The butterfly subfamily Pseudopontiinae is not monobasic: marked genetic diversity and morphology reveal three new species of Pseudopontia (Lepidoptera: Pieridae)



The Afrotropical butterfly subfamily Pseudopontiinae (Pieridae) was traditionally thought to comprise one species, with two subspecies (Pseudopontia paradoxa paradoxa Felder & Felder and Pseudopontia paradoxa australis Dixey) differing in a single detail of a hindwing vein. The two subspecies also differ in their known geographic distributions (mainly north of versus south of the equator). Unlike most butterflies, Pseudopontia is white with no visible wing or body markings. We now report that males of P. paradoxa australis have an area of ultraviolet-reflecting scales along the anal vein of the forewing, whereas males of P. paradoxa paradoxa and all females do not. A total of 21 individuals of the northern subspecies, which were collected in three localities south of the equator, were found in the collection of the Royal Museum for Central Africa, indicating sympatry of the two traditional subspecies in the Congo River basin. To determine if additional cryptic species might be present, we sequenced three nuclear genes (CAD, DDC and wingless) as well as cytochrome oxidase I (COI), examined amplified fragment-length polymorphisms, and re-examined wing and genitalic morphology, using recently collected specimens from several regions of Africa. Phylogenetic analyses of the COI sequences and amplified fragment-length polymorphism data concur, and indicate the existence of at least five monophyletic, non-interbreeding populations, with a particularly deep divergence between three populations of P. paradoxa paradoxa and two of P. paradoxa australis. Despite the slow rate of evolution of the nuclear genes studied, individual gene trees and a concatenated three-gene tree demonstrate, with high bootstrap support, clear divergence among the five populations of Pseudopontia. In addition, consistent variations in details of wing vein stalks were found among four of the genetically distinct populations, which supports the hypothesis of multiple species. Division of Pseudopontia into five phylogenetic species is proposed, including the elevation of ssp. australis to species rank and the description of Pseudopontia mabira, Pseudopontia gola and Pseudopontia zambezi