Highlighted Student ResearchThe role of distyly in pollen flow of the hummingbird-pollinated Palicourea rigida (Rubiaceae)
Introduction
Heterostyly is a floral polymorphism characterized by the presence of two (distyly) or three (tristyly) floral morphs in the same population of a species (Barrett and Shore, 2008). The most common form, distyly, has been described from 26 angiosperm families (Naiki, 2012). Distylous populations are characterized by individuals producing flowers with short styles and long stamens, the S-morph, and others producing flowers with long styles and short stamens, the L-morph (Bahadur, 1968; Ganders, 1979). The height of the anthers in one morph is the same as that of the stigmas in the other morph, a condition known as reciprocal herkogamy (RH) (Barrett, 2002; Lloyd and Webb, 1992). RH is a mechanism that is thought to facilitate accurate pollen transfer between the sexual organs of different floral morphs (legitimate or disassortative pollination) and decrease gamete loss (Barrett and Shore, 2008; Darwin, 1877). RH also reduces the chances of self-interference since the herkogamy of each morph decreases interference between pollen deposition and removal within the same flower (Barrett and Shore, 2008). Distylous species often have a set of ancillary polymorphisms, such as differences in size and number of pollen grains and size of stigmas between morphs (Dulberger, 1992; Ganders, 1979; Halbritter et al., 2018). In addition to RH and ancillary polymorphisms, distylous species usually present a heteromorphic self-incompatibility system that prevents selfing and intra-morph mating so that fruits and seeds are formed following crosses between different morphs (Barrett, 2002; Barrett and Shore, 2008; Bawa and Beach, 1983). At equilibrium, populations of distylous species are expected to show a 1:1 ratio of floral morphs, a condition known as isoplethy (Pannell et al., 2005). The function of distylous traits to promote compatible pollination between morphs was first proposed by Darwin (1877). Later the disassortative pollination hypothesis was proposed postulating that RH in distylous flowers enhances legitimate pollination, in comparison to pollen transfer within flowers and between flowers of the same morph (illegitimate pollination) (Barrett, 2002; Lau and Bosque, 2003). Ideally, this is realized when pollinators pick up pollen of the S- and L-morph on different parts of their bodies, which then contact the stigmas of the opposite morph (Barrett, 2002; Darwin, 1877; Deschepper et al., 2018). Several studies have pointed out that RH is an important factor in the functioning and evolutionary maintenance of distyly since pollination represents a fundamental step in the reproduction and viability of distylous populations (Darwin, 1877; Ferrero et al., 2011; Hodgins and Barrett, 2006; Lloyd and Webb, 1992).
Floral herkogamy in distylous species is considered a strategy to reduce sexual interference between male and female organs within the same flower, because the presence of the two sexual organs may potentially compromise both maternal and/or paternal fitness (Barrett and Shore, 2008; Cesaro et al., 2004; Lloyd and Yates, 1982; Webb and Lloyd, 1986). Specifically, for distylous flowers, sexual conflict is differentiated between the morphs due to the difference in the arrangement of the organs. For S flowers, female fitness may be negatively affected in two ways: 1) anthers may obstruct pollen deposition on stigma; 2) since the stigma is located below the anthers, the deposition of self-pollen may cause stigma clogging and reduce the growth of pollen tubes. For L flowers, male fitness may be negatively affected in a single way: reduced pollen dispersal when the pistil obstructs access to the anthers (Cesaro et al., 2004; Kohn and Barrett, 1992). Some studies of Palicourea Aubl. (Rubiaceae) show that different floral characteristics, such as a larger corolla-tube entrance width and a greater herkogamy on S flowers, can reduce such problems (Hernández and Ornelas, 2007; Valois-Cuesta et al., 2011). However, for some species of the same genus, like Palicourea rigida Kunth., there is no difference in the width and length of the corolla between the morphs and when there is a difference in herkogamy, it is greater in the L-morph (Machado et al., 2010). Another interesting floral trait in some Rubiaceae species, such as Palicourea rigida, Psychotria deflexa DC. and Psychotria nitidula Cham. & Schltdl., is that the stamens exerted from S flowers are aggregated in the upper portion of the corolla (Furtado et al., 2020; Matias et al., 2016b), which reduces the obstruction in the deposition of pollen in the S stigmas. At the same time, stamens from L flowers are distributed around the pistil (Furtado et al., 2020; Matias et al., 2016b), which avoids physical obstruction of access to the pollen.
Several studies of distylous species have demonstrated deviations from perfect reciprocity, potentially affecting pollen deposition patterns by the action of pollinators (Brys and Jacquemyn, 2019; Deschepper et al., 2018; García-Robledo, 2008; Hernández and Ornelas, 2007; Jacquemyn et al., 2018; Lau and Bosque, 2003; Valois-Cuesta et al., 2011). These studies have revealed asymmetries in the probability of legitimate and illegitimate pollen transfer between morphs and interpreted the results in the context of variations in reciprocity. Recent observations of Palicourea species have shown greater reciprocity in low organs (L anthers and S stigmas) than in high organs (S anthers and L stigmas) (Furtado et al., 2020). These results largely confirm observations of other distylous genera, showing that most of the inaccuracy in reciprocity is due to higher variation in the high organs (Armbruster et al., 2017; Furtado et al., 2020; Jacquemyn et al., 2018; Matias et al., 2020). This greater reciprocity in low organs results in a greater transfer of legitimate pollen compared to high organs (Barrett, 2002; Furtado et al., 2020; Hodgins and Barrett, 2006; Jacquemyn et al., 2018). This reinforces the idea that when reciprocity is greater, the deposition of legitimate pollen grains is also expected to be larger. However, legitimate pollination is not only the result of numerical precision of reciprocity but also of the interaction between the pollinators and floral morphology, as the larger corolla-tube entrance width observed in S flowers of some Palicourea species and different types of pollinators that may vary in size, morphology and behavior, resulting in variable patterns of capture and deposition of pollen grains (Wolfe and Barrett, 1989). Flowers of the distylous Primula secundiflora Franch. (Primulaceae), for example, are visited by nectar-robbing bumblebees and pollen-collecting syrphid flies (Zhu et al., 2015). Bumblebees only pollinate S flowers, and syrphid flies pollinate mostly L flowers, resulting in two kinds of asymmetrical disassortative pollination. Research on the distylous Pulmonaria officinalis L. (Boraginaceae) has shown that its flowers are visited by several insect species, mainly bumblebees, and honey bees, but that the effectiveness of pollination in this species strongly depends on the length of the proboscis (Brys et al., 2008). The efficiency in the capture and deposition of pollen grains on the pollinator's body can have a great impact on pollen transfer patterns and, therefore, on the optimization of function of distylous plants (Deschepper et al., 2018; Wolfe and Barrett, 1989).
Many studies of heterostylous species have suggested that pollen deposition on the two morphs is stratified on different parts of the pollinator's body. However, few studies have provided experimental evidence of this idea (e.g. Buzato et al., 2006; Lopes, 2002; Massinga et al., 2005; Wolfe and Barrett, 1989), especially for hummingbird-pollinated distylous species (Ornelas et al., 2004). Studies that have investigated pollen deposition on pollinator bodies have identified the expected tendency for L and S pollen to be placed on distinct parts of the pollinator's body (see Brys et al., 2008; Deschepper et al., 2018; Keller et al., 2014; Massinga et al., 2005). In Primula elatior (L.) Hill and P. vulgaris Huds., RH promotes the differential placement of pollen on different parts of the proboscis of bees, thus effecting larger deposits of legitimate rather than illegitimate pollen on the stigmas and confirming the key predictions of the disassortative pollination hypothesis (Keller et al., 2014). In Pentanisia prunelloides (Klotzsch ex Eckl. & Zeyh.) Walp. (Rubiaceae), butterflies carried pollen from the S- and L-morphs primarily on the head and proboscis, respectively, promoting legitimate pollen transfer (Massinga et al., 2005). To our knowledge, the function of RH in the deposition of pollen grains on specific parts of the pollinator's body remains to be tested among other groups of pollinators such as hummingbirds, since most studies have investigated pollen deposition among insects pollinators. Differences in pollen size between the L and S flowers of distylous species provides a unique opportunity to examine pollen loads from L- and S-morphs on both pollinators and stigmas, and thus test Darwin's hypothesis that RH promotes mechanical efficiency of pollen transfer in heterostylous plants (Darwin, 1877).
The present study examined a widespread and abundant shrub species of the Neotropical savanna, Palicourea rigida, to investigate the morphological functioning of distyly through the deposition of S and L pollen grains on the pollinator's body and the patterns of legitimate and illegitimate pollen deposition (Darwin, 1877; Lloyd and Webb, 1992). This species constitutes a useful model for this investigation because information on its reproductive biology, such as the presence of two floral morphs, heteromorphic self-incompatibility system, pollen dimorphism, and pollinators, is available in the literature (Furtado et al., 2020; Justino et al., 2012; Machado et al., 2010; Maruyama et al., 2013; Matias et al., 2016b; Silva, 1995). Palicourea rigida has floral traits adapted to pollination by hummingbirds, which have been confirmed as pollinators of this species (Furtado et al., 2020; Justino et al., 2012; Machado et al., 2010; Maruyama et al., 2013; Matias et al., 2016a; Silva, 1995). In the present study, we investigated sexual organ reciprocity, pollen dimorphism, legitimate and illegitimate pollen flow, and pollen deposition on the hummingbird's bill in three populations of P. rigida. Four associated predictions were tested: (1) high and low sexual organs differ in the degree of reciprocity; (2) the reciprocal placement of anthers and stigmas between floral morphs in distylous species promotes more legitimate than illegitimate pollen deposited on stigmas; (3) the sexual organs with greater reciprocity have larger quantities of legitimate pollen deposited on stigmas than those with lower reciprocity; (4) the deposition of L and S pollen occurs in a stratified manner on the hummingbird's bill.
Section snippets
Study areas and species
The study was conducted in 2017 and 2018, during the flowering period of the Palicourea rigida, between September and March. Data were collected in three areas: the Urban Natural Vegetation Area Pasto do Pedrinho in Catalão-GO (hereafter: population A; 18°09’46” S and 47°56’31” W), Pé-do-Morro Farm in Catalão-GO (hereafter: population B; 18°06’51.9” S and 47°59’55.6” W) and IBGE Ecological Reserve in Brasília-DF (hereafter: population C; 15º57’09” S and 47º52’11” W). In all areas, the study was
Floral morphology and reciprocal herkogamy
In general, we found differences in anther and stigma heights, stigma lobe length, and herkogamy between morphs (Table 1); however, at the population level, both morphs showed similar levels of herkogamy within the population C (Table 2). There were no differences in the parameters of each sexual organ between populations, except for herkogamy, and only for anther height and herkogamy there was a significant morph * population interaction (Table 1). Total inaccuracy in populations varied
Discussion
In our study, we investigated the functioning of distyly in Palicourea rigida populations, analyzing reciprocal herkogamy, pollen flow and pollen deposition on the hummingbird's bill. Specifically, we tested the predictions that: (1) the high and low sexual organs differ in the degree of reciprocity. Our results showed that low organs were more reciprocal in all three populations studied (Table 3); (2) RH promotes more legitimate (than illegitimate) pollen deposit on stigmas. Our results
Conclusion
We found greater reciprocity in low organs, which helped to explain their larger legitimate pollen flow compared to less reciprocal high organs. These findings indicate that small changes in the level of reciprocity can lead to differences in pollen flow. In all three populations, the amount of legitimate pollen deposited on stigmas was larger than the amount of illegitimate pollen. This suggested that the reciprocal arrangement of stigmas and anthers promotes legitimate pollen flow, even with
CRediT authorship contribution statement
Paola Pisetta Raupp: Investigation, Methodology, Formal analysis, Writing - original draft. Raphael Matias: Conceptualization, Writing - review & editing, Methodology, Supervision. Marco Túlio Furtado: Writing - review & editing, Formal analysis. Hélder Consolaro: Conceptualization, Methodology, Writing - review & editing, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors wish to thank the people from the Reproductive Biology Lab at the Federal University of Catalão for the incentive and support in the development of the study, Paulo Eugênio Oliveira for his relevant considerations, Miguel Marini for the loan of the hummingbird, and Lorena de Paula by the hummingbirds’ illustration in Fig. 3. We also thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financing the Scientific Initiation Scholarship of P.P. Raupp
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