Data for: Kin recognition for incest avoidance in Damaraland mole-rats, Fukomys damarensis

Across taxa, breeding among close relatives is usually avoided because it incurs fitness costs to offspring. Incest is often averted through the dispersal of either sex from the natal area to breed. In some philopatric species, association among relatives extends into adulthood, and an ability to discriminate kin may be required for individuals to reduce inbreeding risk. Here, we aim to determine the mechanism of kin recognition for incest avoidance in the Damaraland mole-rat Fukomys damarensis, a cooperative breeder characterized by extreme reproductive skew. Pairs of opposite-sex adults were formed in the laboratory and, within pairs, genetic relatedness and degree of familiarity were manipulated through cross-fostering experiments. We found that unfamiliar pairs were more likely to engage in sexual behaviours and bred more successfully than familiar pairs, regardless of their genetic similarity.  Females paired with unfamiliar males were also more likely to exhibit reproductive activation, characterized by increased levels of oestradiol and progesterone. This study shows that in Damaraland mole-rats, inbreeding avoidance can be achieved through a discrimination mechanism that relies on association during rearing, and that ovulation is induced by mating. This study advances our understanding of incest avoidance in species with constrained dispersal.

Methods
The experiment was initially carried out on 32 pairs of opposite-sex individuals that were either: (i) familiar kin (n = 8); (ii) familiar non-kin (n = 8); (iii) unfamiliar kin (n = 8); or (iv) unfamiliar non-kin (n = 8), in a full factorial treatment design. 

Behavioural observations were carried out to quantify copulation, defined as one individual mounting another and attempting intromission with pelvic thrusts, and sex foreplay, defined as the rapid succession of bites, sparring, sniffs, passes, and drumming. A full ethogram of these and other observed mole-rat behaviours are presented in Supplementary Table S1A. Behavioural observations consisted of focal and scan sampling. Focals were carried out on the female. One two-hour focal session was carried out immediately after pairing (day 0, approximately 1000 SAST), and another one-hour focal session the following day (day 1, approximately 0800 SAST). Focal behaviours were sampled as ‘states’, recorded with a start and an end time, or ‘events’, recorded at observation without a duration (Supplementary Table S1B). Weekly 12-hour scan sessions were carried out for eight weeks, starting 2-8 days after pairing (approximately 0700 SAST). Four pairs were observed concurrently during each session (n = 8 individuals). Scan sessions comprised a combination of instantaneous and continuous sampling (Supplementary Table S1C). Behavioural states were recorded every four minutes, generating 180 instantaneous samples per individual. In between instantaneous sampling, events and states of short duration were recorded continuously. For both focals and scans, observations were recorded using Observer 11XT pocket version 3.2.

Urine samples were collected to quantify oestradiol (E2) and progesterone (P4) and determine the effect of treatment on reproductive activation. Samples were collected 2-4 days before pairing to establish baseline E2 and P4 levels. Samples were subsequently collected on day 1, then every 3 days until day 90, and every 7 days between day 90 and day 270. E2 and P4 levels were quantified using high-performance liquid chromatography-tandem mass-spectrometry (Supplementary Material S2).

All pairs were closely monitored for nine months following pairing. To investigate reproductive success, we tested the effect of treatment on the: (i) probability of successful gestation following reproductive activation; (ii) total number of pups produced; and (iii) total number of litters produced.

跨分类群生物通常会避免近亲繁殖，因为这会对子代产生适合度代价。近交规避往往通过两性之一从出生栖息地扩散至别处繁殖来实现。在部分具有恋巢性（philopatric）的物种中，亲属间的社交关联会延续至成年，个体需具备亲属识别能力以降低近交风险。本研究旨在探究达马拉兰鼹形鼠（Fukomys damarensis）——一种以极端繁殖偏斜为特征的合作繁殖物种——的近交规避亲属识别机制。我们在实验室中构建了异性成年个体配对，并通过交叉抚育（cross-fostering）实验操控配对个体间的遗传亲缘关系与熟悉程度。结果发现，无论遗传相似性如何，不熟悉的配对个体更易发生性行为，且繁殖成功率更高；与不熟悉雄性配对的雌性，其生殖激活概率也更高，表现为雌二醇（oestradiol）与孕酮（progesterone）水平升高。本研究表明，达马拉兰鼹形鼠可通过依赖抚育期社交关联的识别机制实现近交规避，且排卵由交配诱导。本研究增进了我们对扩散受限物种近交规避策略的理解。

方法
本实验初始设置32组异性个体配对，采用完全因子实验设计，分为四组处理：(i) 熟悉亲属组（n=8）；(ii) 熟悉非亲属组（n=8）；(iii) 不熟悉亲属组（n=8）；(iv) 不熟悉非亲属组（n=8）。

行为观察用于量化交配行为与性前戏行为：交配行为定义为一方骑乘另一方并通过骨盆冲刺尝试插入，性前戏行为定义为快速连续的啃咬、争斗、嗅探、穿梭与叩击。上述行为及其他观测到的鼹形鼠行为的完整行为谱（ethogram）见补充表S1A。行为观察采用焦点取样（focal sampling）与扫描取样（scan sampling）结合的方式：焦点取样以雌性为观测对象，配对后即刻（第0天，约南非标准时间10:00）进行一次2小时的焦点观测，次日（第1天，约南非标准时间08:00）进行一次1小时的焦点观测。焦点行为记录分为“状态事件”（记录起始与结束时间）与“瞬时事件”（观测时直接记录无持续时长），具体规则见补充表S1B。配对后2-8天开始，每周进行1次12小时的扫描观测，持续8周（观测时间约为南非标准时间07:00），每次同时观测4组（共8只个体）。扫描观测结合瞬时取样与连续取样方式（补充表S1C）：每4分钟记录一次行为状态，每只个体可获得180次瞬时样本；在瞬时取样间隔期间，持续记录短时发生的事件与状态。所有焦点与扫描观测均使用Observer 11XT pocket version 3.2软件进行记录。

采集尿液样本以量化雌二醇（oestradiol, E2）与孕酮（progesterone, P4）水平，探究实验处理对生殖激活的影响。样本采集于配对前2-4天，用于建立雌二醇与孕酮的基线水平；后续分别于配对后第1天采集样本，之后每3天采集一次直至第90天，第90天至第270天期间每7天采集一次。雌二醇与孕酮水平采用高效液相色谱-串联质谱法（high-performance liquid chromatography-tandem mass-spectrometry）进行定量，详细方法见补充材料S2。

所有配对组在配对后均被密切监测9个月。为探究繁殖成功率，我们分析了实验处理对以下三项指标的影响：(i) 生殖激活后成功妊娠的概率；(ii) 总产仔数；(iii) 总产仔窝数。