Abstract
Identifying early life stages of Neotropical fishes using morphological traits remains challenging. DNA metabarcoding can overcome this limitation, enabling precise taxonomic resolution for ichthyoplankton. This study characterized the taxonomic composition and spatial and ontogenic patterns of ichthyoplankton across plateau and transitional floodplain regions of the Paraguay River Basin, aiming to identify river reaches associated with reproductive activity and early-life transport, especially for migratory fish. Ichthyoplankton was sampled monthly over two reproductive periods at 38 strategic sites across five sub-basins. DNA was extracted from eggs and larvae and sequenced on the Illumina MiSeq platform targeting the COI gene. We identified 53 species from 210,319 valid sequence reads. Although mean species richness did not vary significantly among sub-basins, taxonomic composition exhibited significant spatial variation. The Piquiri and Cuiabá sub-basins supported distinct assemblages and presented significant indicator species, such as Sorubim lima and Hoplias malabaricus, respectively. The detection of ontogenetic phases was complementary, with 17 species identified exclusively as eggs and 18 as larvae, highlighting the importance of analyzing both stages. Their spatial distributions indicated that the sampled sub-basins are associated with both reproduction and ichthyoplankton transport. Migratory species demonstrated broad larval dispersal but localized spawning events, reinforcing their dependence on passive drift and longitudinal connectivity. The spatial segregation between embryonic and larval periods indicates that these sub-basins act as an integrated environmental mosaic. Maintaining the free-flowing condition and longitudinal connectivity of these tributaries is therefore essential for conserving fish reproductive processes and mitigating potential future impacts on the regional ichthyofauna.