Leech iDNA as a tool to monitor seasonal shifts in vertebrate diversity in a semi-urban river of China

Aim: To assess the effectiveness of leech-derived invertebrate DNA (iDNA) for monitoring seasonal variation in vertebrate diversity and to identify indicator species that reflect temporal dynamics in freshwater ecosystems. Location: A semi-urban section of the Chaobai River, Tianjin, China. Methods: Aquatic blood-feeding leeches (Hirudo tianjinensis) were collected across four seasons from multiple river sites. DNA from leech blood meals was extracted, amplified with vertebrate-specific primers, and sequenced using Illumina NovaSeq. Operational taxonomic units (OTUs) were assigned using BLAST against the NCBI database. Alpha diversity, species composition, and seasonal indicator taxa were analyzed using Hill numbers, ANOSIM, and Linear Discriminant Analysis Effect Size (LEfSe). Results: A total of 65 vertebrate species were identified, including 31 fish, 14 amphibians, and 13 mammals, with Chordata accounting for ~88% of all taxa. Significant seasonal differences in alpha diversity and species composition were observed, with highest richness in spring and lowest in summer. Among 110 taxa showing seasonal variation, 57 species exhibited strong seasonal associations. Co-occurrence network analysis highlighted Macropodus concolor, Carnegiella marthae, Rectoris posehensis, Hypoptopoma inexspectatum, and Sclerophrys brauni as potential keystone indicator species. Main conclusions: Leech-derived iDNA is a sensitive, scalable, and cost-effective tool for detecting seasonal shifts in vertebrate diversity, particularly for elusive or transient species. Its integration with high-throughput sequencing provides robust biodiversity monitoring for freshwater ecosystems and offers valuable insights for conservation planning.Table S1. OTU abundance table across all samples (initial identification).Table S2. Filtered OTU abundance table after quality control and taxonomic verification.Table S3. Complete taxonomic annotation for all validated OTUs.Table S4. Metadata and grouping information for all samples used in the study.Figure S1. Sampling locations along the Chaobai River. Map of Tianjin showing major river systems. Red arrows indicate leech sampling sites along upstream and downstream sections of the Chaobai River Wetland Park.Figure S2. Hill number-based rarefaction and extrapolation curves across seasons. Top panel: species richness (q = 0); Middle panel: sample coverage; Bottom panel: species richness vs. coverage. Solid lines indicate rarefied values; dashed lines represent extrapolated estimates.Figure S3. Species diversity accumulation curves for major vertebrate groups. Rarefaction and extrapolation curves for fish, amphibians, and mammals across the four seasons, based on Hill numbers q = 0 (richness), q = 1 (Shannon), and q = 2 (Simpson).Figure S4. Length distribution of sequencing reads. Stacked histogram showing the distribution of read lengths across all samples after quality filtering.