Kin do not always help: testing multiple hypotheses on nest feeding in a cooperatively breeding bird

In cooperatively breeding species, group members may derive multiple benefits from helping to raise other individuals’ offspring, yet not all individuals do so. In this study, we tested several hypotheses to explain why group members feed offspring of breeding placid greenbuls (Phyllastrephus placidus). In accordance with the kin selection hypothesis, all helpers were first-order kin of the breeding female and the presence of helpers was associated with increased survival of the breeding pair. However, the propensity to help varied widely among group members, as 46% of group members related to the breeding female did not feed nestlings. Sex, relatedness to the breeding male, and group size did not explain additional variation in helping propensity or effort, however, younger helpers fed offspring more often than older ones. Overall, our results show mixed support for predictions of the group augmentation hypothesis, while predictions of the pay-to-stay hypothesis and skills hypothesis were not supported. We suggest that costs associated with providing food to nestlings, and benefits of other types of helping behavior (e.g. anti-predator behavior), may jointly explain why members of breeding groups often refrain from helping at the nest.

Methods
Study system and data collection

Data were collected during the breeding seasons of 2012-2018 in the Dabida forest archipelago of the Taita Hills (SE Kenya; 30°25'S, 38°20'E). The landscape consists of a heterogeneous mixture of indigenous cloud forest remnants (<1 ha to 120 ha), exotic plantations and small-scale subsistence agriculture. Eight of these forest remnants contain subpopulations of the placid greenbul, a long-lived insectivorous passerine from East Africa’s moist forests that occupies pair- or group territories year-round (DVL & BA personal observations). Their breeding season coincides with the onset of the short rainy season in November and lasts until March. Typically, breeding females lay and incubate two eggs that hatch synchronously, and pairs generally re-nest after breeding failure and (occasionally) also after successful breeding. Approximately 50% of greenbul offspring in our study area consist of extra-pair young sired by neighboring territory owners (Cousseau, Van de Loock, et al. 2020). Nest failure is mostly due to predation (Spanhove et al. 2014) and larger breeding groups are associated with lower rates of nest predation and higher post-fledgling survival (Van de Loock et al. 2017; Van de Loock 2019). Due to continued ringing and nest monitoring effort since 1996 and 2007, respectively, ca. 75% of the greenbul population is color-banded at any time (based on the ratio of color-banded individuals vs. total traps during mist-netting).

In our study area, the vast majority of subordinate group members are offspring from previous breeding season(s) that delay their natal dispersal, although non-natal individuals may occasionally join breeding groups (Cousseau, Hammers, et al. 2020). Both subordinate males and females show delayed dispersal, but males delay on average longer and eventually start breeding closer to their natal territory than females (Cousseau, Hammers, et al. 2020). While males disperse earlier when born in smaller forest patches compared to larger ones (Cousseau, Hammers, et al. 2020), this is not reflected in spatial variation in the likelihood that a breeding group contains subordinates, neither in the size of these groups (Van de Loock 2019).

Placid greenbul breeding behavior was monitored in all eight known subpopulations of the Dabida forests. Upon detection, each nest was visited every 3 to 5 days until all nestlings had fledged or the nesting attempt failed. At an age of ca. nine days, nestlings were individually color-banded, measured and a blood sample was taken. We recorded group size, identified breeding and subordinate group members, and quantified their helping behavior and individual nestling provisioning rates through a combination of focal observations, targeted mist-netting, and nest video recordings.

We conducted focal observations during incubation by hiding less than 10m from the nest (2012-15 only) as well as opportunistically at each nest visit (all years). We erected mist nets around the nest when nestlings were ca. 5 days old (range 3 – 8 days) or after nest depredation. Upon trapping, birds were measured, a blood and/or a feather sample was taken and the breeding status (cloacal swelling or brood patch) determined (see below). Unringed individuals were metal- and color-banded. Between 2012 and 2015, focal observations and mist-netting were conducted using playback of greenbul distress calls for a maximum duration of 10 min. As group members respond to these playbacks by approach and displays, presumably to distract and lead away potential predators, this method proved to be a rapid and efficient way to assess group membership and trap individuals. Playback was not used when a predator was present. When nestlings were ca. 8 days old (range 6 – 10 days), we video-recorded food provisioning for 5-6 hours continuously between 7 a.m. and 2 p.m. using a HD camera (Sony Corp.) installed on a tripod about 1.5 m from the nest (one video per nest; on average 25 videos taken per year). Besides helping behavior, video recordings allowed us to extract hourly provisioning rates (number of feeds per hour per nestling) of breeders and helpers. We only extracted provisioning rates when we could identify the visiting individuals in at least 70% of all visits over the total recording period.

Each breeding group consisted of the territorial breeding pair (henceforward called ‘breeders’) and all subordinates (if any) observed at a particular nest. Breeders were identified based on cloacal swellings (males), the presence of a brood patch or observed incubation (females). All other individuals present at a nest were assigned as subordinates. A subordinate was classified as a ‘helper’ if it was observed feeding nestlings at least once during the nest video recording. It follows that subordinates that were never observed feeding nestling during this period were classified as ‘non-helping subordinates’. Whether or not a subordinates helped during a breeding event is called ‘helping propensity’. We measured ‘helping effort’ as the number of feeds per hour per nestling. The age of each group member was assessed from its fledging year (when ringed as nestling) or its feather development at first capture (‘juvenile’, ‘immature’ or ‘fully-grown’ based on molt patterns of primaries, secondaries and coverts; Jackson 2005). Individuals were sexed using a set of sex-linked primers P2/P8 (Griffiths et al. 1998) and relatedness to the breeding pair was based on 12 microsatellite loci (see Husemann et al. 2015 and Cousseau, Van de Loock, et al. 2020 for primer details). We used the exclusion method (Jones and Ardren 2003; by using a self-written R script) to identify first-order kin (son or daughter). Previous analysis of known mother-offspring combinations in our study species showed genetic mismatches at a single locus in only 8% of the cases (Cousseau, Van de Loock, et al. 2020). Group members were therefore considered first-order relatives of a male or female breeder if they either showed no mismatch or a mismatch at a single locus, and were otherwise considered non-relatives.

在合作繁殖物种中，群体成员可通过协助抚育其他个体的后代获得多重收益，但并非所有个体都会参与协助抚育。本研究针对繁殖期静绿鹎（Phyllastrephus placidus）群体成员为何会投喂其后代这一问题，检验了多项假说。结果符合亲缘选择假说（kin selection hypothesis）：所有协助者均为繁殖雌鸟的一级亲属，且协助者的存在与繁殖配对的存活率提升显著相关。不过，群体成员的协助意愿差异显著，46%与繁殖雌鸟存在亲缘关系的群体成员并未投喂雏鸟。性别、与繁殖雄鸟的亲缘关系以及群体规模均无法解释协助意愿或抚育投入的额外变异，但较年轻的协助者比年长个体更频繁地投喂后代。总体而言，我们的结果为群体增益假说（group augmentation hypothesis）的预测提供了混合支持，而付费停留假说（pay-to-stay hypothesis）与技能假说（skills hypothesis）的预测未得到支持。我们认为，向雏鸟投喂食物所需付出的代价，以及其他类型协助行为（如反捕食行为）带来的收益，或许可以共同解释为何繁殖群体的成员通常不会在巢中提供协助。

方法

研究系统与数据采集
数据采集于2012-2018年的繁殖季，地点位于肯尼亚东南部塔伊塔山的达比达森林群岛（南纬30°25′，东经38°20′）。该区域景观由异质化的本土云雾林残块（面积0.1~120公顷）、外来人工林以及小规模自给农业用地混合组成。其中8处森林残块栖息有静绿鹎种群：该物种为东非湿润森林中的长寿命食虫雀形目鸟类，全年占据配对或群体领地（DVL与BA个人观测）。其繁殖季与11月的短雨季起始时间重合，持续至次年3月。通常情况下，繁殖雌鸟会产下并孵化2枚同步出壳的鸟卵，繁殖失败后配对通常会重新筑巢，成功繁殖后偶尔也会再次筑巢。本研究区域内约50%的静绿鹎后代为额外配对子代，由邻近领地的所有者育成（Cousseau, Van de Loock等，2020）。巢失败主要源于捕食（Spanhove等，2014），而更大的繁殖群体与更低的巢捕食率以及更高的雏鸟出飞后存活率相关（Van de Loock等，2017；Van de Loock，2019）。由于自1996年起持续进行环志，以及自2007年起持续开展巢监测工作，约75%的静绿鹎种群个体始终带有彩色脚环（基于雾网采样中带彩色脚环个体与总捕获个体的比例计算）。

在本研究区域内，绝大多数从属群体成员为上一个或多个繁殖季产下的后代，它们会延迟出生扩散（natal dispersal），尽管非本土个体偶尔也会加入繁殖群体（Cousseau, Hammers等，2020）。从属个体无论雌雄均会延迟出生扩散，但雄性的延迟时长平均更长，且最终会在比雌性更接近其出生领地的位置开始繁殖（Cousseau, Hammers等，2020）。尽管与较大的森林斑块相比，在较小森林斑块中出生的雄性会更早扩散（Cousseau, Hammers等，2020），但这种差异并未体现在繁殖群体是否拥有从属个体的可能性的空间变异中，也未体现在这些群体的规模上（Van de Loock，2019）。

我们对达比达森林已知的全部8个静绿鹎亚种群的繁殖行为进行了监测。每发现一个鸟巢，便每3~5天探访一次，直至所有雏鸟出飞或繁殖尝试失败。雏鸟约9日龄时，会为其佩戴单独的彩色脚环、测量身体指标并采集血液样本。我们记录了群体规模，确定了繁殖个体与从属群体成员，并通过焦点取样观察（focal observations）、针对性雾网采样以及巢内视频录制相结合的方式，量化了它们的协助行为与单只雏鸟的投喂频次。

我们在孵化期进行焦点取样观察：2012-2015年期间，会在距离巢穴不足10米的位置隐蔽观察；其余年份则在每次探访鸟巢时进行机会性观察。当雏鸟约5日龄时（范围为3~8日龄），或在巢被捕食后，我们会在巢穴周围架设雾网。捕获鸟类后，会测量其身体指标、采集血液或羽毛样本，并确定其繁殖状态（泄殖腔肿胀或育雏斑，详见下文）。未佩戴脚环的个体将被佩戴金属脚环与彩色脚环。2012-2015年期间，我们通过播放静绿鹎的遇险鸣唱进行焦点取样与雾网采样，单次播放时长不超过10分钟。由于群体成员会对这些播放做出响应，靠近巢穴并做出展示行为，以干扰并引开潜在捕食者，该方法被证明是评估群体组成与捕获个体的快速高效手段。当存在捕食者时，不会使用播放手段。当雏鸟约8日龄时（范围为6~10日龄），我们会使用高清摄像机（HD camera，索尼公司（Sony Corp.）生产）在距离巢穴约1.5米的三脚架上进行连续5~6小时的视频录制，录制时间为上午7时至下午2时（每个巢穴录制1段视频，平均每年录制25段视频）。除协助行为外，视频录制还可帮助我们计算繁殖个体与协助者的每小时投喂频次，即每只雏鸟每小时的投喂次数。我们仅在总录制时段内至少70%的到访个体可被识别的情况下，才提取投喂频次数据。

每个繁殖群体由具有领地的繁殖配对（以下简称“繁殖者”）以及在特定巢穴中观察到的所有从属个体（若有）组成。繁殖者的识别依据为：雄性的泄殖腔肿胀、雌性的育斑或已观察到的孵化行为。巢穴中其余所有个体均被归类为从属个体。若某从属个体在巢视频录制期间至少被观察到投喂雏鸟一次，则将其归类为“协助者”；在此期间未被观察到投喂雏鸟的从属个体则被归类为“非协助从属个体”。从属个体在某次繁殖事件中是否提供协助被称为“协助意愿”。我们将“协助投入”定义为每只雏鸟每小时的投喂次数。每个群体成员的年龄通过其出飞年份（若作为雏鸟时已被环志）或首次捕获时的羽毛发育情况进行评估：依据初级飞羽、次级飞羽以及覆羽的换羽模式，分为“幼鸟”“亚成鸟”或“成鸟”（Jackson，2005）。个体性别通过性连锁引物P2/P8进行鉴定（Griffiths等，1998），与繁殖配对的亲缘关系基于12个微卫星位点（microsatellite loci）计算（引物详情详见Husemann等，2015与Cousseau, Van de Loock等，2020）。我们使用排除法（Jones与Ardren，2003；通过自行编写的R脚本（R script）实现）来识别一级亲属（子代）。此前对本研究物种已知母-子代组合的分析显示，仅在8%的案例中出现单个位点的遗传错配（Cousseau, Van de Loock等，2020）。因此，若某群体成员与繁殖雄鸟或雌鸟无位点错配，或仅存在单个位点错配，则将其视为一级亲属，否则视为非亲属。