AbstractThe social environment is often the most dynamic and fitness-relevant environment animals experience. Here we tested whether plasticity arising from variation in social environments can promote signal-preference divergence-a key prediction of recent speciation theory but one that has proven difficult to test in natural systems. Interactions in mixed social aggregations could reduce, create, or enhance signal-preference differences. In the latter case, social plasticity could establish or increase assortative mating. We tested this by rearing two recently diverged species of Enchenopa treehoppers-sap-feeding insects that communicate with plant-borne vibrational signals-in treatments consisting of mixed-species versus own-species aggregations. Social experience with heterospecifics (in the mixed-species treatment) resulted in enhanced signal-preference species differences. For one of the two species, we tested but found no differences in the plastic response between sympatric and allopatric sites, suggesting the absence of reinforcement in the signals and preferences and their plastic response. Our results support the hypothesis that social plasticity can create or enhance signal-preference differences and that this might occur in the absence of long-term selection against hybridization on plastic responses themselves. Such social plasticity may facilitate rapid bursts of diversification.

Membracis mexicana (Hemiptera: Membracidae) is distributed in four biogeographic provinces of Mexico. Field observations indicate that there are different forms of this species, but the distribution of the phenotype and the genetic variation of this species have not been clarified. The aim of this study was to quantify the phenotypic and genetic variation of M. mexicana and determine whether the configuration of biogeographic provinces impacts the distribution of this variation. To achieve this, we analyzed 307 photographs using 19 landmarks and geometric morphometrics to quantify the phenotypic variation in helmets. We sequenced five molecular markers for 205 individuals to describe the phylogeographic pattern. As a result, we identified three morphological configurations of the helmet of M. mexicana and two genetic lineages. The morphotypes are (1) a large and wide helmet with small dorsal spots, (2) a small and narrow helmet with large dorsal spots, and (3) a small and narrow helmet with small spots. Genetic lineages are distributed in southeast and western Mexico. The western lineage corresponds to two helmet morphotypes (1 and 2) and the southeast lineage to morphotype 3. We found that the larger helmets correspond to the western lineage and are distributed in Trans-Mexican Volcanic Belt and Pacific lowlands provinces, whereas the smallest helmets correspond to the southeast lineage and are present in the Veracruzan and Yucatan Peninsula provinces.

Membracids (family: Membracidae), commonly known as treehoppers, are recognizable by their enlarged and often elaborated pronota. Much of the research investigating the development and evolution of this structure has focused on the fifth instar to adult transition, in which the pronotum undergoes the largest transformation as it takes on adult identity. However, little is known about the earlier nymphal stages, the degree to which the pronotum develops at these timepoints, and how development has changed relative to the ancestral state. Here, we studied the nymphal stages and adults of five morphologically distinct membracid species and of Aetalion reticulatum (family: Aetalionidae), the outgroup which was used as an ancestral state proxy. We found that shape differentiation in the pronotum of membracids can start as early as the second instar stage. Most shape differentiation occurs within the nymphal stages and not in the embryo since the shape of the first-instar pronotum did not differ from the outgroup species in all but one species we investigated. We found the anterior-posterior axis of the pronotum elongated at a faster relative rate in membracid species than in A. reticulatum, which contributed to the development of exaggerated pronotal size. Finally, we found differences in the morphogenesis of shape across species. We suggest this is due to the developmental and evolutionary divergence of differential growth patterning of the dorsal surface of the pronotum, not only across species, but also between stages within the same species. This lability may contribute to the evolvability and diversification of the membracid pronotum.

Treehoppers of the insect family Membracidae have evolved enlarged and elaborate pronotal structures, which is hypothesized to involve co-opted expression of genes that are shared with the wings. Here, we investigate the similarity between the pronotum and wings in relation to growth. Our study reveals that the ontogenetic allometry of the pronotum is similar to that of wings in Membracidae, but not the outgroup. Using transcriptomics, we identify genes related to translation and protein synthesis, which are mutually upregulated. These genes are implicated in the eIF2, eIF4/p70S6K and mTOR pathways, and have known roles in regulating cell growth and proliferation. We find that species-specific differential growth patterning of the pronotum begins as early as the third instar, which suggests that expression of appendage patterning genes occurs long before the metamorphic molt. We propose that a network related to growth and size determination is the more likely mechanism shared with wings. However, regulators upstream of the shared genes in pronotum and wings need to be elucidated to substantiate whether co-option has occurred. Finally, we believe it will be helpful to distinguish the mechanisms leading to pronotal size from those regulating pronotal shape as we make sense of this spectacular evolutionary innovation.

Animals may develop mutualistic associations with other species, whereby prey offer resources or services in exchange for protection from predators. Alternatively, prey may offer resources or services directly to their would-be predators in exchange for their lives. The latter may be the case of hemipterans that engage in mutualistic interactions with ants by offering a honeydew reward. We test the extent to which a honeydew offering versus partner recognition may play a role as proximate mechanisms deterring ants from predating upon their hemipteran partners. We showed that, when presented with a choice between a hemipteran partner and an alternative prey type, mutualist ants were less likely to attack and more likely to remain probing their hemipteran partners. This occurred even in the absence of an immediate sugary reward, suggesting either an evolved or learned partner recognition response. To a similar extent, however, ants were also less likely to attack the alternative prey type when laced with honey as a proxy for a honeydew reward. This was the case even after the honey had been depleted, suggesting an ability of ants to recognize new potential sources of sugars. Either possibility suggests a degree of innate or learned partner recognition.

Some insects possess complex three-dimensional (3D) structures that develop under the old cuticle prior to the last imaginal molt. Adult treehoppers (Insecta: Hemiptera: Auchenorrhyncha: Membracidae) have one such complex 3D structure, known as a helmet, on their dorsal side. The adult helmet likely forms inside the nymphal pronotum during the final instar nymphal stage. Previous morphological studies have reported that the adult helmet is a large, bi-layered, plywood-like structure, whereas the nymphal pronotum is a monolayer, sheath-like structure. The adult helmet is much larger than nymphal helmet. Thus, the emergence of the adult helmet involves two structural transitions: a transition from a monolayer, sheath-like pronotum to a bi-layer, plywood-like helmet, and a transition in size from small to large. However, when, how, and in what order these transitions occur within the nymphal cuticle is largely unknown. To determine how adult helmet development occurs under the nymphal cuticle, in the present study we describe the morphology of the final adult helmet and investigate developmental trajectories of the helmet during the final instar nymphal stage. We used micro-CT, scanning electron microscope and paraffin sections for morphological observations, and used Antianthe expansa as a model species. We found that the structural transition (from monolayer, sheath-like structure to bi-layer, roof-like structure) occurs through the formation of a \"miniature\" of the adult helmet during the middle stage of development and that subsequently, extensive folding and furrows form, which account for the increase in size. We suggest that the making of a \"miniature\" is the key developmental step for the formation of various 3D structures of treehopper helmets.

The importance and prevalence of phylogenetic tracking between hosts and dependent organisms caused by co-evolution and shifting between closely related host species have been debated for decades. Most studies of phylogenetic tracking among phytophagous insects and their host plants have been limited to insects feeding on a narrow range of host species. However, narrow host ranges can confound phylogenetic tracking (phylogenetic tracking hypothesis) with host shifting between hosts of intermediate relationship (intermediate hypothesis). Here, we investigated the evolutionary history of the Enchenopa binotata complex of treehoppers. Each species in this complex has high host fidelity, but the entire complex uses hosts across eight plant orders. The phylogenies of E. binotata were reconstructed to evaluate whether (1) tracking host phylogeny; or (2) shifting between intermediately related host plants better explains the evolutionary history of E. binotata. Our results suggest that E. binotata primarily shifted between both distant and intermediate host plants regardless of host phylogeny and less frequently tracked the phylogeny of their hosts. These findings indicate that phytophagous insects with high host fidelity, such as E. binotata, are capable of adaptation not only to closely related host plants but also to novel hosts, likely with diverse phenology and defense mechanisms.