<b>水蛭iDNA作为监测中国半城市河流脊椎动物多样性季节变化的工具</b><b>运行标题：</b><b>水蛭 iDNA 用于</b><b>s</b><b>伊索纳尔</b><b>m</b><b>的 onitoring</b><b>v</b><b>ertebrates</b>

<b>目的</b><b>：</b>评估水蛭来源的无脊椎动物 DNA （iDNA） 在监测脊椎动物多样性季节性变化方面的有效性，并确定反映淡水生态系统时间动态的指示物种。 <b>位置</b><b>：</b>中国天津潮白河的半城市区段。 <b>方法</b><b>：</b>多个河流地点采集水生食血水蛭（Hirudo tianjinensis）四季采集。从水蛭血粉中提取DNA，用脊椎动物特异性引物扩增，并使用Illumina NovaSeq进行测序。使用 BLAST 针对 NCBI 数据库分配作分类单元 （OTU）。使用希尔数、ANOSIM 和线性判别分析效应大小 （LEfSe） 分析了阿尔法多样性、物种组成和季节性指标类群。<b>结果</b><b>：</b>共鉴定出65种脊椎动物，包括31种鱼类、14种两栖动物和13种哺乳动物，脊索动物占所有类群的~88%。α多样性和物种组成存在显著的季节差异，春季丰富度最高，夏季最低。在110个表现出季节变化的类群中，有57个物种表现出较强的季节性关联。共现网络分析突出了 Macropodus concolor、Carnegiella marthae、Rectoris posehensis、Hypoptopoma inexspectatum 和 Sclerophrys brauni 作为潜在的关键指标物种。<b>主要结论</b><b>：</b>水蛭衍生的 iDNA 是一种灵敏、可扩展且经济高效的工具，用于检测脊椎动物多样性的季节性变化，特别是对于难以捉摸或短暂的物种。它与高通量测序的集成为淡水生态系统提供了强大的生物多样性监测，并为保护规划提供了宝贵的见解。S1-S4：补充图表S1-S4：补充表

Purpose: To evaluate the effectiveness of leech-derived invertebrate DNA (iDNA) in monitoring seasonal changes in vertebrate diversity, and to identify indicator species that reflect the temporal dynamics of freshwater ecosystems.
Location: The semi-urban section of the Chaobai River in Tianjin, China.
Method: Aquatic hematophagous leeches (Hirudo tianjinensis) were seasonally collected at multiple river sites. DNA was extracted from the blood meals of the leeches, amplified with vertebrate-specific primers, and sequenced using Illumina NovaSeq. Operational taxonomic units (OTUs) were assigned via 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 species, 14 amphibian species and 13 mammal species, with chordates accounting for approximately 88% of all detected taxa. Significant seasonal differences were observed in both alpha diversity and species composition, with the highest species richness recorded in spring and the lowest in summer. Among the 110 taxa exhibiting seasonal shifts, 57 species displayed strong seasonal associations. Co-occurrence network analysis highlighted Macropodus concolor, Carnegiella marthae, Rectoris posehensis, Hypoptopoma inexspectatum and Sclerophrys brauni as potential key indicator species.
Main Conclusions: Leech-derived iDNA is a sensitive, scalable and cost-effective tool for detecting seasonal changes in vertebrate diversity, particularly for elusive or transient species. Its integration with high-throughput sequencing provides a powerful biodiversity monitoring framework for freshwater ecosystems and offers valuable insights for conservation planning.
S1–S4: Supplementary Figures S1–S4; Supplementary Tables