supplementary files for manuscript "Intraspecific variation in the <i>hirudin</i> gene family of the Asian buffalo leech (<i>Hirudinaria manillensis</i>)"

Hirudin is a natural thrombin inhibitor unique to leeches and has important research and development value. The Asian buffalo leech (Hirudinaria manillensis), an important antithrombotic traditional Chinese medicine, contains five hirudin genes, and the study of the variation patterns of these genes will help to understand the pharmacological mechanism of the Asian buffalo leech. In this study, a total of 70 samples were collected from seven populations of the Asian buffalo leech, and the sequence variation, selection pressure and gene expression characteristics of hirudin genes at the intraspecific level were systematically analyzed based on genome resequencing and transcriptome sequencing data. The results showed that all five hirudin genes were rich in intraspecific variation, with hirudin1 and hirudin2 having high rates of variation, followed by hirudin3 and hirudin5, and hirudin4 being relatively conserved. hirudin5 had two transcript types, S-type (short) and L-type (long), with four and six exons, respectively. In terms of the median selection pressure index (dN/dS), hirudin2 was strongly positively selected (dN/dS = 3.288), hirudin5 was weakly positively selected (dN/dS = 1.110), and the remaining three genes were overall subjected to purified selection (dN/dS &lt; 1). The overall expression differences among the five hirudin genes were highly significant (P &lt; 0.001), with hirudin2 having the highest expression, followed by hirudin1, and the remaining three genes being significantly lower. hirudin2 and hirudin5 showed highly significant differences in expression among the seven populations (P &lt; 0.01), while the remaining three genes did not show any differences between populations. The results of this study indicate that the five hirudins of the Asian buffalo leech are dramatically different in terms of sequence variation, selective pressure, and gene expression characteristics, providing rich genetic resources for the study of hirudin evolutionary patterns and drug development.水蛭素（hirudin）是水蛭特有的天然凝血酶抑制剂，具有重要的研发价值。亚洲牛蛭（Hirudinaria manillensis）作为重要的抗血栓传统中药，体内含有5个水蛭素基因，对这些基因的变异模式开展研究有助于解析亚洲牛蛭的药理作用机制。本研究共采集该物种7个种群的70份样本，基于基因组重测序与转录组测序数据，系统分析了种内水平下水蛭素基因的序列变异、选择压力及基因表达特征。研究结果显示，5个水蛭素基因均存在丰富的种内变异：hirudin1与hirudin2的变异速率较高，其次为hirudin3与hirudin5，而hirudin4相对保守。hirudin5存在两种转录本类型：S型（短型）与L型（长型），分别包含4个和6个外显子。在选择压力中位数（dN/dS）方面，hirudin2受到强烈正向选择（dN/dS = 3.288），hirudin5受到微弱正向选择（dN/dS = 1.110），其余3个基因整体处于纯化选择状态（dN/dS < 1）。5个水蛭素基因的整体表达差异极显著（P < 0.001），其中hirudin2表达量最高，其次为hirudin1，其余3个基因的表达量显著更低。7个种群间，hirudin2与hirudin5的表达差异极显著（P < 0.01），而其余3个基因在种群间无显著差异。本研究结果表明，亚洲牛蛭的5个水蛭素基因在序列变异、选择压力与基因表达特征上均存在显著分化，为水蛭素进化模式研究与药物开发提供了丰富的遗传资源。