Research Article |
Corresponding author: Javier Guitián ( javier.guitian@usc.es ) Academic editor: Renate Wesselingh
© 2022 Javier Guitián, Pablo Guitián.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Guitián J, Guitián P (2022) The breeding system of Hyacinthoides non-scripta (Asparagaceae): assessing the role of geitonogamy with captive pollinators. Plant Ecology and Evolution 155(2): 182-188. https://doi.org/10.5091/plecevo.85658
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Background and aims – In self-incompatible species, geitonogamous pollen deposition can result in stigma clogging, with negative effects on cross-fertilisation and subsequently reduced fruit and seed set. In this work, using laboratory and field experiments with pollinators in captivity, we describe the reproductive system of Hyacinthoides non-scripta and analyse the effect of the different mechanisms of pollen transfer, trying to quantify the effect of geitonogamy on the reproductive success of the species.
Material and methods – Using laboratory and field experiments with pollinators in captivity we evaluated the effect of the different types of pollen transfer.
Key results – The results show that H. non-scripta is a self-incompatible species and the pollen tubes in autogamous and geitonogamous pollination are rejected at the base of the style. In the field experiments, the presence of geitonogamous pollen on the stigma did not prevent subsequent fertilization by xenogamous pollen, but it reduced seed production.
Conclusion – This study provides evidence that natural rates of geitonogamy significantly reduce female fitness in plants with large numbers of flowers per inflorescence and show how experiments with captive pollinators can help quantify the intensity of this effect.
geitonogamy, Hyacinthoides non-scripta, pollen tube growth, pollinators in captivity, self-incompatibility
Even though mixed-mating systems are frequent in plants, outcrossing and self-pollination represent two alternative reproductive pathways with important genetic and evolutionary consequences (
In many species, the selfing component of mixed mating may represent a non-adaptive cost associated with the large floral displays required to attract animal pollinators. Rates of geitonogamy are expected to be correlated with the number of flowers per individual. In self-compatible species, significant amounts of selfed seed may arise from inter flower pollen transfer (geitonogamy) and provide little benefit to fitness because of strong inbreeding depression and pollen discounting (
Self-incompatibility is the most important mechanism used to prevent inbreeding. There are two major classes of self-incompatibility at the genetic level: gametophytic self-incompatibility and sporophytic self-incompatibility. In gametophytic self-incompatibility, the incompatibility phenotype of the pollen is determined by its haploid genome, whereas in sporophytic self-incompatibility the pollen exhibits the incompatibility phenotype of its diploid parent. Members of the Brassicaceae, Asteraceae, and Convolvulaceae are well-known examples of the sporophytic type, and those of the Solanaceae, Asparagaceae, Poaceae, and Fabaceae are of the gametophytic type.
Our aim is to describe the breeding system and the consequences of geitonogamy in Hyacinthoides non-scripta, using laboratory and field experiments. Traditionally, studies on the reproductive consequences of geitonogamy are based on manual transfers of pollen from a flower of the same plant, accompanied by emasculation of the focal flowers and subsequent bagging to prevent the arrival of xenogamous pollen (
Specifically, we ask the following questions: (1) Is H. non-scripta a self-incompatible species? If so, (2) what is the effect of the different mechanisms of pollen transfer on the pollen tube growth and reproductive success of the species? Additionally, (3) in a field experiment with captive pollinators, we assess the extent to which the reproductive success of the species is affected by pollinator behaviour (xenogamous versus geitonogamous pollen transfer).
Hyacinthoides non-scripta (L.) Chouard ex Rothm. (Asparagaceae), commonly called English bluebell, is a bulbous perennial that is native to open woodland areas of western Europe. Racemes are unilateral (one-sided) with 4–16 flowers. Flowers are made up of six perianth segments (tepals), usually violet-blue, but rarely white. The flowers are bisexual and contains six creamy-white anthers of unequal length and a persistent style. The ovary is made up of three carpels with about eight ovules each.
Hyacinthoides non-scripta has both sexual and asexual reproduction through the formation of small bulbs (
The study was conducted in the Alto do Couto (1400 m a.s.l.), in the Sierra de O Caurel (Lugo province), located in the NW of the Iberian Peninsula.
In late May and early June 2018, we collected 62 plants in population and moved them to the laboratory where they were kept under controlled temperature (day: 20°C; night: 14°C) and natural light conditions in the absence of pollinators. Plants were watered on alternate days throughout the experiment.
The treatments were:
To calculate reproductive success, 46 plants were used: 15 in the xenogamy treatment, 17 in the geitonogamy treatment, and 14 autogamous controls, with a total of 98, 110, and 83 flowers, respectively; 16 plants died during the experimental period. At the time of fruiting, the fruits, seeds, and ovules were counted per treatment.
We calculated the Self-Compatibility Index (SCI) to describe the breeding system (as in
To assess the occurrence of self-pollination and fertilisation, we calculated the Auto-Fertility Index (AFI) by dividing the seed set for spontaneous autogamy by the seed set for facilitated xenogamy. AFI ranges from zero to above one and gives information about the degree of spontaneous autogamy of the species. Low levels of auto-fertility do not necessarily imply lack of self-pollination under natural conditions (
To examine the pollen tubes, we removed several pistils in each treatment 7–9 days after anthesis. The total number of pistils examined was 71:27 in the geitonogamy treatment, 22 in the xenogamy treatment, and 22 in the autogamous controls. We used the epifluorescence technique with aniline blue. The excised pistils were kept in ethanol (70%) for more than 24 hours after soaking in NaOH for 24 hours and stained with aniline blue (0.1% by 0.1 mol/L K2HPO4) also for 24 hours (
To study the effect of pollen transfer we performed an experiment using bumblebees in captivity. We built a cubic metallic structure (2 m × 2 m × 2 m) covered with tulle, in a plot with plants of H. non-scripta, before the beginning of flowering; all plants inside the structure were labelled with metal labels. The flowers were counted and their position on the inflorescence noted. In the vicinity of plots, we located nests of Bombus pratorum, the most frequent pollinator. Bumblebees were captured with a butterfly net at the exit of the nest. Every morning one bumblebee was released into the tulle structure and recaptured at sunset; the tulle remained closed at night, repeating the process over seven days (total 49 hours of observations). During the day, each visit to a plant and flower and the origin/destination of the bumblebee was recorded. We classified the flowers as having received: (1) a single geitonogamous visit; (2) a single xenogamous visit; (3) several geitonogamous + subsequent xenogamous visits; (4) several xenogamous visits; (5) no visits.
The tulle structure remained in place until fruiting time and fruits were collected and transported to the laboratory, where fruit set and seed-ovule ratios were determined.
Differences in fruit production among pollen transfer treatments were analysed with the nonparametric Kruskal-Wallis test; the Bonferroni correction was applied in pairwise comparisons. Treatment effects in seed production were investigated using a nested ANOVA with Tukey post-test comparisons, with cases (plants) nested in treatments, treatment as a fixed factor and plant within treatment as a random factor. Spearman’s rank correlation coefficient was used to identify and test the strength of the relationship between the number of flowers and the number of geitonogamous visits. The effect of the type of pollen transfer on the seed ovule ratio was analysed using a GLM. All analyses were performed using IBM SPSS (
Plants in the geitonogamy and xenogamy treatments produced fruit, but those in the autogamy (control) treatment did not. Analyses showed significant differences between treatments in fruit set: 0.3 ± 0.28 and 0.07 ± 0.13 (mean ± SD) in the xenogamy and geitonogamy treatments respectively (Kruskal-Wallis; H = 17.87; p = 0.0001) (Fig.
Significant differences were found in seed set among the xenogamy, geitonogamy and autogamy treatments (Nested ANOVA; F = 15.29; p < 0.001). Both geitonogamy and autogamy treatments had similar seed set (p = 1), close to zero (Fig.
The value of the self-incompatibility index (SCI = 0.05) shows that the species is clearly self-incompatible. The value of the auto-fertility index was zero.
In the autogamy (control) treatment, the proportion of pollen tubes that germinated on the style was 0.86 ± 2.83; there were no pollen tubes in the middle and at the base of the style.
All flowers in the samples of geitonogamy and xenogamy treatments showed pollen tube growth. In the geitonogamy treatment, pollen tube growth was inhibited the most in the basal half of the style (Fig.
Fluorescence microscope images of different pollen-pistil interactions in Hyacinthoides non-scripta pollination. A. Pistils without pollen tubes from the autogamy treatment. B–C. Pistils with well-developed tubes from cross-pollination treatment. D. Germinated pollen grains. E. Base of style in cross-pollination treatment. F. Base of style in autogamy treatment showing the progressive elimination of pollen tubes.
In experiments with captive pollinators, the number of visits increased with the number of flowers (Spearman rank correlation 0.48; p = 0.08, marginally significant). Flowers without visits did not produce any fruit.
The seed-ovule ratio depended on the number of xenogamous visits (Table
Effect of number of xenogamous visits in seed ovule ratio of Hyacinthoides non-scripta.
Effect | d.f. | SS | F | p |
Intercept | 1 | 21.940 | 197.465 | 0.000 |
N° of visits | 1 | 0.6050 | 5.45 | 0.021 |
Error | 98 | 10.888 |
Hyacinthoides non-scripta is a species pollinated by Hymenoptera that regularly make visits between flowers on the same plant, transferring self-pollen (geitonogamy). The analysis of the reproductive system in the laboratory and the values of the SCI and AFI indices show that the species is self-incompatible and only manual transfer of outcross pollen produced seeds. This result contrasts with those obtained by
In H. non-scripta, pollen rejection from the same genotype does not occur at the level of the stigma, since a high percentage of pollen grains develop pollen tubes; generally, these are arrested in the basal part of the stigma (gametophytic self-incompatibility, GSI).
In the field experiment with Bombus in captivity, the transfer of geitonogamous pollen in H. non-scripta did not produce seeds. A single visit with self-pollen did not prevent the subsequent fertilisation by xenogamous pollen, but it reduced seed production by a third. Similar results have been obtained in the field with the self-incompatible Polemonium viscosum Nutt., in which self-pollen on a stigma reduced germination of subsequently applied compatible pollen by 32% and reduced seed set by 40% (
In animal-pollinated species like H. non-scripta, the abundance and behaviour of pollinators and their interaction with floral design and display are the primary determinants of the relative frequency of geitonogamous selfing (see
The “plant’s dilemma hypothesis” proposes that evolution of inflorescence size is driven by selection for pollinator attraction but constrained by higher rates of geitonogamy experienced by larger inflorescences (
This study provides evidence that natural rates of geitonogamy significantly reduce female fitness in Hyacinthoides non-scripta plants with large inflorescences and show how experiments with captive pollinators can help quantify the intensity of the effect. Further studies should determine to what extent the population structure and the ecological context influence the balance between geitonogamous selfing and outcrossing and its consequences in natural conditions.
The authors thank Paula Domínguez, Tania Veiga, and Javier Valverde for laboratory and field assistance. The editor’s comments and two anonymous reviewers improved the final version of the manuscript. This work was financed by the Plan Nacional de I+D+I, CGL2009-08959 Ministerio de Ciencia.