RT Journal Article SR Electronic T1 Multiple loci control eyespot number variation on the hindwings of Bicyclus anynana butterflies JF bioRxiv FD Cold Spring Harbor Laboratory SP 653451 DO 10.1101/653451 A1 Angel G. Rivera-Colón A1 Erica L. Westerman A1 Steven M. Van Belleghem A1 Antónia Monteiro A1 Riccardo Papa YR 2019 UL http://biorxiv.org/content/early/2019/05/29/653451.abstract AB 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.Data accessibility The Bicyclus anynana PstI RAD-tag sequencing data is available via the Genbank BioProject PRJNA509697. Genotype VCF files will be made available through Figshare upon acceptance.