Journal of Plant Research https://doi.org/10.1007/s10265-020-01226-8 REGULAR PAPER – ECOLOGY/ECOPHYSIOLOGY/ENVIRONMENTAL BIOLOGY Post‑pollination barriers enable coexistence of pollinator‑sharing ornithophilous Erica species Anina Coetzee1 · Claire N. Spottiswoode1,2 · Colleen L. Seymour1,3 Received: 6 June 2020 / Accepted: 23 September 2020 © The Botanical Society of Japan 2020 Abstract Some evolutionary radiations produce a number of closely-related species that continue to coexist. In such plant systems, when pre-pollination barriers are weak, relatively strong post-pollination reproductive barriers are required to maintain spe- cies boundaries. Even when post-pollination barriers are in place, however, reproductive interference and pollinator depend- ence may strengthen selection for pre-pollination barriers. We assessed whether coexistence of species from the unusually speciose Erica genus in the fynbos biome, South Africa, is enabled through pre-pollination or post-pollination barriers. We also tested for reproductive interference and pollinator dependence. We investigated this in natural populations of three bird-pollinated Erica species (Erica plukenetii, E. curvifora and E. coccinea), which form part of a large guild of congeneric species that co-fower and share a single pollinator species (Orange-breasted Sunbird Anthobaphes violacea). At least two of the three pre-pollination barriers assessed (distribution ranges, fowering phenology and fower morphology) were weak in each species pair. Hand-pollination experiments revealed that seed set from heterospecifc pollination (average 8%) was signifcantly lower than seed set from outcross pollination (average 50%), supporting the hypothesis that species boundaries are maintained through post-pollination barriers. Reproductive interference, assessed in one population by applying outcross pollen three hours after applying heterospecifc pollen, signifcantly reduced seed set compared to outcross pollen alone. This may drive selection for traits that enhance pre-pollination barriers, particularly given that two of the three species were self-sterile, and therefore pollinator dependent. This study suggests that post-pollination reproductive barriers could facilitate the coexistence of congeneric species, in a recent radiation with weak pre-pollination reproductive barriers. Keywords Breeding system · Cape foristic region · Ericaceae · Hybrid · Nectariniidae · Pollen interference Introduction Relatively strong reproductive barriers are necessary to maintain species boundaries among such coexisting and Although evolutionary radiations often result in geographi- recently radiated species (Mallet 2005). Reproductive bar- cally separated species, some produce a diversity of coex- riers include premating and postmating isolation barriers isting, closely-related species (Weber and Strauss 2016). (Arceo-Gómez et al. 2016; Nosil et al. 2007). The latter can impose prezygotic isolation (via sperm competition and Electronic supplementary material The online version of this gametic incompatibility) and/or postzygotic isolation (e.g. article (https ://doi.org/10.1007/s1026 5-020-01226 -8) contains via reduced ftness in hybrids) (Rieseberg and Willis 2007). supplementary material, which is available to authorized users. In plants, most species are preserved by multiple reproduc- tive isolation barriers, and prezygotic barriers are thought * Anina Coetzee [email protected] to be about twice as strong as postzygotic barriers (Lowry et al. 2008; Rieseberg and Willis 2007). Thus, understand- 1 FitzPatrick Institute of African Ornithology, DSI-NRF ably, studies on the coexistence of closely-related species Centre of Excellence, University of Cape Town, Private Bag have predominantly focussed on premating barriers (Weber X3, Rondebosch 7701, South Africa and Strauss 2016). However, studies from hummingbird- 2 Department of Zoology, University of Cambridge, Downing pollination systems and other plant communities with high Street, Cambridge CB2 3EJ, UK species diversity suggest that the frequency of heterospecifc 3 South African National Biodiversity Institute, Kirstenbosch pollen transfer can be high (Da Fonseca et al. 2016; Fang Research Centre, Claremont 7753, South Africa Vol.:(0123456789)1 3 Journal of Plant Research and Huang 2013), in which case postmating barriers become predominantly pollinated by a single species, the endemic crucial. Orange-breasted Sunbird Anthobaphes violacea (Nectari- In plants, reproductive barriers are most easily distin- niidae) (Rebelo et al. 1985). In some communities, South- guished as pre- and post-pollination barriers. Pre-pollina- ern Double-collared Sunbird Cinnyris chalybea and Mala- tion barriers include geographical separation, segregation chite Sunbird Nectarinia famosa pollinate these ericas, but of fowering periods or pollinator isolation (Rieseberg and again, pollination is carried out almost exclusively by a Willis 2007). The latter requires diferent pollinator guilds single bird species within these sites (Malan 2013; Rebelo (e.g. birds, insects or wind), pollinator species or pollen et al. 1984, 1985; Turner et al. 2012; Van der Niet et al. placement sites (through diferential fower morphology), 2014). This guild of bird-pollinated ericas often co-fower or can arise when pollinator individuals show fower con- (Rebelo et al. 1984). Almost all have straight or curved stancy (foraging selectively on one plant species) (Grant tubular corollas > 15 mm in length (Rebelo et al. 1985), 1994; Waser 1986). When pre-pollination barriers fail, but vary in the length of their reproductive parts (Anina plants can receive heterospecifc pollen and hybridisation Coetzee, unpublished data). The breeding systems of only could occur (Rieseberg and Willis 2007). This could lead to nine bird-pollinated Erica species have been tested, but of genetic swamping (Rieseberg and Willis 2007), particularly those, three displayed self-compatibility and only one was in closely-related species with little genetic divergence (Lar- partially capable of autonomous self pollination (Angoh combe et al. 2015; Mallet 2005). Coexisting closely-related et al. 2017; Arendse 2015). Self-sterility in this genus species are thus likely to experience reduced plant ftness seems to be a function of a late-acting mechanism within when interspecifc mating occurs, favouring the evolution of the ovule (Arendse 2015): embryo development is arrested post-pollination reproductive barriers (Arceo-Gómez et al. after fertilisation, which prevents wasting resources on 2016), which include premating barriers at the stigma, and selfed seeds but carries the cost of reducing the number pre- and postzygotic barriers downstream of the stigma. of ovules available for cross fertilisation (Gibbs 2014). Even when post-pollination barriers are efcient, the risk Regular natural hybridisations in sunbird-pollinated of reproductive interference remains when pre-pollination orchid and protea species have been reported in the CFR barriers are weak. Post-pollination reproductive interference (Johnson 2018; Mitchell and Holsinger 2018). The success- results in reduced seed set through interactions at the stigma, ful interspecifc crossing and cultivating of many Erica spe- style and/or ovule (Brown and Mitchell 2001; Nishida et al. cies by eighteenth century horticulturists (Nelson and Oliver 2014), which may drive selection for stronger pre-pollina- 2004) reveals that hybridisation is indeed possible between tion barriers (Morales and Traveset 2008). The deposition of certain Erica species, too. Currently, the rate of interspecifc incompatible pollen onto stigmas before, or together with, pollen transfer in the natural system is unknown, but aviary compatible pollen is a common form of post-pollination experiments show that Orange-breasted Sunbirds forage ran- reproductive interference in co-occurring, closely-related domly between diferent Erica fower morphotypes (Heystek species (Weber and Strauss 2016). Furthermore, condi- et al. 2014). If such random foraging occurs between species tions that drive selection for traits that enhance pollinator as well, these ericas would experience at least low levels of efectiveness (e.g. a fower morphology that increases pol- heterospecifc pollen transfer. len placement accuracy or fower constancy) may also, as a Here, we test whether coexistence of bird-pollinated by-product, strengthen pre-pollination barriers (Hopkins and Erica species is enabled through pre-pollination or post- Rausher 2012). Such conditions include pollinator depend- pollination barriers, and whether they experience processes ence. Pollinator dependence results from one or more of an that could drive selection for stronger pre-pollination barri- inability to reproduce (1) asexually, (2) through self pol- ers. We estimate the reduction in potential gene fow through lination (using external vectors) or (3) through autonomous pre-pollination barriers (separation of geographic range, selfng (without an external pollen vector; Lloyd et al. 1992). fowering phenology and reproductive fower parts) and Therefore, an assessment of the conditions that strengthen post-pollination barriers (by assessing seed set from hetero- pre-pollination barriers is also important for understanding specifc pollination relative to outcross pollination). Next, how closely-related species coexist. we use hand-pollination experiments to detect two condi- In the relatively small Cape Floristic Region (CFR, tions (reproductive interference