Supplementary files to the "Cretaceous amber inclusions illuminate the evolutionary origin of tardigrades" paper which include All raw files from the phylogenetic analysis (i.e., t, p, and tree files fromMrBayes, output tree from ASTRAL and IQTree, mcmc files from MCMCTree, and tre and log files from BEAST), RScripts used, and assembled Actinarctus doryphorus genome.

Tardigrades are a diverse phylum of microscopic invertebrates widely known for their extreme survival capabilities. Molecular clocks suggest that tardigrades diverged from other panarthropods before the Cambrian, but their fossil record is extremely sparse. Only the fossil tardigrades Milnesium swolenskyi (Late Cretaceous) and Paradoryphoribius chronocaribbeus (Miocene) have resolved taxonomic positions, restricting the availability of calibration points for estimating for the origin of this phylum. Here, we revise two crown-group tardigrades from Canadian Cretaceous-aged amber using confocal fluorescence microscopy, revealing critical morphological characters that resolve their taxonomic positions. Formal morphological redescription of Beorn leggi reveals that it features Hypsibius-type claws. We also describe Aerobius dactylus gen. et sp. nov. based on its unique combination of claw characters. Phylogenetic analyses indicate that Beo. leggi and Aer. dactylus belong to the eutardigrade superfamily Hypsibioidea, adding a critical fossil calibration point to investigate tardigrade origins. Our molecular clock estimates suggest an early Paleozoic diversification of crown-group Tardigrada and highlight the importance of Beo. leggi as a calibration point that directly impacts estimates of shallow nodes. Our results suggest that independent terrestrialization of eutardigrades and heterotardigrades occurred around the end-Carboniferous and Lower Jurassic, respectively. These estimates also provide minimum ages for convergent acquisition of cryptobiosis.