Phenotypic variation among species is a product of evolutionary changes to developmental programs1,2. However, how these changes generate novel morphological traits remains largely unclear. Here we studied the genomic and developmental basis of the mammalian gliding membrane, or patagium-an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species. Through comparative genomic analysis of 15 marsupial genomes, both from gliding and non-gliding species, we find that the Emx2 locus experienced lineage-specific patterns of accelerated cis-regulatory evolution in gliding species. By combining epigenomics, transcriptomics and in-pouch marsupial transgenics, we show that Emx2 is a critical upstream regulator of patagium development. Moreover, we identify different cis-regulatory elements that may be responsible for driving increased Emx2 expression levels in gliding species. Lastly, using mouse functional experiments, we find evidence that Emx2 expression patterns in gliders may have been modified from a pre-existing program found in all mammals. Together, our results suggest that patagia repeatedly originated through a process of convergent genomic evolution, whereby regulation of Emx2 was altered by distinct cis-regulatory elements in independently evolved species. Thus, different regulatory elements targeting the same key developmental gene may constitute an effective strategy by which natural selection has harnessed regulatory evolution in marsupial genomes to generate phenotypic novelty.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.