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The regressive evolution of eyes has long intrigued biologists yet the genetic underpinnings remain opaque. A system of discrete aquifers in arid Australia provides a powerful comparative means to explore trait regression at the genomic level. Multiple surface ancestors from two tribes of diving beetles (Dytiscidae) repeatedly invaded these calcrete aquifers and convergently evolved eye-less phenotypes. We use this system to assess transcription of opsin photoreceptor genes among the transcriptomes of two surface and three subterranean dytiscid species and test whether these genes have evolved under neutral predictions. Transcripts for  UV ,  long-wavelength  and  ciliary-type  opsins were identified from the surface beetle transcriptomes. Two subterranean beetles showed parallel loss of all opsin transcription, as expected under ‘neutral’ regressive evolution. The third species  Limbodessus palmulaoides  retained transcription of a  long-wavelength opsin  ( lwop ) orthologue, albeit in an aphotic environment. Tests of selection on  lwop  indicated no significant differences between transcripts derived from surface and subterranean habitats, with strong evidence for purifying selection acting on  L. palmulaoides lwop . Retention of sequence integrity and the lack of evidence for neutral evolution raise the question of whether we have identified a novel pleiotropic role for  lwop , or an incipient phase of pseudogene development.

Opsin transcripts of predatory diving beetles: a comparison of surface and subterranean photic niches