and positive GC-skew (0.33). The rrnL subunit is located between trnV and trnL1, and the rrnS is located at the trnV and A + T-rich region.

3.4. A + T-Rich Region

It is well known that the A + T-rich region (control region, CR) acts in transcription initiation, transcription elongation, and DNA replication [57,58]. This non-coding region is 2119 bp in total length, located between rrnS and trnI. The A + T content is 87.54% (Table S5), with positive AT-Skew (0.04) and negative GC-skew (−0.39). There are five tandem repeat sequence units in this region, of which the length and position are provided (Figure 1).

4. Phylogenetic Analysis

The BI tree was constructed based on the best-fit model GTR + F+ I + G4. Based on the BI tree topology, the monophyly of Tenebrionoidea was again confirmed, as all the tenebrionoid species converged together as an independent clade with high-value support (PP = 1) (Figure 5). The Tetratomidae, Boridae, Mycetophagidae, Melandryidae, and Pyrochroidae converged together as an independent clade with high support (PP = 0.98), which formed a sister group of other tenebrionid families. All the Tenebrionidae species formed a clade. The Meloidae was close to Anthicidae and formed a clade. And Ciidae and Mordellidae were clustered together and formed a sister group of Tenebrionidae with high support (PP = 1). The results also showed that the Melandryidae is not monophyletic. So, the interrelationships among the families of Tenebrionoidea still require more data to be determined completely. These questions will be well addressed in the future when sufficient numbers of complete mitogenomes of Tenebrionoidea species are accumulated.

5. Conclusions

In the present study, the mitochondrial genome of Penthe kochi, which is the first complete mitogenome of Penthinae, was sequenced, annotated, and analyzed. It was found to be consistent with the known mitogenome of Tenebrionoidea in genomic organization, the codon usage of 13 PCGs, A + T biased base composition, and the secondary structures of tRNAs. Among the 13 PCGs, the gene nad2 (Pi = 0.282) is the most diverse nucleotide, and cox2 (Pi = 0.154) is the most conserved gene. The A + T-rich region has five tandem repeat sequence units with different lengths. Phylogenetic analyses based on 13 PCGs from 33 species show that Tetratomidae is close to Mycetophagidae.