Gut passage in frugivores enhances the germination of mistletoe seeds Bakerella gonoclada, in a Madagascar rainforest

In Madagascar's rainforests, frugivores influence Bakerella gonoclada mistletoe seed dispersal and germination. Our germination experiments show that mistletoe seeds defecated by lemurs had significantly higher germination success, germination speed, hypocotyl growth, and survival probability than seeds planted directly from mistletoe. These results suggest a mutualistic relationship between mistletoe and lemurs.

Methods
The study was conducted in Talatakely, a region within Ranomafana National Park (RNP), a lowland to montane rainforest located in southeastern Madagascar (47°18’- 47°37’E and 21°02’- 21°25’S). Talatakely is a degraded forest characterized by many shrub species in the families of Rubiaceae and Myrsinaceae, bamboo, epiphytes, and mistletoes (Atsalis, 1999; Turk, 1997); some areas are invaded by the exotic plant strawberry guava (Psidium cattleianum).

To investigate mistletoe biology, we compared the probability and speed of germination of Bakerella seeds defecated by lemurs to seeds pulled directly from mistletoe fruits by the experimenter, hereinafter referred to as ‘hand-removed seeds’, in Petri dish experiments. From January to April 2023, we followed four groups of red-fronted brown lemurs (Eulemur rufifrons) on alternate days from 8:00 am to 4:00 pm. Whenever a group defecated, we noted the age class and sex of individuals. We checked defecated feces for mistletoe seeds; any feces containing Bakerella seeds were wrapped in aluminum foil for lab analysis. Additionally, on January 19, 2023, we searched Talatakely trails for fresh frugivore droppings and collected seeds from one sample of a lemur, which we suspected to come from a greater dwarf lemur (Cheirogaleus major), based on previous direct observations (Fenosoa et al., 2020), because the sample contained only Bakerella seeds wrapped in a viscin thread but no fecal matter. Attempts were made to collect Bakerella seeds from the feces of the brown mouse lemur, Microcebus rufus, using capture-release-recapture methods but were unsuccessful.

Bakerella species in RNP are identified by shape, color, and size (Fenosoa et al., prep.). We found that all defecated mistletoe seeds collected (from Eulemur rufifrons and, putatively, from Cheirogaleus major) were B. gonoclada. To compare the germination of defecated to hand-removed seeds, we picked 30 mature fruits from four individual plants of B. gonoclada. We planted all defecated and hand-removed seeds immediately on the evening of the collection day. Defecated and hand-removed seeds were planted in separate disposable Petri dishes of a 9 cm diameter; seeds from the same individual source were planted in the same dish. The seeds in each Petri dish were set on filter paper with at least 1 cm spacing between the seeds. All dishes were left under the same natural conditions with no direct sunlight. We monitored seed and seedling survival and germination, defined as the first visible appearance of the hypocotyl-radicle tip. We recorded the final length of the hypocotyl-radicle after 12 weeks of observation.

在马达加斯加的热带雨林中，果食性动物会影响Bakerella gonoclada槲寄生的种子传播与萌发过程。本研究的萌发实验结果显示，经狐猴粪便排出的槲寄生种子，其萌发成功率、萌发速率、下胚轴生长情况以及存活概率，均显著高于直接从槲寄生上采集的种子。上述结果表明，槲寄生与狐猴之间存在互利共生关系。

研究方法
本研究于马达加斯加东南部的拉努马法纳国家公园（Ranomafana National Park, RNP）内的塔拉塔凯利（Talatakely）区域开展，该区域属于从低地到山地的热带雨林，地理坐标为东经47°18′-47°37′、南纬21°02′-21°25′。塔拉塔凯利为退化林分，以茜草科（Rubiaceae）、紫金牛科（Myrsinaceae）的多种灌木、竹类、附生植物以及槲寄生为主要植被特征（Atsalis, 1999; Turk, 1997）；部分区域被外来物种草莓番石榴（Psidium cattleianum）入侵。

为探究槲寄生的生物学特性，本研究通过培养皿（Petri dish）实验，对比经狐猴粪便排出的Bakerella属槲寄生种子与实验人员直接从槲寄生果实中摘取的种子（以下简称“手采种子”）的萌发概率与萌发速率。2023年1月至4月期间，研究团队于每日上午8:00至下午4:00间隔采样，追踪4群红额棕狐猴（Eulemur rufifrons）。每当观测到群体排便时，记录个体的年龄阶层与性别。对排出的粪便进行检查以寻找槲寄生种子，所有携带Bakerella属种子的粪便均用铝箔纸包裹，用于后续实验室分析。此外，2023年1月19日，研究团队在塔拉塔凯利的步道上搜寻新鲜的果食性动物粪便，并从一份疑似来自大倭狐猴（Cheirogaleus major）的狐猴粪便样本中采集了种子——该推断基于此前的直接观测记录（Fenosoa et al., 2020），因该样本仅含有被黏丝（viscin thread）包裹的Bakerella属种子，未发现粪便残渣。研究团队尝试通过标记重捕法从棕倭狐猴（Microcebus rufus）的粪便中采集Bakerella属种子，但未获成功。

拉努马法纳国家公园内的Bakerella属物种可通过形态、颜色与尺寸进行鉴定（Fenosoa et al., 待刊）。经鉴定，本次采集的所有经粪便排出的槲寄生种子（取自红额棕狐猴，以及推断的大倭狐猴样本）均为B. gonoclada。为对比粪便排出种子与手采种子的萌发情况，研究团队从4株B. gonoclada植株上采集了30颗成熟果实。所有粪便排出种子与手采种子均于采集当日傍晚即刻播种。粪便排出种子与手采种子分别种植于直径9 cm的一次性培养皿中，同一来源个体的种子种植于同一培养皿内。每个培养皿内的种子均铺设于滤纸上，种子间间距至少为1 cm。所有培养皿均置于相同的自然环境中，避免阳光直射。研究团队对种子与幼苗的存活情况及萌发情况进行监测，萌发定义为下胚轴-胚根尖端首次肉眼可见的状态。在持续12周的观测后，记录下胚轴-胚根的最终长度。