It has reported that the genetic distance between E. scrobiculatus and E. brandti was 0.173, the differentiation time of the two weevils can be traced back to about 3.76 million years ago. It can be speculated that before this time, the two beetles may be the same species. In order to reduce intra-species competition to adapt to changes in certain environments, they form niche differentiation, and groups occupying different niches have barriers to gene communication, and new species are formed through reproductive isolation. In this long-term evolution process, they have formed into different forms to meet different ecological demands. In the previous study we have known, in the long-term evolution process, E. scrobiculatus and E. brandti utilized different oviposition sites to facilitate coexistence on the single host A. altissima. Further observation of oviposition behavior revealed that females must dig a oviposition hole with their rostra before laying eggs[24, 25]. Therefore, we further put forward the hypothesis that the difference in the egg-laying positions of the two kinds of weevils may be due to the difference in the structure of their rostrums. In other words, in order to adapt to different oviposition positions, the two weevils formed different rostrum shapes to ensure the development of offspring.
Regarding the elongated rostrum, the main difference in length between the male and female weevils of some species has been well known. This was mentioned in Kirby & Spence "Introduction to Entomology". Darwin been sufficiently understood, but it must be seen in relation ‘different habits of life and not at all, or only indirectly, to the reproductive functions’ mentioned that sexual dimorphism in weevil rostra and many similar examples had not (in the sense of sexual selection) . Exaggerated female rostra are well documented within the Curculionidae[4, 26, 30–32]. For example, there was obvious external sexual dimorphism in the length of their rostra between the male and female rostra of Rhopalapion longirostre (Coleoptera: Curculionoidea), and the female rostrum was twice as long the male. The elongated female snout of R. longirostre was a response to the requirements of boring egg channels of maximal depth into the buds of the host plant. In our study, we have known E. brandti female rostra were significantly longer than males, this result seemd to be consistent with Gertha's results on sexual dimorphism of R. longirostre. However, the difference was that we found there were also significant differences in female and male body size, and rostrum length was found to be correlated with body size. For E. brandti, the body size and rostrum length between males and females were significantly different, both female body size and rostrum length are greater than male (Figure S1, B). While the slope value of allometric did not differ between E. brandti females and males (Fig. 2, B). Therefore, we suppose that the E. brandti female rostrum length was caused by body size. This result also applied to E. scrobiculatus. For E. scrobiculatus, there was no difference in body size and rostrum length between males and females (Figure S1, A), and the slope value of allometric did not differ between E. scrobiculatus females and males (Fig. 2, A). In addition, comparing the two weevil females also proved that the E. scrobiculatus rostrum was longer than the E. brandti, and E. scrobiculatus 's body was longer than the E. brandti. This showed that larger specimens had proportionally longer rostrum. The slope of the allometric function relating body size and rostrum length was little than one, showing that rostrum length does not increase isometrically with body size. This result was consistent with the speculation. For large Curculio elephas and the small-bodied Curculio glandium co-occur in oak forests, there were no interspecific differences in adult female body size to rostrum length allometric relationships, and rostrum length was equally correlated with body size in either species. The increased rostrum length was probably a by-product of the larger body sizes of individuals emerging from bigger acorns. There was no divergence in adult allometry between the same species of different sex and same sex of different species of E. scrobiculatus and E. brandti (Fig. 2). Therefore, we speculate that the rostrum length of E. scrobiculatus and E. brandti which was caused by body size was not a feature of the two weevil's adaptation to the oviposition site, it was probably a by-product of the larger body sizes. Perhaps the co-vary in body features and exaggerated feeding traits, rather than just exaggerated rostrum length, is one of the performances of the two kinds of weevil's adaptation to the environment.
In addition, we know that exaggerated female rostrum is an important structure in the oviposition process of weevil. Antliarhinus zamiae females use their extremely long rostra to chew through the cones of their host plants, species of the cycad genus Encephalartos (Zamiaceae) and deposit their eggs into the cycad’s ovules[31, 34]. That species of the genus Curculio attack a wide range of host plants is hypothesized to be a result of ecomorphological adaptations to oviposition site, and seed size has been postulated to be responsible for morphological changes in rostrum size. A series of studies show in great detail that the length of the female rostrum is closely related to the relative thickness of the pericarp of the host plant Camellia japonica in different populations of the weevil species Curculio camelliae (Curculionidae) [3–5]. It was argued that natural selection drives the coevolutionary arms race between the weevil and its host plant, and the specific morphological changes of the curculio’s rostrum are the result of ecological morphological adaptation to the oviposition site. Bland compared the mouthparts and sensors of Hypera postica and Hypera brunneipennis and found that the types and number of tapered sensors at the end of the lower jaw were the same, but the shape and size of the mandibles were different.In our study, the gross morphological features of the mouthparts of E. scrobiculatus and E. brandti are similar to those reported for other weevils[32, 35–39], but their fine structure is slightly different. The highly sclerotized left and right mandibles, hinged to the later apical margin of the rostrum by well-developed dorsal and ventral articulations, and coming together medially, are massive and irregularly semi-globular. The asymmetrical mandibles look like palm-shaped pliers, the outer surface protrudes in an arc shape. When laying eggs, the left and right mandibles bite each other to excavate holes. Each mandible of E.scrobiculatus female possesses two teeth, a conspicuous large apical tooth and a small tooth, these two teeth differ greatly in size (Fig. 3, E, G). The bigger tooth is round and blunt. This structure can not only reduce the stress concentration but also enhance the mechanical strength of the biological material to adapt to the environmental conditions and improve the wear resistance under the action of soil abrasives. It can also improve the distribution of soil stress at the end, change the shape of the compacted soil, and reduce soil adhesion. Each mandible of E. brandti female also has two teeth, but the difference in size between the two teeth is not obvious, almost either one occupies half of the top of the mandible (Fig. 3, F, H). These two teeth rubbing against each other is more conducive to biting the bark, and the slender and smooth rostrum is more conducive to females drilling holes on the hard trunk. Forsythe compared the shapes of mouthparts of some ground beetles (Carabidae), find different mandibular structures adapt to different foods and have different feeding habits. In our study we speculate that the difference in the fine structure of the two rostra of the two weevils is an adaptation to the oviposition sites. The rostrum of E. scrobiculatus is stout, densely covered with setae and minute pores, the mandible of mouthparts possesses two teeth, the two teeth are not the same size. Such structural features are more conducive to digging in the soil. While the rostrum of E. brandti female is thin, and smooth, with much fewer setae and pores. Two teeth of little different in size rubbing against each other are more conducive to biting the bark, and the slender and smooth rostrum is more conducive to females drilling holes on the hard trunk.
In addition, we also compared the fine structure of the rostrum between males and females of the same species, but found that the external morphological structure did not differ much in sex, except for the difference between the body length and the rostrum length of E. brandti, there was no sexual dimorphism between males and females. However, a comparison of the internal male and female mandibular muscles of E. scrobiculatus and E. brandti revealed a distinct sexual dimorphism. It can be seen on the slice of the micro-CT, whether E. scrobiculatus or E. brandti, the female muscles (mandible adductor muscle and mandible abductor muscle) are more developed than the male (Figure S2). Based on image segmentation and three-dimensional reconstruction, we compared the proportion of mandibular muscles between males and females of the same species. The results also show that the female muscle volume (mandible adductor muscle and mandible abductor muscle) was much larger than the male (E. scrobiculatus: VadF 5.42% vs VadM 3.76%, VabF 3.3% vs VabM 2.05%; E. brandti: VadF 6.56% vs VadM 4.34%, VabF 2.7% vs VabM 1.48%. adF = the mandible adductor muscle of female; adM = the mandible adductor muscle of male, abF = the mandible abductor muscle of female, abM = the mandible abductor muscle of male). Gertha compared the mandibular muscles of R. longirostre male and female, the results also showed a distinct sexual dimorphism. The female mandibular adductor muscle and the mandibular abductor muscle were significantly larger than that of the male. This difference between male and female muscles indicates that females need more muscle to increase muscle strength for excavating oviposition sites. In our study, because the microscopic sections of male and female weevils can see the difference in muscle volume, we only selected a single sample randomly for comparison and did not conduct an analysis of variance.
Further, we compared the volume of the two weevils’ female muscles to analyze the response to the oviposition sites. In this experiment, unlike the male and female, it was difficult to see the muscle size of the two females on the micro-CT slice level (Figure S2). Therefore, we conducted an analysis of variance by three-dimensionally reconstructing and calculating the muscles of multiple sample females. The results were shown in the Fig. 5, the mandibular adductor muscle of E. brandti females were significantly larger than that of E. scrobiculatus females, but the mandibular abductor muscle was not different between the two females. Previous studies on the oviposition behavior of the two weevil species have revealed that during the oviposition process, the mandibular of the female was continuously opened and closed to excavate an oval oviposition cavity hole at the oviposition substrate[24, 25], and the mandibular is jointly controlled by the adductor and abductor. The adductor muscle controls the mandibular occlusion, and the abductor muscle controls the mandibular extension. E. scrobiculatus females had no difference in abductor muscles than E. brandti females, but the adductor muscles of E. scrobiculatus females were smaller than that of E. brandti females. From this, we can speculate that there was no difference in the amount of external tension between E. scrobiculatus females and E. brandti, but the bite force on the mandibular of E. brandti females was larger than that of E. scrobiculatus females.