Supplementary Material for: Delineating the W sex chromosome in the clam shrimp, Eulimnadia texana

Introduction: Sex chromosomes have evolved independently across various lineages, often showing convergent degradation of the sex-limited chromosome. While extensively studied in model organisms with ancient sex chromosomal systems, the evolution of early-stage sex chromosomes remains poorly understood. Eulimnadia texana, a freshwater crustacean with a unique androdioecious breeding system (ZZ, ZW, and viable WW genotypes), provides a rare opportunity to study early sex chromosome evolution. This study examines E. texana's W chromosome for evidence of a small localized non-recombining region, characterized by a transposable element (TE) "hotspot", low gene density, and low GC content. Methods: Sex-linked markers were mapped onto the W chromosome (Scaffold 1). TEs in the WW genome were identified using RepeatModeler and RepeatMasker. Statistical analyses compared TE distribution between the genome and Scaffold 1, which was then divided into 20 equal-sized "bins" for finer-scale statistical analyses. Gene density and GC content were analyzed across these bins. Results: While no significant TE accumulation was found across the entire W chromosome compared to the remaining genome, a specific region (6.6-8.8 Mb, fourth bin) showed significantly higher TE accumulation. This region also exhibited low gene density and low GC content, indicative of reduced recombination. Conclusion: Our findings suggest that E. texana's W chromosome contains a smaller region of crossover suppression, supporting the hypothesis that it is in a proto-sex chromosome in early evolutionary development. This study provides valuable insights into early sex chromosome evolution and establishes E. texana as an ideal model for further investigation of evolutionary processes driving proto-sex chromosome differentiation.

引言：性染色体在多个演化支中独立起源，限性染色体常呈现趋同退化的演化特征。针对拥有古老性染色体系统的模式生物，相关研究已较为充分，但早期阶段性染色体的演化机制仍有待深入阐明。淡水甲壳类物种枝额虫（Eulimnadia texana）具有独特的雄全同株繁殖系统，其基因型涵盖ZZ、ZW与可存活的WW，为研究早期性染色体演化提供了极为珍贵的模型体系。本研究针对该物种的W染色体展开分析，旨在探寻其存在小型局部非重组区域的证据，这类区域通常具备转座因子（transposable element, TE）“热点”、低基因密度以及低GC含量的特征。
方法：将性连锁标记定位至W染色体（Scaffold 1）。利用RepeatModeler与RepeatMasker对WW基因组中的转座因子进行鉴定与注释。通过统计分析对比全基因组与Scaffold 1的转座因子分布特征，并将Scaffold 1划分为20个等尺寸的“区段”以开展更高分辨率的统计分析。同时对这些区段的基因密度与GC含量进行了量化分析。
结果：尽管相较于基因组其余区域，整条W染色体并未出现显著的转座因子富集现象，但其中一段特定区域（6.6–8.8 Mb，即第4个区段）的转座因子富集程度显著升高。该区域同时呈现低基因密度与低GC含量的特征，提示该区域的重组水平受到显著抑制。
结论：本研究结果表明，枝额虫的W染色体存在一段受交叉抑制的小型区域，支持其处于演化早期的原性染色体（proto-sex chromosome）阶段的假说。本研究为早期性染色体演化机制提供了重要的科学见解，并确立枝额虫作为研究驱动原性染色体分化的演化过程的理想模式生物。