3.5 ∣ Evolutionary metamorphosis
According to gene clustering analyses, 24,923 gene families could be used for evolutionary comparison between WWS and other 15 representative insects (Supplementary Figure 7, Supplementary Table 10). A phylogenetic tree was generated with robust and consistent results on key taxa from extant insect orders representing hemimetabolic, holometabolic and ametabolic species, and estimated divergence dates based on validated fossils (Figure 2c). Regardless of the reference genomes and tree-building methods used, the genome phylogeny strongly supported the interspecific relationships shown in the tree based on the analyses of reference genomes (Figure 2c). Estimation of divergence times suggests that the aphids Acyrthosiphon pisum and Diuraphis noxiaform a sister group and diverged from the WWS in the Middle Triassic (∼218.0 ± 60.7 Mya). Further, our analysis indicated that the last common ancestor of Holometabola and Hemimetabola separated in the Late Carboniferous (∼301.6 ± 9.7 Mya). This differs from a previous report, which found that fossils of holometabolic insects were first seen at the end of Permian (∼252 Mya) and constitute a monophyletic group (Kristensen, 1975; Nel et al., 2013). Our research indicates that complete metamorphosis emerged in the Late Carboniferous (∼301.6 ± 9.7 Mya, Figure 2c), at least 50 million years earlier than the previous study, and followed by a period were terrestrial plants diversified dramatically (Misof et al., 2014). Based on the phylogenetic analysis, ametabolous represent a primitive group located in the basal branch of the tree, whereas hemiptera and holometabola reside in the lower and upper branches separately. WWS represents an important transitional model between hemimetaboly and holometaboly. Our study provides evidence that holometamorphosis derived from hemimetamorphosis in the Late Carboniferous. Clearly, there is an evolutionary trend from ametabolous to hemimetabolous insect development, followed by the emergence of holometabola (Figure 1b, 2c).