Beautiful, complicated—and intelligent? Novel aspects of the thigmonastic stamen movement in Loasaceae

Abstract

In a recent study we investigated the complex mechanisms regulating the pollen release via thigmonastic stamen movement found exclusively in Loasaceae subfamily Loasoideae. We demonstrated that stamen movement is modulated by abiotic (light and temperature) as well as biotic stimuli (pollinator availability and visitation frequency). This is explained as a mechanism to adjust the rate of stamen movement and thus pollen dispensation to different environmental conditions in order to optimize pollen transfer. Stamen movement is rapid and thus a near-immediate response to pollinator visits. However, Loasaceae flowers also show a response to biotic stimuli on a longer time scale, by adjusting the duration of both the staminate and the carpellate phase of the anthesis. We here present two additional data sets on species not previously studied, underscoring the shortening of the staminate phase in the presence of pollinator visits vs. their absence and the shortening of the carpellate phase after pollination. Overall, the plant shows not only a rapid but an “intelligent” reaction to its environment in adjusting anthesis and pollen presentation to a range of factors. The physiological and morphological bases of the stamen movement are poorly understood. Our previous study showed that there is no direct spatial relationship between the place of stimulation in the flower and the stamen bundle activated. We here further show the morphological basis for stamen movement from a reflexed into an erect position: Only the basal part of the filament curves around the receptacle, while the upper part of the filament retains its shape. We hypothesize that the stimulus is transmitted over the entire receptacle and the place of reaction is determined by stamen maturity, not the location of the stimulus.

Stamen Presentation in Loasaceae

A variety of mechanisms have been described to control pollen dispensation schedules in angiosperms in order to optimize male function and increase cross pollination.^1-3 The complex mechanisms found in Loasaceae subfam. Loasoideae are an extreme example of the control plants can exert over pollen dispensation. Most species of Nasa, the largest genus of Loasoideae (c. 130 taxa), are found in the High Andes of South America where successful reproduction via a crossbred offspring is a challenging task due to mostly small populations scattered over often small and fragmented habitats.^4-7 A whole series of mechanisms ensures controlled pollen dispensation and increases the likelihood of cross pollination. In a recently published study we demonstrate the effect of different abiotic factors and simulated pollinator visitation rates over pollen presentation.⁸ The anthesis of individual flowers begins with the staminate phase. The numerous stamens are organized in five stamen bundles that are initially reflexed into the spreading, boat-shaped petals (Fig. 1A–D).^9-11 Initially, the anthers are immature and closed, maturation takes place sequentially, beginning with the stamens that are facing towards the center of the flower (Fig. 1E). Pollen viability is known to be limited,¹² and pollination success thus also depends on the presentation of fresh, viable pollen. As a consequence, the anthers of Loasaceae open only shortly before the stamen is supposed to move or during stamen movement. Once stamen movement is initiated, the stamens quickly (within 1–2 min) perform a movement of 90–120° from inside the petals to the center of the flower (Fig. 1A, B and F). The stamen movement of an individual flower has a minimum rate in the absence of pollinator visits. This “autonomous stamen movement” is dependent on light and temperature.⁸ The stamen movement rates can be dramatically accelerated if pollinators are abundant and interact with the plant. When harvesting nectar, the flower visitors have to manipulate specialized floral structures, so called nectar scales (Fig. 1A), by bending them outwards to access the nectar. The mechanical stimulus of this movement initiates the (thigmonastic) stamen movement. The rate of thigmonastic movement is positively correlated with the frequency of the pollinator visits. And since only mature anthers move and present their pollen in the center of the flower, anther maturation is accelerated or slowed down accordingly. After the end of the staminate phase, i.e., when all stamens of a flower have moved into the center, the style immediately starts to elongate and the stigma becomes receptive within hours after the last stamen movement (Fig. 1F). After successful pollination petals and androecium rapidly wilt and are shed. Individual floral longevity in Nasa macrothyrsa varies between less than two to more than 10 d, depending on the number of flower visits and successful pollination. Most experiments were undertaken with N. macrothyrsa (Urb. and Gilg) Weigend (Fig. 1A), a perennial, shrubby species that serves as our model-organism studying floral ecology in Loasaceae.^13,14 N. macrothyrsa is, however, not a typical species of Loasaceae due to its perennial life-history and shrubby habit. Like many perennial plants, N. macrothyrsa is predominantly outcrossing, so that optimized pollen presentation is crucial.^15-17 If kept under pollinator exclusion and unmanipulated, no fruit-set could be observed (n = 30 flowers, all abortive).¹⁴ Annual plant taxa tend to be (facultative) self-pollinating as a backup mechanism if cross-pollination is not achieved.^18,19

Figure 1. Flowers and floral morphology of Loasaceae. (A) Longitudinal section through a flower of Nasa macrothyrsa with important floral structures labeled. (B) Flower of Nasa poissoniana, note the stamens that are performing a movement from inside the petals towards the center of the flower (arrows). (C) Flower of Nasa dyeri ssp. australis. (D–F) Flower of Nasa poissoniana, note the gross similarities in floral display between these two species. White petals and contrasting yellow-red colored nectar scales is the typical coloration of insect-pollinated taxa of Loasaceae subfam. Loasoideae. (E) Petals removed to get a better view of the five stamen bundles. Note the smaller, mature anthers on top of each bundle, immature anthers (thicker) underneath. (F) Center of the flower at the beginning of the female (carpellate) phase. Note the accumulated stamens that, in absence of pollinators, still carry pollen grains. Some grains are visible, attached to the style that grew through the stamen bundle.

Selfing and Pollen Presentation in Annual Taxa

No data had so far been presented on the numerous annual species of Nasa, so that it is neither known whether they are selfing, nor whether they have the same type of pollen presentation mechanism. Here we present additional data sets from two annual taxa to demonstrate the high degree of selfing and the presence of the same type of pollen presentation mechanism as in perennial N. macrothyrsa, representing a double strategy, ensuring pollination in the absence of pollinators, but making optimal use of pollinators if present. Successful seed production is particularly important for the large proportion of annual taxa (43 of 130 taxa), whose populations would face immediate extinction if reproduction failed. There are no data on pollinator abundance in the respective habitats, but it can be assumed to vary significantly with fluctuating weather conditions, additionally reducing the likelihood of flower visitation. Most importantly, the annual taxa of Nasa are generally colonizers of disturbed sites,^20,21 and require selfing ability for reproductive assurance and build-up of new populations. Our data confirm that the annual species Nasa dyeri (Urb. and Gilg) Weigend ssp. australis Dostert and Weigend,²⁰ (Fig. 1C) and Nasa poissoniana (Urb. and Gilg) Weigend,²¹ (Fig. 1B, D–F) are facultatively selfing. If left unmanipulated under pollinator exclusion (greenhouse experiments) all flowers set fruit (N. dyeri ssp. australis: n = 10 plants, N. poissoniana: n = 5 plants). In these taxa, the two phases of the anthesis vary profoundly if tested under different pollination scenarios (Fig. 2, Table. 1). The staminate phase in N. dyeri ssp. australis has an average duration of 3.4 d (± 0.51 d, n = 19) if pollinators are absent. By imitating pollinator visits at a regular interval (three daily visits, 10:00 a.m., 2:00 p.m. and 6:00 p.m.) the staminate phase is significantly condensed to 2.7 d (± 0.42 d, n = 21). In this experiment, the stamens were allowed to move freely and accumulate in the center of the flower (Fig. 1F). Self-compatible N. dyeri ssp. australis self-pollinates once the style grows through the bundle of stamens presented in the center of the flower unless pollen has been depleted by previous pollinator visits. This, and likely all florally similar species, thus perform mid-anthetic self-pollination in the absence of pollinators, when pollen is not removed in the staminate phase. Conversely, collection of pollen by flower visitors in Loasaceae is known to be very efficient and the flowers are kept virtually empty of pollen in the presence of pollinators,²² thus eliminating the possibility of mid-anthetic selfing. In the presence of pollinators, the male phase is thus shortened and pollen export per unit time maximized. In the absence of pollinators, the duration of the staminate phase is extended, to increase the likelihood of pollen export. The same mechanism is at work in the carpellate phase: The staminate phase in N. poissoniana lasts 5.5 d (± 0.98 d, n = 61) in the absence of pollinators. In all flowers, the anthers of recently opened flowers were carefully cut off so that no pollen was left in the flowers. If hand-pollinated when the stigma becomes receptive, floral organs are shed 2.4 d (± 0.55, n = 35) after pollination. If left unmanipulated, the flowers maintained floral display for 8.2 d (± 0.98, n = 26). Thus the carpellate phase is cut short as soon as pollination has taken place, but can be extended dramatically in order to increase the likelihood of pollination in the absence of pollinators (Fig. 2; Table 1). These features may act in concert with the mechanisms discussed earlier,⁸ to support the radiation of Loasaceae. Selfing is known as a strategy for colonizing weedy plants to establish new subpopulations prior to the successful recruitment of possible pollinators.^23-25 The high degree of control over pollen dispensation in the presence of pollinators (increasing the likelihood of cross-pollination) in combination with facultative selfing likely contributed to the diversification of Nasa as colonizers in disturbed habitats, providing a maximum of colonizing ability while at the same time maintaining high levels of outcrossing, where possible.

Figure 2. Diagram of the length of the anthesis depending on simulated pollinator activity. Only three visits per day significantly reduce the length of the staminate phase in Nasa dyeri ssp. australis (lower bars). The absence of pollinators during the carpellate phases leaded to its extension by more than 300% in Nasa poissoniana (upper bars). Error bars provide SD, only one direction illustrated.

Table 1. Summarized data on the detailed length of the anthesis depending on pollinator activity.

Species	Anthers removed	Treatment	N	Duration of male (staminate) phase in days ± SD		Duration of female (carpellate) phase in days ± SD
Nasa dyeri ssp. australis	no	pollinator visits imitated 3 x per day	21	2.7 ± 0.42	p < 0.001	0.4 ± 0.15	p = 0.21
Nasa dyeri ssp. australis	no	control, unmanipulated	19	3.4 ± 0.51		0.34 ± 0.1
Nasa poissoniana	yes	hand-pollinated when stigma receptive	35			2.37 ± 0.55	p < 0.001
Nasa poissoniana	yes	control, unmanipulated	26	5.5 ± 0.98		8.19 ± 0.98

P-values calculated by t-tests

Mechanism of Movement

The descriptive and functional framework for stamen movement has thus been established, but the morphological and physiological details are largely unknown. Loasoideae seem an ideal model organism to study many aspects of “plant signaling and behavior,” since the individual flower can be repeatedly used for a single experiment and the reaction time and speed of the movement are amenable to experimentation (< 5 min). These plants are thus ideally suited to studying the physiological processes connected with the thigmonastic stamen movement. This movement is exclusively found in the subfamily Loasoideae and there is evidence in the literature that its development was phylogenetically preceded by the development of striking girdling vascular bundles in the receptacle,²⁶ and accompanied by profound changes in the complexity and spatial arrangement of these vascular bundles. A detailed analysis of floral morphology of Loasaceae,¹¹ revealed different types of vascularization of the floral organs between the derived subfamily Loasaceae with thigmonastic stamens and its non-thigmonastic relatives from the subfamily Mentzelioideae. The stamen bundles of Mentzelioideae are vascularized by a single bundle that branches from a trunk that leads to the ovary. The stamens are here all supplied by one bundle that fans out from the innermost stamen to the outer ones and not vascularized individually. In derived Loasoideae, the stamens and staminodial complexes (nectar scales) are directly connected to the vascular girdling bundle (Fig. 3) and are all individually vascularized by strands that branch directly from the androecial vessel ring (see figures 2 and 3 in ref.11). “The androecial vasculature in the Loasoideae is fused to the vascular ring at the top of the ovary and thus there is no “inner” or “outer” but only points along a circumference.”¹¹ So there is strong evidence, that the basis for the complex floral behavior in Loasaceae subfam. Loasoideae is an increased synorganization of receptacular vasculature and a more direct signal transmission between the floral organs.

Figure 3. Longitudinal sections through a flower bud of Nasa vargasii (Nasa poissoniana group, close ally of the species investigated with identical flower morphology). (A) Section through the basis of a stamen bundle. (B) Section through the staminodial complex. Note the girdling bundle (gb, reddish-violet) at the base of both structures. St, stamen; a, anthers; p, petal; ns, nectar/floral scale; fs, free inner staminode; sp, sepal.

Vascular bundles are known to play an important role in the transduction of electric signals in plants.²⁷ The fact that the structures where the stimulus is detected (nectar scales) and those that answer it (stamen bundles) are lined-up on a ring of vascular bundles suggests a direct physiological connection via the vascular ring. We could prove that the stimulus is transmitted to all stamen bundles, regardless of which or how many of the five nectar scales are stimulated.⁸ Hence there must be a measurable electric signal along this vascular ring which, due to the flower size of Loasoideae, has to cover quite a distance. Depending on the frequency of pollinator visits, the stamen movement can be triggered in a very short sequence. And due to the high number of stamens (typically 80 to > 250), signaling quality must be maintained accordingly. In contrast to other plant species with similar thigmo-responses (e.g., Dionaea muscipula L,²⁸ Portulaca grandiflora Hook²⁹), the signaling must therefore not only be quickly but also completely physiologically repeatable without any losses in quantity and quality. Otherwise, the complex behavioral adaptations between the plants and their pollinators would be pointless. Different types of electric signaling are known in higher plants. Besides common action potentials (APs), variation (VPs) or slow wave potentials (SWPs) occur.³⁰ The latter are unique to plants and both use the xylem in the vascular bundles for propagation.³¹ Plants can use these potentials to react on biotic stimuli, e.g., the trap mechanisms of Aldrovanda vesiculosa L. and Dionaea muscipula,³² (for these and other examples see ref.33). But it has also been reported that abiotic stimuli are processed in this way.^32,34,35 Additionally, electric signals have been observed in the context of pollination.^36,37Brenner³¹ stated: “An alternative definition of plant intelligence is an intrinsic ability to process information from both abiotic and biotic stimuli that allows optimal decisions about future activities in a given environment.” Loasaceae are thus highly suitable research objects for “plant intelligence,” since we could demonstrate how abiotic and biotic factors modulate floral performance and thus plant behavior.⁸

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.