Molecular and developmental contributions to divergent pigment patterns in marine and freshwater sticklebacks.

Pigment pattern variation across species or populations offers a tractable framework in which to investigate the evolution of development. Juvenile threespine sticklebacks (Gasterosteus aculeatus) from marine and freshwater environments exhibit divergent pigment patterns that are associated with ecological differences. Juvenile marine sticklebacks have a silvery appearance, whereas sticklebacks from freshwater environments exhibit a pattern of vertical bars. We investigated both the developmental and molecular basis of this population-level variation in pigment pattern. Time course imaging during the transition from larval to juvenile stages revealed differences between marine and freshwater fish in spatial patterns of chromatophore differentiation as well as in pigment amount and dispersal. In freshwater fish, melanophores appear primarily within dark bars whereas iridophores appear within light bars. By contrast, in marine fish, these chromatophores are interspersed across the flank. In addition to spatially segregated chromatophore differentiation, pigment amount and dispersal within melanophores varies spatially across the flank of freshwater, but not marine fish. To gain insight into the molecular pathways that underlie the differences in pigment pattern development, we evaluated differential gene expression in the flanks of developing fish using high-throughput cDNA sequencing (RNA-seq) and quantitative PCR. We identified several genes that were differentially expressed across dark and light bars of freshwater fish, and between freshwater and marine fish. Together, these experiments begin to shed light on the process of pigment pattern evolution in sticklebacks.