DOI: 10.5061/dryad.gxd2547m1
title: Stamen dimorphism in bird-pollinated flowers – investigating alternative hypotheses on the evolution of heteranthery

description: 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.
description: 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 (<i>Axinaea confusa
 </i>E. Cotton, <i>A. costaricensis </i>Cogn.), to study
 patterns of stamen removal. We conducted fieldwork in Ecuador (<i>A.
 confusa</i>, forests around Estación Científica San Francisco, Loja
 province, September 2016) and Costa Rica (<i>A.
 costaricensis</i>, 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.,
 <i>in press</i>). From these previous investigations, we know
 that in <i>A. confusa</i>, 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 <i>A. costaricensis</i>, 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., <i>in press</i>). For the
 present study, we randomly collected 50 anthetic flowers in the population
 of <i>A. confusa</i> and 50 anthetic flowers in the population
 of <i>A. costaricensis</i>. 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
 <i>Axinaea</i> 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., <i>in press</i>), 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 <i>A. confusa</i>, we could
 separate stamens into the two distinct stamen whorls, for <i>A.
 costaricensis</i>, 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).
description: 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