Research Article Evidence for passerine bird pollination in Rhododendron species Zhi-Huan Huang1, Yun-Peng Song2 and Shuang-Quan Huang2* 1Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China 2Institute of Evolution and Ecology, School of Life Sciences, Central China Normal University, Wuhan 430079, China Received: 16 May 2017 Editorial decision: 3 November 2017 Accepted: 7 November 2017 Published: 9 November 2017 Associate Editor: Karina Boege Citation: Huang Z-H, Song Y-P, Huang S-Q. 2017. Evidence for passerine bird pollination in Rhododendron species. AoB PLANTS 9: plx062; doi: 10.1093/aobpla/plx062 Abstract. When insect activity is limited at low temperature, birds may be comparatively more important pol- linators than insects for flowering plants. It has been thought that many large-flowered Rhododendron species are pollinated by local birds in the Himalayan regions because most of these species flower in spring at high elevation with cool atmospheric temperature. However, experimental evidence for the role of bird pollination in this hyper- diverse genus remains scarce. To examine the role of birds and insects in pollination, we observed floral visitors to 15 Rhododendron species with different floral sizes and abundant flowering individuals in the eastern Himalayas, Southwest China. To examine the role of birds and insects in female reproductive success in each species, cages were used to exclude birds but not insects from visiting flowers and net bags were used to exclude all floral visi- tors. Inflorescences where visitation was excluded did not produce fruits in any of the Rhododendron species, indi- cating that sexual reproduction in these species depended on pollinator visitation. Bird visits were generally less frequent than bee visits in the studied species. However, in the nine species on which bird visitors were observed, fruit and/or seed set were greatly reduced in inflorescences caged to exclude birds but not bees, compared to open- pollinated inflorescences. In the other six species on which bird visitation was not observed, fruit and seed set did not differ significantly between caged and open inflorescences except in one species (R. wardii). Manipulations to achieve selective exclusion of visitors demonstrated that birds could be effective pollinators for 10 out of 15 studied Rhododendron species in the eastern Himalayas. Floral characteristics of these Rhododendron species and weather conditions might favour the evolution of bird pollination systems in the East Himalayas. Keywords: Bee pollination; bird pollination; flower size; fruit/seed production; low temperature; pollinator exclusion experiment; Rhododendron. Introduction (Cronk and Ojeda 2008). Previous comparative studies have shown that shifts from bee pollination to bird pol- It has been estimated that 87.5 % of flowering plant spe- lination have occurred independently in numerous line- cies rely on animals for pollination (Ollerton et al. 2011). ages of flowering plants (Kay et al. 2005; Specht 2006; Most pollinators are insects but birds are important Whittall and Hodges 2007; Wilson et al. 2007; Sakai et al. pollinators for some species in about 65 plant families 2013), whereas reversal is much less common (Wolfe *Corresponding author’s email address: [email protected] © The Author(s) 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. AoB PLANTS https://academic.oup.com/aobpla © The Authors 2017 1 Huang et al. – Bird pollination in Rhododendron et al. 2006; Wilson et al. 2007). Of these two groups anecdotal observations showed that sunbirds and pas- of pollinators, birds tend to be larger and can be more seriforms feed on nectar in numerous species (Huang active in cool temperature than insects. It has been pro- 2011; Georgian et al. 2015). However, there has been no posed that bird pollination enhances plant reproductive experimental examination of the effectiveness of bird success at low atmospheric temperature when weather pollination in this well-known genus. conditions are unfavourable for bees (Cruden 1972). To test the effectiveness of birds and insects as pol- One hypothesis for the shift from bee to bird pollina- linators of 15 Rhododendron species, we addressed the tion is the high ratio of pollen receipt to pollen removal following specific questions: (i) Given that low tempera- (Wilson et al. 2007). A successful pollination depends on ture limits insect activity, do warm-blooded birds act as a vector removing pollen from one flower and delivering floral visitors in someRhododendron species? (ii) How pollen to another flower. Unlike bird foraging behaviour, do birds contribute to pollination in these species? (iii) which involves little grooming, bees (e.g. bumblebees) Given that a morphological fit between floral morph- usually groom pollen from their body into corbiculae ology and pollinator body could facilitate pollen transfer where it is unavailable for pollen transfer to the next (see Stevens 1976), do birds tend to visit relatively large flower visited. Empirical studies have shown that polli- Rhododendron flowers? We investigated pollinator spe- nators that rarely exhibit grooming behaviour are more cies and their visitation frequency in 15 Rhododendron efficient at delivering pollen (Muchhala and Thomson species that had abundant flowering individuals with a 2010). In addition, birds are considered to become wide variation in flower size. We conducted two types important supplemental pollinators in habitats where of pollinator experiments by excluding all pollinators, or insect activity is limited. For example, high mountains, just birds, to compare the pollination role of birds and where cold and/or rainy weather conditions are fre- insects in these Rhododendron species. quent, are less conducive to insect activity (Stiles 1971, 1978; Cruden 1972). Furthermore, insects are generally short-lived and overwinter as immature stages or in Methods hibernation (i.e. many bees, see Goulson 2003), and are rarely available for plants that flower in winter or early spring when the temperature is low. Species and study area Rhododendron (Ericaceae) is one of the largest plant Rhododendron (Ericaceae) is a species-rich genus of per- genera with around 1000 evergreen or deciduous spe- ennial shrubs or trees. Because their flowers are usu- cies mainly in Asia (Chamberlain et al. 1996), includ- ally large and brightly coloured and flower in spring, ing many endemic species in the Himalayan region. Rhododendron species are important horticultural Most species in this region have conspicuous flowers. plants worldwide. The diversity of Rhododendron is es- Rhododendron species usually blossom from late win- pecially high in East Asia, with over 500 species endemic ter to early summer when the temperature is low and to Southwest China. Floral and vegetative traits are the activity of insects is restricted. Rhododendron can highly variable, but Rhododendron flowers are charac- be a model genus in which to examine the divergent terized by an open-shaped funnelform, campanulate or evolution of floral traits related to pollinator shifts tubular corolla which is formed by petal fusion. There are because the flower morphology varies widely in the usually 5–10 stamens surrounding one exserted style same region. In diverse plant groups, selection on with a capitate stigma. Pollen grains released from the floral morphology has been shown to be mediated in poricidal anthers are connected together by sticky viscin part by morphometric interactions with the mouth or threads. body part length of pollinators across regional floras The field survey was conducted in Cangshan National (Inouye 1980; Inouye and Pyke 1988; Nilsson 1988; Nature Reserve (N 25°43′; E 100°01′) and Laojunshan Alexandersson and Johnson 2002; Moré et al. 2012). Nature Reserve (N 26°39′; E 99°44′), Yunnan Province, Based on morphological studies of nearly 300 tropi- Southwest China. These areas are in the Hengduan cal species from the Malayan region (Southeast Asia Mountains, East Himalayas, with elevations from 1800 to Northern Australia), Stevens (1976) estimated that to 4200 m (see details in Table 1), usually comprising a one-third of Rhododendron species may have evolved mosaic of deciduous broad-leaved and coniferous for- bird pollination, particularly large-flowered species at ests, pastures and scrubby stream mountain vegetation. high elevations. Although insects have been observed We investigated floral traits of 15 Rhododendron species to be effective pollinators in several Rhododendron spe- in which abundant flowering individuals were available cies (Stout et al. 2006; Stout 2007; Tagane et al. 2008; and conducted pollinator selective exclusion experi- Ma et al. 2010, 2015; Kudo et al. 2011; Epps et al. 2015), ments on these species. 2 AoB PLANTS https://academic.oup.com/aobpla © The Authors 2017 Huang et al. – Bird pollination in Rhododendron Table 1. Details of field populations of 15 Rhododendron species, pollinator observation censuses and species of bird visitors that were observed in Cangshan and Laojunshan Mountains, Yunnan Province, Southwest China. The two sunbirds are Aethopyga gouldiae