Comparative mitogenomics of Braconidae (Insecta: Hymenoptera) and the phylogenetic utility of mitochondrial genomes with special reference to Holometabolous insects

Abstract

Background: Animal mitochondrial genomes are potential models for molecular evolution and markers for phylogenetic and population studies. Previous research has shown interesting features in hymenopteran mitochondrial genomes. Here, we conducted a comparative study of mitochondrial genomes of the family Braconidae, one of the largest families of Hymenoptera, and assessed the utility of mitochondrial genomic data for phylogenetic inference at three different hierarchical levels, i.e., Braconidae, Hymenoptera, and Holometabola.

Results: Seven mitochondrial genomes from seven subfamilies of Braconidae were sequenced. Three of the four sequenced A+T-rich regions are shown to be inverted. Furthermore, all species showed reversal of strand asymmetry, suggesting that inversion of the A+T-rich region might be a synapomorphy of the Braconidae. Gene rearrangement events occurred in all braconid species, but gene rearrangement rates were not taxonomically correlated. Most rearranged genes were tRNAs, except those of Cotesia vestalis, in which 13 protein-coding genes and 14 tRNA genes changed positions or/and directions through three kinds of gene rearrangement events. Remote inversion is posited to be the result of two independent recombination events. Evolutionary rates were lower in species of the cyclostome group than those of noncyclostomes. Phylogenetic analyses based on complete mitochondrial genomes and secondary structure of rrnS supported a sister-group relationship between Aphidiinae and cyclostomes. Many well accepted relationships within Hymenoptera, such as paraphyly of Symphyta and Evaniomorpha, a sister-group relationship between Orussoidea and Apocrita, and monophyly of Proctotrupomorpha, Ichneumonoidea and Aculeata were robustly confirmed. New hypotheses, such as a sister-group relationship between Evanioidea and Aculeata, were generated. Among holometabolous insects, Hymenoptera was shown to be the sister to all other orders. Mecoptera was recovered as the sister-group of Diptera. Neuropterida (Neuroptera + Megaloptera), and a sister-group relationship with (Diptera + Mecoptera) were supported across all analyses.

Conclusions: Our comparative studies indicate that mitochondrial genomes are a useful phylogenetic tool at the ordinal level within Holometabola, at the superfamily within Hymenoptera and at the subfamily level within Braconidae. Variation at all of these hierarchical levels suggests that the utility of mitochondrial genomes is likely to be a valuable tool for systematics in other groups of arthropods.

Figure 1

Predicted phylogenetic relationships among five braconid species based on the secondary structures of H39, H47 and H367 in Domain I of rrnS. Base-pairing is indicated as follows: Watson-Crick pairs by lines, wobble GU pairs by dots and other noncanonical pairs by circles.

Figure 2

Structural elements of A+T-rich region in three braconid mitochondrial genomes. (A) Structure of Spathius agrili mitochondrial A+T-rich region. Three repeat sequences are aligned. (B) Structure of Diachasmimorpha longicaudata mitochondrial A+T-rich region. Five repeat sequences including three elements were aligned. (C) Structure of Cotesia vestalis mitochondrial A+T-rich region. PolyT stretches were compared in C. vestalis and Bombus ignitus. Short dashes indicate gaps; underlines of the PolyT stretch sequence indicate conserved region between C. vestalis and B. ignitus.

Figure 3

Gene arrangement of seven braconid mitochondrial genomes sequenced in this study. Abbreviations for the genes are as follows: cox1, cox2, and cox3 refer to the cytochrome oxidase subunits, cob refers to cytochrome b, and nad1-nad6 refer to NADH dehydrogenase components, rrnL and rrnS refer to ribosomal RNAs. Transfer RNA genes are denoted by one letter symbols according to the IPUC-IUB one-letter amino acid codes. L1, L2, S1, S2 denote tRNA^Leu(CUN), tRNA^Leu(UUR), tRNA^Ser(AGY), tRNA^Ser(UCN), respectively. Boxes with underscores indicate that the gene is encoded in minority strand. Shaded boxes indicate that the gene was rearranged compared with ancestral arrangement of Hexapoda.

Figure 4

Mechanism of trnH remote inversion in Spathius agrili mitochondrial genome. (A) Presumed pseudo-trnH sequence (HO) and 10 hymenopteran trnH sequences are aligned according to their secondary structures. AM: Accepter arm, DA: D-loop arm, DL: D-loop, AA: Anticodon arm, AL: Anticodon loop, VL: Variable loop, TA: T Ψ C arm, TL: T Ψ C loop. (B) Secondary structure trnH is predicted in tRNAscan-SE search server [82] and HO is predicted manually. The inserted uracil in the anticodon is showed by a circle. (C) Recombination of two strands with opposite orientations leads the inversion of trnH, and the following recombination events lead the duplication of trnH. DS: Diadegma semiclausum, PS: Primeuchroeus spp., BI: Bombus ignites; PH: Polistes humilis, CV: Cotesia vestalis, VE: Vanhornia eucnemidarum, MB: Melipona bicolor, PC: Perga condei, NG: Nasonia giraulti, AM: Apis mellifera, SA: Spathius agrili.

Figure 5

Putative gene rearrangement events in Cotesia vestalis mitochondrial genome. Four regions underwent gene rearrangements. Rearranged genes are shown in gray. The region from cox3 to cob experienced protein-coding gene rearrangements, and three types of rearrangement events might happen: large-scale inversion, tRNA rearrangement and small region rearrangement. The intermediate statuses are used to show different types of gene rearrangement events, but not the rearrangement process.

Figure 6

Evolutionary rates of braconid mitochondrial genomes. The ration of the number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) for each braconid mitochondrial genomes, using that of Diadegma semiclausum or Enicospilus sp. as reference sequences.

Figure 7

Braconidae phylogeny based on complete mitochondrial genome sequences. Bayes phylogenetic trees for all seven braconid species based on amino acid sequence (aa) and nucleotide sequences of first and second codon positions (Pos12-BI) of all protein-coding genes except nad2, for species without Macrocentrus camphoraphilus (Pos12-noMC-BI) or Phanerotoma flava (Pos12-noPF-BI) were present. Bootstrap support values followed by Bayesian posterior probabilities (BPP) are shown at the right of respective nodes.

Figure 8

A combined holometabolous phylogenetic tree based on all mitochondrial protein-coding genes. Bootstrap support values for the nodes inferred from four analyses (by Bayes inference method based on first, second and RY-coded third codon positions, first and second codon positions, and amino acid sequences, and by most likelihood method based on first, second and RY-coded third codon positions of all mitochondrial protein-coding genes) were shown sequentially separated by "/". "*" indicates that the node were fully supported by all four inferences; "-" indicates that the node was not recovered by the corresponding inference.