From egg to adult: combining morphology, bulk metabarcoding and eDNA for comprehensive monthly monitoring of ichthyoplankton in a temperate estuary

Monitoring fish species through ichthyoplankton (fish eggs and larvae) surveys provides important information for fish stock assessment and management, with molecular tools complementing morphology by enabling more accurate identifications. To test the effectiveness of DNA metabarcoding to identify fish species and to capture seasonal variations in local ichthyofauna, monthly ichthyoplankton and 2 L water samples were collected over 13 months in the lower section of the Guadiana River Estuary in southeast Portugal. Both sample types underwent high-throughput sequencing for three genetic markers (COI, 12S, 16S), with morphological identification also performed for ichthyoplankton. Bulk and water samples identified a total of 118 fish taxa (26 orders and 45 families) throughout the year. DNA metabarcoding demonstrated higher taxonomic resolution and diversity detection, with 97 species recovered, while morphology identified only 23 species. Ichthyoplankton metabarcoding also detected 32% more fish than water eDNA, suggesting that DNA from small eggs and larvae is more difficult to capture in environmental samples. The integration of multiple molecular markers was crucial to maximize diversity detection in both DNA-based methods. In addition, DNA metabarcoding was able to identify ichthyofaunal spawning periods and captured significant seasonal variations in fish community. Nearly 68% of the taxa historically recorded in the ichthyoplankton were detected, and first records of developmental stages in the estuary were obtained for 67 species. These findings demonstrate the capability of (e)DNA metabarcoding to uncover seasonal variations in the regional fish community, provided new insights on the ichthyofauna of the Guadiana Estuary, and revealed the need for more in-depth studies to improve the efficiency of multiple sampling methods for fish species identification.