Heteranthery, the presence of distinct stamen types within a flower, is commonly explained as functional adaptation to alleviate the ‘pollen dilemma’, defined as the dual and conflicting function of pollen as pollinator food resource and male reproductive agent. A single primary hypothesis, ‘division of labour’, has been central in studies on heteranthery. This hypothesis postulates that one stamen type functions in rewarding pollen-collecting pollinators and the other in reproduction, thereby minimizing pollen loss. Only recently, alternative functions (i.e. staggered pollen release), were proposed, but comparative and experimental investigations are lagging behind.

Here, we use 63 species of the tribe Merianieae (Melastomataceae) to demonstrate that, against theory, heteranthery occurs in flowers offering rewards other than pollen, such as staminal food bodies or nectar. Although shifts in reward type released species from the ‘pollen dilemma’, heteranthery has evolved repeatedly de novo in food-body-rewarding, passerine-pollinated flowers. We use field investigations to show that foraging passerines discriminated between stamen types and removed large stamens more quickly than small stamens. Passerines removed small stamens on separate visits towards the end of flower anthesis. We propose that the staggered increase in nutritive content of small stamens functions to increase chances for outcross-pollen transfer.

Heteranthery across Merianieae:

Here, we extracted information on heteranthery from Dellinger et al. (2019b), who had investigated ethanol-preserved floral material and field images, and coded for general differences in size (i.e. anther length, appendage dimensions), shape (i.e. appendage shape), structure (i.e. thecal wall structure) and colour (as perceived by humans) between stamen whorls (Table S1). We considered species as “strongly dimorphic” when stamen whorls differed in all three parameters; when whorls differed in one or two parameters only (e.g. size), we considered them as “weakly dimorphic”; and when stamen whorls presented no clear differences in any of these parameters, we coded them as “isomorphic”. Please note that none of the investigated species differed in colour only; we may hence rule out erroneous classification as strongly or weakly dimorphic based on human colour vision.

We also extracted information on reward types from Dellinger et al. (2019b), who had performed detailed studies on stamens to reliably determine the reward type. In nectar-rewarding species, nectar is secreted through distinct ruptures on the filaments, visible by eye or using Scanning Electron Microscopy, and nectar droplets are visible when fresh flowers are observed in the field (Dellinger et al. 2019c). In food-body-rewarding species, the foraging passerine birds rip the bulbously inflated (food body reward) stamens out of the flower (Dellinger et al. 2014), so that anthetic flowers are often missing one or several entire stamens. This reward type is hence easily identified when assessing photos, herbarium vouchers or ethanol-preserved material. Finally, pollen-rewarding species are characterized by all stamens being present throughout anthesis, non-bulbous stamen appendages and smooth filaments, lacking conspicuous ruptures (Dellinger et al. 2019b). As a side note, empirical pollinator observations are available for 25 of the 63 Merianieae species studied here (Table S1).

Empirical field dataset:

Given the frequent occurrence of heteranthery in food-body-rewarding flowers (Fig. 1A), we aimed to better resolve the possible function of heteranthery in passerine-pollinated flowers. From previous work, we knew that passerines usually do not remove all ten stamens available per flower during the first visit (Dellinger et al. 2014). We had not investigated, however, whether passerines discriminate between the two stamen whorls and were more likely to remove stamens of one whorl first. Documenting foraging behaviour, however, is crucial to determine possible functional adaptations of heteranthery in non-pollen-rewarding flowers.

We selected two passerine-pollinated species (Axinaea confusa E. Cotton, A. costaricensis Cogn.), to study patterns of stamen removal. We conducted fieldwork in Ecuador (A. confusa, forests around Estación Científica San Francisco, Loja province, September 2016) and Costa Rica (A. costaricensis, forests around Finca Truchas Selva Madre, Alajuela province, March 2020). In both populations, we had monitored pollinators previously, and found different species of passerines (tanagers) as the only pollinators (i.e. capable of activating the bellows mechanism and touching stigmas while foraging, Dellinger et al., in press). From these previous investigations, we know that in A. confusa, 6 (+-4) stamens get removed on the first day of anthesis, 8 (+-2) are removed by the end of the second day or later; in A. costaricensis, 3 (+-3) stamens get removed on the first day of anthesis, 5 (+-3) are removed by the end of the second day or later (Dellinger et al., in press). For the present study, we randomly collected 50 anthetic flowers in the population of A. confusa and 50 anthetic flowers in the population of A. costaricensis. We chose a broad sample of different flower ages, including both young (urceolate corolla) and older (more opened corolla) flowers, and without a priori checking the number of stamens present. For each flower, we then counted how many stamens had been removed per whorl.

The bulbous stamen appendages of passerine-pollinated Merianieae serve as food body reward. To determine whether differences in the caloric content of stamen appendages may cause differences in removal patterns, we collected single stamens of both Axinaea species. Further, we wanted to test whether the caloric content changes across anthesis, and hence separated stamens from young flowers (first day of anthesis) from old flowers (second day or later). From closely monitoring the duration of anthesis (ca. four days) in these species (Dellinger et al., in press), we could easily distinguish young flowers from old flowers by their strongly urceolate corolla with a small opening only, while corollas openings are considerably wider in old flowers. We aimed to collect at least 0.1 g dry weight per stamen whorl. For A. confusa, we could separate stamens into the two distinct stamen whorls, for A. costaricensis, we pooled both whorls and just separated young from old stamens since dry weight of young small stamens was below 0.05g. We microwave-shock-dried stamens (2.5 minutes at 600 watt) to conserve sugars, then slowly dried them in a drying oven overnight at 40° and stored them in silica gel (Dellinger et al. 2014). We then removed the filaments and anthers from the stamens, pulverized the appendages using a mortar and compressed the pulverized appendage material into a pellet. We analysed the six samples in an IKA Calorimeter C 2000 basic version 1 (IKA-Werke GmbH & Co. KG, Germany).

dataset information:

binary_states.csv: binarized states for "reward type" (p) and dimorphism (d)

cor_ciscrete.csv: three factor levels per trait, reward type ("poll" - pollen - 0, food body - 1, nectar - 2) and dimorphism ("het" - none - 0, weak - 1, strong - 2)

het_binary.csv: binarized dimorphism

poll_binary.csv: binarized reward type

Axconf_starem.csv - stamen removal in Axinaea confusa

Axcost_stamenremoval.csv - stamen removal in Axinaea costaricensis

prop2.csv - proportion of viable/unviable pollen for the two Axinaea species as assessed through staining using 1% acetocarmine