Evolution of defensive strategies in the treehopper tribe Darnini (Hemiptera: Membracidae) revisited using anchored hybrid enrichment data

The Neotropical tribe Darnini (Hemiptera: Membracidae) includes approximately 102 species classified in 18 genera. Darnini displays marked morphological heterogeneity among its genera, and the tribe has been poorly studied compared with other treehopper groups. The tribe has been considered monophyletic due to the presence of cucullate setae on the ventral sides of the femora. A previous morphology-based analysis placed the genera of Darnini into three groups that differ in pronotal shape, suggesting that the common ancestor of each group acquired a different defensive strategy, mimicking either bird droppings, thorns, or raindrops. To test this hypothesis, we compiled the most taxon- and character-rich dataset for Darnini and related groups to date, using anchored hybrid enrichment to obtain data for 492 genetic loci comprising >133,855 nucleotide positions for a total of 51 taxa (31 Darnini species, 11 representatives of other tribes of Darninae and 9 taxa representing other subfamilies). Phylogenetic analysis of the concatenated nucleotide sequence data using Maximum Likelihood and coalescent gene tree (ASTRAL) analyses yielded similar topologies, with most branches having maximum support. The results are consistent with the hypothesis of a single acquisition of each of the three defensive pronotal syndromes early in the evolution of Darnini, but also indicate that two genera of the tribe Hemikypthini (Hemikyptha and Atypa) are derived within the ‘thorny’ and ‘raindrop’ groups of Darnini, consistent with their pronotal shapes. This indicates that Hemikypthini is polyphyletic and that the characters of the leg chaetotaxy used to diagnose both tribes are homoplasious. Therefore, we treat Darnini and Hemikypthini as synonyms.