CO1 and ITS alignments of bladder grasshopper species

Polymorphism in one sex often occurs amongst arthropods, and is often linked with alternate mating strategies. Bladder grasshoppers show distinct polymorphism resulting in two different male morphs utilizing two different mating strategies. Primary males are larger in size and make use of acoustic communication to attract mates, while secondary (alternate) males are significantly smaller in size, flightless, and do not call, making use of a “sneaker” or satellite strategy. Three species of bladder grasshoppers have been described (Parabullacris vansoni, Paraphysemacris spinosus, and Pneumoracris browni) that only have an alternate male morph and no primary male, whereas all other species in the family are described as having only a primary male. However, it is now known that both types of males occur in at least three species (Bullacris discolor, B. membracioides, and B. obliqua). Thus, the validity of the taxonomic descriptions for species with no identified primary male has come into question. We therefore conducted a phylogenetic analysis to examine the genetic relationships between primary and secondary males for all species where secondary males are documented or suspected to occur. This was done using both mitochondrial (COI) and nuclear (ITS) DNA markers. We confirm that dimorphic males are indeed conspecific for the species observed to have both primary and alternate males. We also demonstrate that Parabullacris vansoni, Pneumoracris browni and Paraphysemacris spinosus are invalid taxa as they are conspecific alternate male morphs of B. unicolor, Peringueyacris namaqua and Physemacris variolosa, respectively.

Methods
Specimens used for this study were from previous collections that had been preserved in 90–100% ethanol. Collection dates span many years. 

DNA extraction

PCR and gel electrophoresis

Sequences alignments and phylogenetic analysis