A Paleozoic stem group to mite harvestmen revealed through integration of phylogenetics and development

Successfully placing fossils in phylogenies is integral to understanding the tree of life. Crown-group Paleozoic members of the arachnid order Opiliones are indicative of ancient origins and one of the earliest arthropod terrestrialization events [1, 2]. Opiliones epitomize morphological stasis, and all known fossils have been placed within the four extant suborders [3-5]. Here we report a Carboniferous harvestman species, Hastocularis argusgen. nov., sp. nov., reconstructed with microtomography (microCT). Phylogenetic analysis recovers this species, and the Devonian Eophalangium sheari, as members of an extinct harvestman clade. We establish the suborder Tetrophthalmi subordo nov., which bore four eyes, to accommodate H. argus and E. sheari, the latter previously considered to be a phalangid [6-9]. Furthermore, embryonic gene expression in the extant species Phalangium opilio demonstrates vestiges of lateral eye tubercles. These lateral eyes are lost in all crown-group Phalangida, but are observed in both our fossil and outgroup chelicerate orders. These data independently corroborate the diagnosis of two eye pairs in the fossil and demonstrate retention of eyes of separate evolutionary origins in modern harvestmen [10-12]. The discovery of Tetrophthalmi alters molecular divergence time estimates, supporting Carboniferous rather than Devonian diversification for extant suborders and directly impacting inferences of terrestrialization history and biogeography. Multidisciplinary approaches integrating fossil and neontological data increase confidence in phylogenies and elucidate evolutionary history.