Understanding the origin and early diversification of vertebrates has always been a challenge because of the ambiguous and conflicting interpretations of the soft-bodied, pre-biomineralisation fossil record. Here, we apply synchrotron radiation techniques to Jamoytius and Lasanius, two soft-bodied Silurian vertebrates, key taxa for discerning vertebrate bone evolution due to highly localised, but debated, biomineralisation. We map soft-tissue structures and quantify details of biochemical residue impossible to resolve with traditional methods. We present the first unequivocal evidence for biomineralised apatitic scales in Jamoytius by combining Synchrotron Rapid Scanning X-ray Fluorescence (SRS-XRF) and Fourier Transform Infra-Red (FTIR) spectroscopy (elevated Ca 37% and P 21%). This approach also recovers robust evidence for apatitic biomineralisation in Lasanius. Chemical mapping of the optical anatomy of Jamoytius recovers a close correlation with Zn and Cu distribution, providing evidence for a retinal pigmented epithelium and complex eyes. In both taxa, chemical maps reveal original anatomical details not apparent in visible light, including potential evidence of other sensory anatomy in Jamoytius.

Our work resolves long-standing fundamental anatomical debates, indicating stem-group origins for bone and complex eyes in vertebrates. We highlight the potential of using a powerful combination of analytical techniques to unlock otherwise inaccessible data in problematic fossils.