Speciation in North American Junonia from a genomic perspective

Delineating species boundaries in phylogenetic groups undergoing recent radiation is a daunting challenge akin to discretizing continuity. Here, we propose a general approach exemplified by American butterflies from the genus Junonia notorious for the variety of similar phenotypes, ease of hybridization, and the lack of consensus about their classification. Due to this formidable complexity, anything but a genome-wide scan seems futile. Thus, we obtain whole genome shotgun sequences of nearly 200 specimens. We reason that discreteness emerges from continuity by means of a small number of key players, and search for the proteins that diverged markedly between sympatric populations of different species, while keeping low polymorphism within these species. Being a quarter of percent of the total number, these three dozen 'speciation' proteins indeed partition pairs of Junonia populations into two clusters with a prominent break in between, while all proteins taken together fail to reveal this discontinuity. Population with larger divergence from each other, comparable to that between two sympatric species, correspond to different species. The other cluster is characterized by smaller divergence similar to that between allopatric populations of the same species and is comprised of conspecific pairs. Using this method, we conclude that J. genoveva, J. litoralis, J. evarete, and J. divaricata are restricted to South America. We find that 6 species of Junonia are present in the United States, one of which is new: Junonia stemosa Grishin, sp. n. (1), restricted to south Texas and phenotypically closest to J. nigrosuffusa (2) in its dark appearance. In the pale nudum of antennal club, these two species resemble J. zonalis (3) from Florida and the Caribbean Islands. The pair of sister species J. grisea (4) and J. coenia (5) represent the classic west/east USA split. The mangrove feeder (as caterpillar), dark nudum J. neildi (6) enters south Texas as a new subspecies Junonia neildi varia Grishin ssp. n. characterized by more extensive hybridization with and introgression from J. coenia, and, as a consequence, more variable wing patterns compared to the nominal J. n. neildi in Florida. Furthermore, a new mangrove-feeding species from the Pacific Coast of Mexico is described as Junonia pacoma Grishin sp. n. Finally, genomic analysis suggests that J. nigrosuffusa is a hybrid species formed by the ancestors of J. grisea and J. stemosa sp. n.