Multiple loci control eyespot number variation on the hindwings of Bicyclus anynana butterflies

The underlying genetic changes that regulate the appearance and disappearance of repeated traits, or serial homologs, remain poorly understood. One hypothesis is that variation in genomic regions flanking master regulatory genes, also known as input-output genes, controls variation in trait number, making the locus of evolution almost predictable. Other hypotheses implicate genetic variation in up-stream or downstream loci of master control genes. Here, we use the butterfly Bicyclus anynana , a species which exhibits natural variation in eyespot number on the dorsal hindwing, to test these two hypotheses. We first estimated the heritability of dorsal hindwing eyespot number by breeding multiple butterfly families differing in eyespot number, and regressing eyespot number of offspring on mid-parent values. We then estimated the number and identity of independent genetic loci contributing to eyespot number variation by performing a genome-wide association study with restriction site-associated DNA Sequencing (RAD-seq) from multiple individuals varying in number of eyespots sampled across a freely breeding lab population. We found that dorsal hindwing eyespot number has a moderately high heritability of approximately 0.50. In addition, multiple loci near previously identified genes involved in eyespot development display high association with dorsal hindwing eyespot number, suggesting that homolog number variation is likely determined by regulatory changes at multiple loci that build the trait and not by variation at single master regulators or input-output genes.