Raw Genotyping-by-sequencing reads of Petunia axillaris subsp. subandina, Petunia exserta, and Petunia sp3 (natural hybrid between P. exserta and P. secreta). Raw sequence reads

Delimiting species is a complex yet crucial task for understanding biodiversity. However, this challenge intensifies when species boundaries are blurred by hybridization, as most species delimitation methods do not directly account for gene flow. The plant genus Petunia offers a unique opportunity to investigate the impact of gene flow on species diversification and delimitation due to diverse hybrid classes found in nature, even between species with distinct pollination syndromes. This study aimed to reconstruct phylogenetic relationships among multiple Petunia lineages within the long corolla tube clade and assess the effect of sampling admixed individuals on species delimitation analyses. We used genotyping-by-sequencing (GBS) data from 315 individuals spanning different lineages and contact zones. Our findings unveil rapid diversification and substantial levels of incomplete lineage sorting among these taxa. Moreover, these species have undergone extensive ancient and recent hybridization, even between species with distinct pollination syndromes. Using BFD* to delimit species, we found that species count is overestimated regardless of the presence of admixed individuals. Our conclusion highlights the persistent challenge of delineating species boundaries, indicating that even with genomic advancements, integrative taxonomy remains essential for delimiting species, particularly in hybridization-prone taxa.

界定物种是理解生物多样性的一项复杂却至关重要的任务。然而，当物种边界因杂交作用变得模糊时，这一挑战会进一步加剧——因为绝大多数物种界定方法无法直接考量基因流。矮牵牛属（Petunia）为探究基因流对物种分化与物种界定的影响提供了独特契机，因其在自然界中存在多样的杂交类群，即便在具有截然不同传粉综合征的物种之间亦是如此。本研究旨在重构长花冠管演化支内多个矮牵牛谱系的系统发育关系，并评估采样混合遗传背景个体对物种界定分析的影响。我们使用了覆盖不同谱系与接触带的315份样本的基因型分型测序（genotyping-by-sequencing, GBS）数据。研究结果揭示了这些类群间快速的物种分化与显著水平的不完全谱系分选现象。此外，这些物种经历了广泛的古代与近代杂交事件，即便在具有迥异传粉综合征的物种之间亦不例外。借助BFD*进行物种界定，我们发现无论是否存在混合遗传背景个体，物种数量均会被高估。本研究结论凸显了界定物种边界的长期挑战，表明即便借助基因组学技术的进步，整合分类学在物种界定工作中仍不可或缺，对于易发生杂交的类群而言尤为如此。