General features of the mitogenome
The mitochondrial sequence of A. fulloi has been submitted to GenBank with the accession number OK545741. The nearly complete mitogenome was 15,692 bp in length, within the range of other hymenopterous mitogenomes: from 15,137 (Idris sp.) to 20,370 bp (Trachelus iudaicus)34,35. The A. fulloi mitogenome contained 37 genes (13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes) and a CR (Table 1 and Figure 1). This is common for most hymenopterous mitogenomes17,36,37. However, extra tRNA genes and CRs are also found in parasitoids such as Pteromalus puparum, Ibalia leucospoides, and Aphidius gifuensis26,38,39. These duplications may be attributed to ongoing gene rearrangement39.
Table 1
Characteristics of the mitochondrial genome of A. fulloi.
Feature | Strand | Start sites | Stop sites | Size(bp) | Anticodon | Start codon | End codon | Intergenic nucleotides |
trnV | + | 1 | 66 | 66 | UAC | | | -2 |
rrnS | + | 65 | 834 | 770 | | | | 5 |
trnA | + | 840 | 904 | 65 | UGC | | | -18 |
rrnL | + | 887 | 2252 | 1366 | | | | 1 |
trnL1 | + | 2254 | 2320 | 67 | UAG | | | -3 |
nad1 | + | 2318 | 3265 | 948 | | ATA | TAA | 9 |
trnS2 | - | 3275 | 3342 | 68 | UGA | | | 2 |
cytb | - | 3345 | 4481 | 1137 | | ATG | TAA | 10 |
nad6 | - | 4492 | 5061 | 570 | | AAC | TAA | -2 |
trnT | - | 5060 | 5127 | 68 | UGU | | | 12 |
trnP | + | 5140 | 5206 | 67 | UGG | | | 4 |
nad4l | + | 5211 | 5498 | 288 | | ATT | TAA | -7 |
nad4 | + | 5492 | 6838 | 1347 | | ATG | TAA | 0 |
trnH | + | 6839 | 6906 | 68 | GUG | | | -3 |
nad5 | + | 6904 | 8581 | 1678 | | ATA | T | -3 |
trnF | + | 8579 | 8648 | 70 | GAA | | | 16 |
trnE | - | 8665 | 8732 | 68 | UUC | | | 6 |
cox1 | + | 8739 | 10280 | 1542 | | ATA | TAA | 32 |
trnL2 | + | 10313 | 10379 | 67 | UAA | | | 0 |
cox2 | + | 10380 | 11102 | 723 | | ATT | TAA | 9 |
trnK | - | 11112 | 11182 | 71 | UUU | | | 13 |
trnD | + | 11196 | 11266 | 71 | GUC | | | 30 |
atp8 | + | 11297 | 11458 | 162 | | ATT | TAA | -7 |
atp6 | + | 11452 | 12123 | 672 | | ATG | TAA | -1 |
cox3 | + | 12123 | 12914 | 792 | | ATT | TAA | 43 |
trnG | + | 12958 | 13022 | 65 | UCC | | | 18 |
nad3 | + | 13041 | 13409 | 369 | | ATA | TAG | 6 |
trnS1 | - | 13416 | 13475 | 60 | UCU | | | 3 |
trnY | + | 13479 | 13549 | 71 | GUA | | | 82 |
trnN | + | 13632 | 13698 | 67 | GUU | | | 129 |
trnC | - | 13828 | 13889 | 62 | GCA | | | 53 |
trnR | - | 13943 | 14007 | 65 | UCG | | | 6 |
trnQ | - | 14014 | 14082 | 69 | UUG | | | -2 |
trnW | - | 14081 | 14145 | 65 | UCA | | | 2 |
nad2 | - | 14148 | 15164 | 1017 | | ATA | TAA | 29 |
trnI | + | 15194 | 15263 | 70 | GAU | | | 11 |
trnM | - | 15275 | 15345 | 71 | CAU | | | 0 |
CR | | 15346 | 15692 | 347 | | | | |
Note: + represents the majority strand; - represents the minority strand. |
Overlapping and intergenic regions are usually detected in hymenopterous mitogenomes18,20,40. In general, overlapping nucleotides are smaller in size than intergenic regions and are exclusively found among those lacking rearrangement mitogens, which may assure their function19,41,42. In the mitogenome of A. fulloi, a total of 48 overlapping sites were identified in 10 locations, ranging in size from 1 to 18 bp (Table 1). The longest overlapping region was found between trnA and rrnL. The A. fulloi mitogenome also contained 24 intergenic spacers with a total length of 531 bp. These intergenic spacers range from 1 to 129 bp (Table 1). The longest gene spacer was located between trnC and trnN.
The nucleotides of the A. fulloi mitogenome comprise A (39.06%), T (44.77%), C (6.02%), and G (10.15%) (Table 2). The A. fulloi mitogenome was highly biased towards A and T nucleotides with an A + T content of 83.83%. The mitogenome of A. fulloi had a negative AT skew (-0.069) and a positive GC skew (0.255) (Table 2). These results are identical to those reported for other hymenopteran mitogenomes20,43.
Table 2
Nucleotide composition of A. fulloi mitochondrial genome.
Feature | A% | T% | C% | G% | AT% | GC% | AT Skew | GC Skew |
protein-coding genes | 35.13 | 47.05 | 8.00 | 9.82 | 82.18 | 17.82 | -0.145 | 0.102 |
1st codon position | 38.92 | 38.92 | 7.92 | 14.24 | 77.84 | 22.16 | 0.000 | 0.285 |
2nd codon position | 22.04 | 52.62 | 13.71 | 11.63 | 74.66 | 25.34 | -0.410 | -0.082 |
3rd codon position | 44.42 | 49.63 | 2.37 | 3.58 | 94.05 | 5.95 | -0.055 | 0.203 |
tRNAs | 43.08 | 44.50 | 4.59 | 7.83 | 87.58 | 12.42 | -0.016 | 0.261 |
rRNAs | 43.59 | 43.68 | 4.03 | 8.71 | 87.27 | 12.74 | -0.001 | 0.367 |
CR | 41.21 | 40.92 | 6.63 | 11.24 | 82.13 | 17.87 | 0.004 | 0.258 |
whole mitogenome | 39.06 | 44.77 | 6.02 | 10.15 | 83.83 | 16.17 | -0.069 | 0.255 |
PCGs
The total length of 13 PCGs was 11,245 bp, accounting for 71.66% of the whole mitogenome. This is identical to other hymenopteran mitogenomes17,27. This set of PCGs is conserved in animal mitogenomes, with the exception of nematodes and a bivalve that lack atp815. These PCGs range in size from 162 bp (atp8) to 1678 bp (nad5). The A + T content of all PCGs was 82.18% (Table 2). A remarkably high A + T content (94.05%) was found at the third codon sites of these PCGs, which may result from the high bias towards A and T nucleotides in the mitogenome19,44,45. Meanwhile, the AT skew and GC skew of the PCGs were -0.145 and 0.102, respectively. Of the 13 PCGs, 10 were encoded on the majority strand, whereas cytb, nad2, and nad6 were encoded on the minority strand. In insect mitogenomes, PCGs usually start with ATN codons (ATA, ATT, ATC, and ATG) and terminate with TAA or TAG46–48. However, unusual start- and termination codons were simultaneously and exclusively found, such as the start codons of TTG, CGA, GTG, and the incomplete stop codon of T49–51. In the A. fulloi mitogenome, most PCGs started with ATN, including ATA (nad1-3, nad5, and cox1), ATT (atp8, cox2, cox3, and nad4l) and ATG (atp6 and nad4). However, nad6 used an atypical starting codon of AAC, which has also been found in Cheirotonus jansoni and Prosopocoilus gracilis52,53. All PCGs terminated with TAA except nad3 (TAG) and nad5 (incomplete codon T). Previous studies have inferred that the incomplete termination codon could be completed by posttranscriptional polyadenylation15,54,55. The codon usage of PCGs was assessed by the relative synonymous codon usage (RSCU) value (Figure 2). Of the 3736 amino acid-encoding codons, TTA (Leu1), CGA (Arg), TCA (Ser2), and TCT (Ser2) were predominant. Codons such as CGC, AGC, and CTG were not presented.
tRNAs, rRNAs and the control region
The total length of 22 tRNAs was 1481 bp, accounting for 9.44% of the whole A. fulloi mitogenome (Table 2). These tRNAs range from 60 to 71 bp, within the range reported for other hymenopteran parasitoids17,27,36 (Table 1). Among these tRNAs, 12 tRNAs were coded in the majority strand, and the remaining 10 tRNAs were identified in the minority strand, which is inconsistent with other parasitoid species due to their high rates of rearrangement19,34,56. These tRNAs had a high A + T content of 87.58%, a slightly negative AT skew value (-0.016), and a positive GC skew value (0.261). Except for the absence of a dihydrouridine (DHU) arm in trnS1, most tRNAs had a cloverleaf secondary structure (Figure 3). The loss of the DHU arm in trnS1 is normal in insects5,57.
Two rRNAs, i.e., the small ribosomal RNA (rrnS) and large ribosomal RNA (rrnL), were 770 bp and 1366 bp in length, respectively. These lengths are similar to those of most reported hymenopteran insects27,58. These rRNAs were located on the majority strand and were separated by trnA. They consisted of A (43.59%), T (43.68%), C (4.03%), and G (8.71%), with an A + T content of 87.27%. The AT skew and GC skew were -0.001 and 0.367, respectively.
In insect mitogenomes, the control region, i.e., the A + T-rich region, is variable not only in size but also in base composition59. The CR was 3,308 bp with an A + T content of 85.9% in Pteromalus puparum39 but 578 bp with an A + T content of 93.6% in Spathius agrili18. This region is associated with replication and transcription60,61. Currently, the long and complex control region still presents challenges to obtaining complete mitogenomes in certain groups of parasitoids19,20,27,62−64. In this study, we failed to obtain the complete segment of the CR by next generation sequencing or Sanger sequencing. The partial control region obtained for the A. fulloi mitogenome was 347 bp with an A + T content of 82.13% and was located between trnM and trnV.
Gene rearrangement
Gene order is widely considered to be important phylogenetic information for insects5,65. The gene orders of parasitoid mitogenomes assigned to 6 families are shown in Figure 4. Compared with the gene order in the ancestral insect mitogenome, parasitoid mitogenomes exhibit large-scale rearrangement events for tRNA genes and PCGs, as reported in other studies of parasitoid mitogenomes17,27,66. In addition, the gene orders of these mitogenomes from different families are variable. Within Eupelmidae, mitogenomes of A. fulloi and Eupelmus sp. also exhibit different gene orders that are attributed to the rearrangement of tRNAs. Species from the same genus, such as species of Trichogramma and or Encyrtus, showed the same gene order in mitogenomes. This indicates that the mitochondrial gene arrangement of parasitoids from Chalcidoidea might be useful for phylogenetic analysis19,20.
CREx analysis demonstrated that the gene order of the mitogenome of A. fulloi is novel. Compared with the gene order in the ancestral insect mitogenome, the segment between cox1 and nad3 was relatively conserved except for the inversion of trnK, which is the same as Metaphycus eriococci, Encyrtus sasakii, and Chouioia cunea67 (Figure 5). It has been reported that the segment “trnE -trnF -nad5 -trnH -nad4 -nad4l trnT -trnP nad6 cytb” is conserved between Megraphragma and Philotrypesis40,68. However, in the mitogenome of A. fulloi, the segment “trnE -trnF -nad5 -trnH -nad4 -nad4l trnT -trnP nad6 cytb trnS2 -nad1 -trnL1 -rrnL” was inversed, and trnT -trnP have exchanged their positions. The segment “-trnV -rrnS CR trnI -trnQ trnM nad2 trnW” was highly rearranged, including the inversions of trnV, rrnS, trnM, nad2 and trnW, position exchanges of trnV and rrnS, trnM and trnI, and the transposition of trnQ (Fig. 5). In addition, trnC and trnY were transported to the segment “trnR trnN trnS1”. The gene order of these tRNA genes was changed to “-trnS1 trnY trnN -trnC -trnR”.
Phylogenetic analyses
The phylogenetic relationships of 20 parasitoids within Chalcidoidea were analysed and are displayed in Figure 6. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees were constructed based on nucleotide sequences of 13 PCGs of these mitogenomes in CIPRES69. Although some clades exhibited low support values, the same topological structures were found in two phylogenetic trees. Two species within Eupelmidae, A. fulloi and Eupelmus sp., were clustered together. Other species within the same families were grouped and separated from other parasitoids in different families. In Chalcidoidea, Eupelmidae is considered to be closely related to Encyrtidae, and to share several of the same features, such as an expanded acropleuron70,71. However, this family is not monophyletic, representing a grade rather than a clade24,72,73. Some species may show a close relationship to Pteromalidae71,72. In the present study, the phylogenetic relationship of parasitoids within Chalcidoidea can be presented as follows: Mymaridae + (Eupelmidae + (Encyrtidae + (Trichogrammatidae + (Pteromalidae + Eulophidae)))). This result is consistent with other reports27,37.