Description of Nearly Completed Mitochondrial Genome Sequences of the Garden Chafer Polyphylla laticollis manchurica, Endangered in Korea (Insecta: Coleoptera)

  • cc icon
  • ABSTRACT

    In this study, we present the nearly complete mitogenome sequences of the garden chafer, Polyphylla laticollis manchurica, which is listed as an endangered species in Korea. The P. l. manchurica mitogenome, which includes unfinished whole A+T-rich region and a partial srRNA was 14,473-bp long, possessing typical sets of genes (13 PCGs, 22 tRNA genes, and 2 rRNA genes). Gene arrangement of the P. l. manchurica mitogenome was identical to the common one found in the majority of insects. The 5 bp-long motif sequence (TAGTA) that has been suggested to be the possible binding site for the transcription termination peptide for the major-strand was also found in the P. l. manchurica mitogenome between tRNASer(UCN) and ND1. The start codon for COI gene and ATPase8 was designated as a typical TTG. All tRNAs of the P. l. manchurica showed a stable canonical clover-leaf structure of other mt tRNAs, except for tRNASer(AGN), DHU arm of which could not form stable stemloop structure. As has been previously determined, the high A/T content was unanimously observed in P. l. manchurica in terms of A/T bias in the third codon position (73.5%) compared with the first (66.4%) and second codon position (66.2%). The PCGs encoded in major-strands are slightly T-skewed, whereas those of the minor-strand are A-skewed, indicating strand asymmetry in nucleotide composition in the Coleoptera including P. l. manchurica.


  • KEYWORD

    Mitochondrial genome , Polyphylla laticollis manchurica , Garden chafer , A/T bias

  • Introduction

    Animal mitochondrial genomes (mitogenomes) are approximately 16~20 kbp, and are encoded with a remarkably conserved set of 37 genes: 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes, and one major non-coding sequence, which is termed the control region (Boore, 1999). This control region in insect instead is called as the A+T-rich region due to the high adenine and thymine (A/T) content, and in fact, this region contains the highest A/T content of any region of the mitogenomes in insects (Kim et al., 2010).

    The mitogenome information has greatly been devoted to our understanding of several fields of biology (i.e., comparative and evolutionary genomics, molecular evolution, and phylogenetics). However, still newly sequenced insect mitogenome information provides us with new insights into genomic structures (Wan et al., 2012) and the evolutionary relationships of several levels of taxonomic groups (Kim et al., 2011; Cameron et al., 2009), and gene arrangement (Wang et al., 2013).

    Up to now, more than 250 mitogenome sequences have been determined from a variety of insects, but this list includes only ~34 coleopteran species (http://www.ncbi.nlm.nih.gov/genomes/ORGANELLES). Considering that the suborder Polyphaga contains the vast majority of beetle diversity, with at least 300,000 described species (90% of the beetles) belonging to more than 100 families in four infraorders (Hammond, 1992), genomic information is extremely limited. In particular, the complete mitogenome sequence of the infraorder Scarabaeiformia in Polyphaga is available only for two species (Cameron et al., 2009; Sheffield et al., 2009). Recently, Kim et al. (2013a, 2013b) additionally reported two complete mitogenome sequences of Scarabaeiformia, the whiter-spotted flower chafer, Protaetia brevitarsis (Scarabaeidae) and the two-spotted stag beetle, M. blanchardi (Lucanidae).

    In this paper, we report the mitogenome sequence of Polyphylla laticollis manchurica to describe the genome via comparison to those of pre-existing coleopteran insects in terms of whole genome organization, arrangement, and the major characteristics of individual genes. P. l. manchurica is distributed throughout Korea, including Jeju Island, and also in Mongolia and japan, as well as Eastern China (Won et al., 1998). Due to the rarity the species is listed as an endangered species in Korea (Won et al., 1998).

    Materials and Methods

      >  Specimen collection and genomic DNA extraction

    The garden chafer, Polyphylla laticollis manchurica (Scarabaeiformia: Scarabaeidae), was collected in Yeongwol, Gwangwondo-Province in Korea. P. l. manchurica is listed as a first degree endangered species in Korea, and thus, proper permission was obtained from relevant supervisory office before collection. Total genomic DNA was extracted using the Wizard™ Genomic DNA Purification kit (Promega, USA) following the manufacturer's instruction.

      >  PCR amplification, cloning, and sequencing

    Three short fragments, corresponding each about 500~700 bp were amplified from three genes, such as COI (SF1), CytB (SF2), and srRNA (SF3) (Fig. 1). Primers for these

    short fragments were designed via the alignment of several coleopteran mitogenomes sequenced in their entirety. These short fragments were amplified with AccuPower® PCR PreMix (Bioneer, Korea) using an initial denaturation at 94℃ for 4 min, followed by 35 cycles of 30 s denaturation at 94℃, 40 sec annealing at 50-55℃, and a 60 sec extension at 72℃. The final extension step was continued for 8 min.

    Using the sequence information obtained from the short fragments, three primer sets specific to each species were designed to amplify three long fragments (LF1 ~ LF3) which overlapped with the short fragments (SF1 ~ SF3). PCR cycles were as follows: denaturation for 2 min at 96℃, followed by 30 cycles of 10 sec at 98℃ and 15 min at 58-65℃, and a final 10 min extension at 72℃. In order to sequence the long fragments, both primer walking and shotgun approaches were used, because the success of sequencing varied depending on fragments. Nevertheless, both approaches were unsuccessful for LF3, and, thus, the partial LF3 that encompasses the 5’-end of the srRNA and the whole A+T-rich region was unfinished. We believe this may have happened because this region is exceptionally long, containing unexpectedly long repeat regions and a high A/T content, considering previous sequence results of other coleopteran species, such as P. brevitarsis and M. blanchardi (Kim et al., 2013a, 2013b). Primer information for each short fragment, long fragment, and internal primers for primer walking are provided (Table 1).

    For the primer walking method, internal primers were directly used to complete the sequences of the long fragment subsequent to purification with OIAquick PCR Purification Kit (Qiagen, USA). For the shotgun approach the long PCR fragments were subjected to shearing into 1~5 kb fragments (Gene Machine, USA) and were cloned into the pUC118 vector (Takara Biomedical, Japan). Each resultant plasmid DNA was isolated using a Wizard Plus SV Minipreps DNA Purification System (Promega, USA). DNA sequencing was conducted using the ABI PRISM® BigDye® Terminator v3.1 Cycle Sequencing Kit and the ABI PRISM® 3100 Genetic Analyzer (PE Applied Biosystems, USA).

      >  Gene identification and structure

    The boundary of individual thirteen mitochondrial protein-coding genes (PCGs) and individual two rRNAs were determined through the alignment of the homologous sequences of known full-length coleopteran mitochondrial genome sequences using the CLUSTAL X program (Thompson et al., 1997). The nucleotide sequences of the PCGs were translated on the basis of the invertebrate mtDNA genetic code (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/index.cgi?chapter=cgencodes). The translated amino acid sequence was further utilized to identify and delimitate the 13 PCGs. The tRNA genes were identified by their predicted cloverleaf secondary structure and anticodon sequences using the tRNAscan-SE version 1.1 with the option of invertebrate codon predictors and a cove score cut off of 1 (Lowe and Eddy 1997). The tRNASer(AGN) and tRNAAsn were initially identified through alignment of the nucleotide sequences of the tRNA genes of known full-length coleopteran mtDNA sequences, and were further confirmed by hand-drawing their secondary cloverleaf structure by inspection of respective anticodon sequences. The sequence data determined in this study have been deposited to GenBank under the accession number KF544959.

      >  Comparative mitochondrial gene analyses

    We compared 35 species of coleopteran insects including the currently sequenced P. l. manchurica. Nucleotide composition of each gene, whole genome, and each codon position of PCGs were calculated using DNASTAR (Burland, 2000). Frequencies of both codon and amino acid were calculated using SWAAP ver. 1.0.3 (http://www.bacteriamuseum.org/SWAAP/SwaapPage.htm). Gene overlap and intergenic-space sequences were handcounted. Nucleotide composition, termed “compositional skew” was calculated for the PCGs between two strands, and the whole genome with the EditSeq program included in the Lasergene software package (www.dnastar.com) using the following formula proposed by Perna and Kocher (1995): GC-skew = (G-C)/(G+C) and AT-skew = (A-T)/(A+T), where C, G, A, and T are the frequencies of the four bases.

    Results and discussion

      >  General features

    The Polyphylla laticollis manchurica mitogenome contains typical sets of genes, such as 13 PCGs, 22 tRNA genes, and 2 rRNA genes (Table 2). Typically, coleopteran insects contain one large non-coding region, named the A+T-rich region in insects, but we were unsuccessful in sequencing this region and the neighboring partial 5’-region of srRNA. In GenBank, a substantial number of incomplete mitogenome sequences lacking the A+T-rich region and neighboring sequences are registered and published as incomplete mitogenomes (Sheffield et al., 2008). Possible reasons for such failure may be the presence of secondary structural motifs and the excessive length of the A+T region. Several coleopteran species have been reported to have an extraordinary long A+T-rich region and an extra non-coding sequences neighboring to the region. For example, the seven-spotted lady beetle has been reported to have a 4,469 bp-long A+T-rich region which is composed of a 2,214 bp of non-repeat region and a 2,255 bp repeat region (Kim et al., 2012). Furthermore, the whiter-spotted flower chafer, P. brevitarsis, has a 5,646-bp long A+T-rich region which is composed of a 1,804 bp non-repeat region and a 3,841 bp repeat region (Kim et al., 2013a). In addition, insect mitogenomes infrequently contain substantially longer intergenic spacer sequences which are composed of tandem repeat unit located neighboring to the A+T-rich region. For example, the Korean firefly, Pyrocoelia rufa, has a 1724-bp long intergenic spacer (Bae et al., 2004). More extreme case is the two-spotted stag beetle,

    which contains a 4,051-bp long, large non-coding sequence located between tRNAIle and tRNAGln, along with a 3,100-bp long A+T-rich region, resulting in a genome size of 21,628 bp, which is at least 5 kb larger than typical animal mitogenomes (Kim et al., 2013b). Considering these, it would be a reasonable speculation that the unfinished region is unusually large. The secondary structural motifs in the P. l. manchurica mitogenome might be another source that prevented the polymerases correctly passing across this region, resulting in casual amplification and making subsequent sequencing impossible (Carapelli et al., 2006). Several coleopteran mitogenomes which have extraordinary long A+T-rich region also have several long repeat sequences (Kim et al., 2012; 2013a, 2013b). These repeat sequences can easily form stem-andloop structures (Zhang and Hewitt, 1997; Kim et al., 2007), and thus, the unfinished A+T-rich region, along with the neighboring region of the P. l. manchurica mitogenome is highly likely to have such motifs. Nevertheless, this failure was not fully understood, considering our previous success in sequencing this region from several coleopteran insects (Kim et al., 2012, 2013a, 2013b).

    The P. l. manchurica mitogenome has an identical gene order that has been found in the majority of insect species (Boore, 1998). Among 35 complete or nearly complete coleopteran mitogenomes (Table 3), only Tribolium castaneum differs from the most common type, by the movement of the tRNAGlu to a position 3’-downstream of tRNAPhe, thus resulting in an order of tRNAPhe and tRNAGlu, rather than the order tRNAGlu and tRNAPhe (Friedrich and Muquim 2003).

      >  Overlapping and intergenic spacer sequences

    The P. l. manchurica mitochondrial genes harbor a total of 47 bp of intergenic spacer sequences, which are spread over six regions, ranging in size from 1 to 20 bp, with the longest being located between tRNASer(UCN) and ND1 (Table 2). Similar sized intergenic spacer sequences are found in the majority of sequenced coleopteran insects (16 ~ 22 bp), except for those of Rhagophthalmus lufengensis and R. ohbai, which are only 5-bp long (Li et al., 2007). Previously, it has been shown that the region contains a 5 bp-long motif sequence (TAGTA) in the coleopteran insects (Sheffield et al., 2008). Our additional analysis, including all available coleopteran species and P. l. manchurica, consistently revealed a motif sequence in the region (Fig. 2). This 5-bp consensus sequence was suggested to be the possible binding site for mtTERM, the transcription termination peptide, with the consideration that the intergenic spacer sequence is detected at the end site of the major-strand coding region in the circular mtDNA (Taanman, 1999).

    Next longer intergenic spacer sequence is located between ND2 and tRNATrp as 14 bp (Table 2). A search on this region from other coleopteran insects has shown fluctuating length: only a few spacer sequences in most species (e.g., 4 bp in Pyrophorus divergens ; Arnoldi et al., 2007), abuttal between the two genes (e.g., Psacothea hilaris ; Kim et al., 2009), and overlapping between the two genes (2 bp in Tribolium castaneum ; Friedrich and Muqim, 2003). An exceptional case is that of P. rufa, which has a 1,724-bp long tandem repeat unit between the two genes (Bae et al., 2004). The P. l. manchurica mitochondrial genes overlap in a total of 33 bp at 12 locations, with the longest overlap measuring 8 bp, and located between tRNATrp and tRNACys. Similarly-sized overlapping sequences are also detected in several coleopteran insects (e.g., P. brevitarsis; Kim et al., 2013a; Metopodontus blanchardi ; Kim et al., 2013b), but abuttal (P. rufa ; Bae et al., 2004) and overlapping regions (8 bp in Pyrophorus divergens ; Arnoldi et al., 2007) are also found between the two tRNAs.

      >  Start and stop codon

    The P. l. manchurica mitogenomes harbors 3,704 codons, excluding termination codons, and this number is identical to those of Naupactus xanthographus (Song et al., 2010) and most similar to those of Sphenophorus sp. (3,705 codons; Song et al., 2010) and Tetraphalerus bruch (3705 codons; Sheffield et al., 2008).

    All PCGs, with the exception of COI and ATPase8 of the P. l. manchurica mitogenomes have the typical ATN codon (Table 2). The initiation site for the COI gene and the precedent tRNATyr does not harbor the typical start codon, except for the infrequent, but typical invertebrate start codon TTG that is located at the beginning region of the COI, overlapping one nucleotide with the 5’-end of the tRNATyr (Fig. 3). Thus, we designated the TTA as the COI start codon. Only a few other sequenced coleopteran insects have a typical ATN start codon, but these are all located inside neighboring tRNATyr (Fig. 3). Thus, Sheffield et al. (2008) previously suggested that start codon for COI genes should be chosen to minimize

    intergenic spacer sequences and gene overlaps. In this regard, they proposed asparagines (AAT or AAC) as the start codon for COI gene, because those are the first non-overlapping inframe codons, and found at the corresponding position in all sequenced Polyphaga in Coleoptera (Fig. 3; Sheffield et al., 2008). Furthermore, they hypothesized that asparagines may function as a molecular synapomorphy for Polyphaga (Sheffield et al., 2008). However, our newly sequenced P. l. manchurica, which belongs to Polyphaga, instead have AAG (Lysine) at the corresponding position and have the typical TTG precedent to AAG. Thus, our P. l. manchurica is the only example that does not follow the unanimous COI start codon in Polyphaga. The P. l. manchurica ATPase8 also has the TTG start codon (Fig. 4). Previously, the TTG codon was found in ND1 gene for coleopteran P. rufa (Bae et al., 2004) and dipteran Anopheles quadrimaculatus (Mitchell et al., 1993), and COI gene for lepidopteran Caligula boisduvalii (Hong et al., 2008), but has never been found for the start codon for ATPase8 in Coleoptera (data not shown).

    Eleven of the 13 PCGs have a complete termination codon of TAA or TAG, but the COII and COIII genes harbor the incomplete termination codon, T (Table 2). The most common interpretation of this phenomenon is that TAA termini are created via post-transcriptional polyadenylation (Ojala et al., 1981).

      >  tRNA and rRNA genes

    The P. l. manchurica mitogenomes harbors 22 tRNA genes that are interspersed thorough genomes (Fig. 5). Except for tRNASer(AGN), the dihydrouridine (DHU) arm of which forms a simple loop, all tRNAs were folded into the typical clover-leaf structure. The aberrant tRNASer(AGN) has been reported in many

    metazoan species, including insects (Wolstenholme 1992; Garey and Wolstenholme, 1989). For the proper function of a tRNA the DHU arm, which is involved in tertiary interaction requires proper folding (Rich and RajBhandary, 1976). The nuclear magnetic resonance analysis from nematodes has shown that the aberrant tRNASer(AGN) also functions in a similar way to that of usual tRNAs by structural adjustment to fit in the ribosome (Ohtsuki et al., 2002).

    The size of tRNAs ranged from 61 (tRNACys) to 71 bp (tRNALys) in P. l. manchurica (Table 2). Interestingly, tRNALys is often slightly larger than tRNAs in many other coleopteran mitogenomes. In fact, 29 among 35 coleopteran species analyzed in this study ranked the tRNALys as the longest tRNA at 71 bp (data not shown). In the varying tRNA size, the size of aminocyle stem as 7 bp, anticodon loops as 7 bp, and anticodon stem as 5 bp were all well conserved in all P. l manchurica tRNAs. Most of size variation among tRNAs stemmed from the length variation in DHU and TΨC arms, within which loop sizes (3 ~ 13 bp) are more variable than stem sizes (3 ~ 7 bp). With the numerous number of G-U base pairs, which form a weak bond in the tRNAs the P. l manchurica tRNAs contained 11 mismatches (three U-C, and each two A-C, U-U, G-A, and A-A mismatches) (Fig. 5). As with all other insect mitogenome sequences, two rRNA genes were detected in P. l manchurica. The lrRNA is located between tRNALeu(CUN) and tRNAVal and the srRNA is located between tRNAVal and the presumable A+T-rich region, respectively (Fig. 1).

      >  Nucleotide composition, codon usage, and skewness

    The nucleotide composition of the mitogenome of P. l. manchurica is also biased toward A/T content at 69.9% (Table 3). This value is well within the range found in the sequenced

    coleopteran insects, where these values range from 67.2% in Apatides fortis to 80.8% in Sphaerius sp. (Table 3). To evaluate the degree of the base bias, the AT-skew and GC-skew each in the whole genome and whole PCGs from the major strand and each major- and minor-strand PCGs was measured from coleopteran insects, including P. l. manchurica (Table 4). Overall, whole genomes of all coleopteran species including the P. l. manchurica are obviously A- and C-skewed, whereas whole PCGs are T- and C-skewed, indicating that Ts are clearly more favored over As in the PCGs, and the evolutionary pattern of PCGs differ from the remaining genes. In the majority of coleopteran species, including P. l. manchurica, the major-strand, in which nine PCGs (ND2,

    ND3, ND6, COI, COII, COIII, ATPase6, ATPase8, and CytB) are encoded is slightly T-skewed (AT skew = -0.129 ~ 0.153), whereas the minor-strand, in which four PCGs (ND1, ND4, ND4L, and ND5) are encoded is obviously A-skewed (AT skew = 0.163 ~ 0.460), although both strands are C-skewed (Table 4). Thus, the two strands are sharply distinct in A/T-skewness, indicating that mutational pressures that favor Ts or As are starkly different between the two strands. It has been suggested that the lagging strand, equivalent to the major strand, should be more prone to chemical conversion of As to Gs and Cs to Ts, by the mechanism called deamination than the leading strand, and this may have resulted in the enriched Ts and Gs in the lagging strand, and As and Cs in the leading strand (Reyes et al., 1998). Nevertheless, current coleopteran mitogenomes pretty strongly display C-skewness rather than G-skewness in the major strand (Table 4). Thus, the strand-based inequality has yet to be clearly understood.

    The genome-wise A/T bias is also reflected in the codon usage of P. l. manchurica mitogenomes (Table 5). The codons TTA (Leu), ATT (Ile), TTT (Phe), and ATA (Met) are the four most frequently used codons in the P. l. manchurica PCGs, accounting for 28.22% and on average these four codons accounted for 31.98% in Coleoptera (Table 5). Considering 60 codons are available, excluding each two start and stop codons (ATA, ATG, TAA, and TAG) the overuse of these four codons is obvious. These four codons are all comprised of A or T nucleotides, thus indicating the biased usage of A and T nucleotides in the coleopteran insects including P. l. manchurica PCGs.

    The analysis of the base composition at each codon position of the concatenated 13 PCGs of P. l. manchurica showed 68.6% of the A/T content in the third codon position. This value is higher than that of the first (66.4%) and second (66.2%) codon positions, and on average, the A/T content in the third codon position was 74.9%, ranging from 65.4% to 82.1% in Coleoptera (Table 6). In a comparison among species-diverse insect orders, Hymenoptera is the highest on average at 93.1% (Hong et al., 2008), Lepidoptera is next at 92.1% (Kim et al., 2010), and Diptera rank third at 92.0% (Cameron et al., 2009), indicating that the Coleoptera is the least A/T-biased. Probably, this aspect has implications for phylogenetic analysis because of the different degrees of A/T bias, resulting in compositional heterogeneity among insect orders as a major source of systematic bias in phylogeny (Jermiin et al., 2004; Sheffield et al., 2009).

  • 1. Arnoldi FG, Ogoh K, Ohmiya Y, Viviani VR (2007) Mitochondrial genome sequence of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae): Mitochondrial genes utility to investigate the evolutionary history of Coleoptera and its bioluminescence. [Gene] Vol.405 P.1-9 google doi
  • 2. Bae JS, Kim I, Sohn HD, Jin BR (2004) The mitochondrial genome of the firefly, Pyrocoelia rufa: complete DNA sequence, genome organization, and phylogenetic analysis with other insects. [Mol Phylogenet Evol] Vol.32 P.978-985 google doi
  • 3. Boore JL (1999) Animal mitochondrial genomes. [Nucleic Acids Res] Vol.27 P.1767-1780 google doi
  • 4. Burland TG (2000) DNASTAR's Lasergene sequence analysis software. [Methods Mol Biol] Vol.132 P.71-91 google
  • 5. Cameron SL, Sullivan J, Song H, Miller KB, Whiting FW (2009) mitochondrial genome phylogeny of the Neuropterida (lacewings, alderflies and snakeflies) and their relationship to the other holometabolous insect orders. [Zool Scr] Vol.38 P.575-590 google doi
  • 6. Carapelli A, Vannini L, Bardi F, Boore JL, Beani L, Dallai R, Frati F (2006) The mitochondrial genome of the entomophagous endoparasite Xenos vesparum (Insecta: Strepsiptera). [Gene] Vol.376 P.248-259 google doi
  • 7. Friedrich M, Muquim N (2003) Sequence and phylogenetic analysis of the complete mitochondrial genome of the flour beetle Tribolium castanaeum. [Mol Phylogenet Evol] Vol.26 P.502-512 google doi
  • 8. Garey JR, Wolstenholme DR (1989) Platyhelminth mitochondrial DNA: evidence for early evolutionary origin of a tRNAser(AGN) that contains a dihydrouridine arm replacement loop, and of serinespecifying AGA and AGG codons. [J Mol Evol] Vol.28 P.374-387 google doi
  • 9. Hammond PM (1992) Species inventory; in Global biodiversity, status of the earth’s living resources. Grrombridge B (ed.) P.17-39 google
  • 10. Hong MY, Jeong HC, Kim MJ, Jeong HU, Lee SH, Kim I (2009) Complete mitogenome sequence of the jewel beetle, Chrysochroa fulgidissima (Coleoptera: Buprestidae). [Mitochondrial DNA] Vol.20 P.46-60 google doi
  • 11. Hong MY, Lee EM, Jo YH, Park HC, Kim SR, Hwang JS, Jin BR, Kang PD, Kim KG, Han YS, Kim I (2008) Complete nucleotide sequence and organization of the mitogenome of the silk moth Caligula boisduvalii (Lepidoptera: Saturniidae) and comparison with other lepidopteran insects. [Gene] Vol.413 P.49-57 google doi
  • 12. Jermiin L, Ho SY, Ababneh F, Robinson J, Larkum AW (2004) The biasing effect of compositional heterogeneity on phylogenetic estimates may be underestimated. [Syst Biol] Vol.53 P.638-43 google doi
  • 13. Kim KG, Hong MY, Kim MJ, Im HH, Kim MI, Seo SJ, Lee SH, Kim I (2009) Complete mitochondrial genome sequence of the yellow-spotted long-horned beetle Psacothea hilaris (Coleoptera: Cerambycidae) and phylogenetic analysis among coleopteran insects. [Mol Cells] Vol.27 P.429-441 google doi
  • 14. Kim I, Cha SY, Kim MA, Lee YS, Lee KS, Choi YS, Hwang JS, Jin BR, Han YS (2007) Polymorphism and genomic structure of the A?T-rich region of mitochondrial DNA in the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae). [Biochem Genet] Vol.45 P.589-610 google doi
  • 15. Kim MJ, Im HH, Lee KY, Han YS, Kim I (2013a) Complete mitochondrial genome of the whiter-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Scarabaeidae). [Mitochondrial DNA In press] google doi
  • 16. Kim MJ, Kang AR, Jeong HC, Kim K-G, Kim I (2011) Reconstructing intraordinal relationships in Lepidoptera using mitochondrial genome data with the description of two newly sequenced lycaenids, Spindasis takanonis and Protantigius superans (Lepidoptera: Lycaenidae). [Mol Phylogenet Evol] Vol.61 P.436-445 google doi
  • 17. Kim MJ, Kim K-G, Kim SR, Kim I (2013b) Complete mitochondrial genome of the two-spotted stag beetle, Metopodontus blanchardi (Coleoptera: Lucanidae). [Mitochondrial DNA In press] google doi
  • 18. Kim MJ, Wan X, Kim I (2012) Complete mitochondrial genome of the seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae) [Mitochondrial DNA] Vol.23 P.179-181 google doi
  • 19. Kim MJ, Wan X, Kim KG, Hwang JS, Kim I (2010) Complete nucleotide sequence and organization of the mitogenome of endangered Eumenis autonoe (Lepidoptera: Nymphalidae). [Afr J Biotechnol] Vol.9 P.735-54 google
  • 20. Lewis DL, Farr CL, Farquhar AL, Kaguni LS (1994) Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA. [Mol Biol Evol] Vol.11 P.523-538 google
  • 21. Li X, Ogoh K, Ohba N, Liang X, Ohmiya Y (2007) Mitochondrial genomes of two luminous beetles, Rhagophthalmus lufengensis and R. ohbai (Arthropoda, Insecta, Coleoptera). [Gene] Vol.392 P.196-205 google doi
  • 22. Lowe TM, Eddy SR (1997) tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. [Nucleic Acids Res] Vol.25 P.955-964 google
  • 23. Mitchell SE, Cockburn AF, Seawright JA (1990) The mitochondrial genome of Anopheles quadrimaculatus species A: complete nucleotide sequence and gene organization. [Genome] Vol.36 P.1058-1073 google doi
  • 24. Ohtsuki T, Sato A, Watanabe Y, Watanabe K (2002) A unique serinespecific elongation factor Tu found in nematode mitochondria. [Nat Struct Biol] Vol.9 P.669-673 google doi
  • 25. Ojala D, Montoya J, Attardi G (1981) tRNA punctuation model of RNA processing in human mitochondria. [Nature] Vol.290 P.470-474 google doi
  • 26. Perna NT, Kocher TD (1995) Patterns of nucleotide composition at four fold degenerate sites of animal mitochondrial genomes. [J Mol Evol] Vol.41 P.353-358 google doi
  • 27. Pons J, Ribera I, Bertranpetit J, Balke M (2010) Nucleotide substitution rates for the full set of mitochondrial protein-coding genes in Coleoptera. [Mol Phylogenet Evol] Vol.56 P.796-807 google doi
  • 28. Reyes A, Gissi C, Pesole G, Saccone C (1998) Asymmetrical directional mutation pressure in the mitochondrial genome of mammals. [Mol Biol Evol] Vol.15 P.957-966 google doi
  • 29. Sheffield NC, Song H, Cameron SL, Whiting MF (2008) A comparative analysis of mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles. [Mol Biol Evol] Vol.25 P.2499-2509 google doi
  • 30. Sheffield NC, Song H, Cameron SL, Whiting MF (2009) Nonstationary evolution and compositional heterogeneity in beetle mitochondrial phylogenomics. [Syst Biol] Vol.58 P.381-394 google doi
  • 31. Song H, Sheffield NC, Cameron SL, Miller KB, Whiting MF (2010) When phylogenetic assumptions are violated: base compositional heterogeneity and among-site rate variation in beetle mitochondrial phylogenomics. [Syst Entomol] Vol.35 P.429-448 google doi
  • 32. Stewart JB, Beckenbach AT (2003) Phylogenetic and genomic analysis of the complete mitochondrial DNA sequence of the spotted asparagus beetle Crioceris duodecimpunctata. [Mol Phylogenet Evol] Vol.26 P.513-526 google doi
  • 33. Taanman JW (1999) The mitochondrial genome: structure, transcription, translation and replication. [Biochim Biophys Acta] Vol.1410 P.103-123 google doi
  • 34. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. [Nucleic Acids Res] Vol.24 P.173-216 google
  • 35. Wan X, Kim MI, Kim MJ, Kim I (2012) Complete mitochondrial genome of the free-living earwig, Challia fletcheri (Dermaptera: Pygidicranidae) and phylogeny of Polyneoptera. [PLoS ONE] Vol.7 P.e42056 google doi
  • 36. Wang AR, Kim MJ, Park JS, Choi YS, Thapa R, Lee KY, Kim I (2013) Complete mitochondrial genome of the dwarf honeybee, Apis florea (Hymenoptera: Apidae). [Mitochondrial DNA] Vol.24 P.208-210 google doi
  • 37. Wolstenholme DR (1992) Animal mitochondrial DNA: structure and evolution; in International Review of Cytology Wolstenholme DR, Jeon KW (eds.) P.173-216 google
  • 38. Won BH, Kwon YJ, Kim SS, Kim W (1998) Endangered wild species in Korea. google
  • 39. Zhang DX, Hewitt GM (1997) Insect mitochondrial control region: A review of its structure, evolution and usefulness in evolutionary studies. [Biochem Syst Evol] Vol.25 P.99-120 google doi
  • 40. Zhou Z, Huang Y, Shi F (2007) The mitochondrial genome of Ruspolia dubia (Orthoptera: Conocephalidae) contains a short A+T-rich region of 70 bp in length. [Genome] Vol.50 P.855-866 google doi
  • [Fig. 1.] Circular map of the mitochondrial genomes of Polyphylla laticollis manchurica. The abbreviations for the genes are as follows: COI, COII, and COIII refer to the cytochrome oxidase subunits, CytB refers to cytochrome B, ATP6 and ATP8 refer to subunits 6 and 8 of F0 ATPase, and ND1 ~ 6 refer to components of NADH dehydrogenase. tRNAs are denoted as one-letter symbols consistent with the IUPACIUB single letter amino acid codes. The one-letter symbols L, L*, S and S* denote tRNALeu(CUN), tRNALeu(UUR), tRNASer(AGN), and tRNASer(UCN), respectively. Gene names that are not underlined indicate a clockwise transcriptional direction, whereas underlined genes indicate a counter-clockwise transcriptional direction. The P. l. manchurica mitogenome was amplified each from three short (SF1, SF2, and SF3) and three long (LF1, LF2, and LF3) overlapping fragments, shown as single lines within a circle. The unknown region that possibly contains partial srRNA and the A+T-rich region is shaded.
    Circular map of the mitochondrial genomes of Polyphylla laticollis manchurica. The abbreviations for the genes are as follows:
COI, COII, and COIII refer to the cytochrome oxidase subunits, CytB
refers to cytochrome B, ATP6 and ATP8 refer to subunits 6 and 8 of F0
ATPase, and ND1 ~ 6 refer to components of NADH dehydrogenase.
tRNAs are denoted as one-letter symbols consistent with the IUPACIUB
single letter amino acid codes. The one-letter symbols L, L*, S
and S* denote tRNALeu(CUN), tRNALeu(UUR), tRNASer(AGN), and
tRNASer(UCN), respectively. Gene names that are not underlined
indicate a clockwise transcriptional direction, whereas underlined
genes indicate a counter-clockwise transcriptional direction. The P. l. manchurica mitogenome was amplified each from three short (SF1,
SF2, and SF3) and three long (LF1, LF2, and LF3) overlapping
fragments, shown as single lines within a circle. The unknown region
that possibly contains partial srRNA and the A+T-rich region is shaded.
  • [Table 1.] List of primers used to amplify and sequence the mitochondrial genomes of Polyphylla laticollis manchurica
    List of primers used to amplify and sequence the mitochondrial genomes of Polyphylla laticollis manchurica
  • [Table 2.] Summary of the mitochondrial genome of Polyphylla laticollis manchurica
    Summary of the mitochondrial genome of Polyphylla laticollis manchurica
  • [Table 3 .] Characteristics of the coleopteran mitochondrial genomes sequenced in their entirety and near entirety
    Characteristics of the coleopteran mitochondrial genomes sequenced in their entirety and near entirety
  • [Fig. 2.] Alignment of the internal spacer region located between ND1 and tRNASer(UCN) from coleopteran species, including Polyphylla laticollis manchurica. The boxed nucleotides indicate the conserved pentanucleotide region (TAGTA) detected in coleopteran insects. Underlined and dotted nucleotides, respectively, indicate the adjacent partial sequences of the ND1 gene and tRNASer(UCN) gene. The arrows indicate the transcriptional direction.
    Alignment of the internal spacer region located between ND1 and tRNASer(UCN) from coleopteran species, including Polyphylla laticollis manchurica. The boxed nucleotides indicate the conserved pentanucleotide region (TAGTA) detected in coleopteran insects.
Underlined and dotted nucleotides, respectively, indicate the adjacent partial sequences of the ND1 gene and tRNASer(UCN) gene. The
arrows indicate the transcriptional direction.
  • [Fig. 3.] Alignment of the initiation context of COI genes of coleopteran insects including that of Polyphylla laticollis manchurica. The first four to seven codons are shown in uppercase letters on the right-hand side of the figure. Underlined nucleotides indicate the adjacent partial sequence of tRNATyr. Arrows indicate the direction of transcription. Boxed nucleotides indicate currently known translation initiators for the COI of coleopteran. The start codon for P. l. manchurica was designated respectively as TTG.
    Alignment of the initiation context of COI genes of coleopteran insects including that of Polyphylla laticollis manchurica. The first
four to seven codons are shown in uppercase letters on the right-hand side of the figure. Underlined nucleotides indicate the adjacent partial
sequence of tRNATyr. Arrows indicate the direction of transcription. Boxed nucleotides indicate currently known translation initiators for the
COI of coleopteran. The start codon for P. l. manchurica was designated respectively as TTG.
  • [Fig. 4.] Alignment of the initiation context of ATPase8 gene of coleopteran insects, including that of Polyphylla laticollis manchurica. The first five codons are shown in uppercase letters on the right-hand side of the figure. Underlined nucleotides indicate the adjacent partial sequence of tRNAAsp. Arrows indicate the direction of transcription. Boxed nucleotides indicate currently known translation initiators for the ATPase8 gene of coleopteran insects. The start codon for P. l. manchurica was designated respectively as TTG.
    Alignment of the initiation context of ATPase8 gene of coleopteran insects, including that of Polyphylla laticollis manchurica. The
first five codons are shown in uppercase letters on the right-hand side of the figure. Underlined nucleotides indicate the adjacent partial
sequence of tRNAAsp. Arrows indicate the direction of transcription. Boxed nucleotides indicate currently known translation initiators for the
ATPase8 gene of coleopteran insects. The start codon for P. l. manchurica was designated respectively as TTG.
  • [Fig. 5.] Predicted secondary clover-leaf structures for the 22 tRNA genes of Polyphylla laticollis manchurica. The tRNAs are labeled with the abbreviations of their corresponding amino acids. The one-letter symbols L, L*, S and S* denote tRNALeu(CUN), tRNALeu(UUR), tRNASer(AGN), and tRNASer(UCN), respectively. Arms of tRNAs (clockwise from top) are the amino acid acceptor (AA) arm, TΨC (T) arm, the anticodon (AC) arm, and the dihydrouridine (DHU) arm. Nucleotide sequences from 5’ to 3’ are indicated from the left side of the amino acid stem. Dashes (-) indicate Watson-Crick base-pairing, and centered asterisks (*) indicate G-U base-pairing.
    Predicted secondary clover-leaf structures for the 22 tRNA genes of Polyphylla laticollis manchurica. The tRNAs are labeled with
the abbreviations of their corresponding amino acids. The one-letter symbols L, L*, S and S* denote tRNALeu(CUN), tRNALeu(UUR),
tRNASer(AGN), and tRNASer(UCN), respectively. Arms of tRNAs (clockwise from top) are the amino acid acceptor (AA) arm, TΨC (T) arm,
the anticodon (AC) arm, and the dihydrouridine (DHU) arm. Nucleotide sequences from 5’ to 3’ are indicated from the left side of the amino
acid stem. Dashes (-) indicate Watson-Crick base-pairing, and centered asterisks (*) indicate G-U base-pairing.
  • [Table 4.] Composition and skewness in the coleoptera mitochondrial genomes
    Composition and skewness in the coleoptera mitochondrial genomes
  • [Table 5.] Frequency of four most frequent codons of coleopteran insects
    Frequency of four most frequent codons of coleopteran insects
  • [Table 6.] Summary of base composition at each codon position of the concatenated 13 PCGs in coleopteran mitochondrial genomes
    Summary of base composition at each codon position of the concatenated 13 PCGs in coleopteran mitochondrial genomes