Comparative Mitogenomic Analyses of Darkling Beetles (Coleoptera: Tenebrionidae) Provide Evolutionary Insights into tRNA-like Sequences

Abstract

Tenebrionidae is widely recognized owing to its species diversity and economic importance. Here, we determined the mitochondrial genomes (mitogenomes) of three Tenebrionidae species (Melanesthes exilidentata, Anatolica potanini, and Myladina unguiculina) and performed a comparative mitogenomic analysis to characterize the evolutionary characteristics of the family. The tenebrionid mitogenomes were highly conserved with respect to genome size, gene arrangement, base composition, and codon usage. All protein-coding genes evolved under purifying selection. The largest non-coding region (i.e., control region) showed several unusual features, including several conserved repetitive fragments (e.g., A+T-rich regions, G+C-rich regions, Poly-T tracts, TATA repeat units, and longer repetitive fragments) and tRNA-like structures. These tRNA-like structures can bind to the appropriate anticodon to form a cloverleaf structure, although base-pairing is not complete. We summarized the quantity, types, and conservation of tRNA-like sequences and performed functional and evolutionary analyses of tRNA-like sequences with various anticodons. Phylogenetic analyses based on three mitogenomic datasets and two tree inference methods largely supported the monophyly of each of the three subfamilies (Stenochiinae, Pimeliinae, and Lagriinae), whereas both Tenebrioninae and Diaperinae were consistently recovered as polyphyletic. We obtained a tenebrionid mitogenomic phylogeny: (Lagriinae, (Pimeliinae, ((Tenebrioninae + Diaperinae), Stenochiinae))). Our results provide insights into the evolution and function of tRNA-like sequences in tenebrionid mitogenomes and contribute to our general understanding of the evolution of Tenebrionidae.

Keywords: Tenebrionidae; control region; mitochondrial genome; phylogenetic analysis; tRNA-like sequence.

Figure 1

Sizes of PCGs, tRNAs, rrnL, rrnS, and CR in Tenebrioidae mitogenomes. Species are abbreviated as follows: Adelium sp, Ap; Alphitobius diaperinus, Ad; Amarygmini sp, As; Asbolus verrucosus, Av; Blaps rhynchoptera, Br; Cerogria popularis, Cp; Gonocephalum sp, Gs; Machla setosa, Mse; Morphostenophanes sinicus, Msi; Morphostenophanes yunnanus, My; Nalassus laevioctostriatus, Nl; Opatrum sabulosum, Os; Pelecyphorus contortus, Pc; Pelecyphorus foveolatus, Pf; Philolithus aegrotus, Pa; Philolithus sp1, Ps1; Philolithus sp2, Ps2; Platydema sp, Pls; Promethis valgipes, Pv; Stenomorpha consobrina, Sc; Stenomorpha obovata, So; T. molitor, Tm; Tenebrio obscurus, To; Tribolium audax, Ta; Tribolium castaneum, Tca; Tribolium confusum, Tco; Uloma sp, Us; Ulomoides dermestoides, Ud; Zophobas atratus, Za; M. exilidentata, Me; A. potanini, Anp; M. unguiculina, Mu; Strongylium suspicax, Ss.

Figure 2

Putative secondary structures of 22 tRNA genes identified in the mitogenome of M. unguiculina. All tRNAs are shown in the order of occurrence in the mitogenome starting from trnL2. Bars indicate Waston-Crick base pairings, and dots between G and U pairs mark canonical base pairings in tRNA. Different colors represent different levels of base conservation. Green indicates conservation among all Tenebrionidae, orange indicates conservation among subfamilies, purple indicates conservation among the three newly sequenced species, and blue indicates a lack of conservation among the three newly sequenced species.

Figure 3

A+T% of the mitochondrial protein-encoding genes in the three groups within the 33 Tenebrionidae species. The dashed line separates the different subfamilies.

Figure 4

AT% vs. AT-skew (A) and GC% vs. GC-skew (B) in the 33 mitogenomes of the Tenebrionidae and one mitogenome of the outgroup. Measured in bp percentage (Y-axis) and level of nucleotide skew (X-axis). Values are calculated on J-strands for full-length mitochondrial genomes.

Figure 5

Evaluation of codon bias in the mitochondrial genomes of 33 Tenebrionidae species. (A) the correlation between ENC (effective number of codons) and the G + C content of the 3rd codon positions. (B) the correlation between CBI (codon bias index) and the 3rd codon positions. (C) the correlation between ENC and the G + C content of all codons. (D) the correlation between CBI and the G + C content of all codons. (E) the correlation between ENC and CBI.

Figure 6

(A) Correlations between the effective number of codons (ENC) and G+C content of the third codon positions (GC3) for the 33 Tenebrionidae species. The solid line represents the relationship between the ENC* (2 + GC3 + (29/[(GC3)^² + (1 − GC3)^²]) and the GC3. (B) The solid line represents the relationship between the GC3 and G+C content of the first and second positions (GC12), whereas the dotted line indicates y = x. Each color dot represents a tenebrionid species and colors match those in Figure 4.

Figure 7

Organization of the control regions in the Tenebrionidae mitogenomes. Location and copy number of tandem repeats are shown in orange and three colors (pink, purple, and grey) with Arabic numbers inside. Sky blue boxes represent interval sequences (positive numbers) or overlap (negative numbers) between two elements. Green boxes represent TA tandem repeat sequences. See Figure 1 for the full names of species.

Figure 8

Five tRNA-like secondary structures with a match of 65% or more were selected from the A+T-rich regions of 33 Tenebrionidae species, which were involved in the construction of the phylogenetic tree. (A) Three trnE-like sequences. (B) one trnS1-like sequence. (C) one trnS2-like sequence. The tRNAs are labeled with the abbreviations of their corresponding amino acids. A dash (-) indicates Watson-Crick base-pairing. Blue, purple, green, and orange represent U, A, G, and C bases, respectively. Percentages represent the consensus sequences of tRNA-like sequences compared to each corresponding conventional tRNA.

Figure 9

Mitogenome-based phylogenetic relationships among 33 Tenebrionidae species based on P123 datasets (protein-coding genes [PCGs] with all codon positions) using Bayesian inference (BI). Posterior probabilities for BI are shown on corresponding nodes in the topology of the BI tree. Different colors of species name blocks represent the different species, tribes, and subfamilies.