Data from: Extreme fertilisation bias towards freshly inseminated sperm in a species exhibiting prolonged female sperm storage

The storage of sperm by females across successive reproductive cycles is well documented in internal fertilisers, yet the fate of stored sperm when they compete with ‘new’ sperm to fertilise a female’s eggs has rarely been considered. This gap in our understanding is likely due to the logistical difficulties of controlling behavioural interactions during or after mating, which in turn may influence how many sperm are inseminated and how stored sperm are ultimately utilised during successive bouts of sperm competition with freshly inseminated sperm. Here, we use artificial insemination (AI) in guppies (Poecilia reticulata), a polyandrous livebearing poeciliid fish exhibiting prolonged sperm storage by females, to overcome these challenges. The use of AI enables us to control potential differential maternal effects (e.g. behaviourally mediated cryptic female choice) and specifically test for post-copulatory paternity biases that favour either stored or fresh sperm when they compete to fertilise eggs. Our paternity analyses revealed the almost complete dominance of freshly inseminated sperm over stored sperm, supporting previous studies reporting similar patterns following natural matings across successive brood cycles. However, our use artificial inseminations, which excluded behavioural interactions between males and females, most likely generated a far stronger pattern of fresh sperm precedence compared to those reported in previous studies, possibly implicating ‘cryptic’ forms of selection by females that may sometimes bolster the success of stored sperm.

在体内受精类群中，雌性在连续生殖周期内储存精子的现象已有充分研究记录，但当储存精子与“新鲜”精子竞争以完成雌性卵子的受精时，储存精子的最终去向却极少被探讨。我们认知中的这一空白，大概率源于难以在交配过程中及交配后控制雌雄个体间的行为互动——这一实验操作难点又会影响受精时注入的精子数量，以及在与新鲜注入精子接连开展的多轮精子竞争中，储存精子的最终利用模式。本研究以孔雀花鳉（*Poecilia reticulata*）为研究对象，该物种为一妻多夫制的胎生花鳉科鱼类，雌性具备长期储存精子的能力；本研究采用人工授精 AI (artificial insemination) 技术以克服上述研究难点。借助人工授精技术，我们得以控制潜在的母体差异效应（例如由行为介导的雌性隐蔽选择），并专门检验当储存精子与新鲜精子竞争受精时，是否存在偏向储存精子或新鲜精子的交配后父权偏倚。我们的父权分析结果显示，新鲜注入精子几乎完全占据了对储存精子的优势地位，这与此前针对自然交配后连续育雏周期的研究结果一致。不过，由于本研究采用的人工授精技术排除了雌雄个体间的行为互动，相较于此前的研究，本研究更清晰地展现了新鲜精子优先的模式；这一结果或暗示，雌性的“隐蔽”选择机制有时反而会提升储存精子的受精成功率。