Rajee Ganesan 1 , Andrew Wang 2 , BaDoi N. Phan 2,3 , Michael J. Leone 2,3 , Heather Sestili Harper 2 , Andreas Pfenning 2,4

Vocal production learning is the ability to imitate sounds through social exposure. The rare trait is believed to have independently evolved three times in birds and five times in mammals, a prime example of convergent evolution. All studied vocal learning species have evolved a specialized forebrain sensorimotor learning circuit that is either absent or rudimentary in their closer, vocal non-learning relatives. Previous work within the lab has found that the “regulatory code” linking genome sequence to cell-type-specific function is highly conserved across mammals. In this study, we leverage this principle to identify candidate enhancers from publicly available Zoonomia datasets, and trained machine learning models predicting open and closed chromatin across all 240 mammalian motor cortex regions. We screened for differences in regulation between vocal learning and non-learning species by applying the Tissue-Aware Conservation Inference Toolkit, a machine learning approach to study how enhancer activity conservation relates to phenotype evolution. These machine learning models can learn sequence patterns of enhancers that robustly predict conserved activity across evolutionary distances, allowing us to test whether enhancer conservation patterns are associated with the evolution of vocal learning. Our results revealed lineage-specific gains and losses of regulatory elements, and we show that L6 corticothalamic neurons and oligodendrocytes had the strongest enrichment with the vocal learning phenotype, suggesting key roles in vocalization-related traits through motor learning and synaptic plasticity. Future studies will include single cell integration across species to identify orthologous cell populations and to better understand molecular mechanisms associated with the evolution of vocal learning.

1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA 2 Computational Biology Department, Carnegie Mellon University, Pittsburgh, PA, USA 3 Medical Scientist Training Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 4 Neuroscience Institute, Carnegie Mellon University, Pittsburgh, PA, USA