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Unionid bivalves are a common and widely distributed group of freshwater mussels, presumably known since the Triassic. One peculiar trait is their life cycle, with brooding of eggs in portions of the gills and release of usually fish-parasitizing larvae. The morphology of these larvae (glochidia or lasidia) is an important character for unionid classification and hence for phylogenetic reconstructions. Dispersal capacity and success of unionids through geological times is assumed to be largely affected by the evolution of temporary parasitism. Nevertheless, pre-Quaternary fossil proof of unionid larvae has been lacking until now, which renders some palaeobiological, phylogenetic and palaeobiogeographical scenarios arguable. Here we document late middle Miocene glochidia from the Pebas Formation (Porvenir, north-eastern Peru). Based on morphological comparisons with Recent glochidia and the accompanying fossil record, these larvae are related to the Hyriidae genus Diplodon Spix, 1827. Due to the lack of 'hooks' (inward-directed projections on the ventral edge of the glochidium) the larvae probably did not parasitize (fish) hosts, which restricted their dispersal ability. In this case, 'unhooked' is presumably a derived feature related to direct larval development. The strata containing these glochidia have been interpreted as marginal marine deposits with high-frequency salinity fluctuations. The associated mollusc and ostracod fauna is strongly dominated by euryhaline, endemic species. Only subordinately 'freshwater' and 'marginal marine' taxa co-occur, as well as euryhaline foraminifers. Stable isotope analyses (δ18O, δ13C) performed on ostracods and foraminifers furnished very light ratios, indicative of freshwater conditions. It remains unresolved if some marine-derived ostracods and also foraminifers were able to adapt to freshwater settings or if marine incursions affected western Amazonia in late middle Miocene times. Possibly the combination of, for example, fluctuating oxygenation and/or salinity and the complex reproductive strategy diminished the competitiveness of unionids within the Pebas wetlands. [ABSTRACT FROM AUTHOR]