Morphology and Development of Reproductive Organs in the Acoel Hofstenia atroviridis

Reconstructing ancestral reproductive systems is crucial for understanding the evolution of bilaterian body plans. Yet, despite extensive research on some organ systems of the last common bilaterian ancestor, the evolution and development of reproductive systems remain poorly understood. To address this gap, we investigated postembryonic sexual development in the xenacoelomorph Hofstenia atroviridis, a basal acoel species belonging to the infraorder Prosopharyngida. We reared H. atroviridis from eggs to adults and described the ontogeny of its reproductive organs using histology and immunohistochemistry with muscle and neural markers, along with observations of mating behaviors in each size class. Hofstenia atroviridis is a protandrous simultaneous hermaphrodite whose sexual maturity correlates with body size rather than age, as observed in Hofstenia miamia. The male copulatory system comprises a seminal vesicle, granular vesicle, penis, and a single copulatory stylet, surrounded by regionally specialized musculature and innervation. In contrast, the female system consists of paired, asaccate ovaries with large, follicle cell-bound oocytes, lacking a discrete gonopore or copulatory organ. Fertilization occurs internally through traumatic insemination, and eggs are likely released through the mouth. Our findings reveal that the male reproductive system of H. atroviridis exhibits unexpected complexity and structural differentiation despite the organism's overall simplicity. We propose that sexual selection acting on a hermaphroditic reproductive system may have driven the evolution of a multifunctional copulatory organ in the Urbilateria. This study establishes H. atroviridis as a promising and comparative model for exploring the evolutionary origins of animal reproductive systems.