Analysis of the Mitogenomes of Two Helotid Species Provides New Insights into the Phylogenetic Relationship of the Basal Cucujoidea (Insecta: Coleoptera)

Simple Summary The family Helotidae represents a unique and primitive group of Cucujoidea, with important implications in understanding the phylogeny of beetles. To better understand the characteristics of the helotid mitochondrial (mt) genome and the evolution of Cucujoidea, we sequenced and compared the first recorded Helotidae mt genomes to reveal their characteristics and reconstruct the phylogenetic relationships of 13 basal families of this group. Phylogenetic analysis of the mt genomes indicated the positions of seven families within Cucujoidea but did not statistically support the presence of the Erotylid series and the Nitidulid series as distinct groups in this superfamily. In the phylogenetic results, Helotidae and Protocucujidae are sister groups. This study provides a new phylogenetic hypothesis regarding the basal relations of Cucujoidea. Abstract Helotid beetles are commonly found in places where sap flows from tree trunks and in crevices in bark. The Helotidae family is a rare and primitive group of Cucujoidea. To date, no complete mitochondrial (mt) genome has been sequenced for this family. To better understand the characteristics of the mt genome and the evolution of Cucujoidea, we sequenced and annotated the complete mt genomes of Helota thoracica (Ritsema, 1895) and Helota yehi Lee, 2017 using next-generation sequencing. These are the first record of Helotidae mt genomes. The RNA secondary structures of both species were also predicted in this study. The mt genomes of H. thoracica and H. yehi are circular, with total lengths of 16,112 bp and 16,401 bp, respectively. After comparing the mt genomes of H. thoracica and H. yehi, we observed the gene arrangement, codon usage patterns, base content, and RNA secondary structures of both species to be similar, which has also been noted in other Coleoptera insects. The nucleotide sequence of the coding regions and the control region has small differences. The phylogenetic analysis indicated that Helotidae and Protocucujidae are sister groups and revealed the relationship between seven families; however, the validity of the two series (Erotylid series and Nitidulid series) as larger groups in the superfamily was not supported. The mt phylogenomic relationships have strong statistical support. Therefore, the division of Cucujoidea into series should be re-examined. Our results will provide a better understanding of the mt genome and phylogeny of Helotidae and Cucujoidea and will provide valuable molecular markers for further genetic studies.


Introduction
As a small family in the superfamily Cucujoidea, the Helotidae is distributed in the Palearctic, Oriental, and Afrotropical regions [1,2]. The earliest fossil record of this family was found in the Early Cretaceous [3] and was regarded as one of the earliest diverging members of Cucujoidea [4]. Members of Helotidae can be distinguished from other families by the wide pronotum base, distinctly convex posterior angles, and the yellow spot on each

Phylogenetic Analyses
The phylogenetic analyses of these families were conducted based on 29 mt genomes from GenBank (http://www.ncbi.nlm.nih.gov (accessed on 10 November 2022) including the two newly sequenced genomes. The ingroup taxa included 28 species from Cucujoidea, representing 13 families. The outgroup Meloidae has a close relationship with Cucujoidea [20] (Table 1). 13 protein-coding genes were used to construct the dataset. The DNA alignment was performed from the amino acid alignment of the PCGs using the software Clustal_X v1.8.0 [49]. We connected all alignment sequences using MEGA v7.0.
The phylogenetic trees were reconstructed using different datasets under homogeneous and heterogeneous models. The homogeneous trees were reconstructed with maximum likelihood (ML) using IQ-Tree v1.6.8 [50] based on the dataset of PCG12. The PCG12 dataset includes 7190 sites for the first and second sites of the codon of 13 PCGs. Model Finder was used to select the model [51] (Table S1). Branch supports were evaluated using the ultra-fast bootstrapping method with 1000 replicates [52]. In addition, the heteroge-Biology 2023, 12, 135 4 of 17 neous tree was reconstructed using PhyloBayes v3.2 based on the dataset of PCG with the CAT-GTR model. The PCG dataset includes 10,785 sites for 13 PCGs. Two Markov chain Monte Carlo (MCMC) chains were employed [53]. FigTree v1.4.3 [54] was used to view and illustrate the inferred phylogenetic trees. The approximately unbiased (AU) test and Shimodaira-Hasegawa (SH) test were used to evaluate the alternative phylogeny hypotheses, and CONSEL v0.1j and RaxML v8.2.4 were used for phylogenetic hypothesis testing. The per site log-likelihood was calculated with RaxML-master 8.2.4 using -f G (g). The p-values for each alternative hypothesis were estimated using the AU test and SH test implemented in CONSEL v0.1j [55][56][57].
The complete mt genome records for these two species are the first for Helotidae. The annotated sequences of the two mt genomes were registered in GenBank with accession numbers OP964453 (H. thoracica) and OP964454 (H. yehi).

Base Composition
Helota thoracica and H. yehi were highly consistent in the analysis of A+T content, ATskew, and GC-skew. The base composition and strand bias of these two species are shown in Table 2. The Helotidae mt genomes exhibited a significant bias towards A and T, with In these two mt genomes, there were overlapping nucleotides and non-coding regions. The conserved overlapping regions were located between ATP8/ATP6 (4/7 bp), ATP6/COX3 (1 bp), and ND4/ND4L (7 bp). Except the CR, in the non-coding regions of the mt genomes of the two species, H. thoracica has 53 bp of non-coding bases, while H. yehi includes 108 bp of non-coding bases (Table S1).
The complete mt genome records for these two species are the first for Helotidae. The annotated sequences of the two mt genomes were registered in GenBank with accession numbers OP964453 (H. thoracica) and OP964454 (H. yehi).

Base Composition
Helota thoracica and H. yehi were highly consistent in the analysis of A+T content, AT-skew, and GC-skew. The base composition and strand bias of these two species are shown in Table 2. The Helotidae mt genomes exhibited a significant bias towards A and T, with nucleotide compositions of A = 39.0%, C = 14.9%, G = 9.9%, and T = 36.2% for H. thoracica, and A = 39.6%, C = 13.4%, G = 9.4%, and T = 37.6% for H. yehi.  The nucleotide compositions of A and T in total ranging were from 77.00% in H. thoracica to 77.91% in H. yehi. The AT-skew was 0.05/−0.03 and the GC-skew was −0.23/−0.20. However, the content of A+T was the lowest in the PCGs, ranging from 74.97% in H. thoracica and 75.84% in H. yehi. Similar to other Coleoptera mt genomes, the content of A+T was the highest in CR, far exceeding the other features. The A+T contents of rRNA were second, and, in the two rRNAs, the A+T content of rrnL was significantly higher than that of rrnS.
Similarly, the mt genomes of H. thoracica and H. yehi exhibited positive AT-skews in tRNAs, CR and negative AT-skews in PCGs, rRNAs, positive GC-skews in all RNAs, and negative GC-skews in PCGs and CR. The PCGs, rRNAs, tRNAs, and CR had different AT-skews and GC-skews.

Protein-Coding Genes
The size of the 13 PCGs of H. thoracica was 11,120 bp. All the PCGs could be translated into 3698 amino acid residues. For H. yehi, the total size was 11,102 bp, which include 3691 amino acid residues. Similar to the CG, the PCGs exhibited a lower A+T content (74.97-75.84%). The AT-skew and GC-skew were both negative for the PCGs, reflecting a bias towards nucleotides T and C, as compared to their counterparts.
The majority of the PCGs started with ATN, except ND1 in H. thoracica and H. yehi, which started with TTG. All PCGs stopped with TAA/TAG or truncated termination codons with T/TA-tRNA.
As shown in Figure 2, the most frequently used aa were Leucine (Leu), Isoleucine (Ile), Phenylalanine (Phe) and Methionine (Met), and the four most frequently used codons were TTA, ATT, TTT, and ATA. The RSCU values of the PCGs revealed that the frequency of A and U in the third site of these two species was higher than the frequency of C and G, which indicated the preference for the nucleotide composition A/T.

Transfer RNAs
The secondary structures of tRNAs were predicted in H. thoracica and H. yehi, which are shown in Figure 5. The 22 tRNAs of these two species were both typical and included all 20 types of amino acids. Most tRNAs were highly consistent between H. thoracica and H. yehi. As a result of the two species being relatively similar and the tRNA genes being relatively conservative, the tRNAs of these two mt genomes were almost identical. The tRNA sizes ranged from 62 to 71 bp in H. thoracica and H. yehi.
Almost all of tRNAs could be folded into clover-leaf secondary structures, except tRNA Ser(AGN) whose DHU arm simply formed a loop. The anticodon of tRNA Ser (AGN) was UCU instead of GCU, which was used as the anticodon for metazoans.
In all predicted tRNA secondary structures, H. thoracica and H. yehi were highly Similarly, the lowest genetic distance was not observed in COX1, with the genetic distance of COX1 being 0.140. This result possibly indicates that COX1 was not the most conservative gene in relation to PCGs in Helotidae. Average non-synonymous (Ka)/synonymous (Ks) ratios were estimated to investigate the evolutionary rates of mt genome PCGs [48]. We calculated the Ka/Ks ratios for each PCG of H. thoracica and H. yehi (Figure 4). The ratios ranged from 0.024 for COX1 to 0.194 for ND6, in the following order: COX1 < COX2 < CYTB < ATP6 < COX3 < ND3 < ATP8 < ND1 < ND2 < ND5 < ND4L < ND4 < ND6. The average Ka/Ks of the 13 PCGs of these two species were all less than 1, which indicates that all the PCGs were under purifying selection. Purifying selection was particularly strong (Ka/Ks < 0.1) in the first nine coding regions of the order presented, with greater emphasis on the genes of complex III (CYTB) and IV (COX1, COX2, and COX3) in the mitogenomes. In particular, COX1 (0.024) and COX2 (0.030) were under the strongest purifying selection. The complex I genes (NADH) exhibited higher Ka/Ks proportions, especially in ND6 (0.194) and ND4 (0.151), which indicates the presence of less conservative evolutionary restrictions in these regions, which exhibited relaxed purifying selection. The results confirmed the pattern observed in previous studies, which also demonstrated heterogeneity among the evolutionary rates of different complexes encoding the mt genome.

Transfer RNAs
The secondary structures of tRNAs were predicted in H. thoracica and H. yehi, which are shown in Figure 5. The 22 tRNAs of these two species were both typical and included all 20 types of amino acids. Most tRNAs were highly consistent between H. thoracica and H. yehi. As a result of the two species being relatively similar and the tRNA genes being relatively conservative, the tRNAs of these two mt genomes were almost identical. The tRNA sizes ranged from 62 to 71 bp in H. thoracica and H. yehi.

Ribosomal RNAs
The rrnL was located in the tRNA Leu(CUN) and tRNA Val , and the length of rrnL ranged from 1258 (H. yehi) to 1286 bp (H. thoracica). The rrnS was located in the tRNA Val and the CR, and its length ranged from 759 (H. yehi) to 786 bp (H. thoracica). These rRNA (rrnL, rrnS) subunits were encoded on the N-strand.
The AT content ranged from 82.11% to 82.50% in rrnL and 79.30% to 79.97% in rrnS, which exhibited a high AT bias. The highest AT content in rrnL was found in H. thoracica, but the higher AT content in rrnS was found in H. yehi.
The secondary structures of rrnL and rrnS were predicted and are shown in Figures Almost all of tRNAs could be folded into clover-leaf secondary structures, except tRNA Ser(AGN) whose DHU arm simply formed a loop. The anticodon of tRNA Ser (AGN) was UCU instead of GCU, which was used as the anticodon for metazoans.
In all predicted tRNA secondary structures, H. thoracica and H. yehi were highly consistent in terms of amino acid acceptor arm and loop, TψC arm and loop, anticodon (AC) arm and loop, and the dihydorouridine (DHU) arm and loop. Among them, these sec-ondary structures of tRNA Leu(CUN) and tRNA Ser(UCN) were identical. The tRNA Tyr , tRNA Thr , tRNA Trp , and tRNA Met only exhibited a single base variation between H. thoracica and H. yehi. The aminoacyl (AA) stem length was 7 bp, which is conservative. The anticodon (AC) arm length was 5 bp, except for tRNA His and tRNA Leu(UUR) , and the AC arm was 4 bp. Almost all tRNAs had the same anticodon (AC) loop length (seven nucleotides), except for tRNA His and tRNA Leu(UUR) (nine nucleotides). The length of the TψC arm varied from 3 to 6 bp and the TψC loop from 3 to 8 nucleotides. The dihydrouridine (DHU) stem varied from 3 to 4 bp, except for tRNA Ser(AGN) , and DHU loop varied from 3 to 8 bp.
There are also base pair mismatches in both H. thoracica and H. yehi. Among them, the number of G-U mismatch pairs in the two species was the same, i.e., 15 G-U pairs, which form weak attraction and constitute bonds situated at the TψC arm (3 bp), the AA arm (3 bp), the AC arm (6 bp), and the DHU arm (3 bp).

Ribosomal RNAs
The rrnL was located in the tRNA Leu(CUN) and tRNA Val , and the length of rrnL ranged from 1258 (H. yehi) to 1286 bp (H. thoracica). The rrnS was located in the tRNA Val and the CR, and its length ranged from 759 (H. yehi) to 786 bp (H. thoracica). These rRNA (rrnL, rrnS) subunits were encoded on the N-strand.
The AT content ranged from 82.11% to 82.50% in rrnL and 79.30% to 79.97% in rrnS, which exhibited a high AT bias. The highest AT content in rrnL was found in H. thoracica, but the higher AT content in rrnS was found in H. yehi.
The secondary structures of rrnL and rrnS were predicted and are shown in Figures 6  and 7, respectively. The rrnL had 35 helices in five structural domains. The rrnL had five domains (I-II, IV-VI), except domain III, as is the case in Coleoptera insects [59].

Control Region
The control region plays an indispensable role in the analysis of molecular evolution, transcription, and contains regulatory functions for replication.
In Helotidae, the control regions were not conserved, but both were located between rrnS and tRNA Ile . The lengths of the CR in the two mt genomes were 1474 bp in H. thoracica and 1766 bp in H. yehi. The A+T content was 84.87% in H. thoracica and 85.73% in H. yehi. The A+T content of CRs was the highest, and both H. thoracica and H. yehi had positive AT-skews and negative GC-skews, which confirmed the characteristic in the Coleoptera mt genome.
The Helotidae mt genomes had 3-5 types of tandem repeat units, ranging from 17 to 102 bp (Figure 8). Five tandem repeat units were found in the CR of the H. thoracica mt genome. They were a 29 bp, 19 bp, 24 bp, and 102 bp sequence tandemly repeated twice, and a 23 bp sequence tandemly repeated four times. In addition, the three tandem repeats in the H. yehi mt genome were a 68 bp tandemly repeated twice, a 17 bp tandemly repeated five times, and a 21 bp tandemly repeated three times.
There was conserved poly-A in the CR of both H. thoracica and H. yehi, upstream of tRNA Ile . The lengths of the poly-thymidine (Poly-T) structures were 13 bp in H. thoracica and 12 bp in H. yehi. The Poly-T stretch was an initiation of transcriptional control and replication. Moreover, there were many microsatellite-like repeat sequences, e.g., (TA) 6, (TA) 8, and (TA) 10, in the CR, and (TA) 10 only appeared in H. thoracica (Figure 9). Both the CRs included many short repeats, which may serve as microsatellites. These may be used to study the differences between individuals in different geographical locations and the phylogeny of Helotidae. The rrnS included three structural domains and 22 helices. However, the nucleotide conservation of two rRNAs was unevenly distributed among different domains. In rrnL, the domains IV and V were more conserved than in other domains, while the stem region of domain III was structurally more conserved in rrnS.

Control Region
The control region plays an indispensable role in the analysis of molecular evolution, transcription, and contains regulatory functions for replication.
In Helotidae, the control regions were not conserved, but both were located between rrnS and tRNA Ile . The lengths of the CR in the two mt genomes were 1474 bp in H. thoracica and 1766 bp in H. yehi. The A+T content was 84.87% in H. thoracica and 85.73% in H. yehi. The A+T content of CRs was the highest, and both H. thoracica and H. yehi had positive AT-skews and negative GC-skews, which confirmed the characteristic in the Coleoptera mt genome.
The Helotidae mt genomes had 3-5 types of tandem repeat units, ranging from 17 to 102 bp (Figure 8). Five tandem repeat units were found in the CR of the H. thoracica mt genome. They were a 29 bp, 19 bp, 24 bp, and 102 bp sequence tandemly repeated twice, and a 23 bp sequence tandemly repeated four times. In addition, the three tandem repeats in the H. yehi mt genome were a 68 bp tandemly repeated twice, a 17 bp tandemly repeated five times, and a 21 bp tandemly repeated three times.   (Figure 9). Both the CRs included many short repeats, which may serve as microsatellites. These may be used to study the differences between individuals in different geographical locations and the phylogeny of Helotidae.

Phylogenetic Analyses
Phylogenetic analyses were performed on the nucleotide datasets (PCG and PCG12). The phylogenetic results are shown in Figure 10. The analyses on the PCG dataset and the PCG12 dataset showed the same topology. Almost all of nodes were highly supported.
The Helotidae was defined as monophyletic and the sister group of Protocucujidae (Bayesian posterior probabilities, PP = 0.93 and ultrafast bootstrap support, BS = 84). Nitidulidae and Monotomidae were sister groups, which was together the sister group of Katertidae, and the PP and BS were mostly high. The sister group relationship between ((Nitidulidae-Monotomidae)-Katertidae) and (Helotidae-Protocucujidae) was highly supported in all analyses. The sister group, Erotylidae and Sphindidae, exhibited an obviously more distant relationship to the other groups. In this study, the monophyly of all these seven families was also supported.

Phylogenetic Analyses
Phylogenetic analyses were performed on the nucleotide datasets (PCG and PCG12). The phylogenetic results are shown in Figure 10. The analyses on the PCG dataset and the PCG12 dataset showed the same topology. Almost all of nodes were highly supported.  The Helotidae was defined as monophyletic and the sister group of Protocucujidae (Bayesian posterior probabilities, PP = 0.93 and ultrafast bootstrap support, BS = 84). Nitidulidae and Monotomidae were sister groups, which was together the sister group of Katertidae, and the PP and BS were mostly high. The sister group relationship between ((Nitidulidae-Monotomidae)-Katertidae) and (Helotidae-Protocucujidae) was highly supported in all analyses. The sister group, Erotylidae and Sphindidae, exhibited an obviously more distant relationship to the other groups. In this study, the monophyly of all these seven families was also supported.
Therefore, after constructing the phylogenetic tree, the ML tree was used to statistically test the inconsistent phylogenetic hypotheses obtained by Zhang Table 3.
Therefore, after constructing the phylogenetic tree, the ML tree was used statistically test the inconsistent phylogenetic hypotheses obtained by Zhang et al. 2 and McKenna et al. 2019 (Hypothesis A), Robertson et al. 2015 (Hypothesis B) and study (Hypothesis C) ( Figure 11). The results are shown in Table 3. Figure 11. Family-level phylogeny hypothesis of seven families [22,23]. The above results show that the p-values of the AU and SH tests were all less t 0.15 in other topologies, except in this study, indicating that there were signifi differences between these studies. Under the mt genome dataset, only the result from study was supported, which demonstrates that, at the mt genome level, the h probability results are consistent with this research. Therefore, the results of this st show that the existence of two series is not supported at the mitogenome level.

Comparative Analysis of the Two Helotid Mitogenomes
Through Illumina DNA sequencing and assembly, the mt genomes of the two hel species H. thoracica and H. yehi were obtained. With the exception of the diversity of nucleotide composition, the mt genomes of these members of Helotidae were simila terms of genome size, organization, arrangement patterns, gene order, aa composit and RSCU to those of other Cucujoidea species [60]. The structural features w conserved. The majority of the PCGs started with ATN. The ND1 of these two spe started with TTG. The TTG initiation has also been reported in other families suc Erotylidae and in other orders [58,61,62].
The analysis of evolutionary patterns showed that ND6 and CYTB exhibited a fa evolution rate, and ATP8 and ND1 exhibited the lowest genetic distance. As comp with non-synonymous substitution, the rates of synonymous substitution w significantly higher in all the PCGs (mainly in COX1) of the mitogenomes of the ge Helota analyzed herein. These were used as references for improved molecular m development [63]. Figure 11. Family-level phylogeny hypothesis of seven families [22,23]. The above results show that the p-values of the AU and SH tests were all less than 0.15 in other topologies, except in this study, indicating that there were significant differences between these studies. Under the mt genome dataset, only the result from our study was supported, which demonstrates that, at the mt genome level, the high-probability results are consistent with this research. Therefore, the results of this study show that the existence of two series is not supported at the mitogenome level.

Comparative Analysis of the Two Helotid Mitogenomes
Through Illumina DNA sequencing and assembly, the mt genomes of the two helotid species H. thoracica and H. yehi were obtained. With the exception of the diversity of the nucleotide composition, the mt genomes of these members of Helotidae were similar in terms of genome size, organization, arrangement patterns, gene order, aa compositions and RSCU to those of other Cucujoidea species [60]. The structural features were conserved. The majority of the PCGs started with ATN. The ND1 of these two species started with TTG. The TTG initiation has also been reported in other families such as Erotylidae and in other orders [58,61,62].
The analysis of evolutionary patterns showed that ND6 and CYTB exhibited a faster evolution rate, and ATP8 and ND1 exhibited the lowest genetic distance. As compared with non-synonymous substitution, the rates of synonymous substitution were significantly higher in all the PCGs (mainly in COX1) of the mitogenomes of the genus Helota analyzed herein. These were used as references for improved molecular mark development [63].
In addition, in the CR, these two mt genomes had a unique type of tandem repeat sequence units, but H. thoracica had two poly-As upstream of tRNA Ile , and H. yehi only had one. These features provide basic information for the further comparative analysis and discussion of Helotidae mt genomes.

Mitochondrial Phylogenomics Provides New Insights into Helotid Evolution
Although recent molecular phylogenetic studies have consistently recovered monophyletic suborders of Coleoptera and provided many new insights into the internal relationships of some suborders, the phylogenetic relationships within series and superfam-ilies of suborder Polyphaga still remain controversial [64]. This is particularly true for the superfamily Cucujoidea, within which the relationships among families were largely unresolved [20][21][22][23]. Our phylogenetic reconstructions at the mitochondrial level are consistent with the previous results of Zhang et al., 2018 [22], McKenna et al., 2019 [23], and Robertson et al., 2015 [20], that all showed that the seven families have a relatively close relationship. The results show that Helotidae forms a sister group to Protocucujidae, while Nitidulidae and Monotomidae are sister groups. Erotylidae and Sphindidae have a distant relationship to the other families. However, the sister groups among the families are in conflict with the results of previous studies. First, in our study, Protocucujidae and Helotidae are sister groups, the sister-group relationship between 'Sphindidae-Protocucujidae' and Helotidae is not supported. Second, although the Nitidulidae, Monotomidae, and Katertidae form a clade, as in previous studies, the sister group relationship is not the same. The research of Zhang et al., 2018 [22] and McKenna et al., 2019 [23] (hypothesis A), and Robertson et al., 2015 [20] (hypothesis B), supported 'Nitidulidae-Katertidae' and Monotomidae as sister groups, but our study suggests that Nitidulidae and Monotomidae are more closely related than Katertidae. Then, the phylogenetic results of mt genomes suggest that Erotylidae and Sphindidae have a close relationship, which also illustrates that our results do not support the existence of the Erotylid and Nitidulid series as divided by Robertson et al., 2015 (hypothesis B) [20]. This result has been statistically tested and does not support previous research based on nuclear protein-coding (NPC) and several molecular markers. This bias may originate from the genes in and the genetic differences between mt and nuclear genes [65,66]. However, one cannot easily reject the existing views based on information from one source of mt genome data. Multiple sources are necessary to make a final judgment. Therefore, we should take a balanced sample of the various taxa involved and conduct an in-depth discussion concerning this problem through the phylogenetic reconstruction of Cucujoidea within a larger sample range.
Compositional heterogeneity and evolution rate variability may be the most common sources of phylogenetic incongruence [67][68][69][70][71]. As a result of heterogeneity in the mt genome, dense taxon sampling and the model of CAT-GTR+G used, Bayesian analyses can produce robust phylogenetic trees though compositional heterogeneity cannot be eliminated [72]. Therefore, we used the CAT-GTR model in PhyloBayes to reconstruct the heterogeneous tree. In the maximum likelihood method, we select discarding/down weighting third codon positions to make the result more accurate [73,74].

Conclusions
In this study, two complete mt genomes of the family Helotidae were sequenced, they represent the first report of Helotidae mitochondrial genomes. This study opens a new phase in the study of genetic diversity among various families within Cucujoidea. At the same time, by reconstructing the phylogenetic relationships of 13 basal families in Cucujoidea, we propose a new view on the Erotylid and Nitidulid series. New insights into the phylogenetic position and evolution of the family Helotidae have also been provided. More data from other genera and species in the family are needed for further phylogenetics studies and to elucidate the molecular evolution of Cucujoidea. The mt genome sequences are important resources for further molecular studies and for the phylogenetic analysis of Helotidae and Cucujoidea.