The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Discovery of distyly in Narcissus (Amaryllidaceae)

There has been disagreement concerning the nature of sexual polymorphisms in Narcissus, a genus of insect-pollinated geophytes native to the Mediterranean. The existence of trimorphic heterostyly in the genus has recently been confirmed, but the occurrence of distyly remains enigmatic. All sexually dimorphic species previously investigated possess two distinct style lengths but anthers of similar height. Stigma-height dimorphism does not qualify as true distyly because of the absence of a reciprocal correspondence in stigma and anther position in the floral morphs. Such reciprocal herkogamy is generally regarded as the defining feature of heterostyly. Here we report on distyly in N. albimarginatus (section Apodanthae), a rare species confined to a single mountain in northwestern Morocco. A population composed of equal numbers of long- and short-styled plants exhibited reciprocal herkogamy with lower anthers of the long-styled morph and upper anthers of the short-styled morph corresponding in height to stigmas of short- and long-styled plants, respectively. The presence of both stigma-height dimorphism and distyly in Narcissus is of general significance to theoretical models of the evolution of heterostyly.     

Ovarian self‐incompatibility in Narcissus papyraceus (Amaryllidaceae) is the result of a pre‐zygotic response

Ovarian self‐incompatibility, including pre‐ and post‐zygotic reactions, is a complex mechanism for which we still lack many details relating to its function and significance. The joint presence of ovarian self‐incompatibility with style polymorphism is a rare combination that is found in the genus Narcissus. Usually, style polymorphic species have heteromorphic (diallelic and linked to style length locus) incompatibility, which prevents fertilization between individuals of the same morph, thereby helping to maintain equal proportions of floral morphs in populations. However, when present, self‐incompatibility in Narcissus is not linked to style polymorphism and cross‐fertilization within each morph is possible. Hence, self‐incompatibility in Narcissus is of particular interest when attempting to unravel the nature of the rejection reaction and aiming to assess possible cryptic differences in the fertilization process in intra‐ and inter‐morph crosses, which might ultimately explain the wide variation of morph‐ratio in the field. We examined the breeding system of Narcissus papyraceus, a style‐dimorphic species that has biased morph ratios in most of its populations. We studied pollen‐tube growth in the pistil and ovule fate after experimentally controlled hand pollinations. The growth of pollen tubes in self‐ and intra‐ and inter‐morph crosses was similar up to the point of micropyle penetration in both morphs but, subsequently, a pre‐zygotic failure appeared to affect male and female gametophytes in selfed pistils. A high proportion of ovules from self‐pollinated flowers showed signs of collapse and self‐pollen tubes were blocked or behaved abnormally before entering the embryo sac. Self‐incompatibility was stronger in the long‐styled morph than in the short‐styled morph. We did not find any conclusive sign of differential functioning between intra‐ and inter‐morph cross‐pollinations in any morph. These results enable us to rule out the possible effects of pollen–pistil interactions in N. papyraceus as a cause of morph‐ratio biases and confirm the exceptional nature of the self‐incompatibility mechanism in this polymorphic species. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 629–643.     

Pollination and late-acting self-incompatibility in Cyrtanthus breviflorus (Amaryllidaceae): implications for seed production

Background and Aims

Animal pollination is typically an uncertain process that interacts with self-incompatibility status to determine reproductive success. Seed set is often pollen-limited, but species with late-acting self-incompatibility (SI) may be particularly vulnerable, if self-pollen deposition results in ovule discounting. Pollination is examined and the occurrence of late-acting SI and ovule discounting assessed in Cyrtanthus breviflorus.

Methods

The pollination system was characterized by observing floral visitors and assessing nectar production and spectral reflectance of flowers. To assess late-acting SI and ovule discounting, growth of self- and cross-pollen tubes, and seed set following open pollination or hand pollination with varying proportions of self- and cross-pollen, were examined.

Key Results

Native honeybees Apis mellifera scutellata pollinated flowers as they actively collected pollen. Most flowers (≥70 %) did not contain nectar, while the rest produced minute volumes of dilute nectar. The flowers which are yellow to humans are visually conspicuous to bees with a strong contrast between UV-reflecting tepals and UV-absorbing anthers and pollen. Plants were self-incompatible, but self-rejection was late-acting and both self- and cross-pollen tubes penetrated ovules. Seed set of open-pollinated flowers was pollen-limited, despite pollen deposition exceeding ovule number by 6-fold. Open-pollinated seed set was similar to that of the cross + self-pollen treatment, but was less than that of the cross-pollen-only treatment.

Conclusions

Flowers of C. breviflorus are pollinated primarily by pollen-collecting bees and possess a late-acting SI system, previously unknown in this clade of the Amaryllidaceae. Pollinators of C. breviflorus deposit mixtures of cross- and self-pollen and, because SI is late-acting, self-pollen disables ovules, reducing female fertility. This study thus contributes to growing evidence that seed production in plants with late-acting SI systems is frequently limited by pollen quality, even when pollinators are abundant.     

Reorientation of daffodil(Narcissus: Amaryllidaceae) flowers inwind: drag reduction andtorsional flexibility

Daffodil flowers extend laterally from the long axes of their stems; as a result, wind on a flower exerts torsional as well as flexural stress on the stem. Stems respond by twisting, and thus flowers reorient to face downwind in moderate winds, in the process reducing their drag by ～30%. This repositioning is facilitated by the stems' relatively low torsional stiffness. Daffodil stems have a ratio of flexural to torsional stiffness of 13.27 ± 0.96 (SD), compared with 8.33 ± 3.20 (SD) for tulip stems, which bear flowers as symmetrical extensions of their long axes, and compared with 1.5 for isotropic, incompressible, circular cylinders.     

Evidence for ovarian self-incompatibility as a cause of self-sterility in the relictual woody angiosperm,Pseudowintera axillaris (Winteraceae)

Species within the genus Pseudowintera exhibit high rates of self-sterility. Self-sterility in the genus has been previously posited-but not confirmed-to be the result of late-acting ovarian self-incompatibility (OSI) functioning within nucellar tissue of the ovule to prevent self pollen tubes from entering the embryo sac. Structural and functional aspects of pollen-carpel interactions and early seed development following cross- and self-pollination were investigated in P. axillaris to determine the site, timing and possible mechanisms of self-sterility. No significant differences were observed between pollen tube growth, ovule penetration and double fertilization following cross- and self-pollination. Pollen tubes exhibited phasic growth in an extracellular matrix composed of proteins and carbohydrates, as well as arabinogalactans/arabinogalactan proteins. A uniform failure in embryo sac development prior to division of the zygote was apparent within 15 d following double fertilization by self gametes. Results indicate that SI mechanisms in P. axillaris do not prevent double fertilization from occurring. Instead, mechanisms of self-sterility affect post-zygotic development of the embryo sac. Although self-sterility may be attributed to inbreeding depression, given the post-zygotic nature of failure in embryo sac development, the possibility of late-acting OSI is discussed.     

Early ovule development following self- and cross-pollinations in Eucalyptus globulus Labill. ssp. globulus

The study was conducted to identify the self-incompatibility mechanism in Eucalyptus globulus ssp. globulus. Controlled self- and cross-pollinations were conducted on individual flowers from three mature trees that had self-incompatibility levels of 76, 99.6 and 100%. Flowers were harvested at 4, 6 and 8 weeks after pollination. Embryology was investigated by bright field microscopy on material harvested at 4 and 6 weeks after pollination. Fertilization had taken place at 4 weeks after pollination with zygotes and free nuclear endosperm visible. There was a greater proportion of healthy, fertilized ovules in the cross- compared with the self-pollination treatment, and approx. half the ovules examined from both pollen treatments were not fertilized or were degenerating. By 6 weeks after pollination a few zygotes were starting to divide. The number of healthy, fertilized ovules was still greater in the cross-pollination treatment, but the number of healthy fertilized ovules was lower in both treatments compared with 4 weeks after pollination, and many ovules were degenerating. Fertilized ovules were significantly larger than non-fertilized or degenerating ovules and this difference was detectable by eye at 6 and 8 weeks after pollination. The mechanism of self-incompatibility appears to have both late pre- and post-zygotic components.     

Variation of pollen limitation in the early flowering Mediterranean geophyte Narcissus assoanus (Amaryllidaceae)

A recent literature review indicates that pollen limitation of female fertility is a common feature of flowering plants. Despite the ecological and evolutionary significance of pollen limitation, most studies have only examined fertility in a single population at one time. Here we investigate pollen limitation of fruit and seed set in five populations of Narcissus assoanus, a self- sterile, insect-pollinated geophyte, over 2–3 years in southern France. In common with many early spring flowering plants, pollinator visitation to N. assoanus is often infrequent. Supplemental hand-pollination of flowers with outcross pollen significantly increased overall fruit and seed set by 11% and 19%, respectively. Four of the five populations experienced some pollen limitation during the study. For a given year, there was significant variation in pollen limitation among populations. Two of the populations were pollen limited in one year but not in other years in which they were studied. Seed:ovule ratios for open- and hand-pollinated flowers averaged 0.29 and 0.33, respectively. While hand pollination significantly increased the seed:ovule ratio, the low value obtained indicates that the majority of ovules in flowers do not mature seeds despite hand pollination. The role of genetic and environmental factors governing low seed:ovule ratios in N. assoanus is discussed. Received: 28 December 1999 / Accepted: 6 April 2000     

The Causes of self-sterility in natural populations of the relictual angiosperm, Illicium floridanum (Illiciaceae)

BACKGROUND AND AIMS: Illicium floridanum, a species belonging to the basal extant angiosperm taxon Illiciaceae, reportedly exhibits self-incompatibility (SI). To date, the site and timing of SI within the carpel of this species remains unidentified. Thus, the objective of this research was to determine the cellular and temporal aspects of SI in I. floridanum. METHODS: Following controlled application of cross- and self-pollen in natural populations of I. floridanum, embryo sac development and temporal aspects of stigma receptivity, as well as pollen tube growth, fertilization, and embryo and endosperm development, were investigated with the aid of light and fluorescence microscopy. KEY RESULTS: Flowers of I. floridanum exhibited complete dichogamy whereby stigmas only supported cross- and self-pollen tube growth prior to anther dehiscence. In contrast to earlier reports of SI in this species, a prezygotic SI resulting in rejection of self-pollen tube growth at the stigma was absent and there were no significant differences between cross- versus self-pollen germination and pollen tube growth within the style and ovary during the first 5 d after pollination. Structural development of the four-celled embryo sac was not differentially influenced by pollen type as noted to occur in other angiosperms with late-acting ovarian SI. The ovule micropyle and embryo sac were penetrated equally by cross- and self-pollen tubes. In addition, there were no statistically significant differences in cross- versus self-fertilization. A resting zygote and multicellular endosperm at a variety of developmental stages was present by 30 d after application of cross- or self-pollen. CONCLUSIONS: In the clear absence of a prezygotic SI that was previously reported to result in differential self-pollen tube growth at the stigma, self- sterility in I. floridanum is likely due to early-acting inbreeding depression, although late-acting post-zygotic ovarian SI cannot be ruled out.     

Post-pollination Events in Species of Chorisia (Bombacaceae) and Tabebuia (Bignoniaceae) with Late-acting Self-incompatibility

The neotropical trees Chorisia chodatii, C. speciosa, Tabebuia caraiba and T. ochracea show late-acting self-incompatibility. Accumulated ovule penetrations following self-pollinations were similar to those in crosses. During the six to eight day period following pollination, in both selfed and crossed ovules, a resting zygote with initial development of the endosperm was formed. Up until the time of rejection of the selfed flowers, no cytological malfunctions were evident in the selfed ovules.     

Organogenesis in plants: the molecular and genetic control of ovule development

The ovule is the site of megagametogenesis and fertilization and hence is of central importance to sexual reproduction in seed plants. In recent years, the ovule has become established as an excellent model system in which to study organogenesis. Progress has been made in several aspects of ovule development: the control of ovule identity, the mechanism of primordium outgrowth, early pattern formation and the regulation of integument morphogenesis.     

Histological study of post-pollination events in Spathodea campanulata beauv. (Bignoniaceae), a species with late-acting self-incompatibility

The reproductive biology of Spathodea campanulata was investigated by means of hand-pollination experiments, observations of pollen tube growth using fluorescence microscopy, and serial sections of ovules in selfed and crossed pistils. Only cross-pollinated flowers developed fruits, and all selfed flowers abscised within 3-4 d. However, self pollen tubes grew successfully to the ovary, penetrating and fertilizing the majority of ovules by 48 h, indicating that S. campanulata is a species with late-acting self-incompatibility. The incidences of ovule penetration, fertilization and endosperm initiation were all significantly slower in selfed vs. crossed pistils, although no other signs of malfunctioning were detected. The possible role of such slow self pollen tube effectiveness as a recognition event is discussed within the context of the slow but not entirely suppressed self pollen tube growth reported for some species with conventional homomorphic self-incompatibility.     

Expression and Possible Role of S4(SSM4) Genes in the Styles of Self-incompatible Pear Cultivar and Its Self-compatible Mutant

Stylar soluble proteins in self-incompatible “Nijisseiki” (S2S4), self-compatible “Osa-Nijisseiki” ( S2SSM4, SM means stylar-part mutant) and its progeny were analyzed by isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE).SSM4-allele associated protein, SSM4-protein, existed in the style of “Osa-Nijisseiki” and its progeny. The SSM4-protein expressed only in the stigma of “Osa-Nijisseiki”, whereas in its original variety “Nijisseiki”, S4-protein expressed in the upper and lower parts of the style as well as in the stigma, and its expression amount decreased from the upper part to the lower part. The protein bands analyzed by IEF-PAGE were subjected to RNase activity staining. The results showed that the S4- and the SSM4-proteins have the similar molecular weights (approximately 30 kD) and RNase activity. The specific-activities measured with yeast RNA were similar, equivalent to approximately 275 U·min-1·mg-1 protein. The S SM4-protein showed almost the same inhibitory effects as the S4-protein on the pollen germination and the pollen tube growth with S4- and SSM4-alleles in vitro . From the above results, the reasons of the self-compatibility of “Osa-Nijisseiki” are considered as (1) low expression of the SSM4-gene and (2) the SSM4-gene expression only in stigma.     

Variation of pollen and ovule parameters among different ploidy levels ofCorydalis (Fumariaceae)

The size and number of pollen grains and ovules are compared between 20 populations of different ploidy levels in two self-incompatible species ofCorydalis (Fumariaceae), to test the presence of ploidy-related variation in these reproductive characters. In both species, higher ploidy levels are associated with larger pollen grains, ovules and corolla, but the number of pollen grains and ovules are not different between ploidy levels. The investment per flower is consequently larger at higher ploidy levels, but the flower number per individual is lower, suggesting that the mode of partition of the investment for sexual reproduction varies between different ploidy levels. InC. orthoceras, sex allocation estimated by pollen:ovule ratios in number and volume is more female-biased in polyploids than in diploids. In spite of these variations, the characters studied can not be used as indicators of ploidy level due to the large overlaps between the ploidy levels.     

Physiology, morphology and phenology of seed dormancy break and germination in the endemic Iberian species Narcissus hispanicus (Amaryllidaceae)

Background and Aims

Only very few studies have been carried out on seed dormancy/germination in the large monocot genus Narcissus. A primary aim of this study was to determine the kind of seed dormancy in Narcissus hispanicus and relate the dormancy breaking and germination requirements to the field situation.

Methods

Embryo growth, radicle emergence and shoot growth were studied by subjecting seeds with and without an emerged radicle to different periods of warm, cold or warm plus cold in natural temperatures outdoors and under controlled laboratory conditions.

Key Results

Mean embryo length in fresh seeds was approx. 1·31 mm, and embryos had to grow to 2·21 mm before radicle emergence. Embryos grew to full size and seeds germinated (radicles emerged) when they were warm stratified for 90 d and then incubated at cool temperatures for 30 d. However, the embryos grew only a little and no seeds germinated when they were incubated at 9/5, 10 or 15/4 °C for 30 d following a moist cold pre-treatment at 5, 9/5 or 10 °C. In the natural habitat of N. hispanicus, seeds are dispersed in late May, the embryo elongates in autumn and radicles emerge (seeds germinate) in early November; however, if the seeds are exposed to low temperatures before embryo growth is completed, they re-enter dormancy (secondary dormancy). The shoot does not emerge until March, after germinated seeds are cold stratified in winter.

Conclusion

Seeds of N. hispanicus have deep simple epicotyl morphophysiological dormancy (MPD), with the dormancy formula C_1bB(root) – C₃(epicotyl). This is the first study on seeds with simple MPD to show that embryos in advanced stages of growth can re-enter dormancy (secondary dormancy).     

Genome size and base composition variation in natural and experimental Narcissus (Amaryllidaceae) hybrids

Background and Aims

Although there is evidence that both allopolyploid and homoploid hybridization lead to rapid genomic changes, much less is known about hybrids from parents with different basic numbers without further chromosome doubling. Two natural hybrids, Narcissus × alentejanus (2n = 19) and N. × perezlarae (2n = 29), originated by one progenitor (N. cavanillesii, 2n = 28) and two others (N. serotinus, 2n = 10 and N. miniatus, 2n = 30, respectively) allow us to study how DNA content and composition varies in such hybrids.

Methods

Flow cytometry measurements with two staining techniques, PI and DAPI, were used to estimate 2C values and base composition (AT/GC ratio) in 390 samples from 54 wild populations of the two natural hybrids and their parental species. In addition, 20 synthetic F₁ hybrid individuals were also studied for comparison.

Key Results

Natural hybrids presented 2C values intermediate between those found in their parental species, although intra-population variance was very high in both hybrids, particularly for PI. Genome size estimated from DAPI was higher in synthetic hybrids than in hybrids from natural populations. In addition, differences for PI 2C values were detected between synthetic reciprocal crosses, attributable to maternal effects, as well as between natural hybrids and those synthetic F₁ hybrids in which N. cavanillesii acted as a mother.

Conclusions

Our results suggest that natural hybrid populations are composed of a mixture of markedly different hybrid genotypes produced either by structural chromosome changes, consistent with classic cytogenetic studies in Narcissus, or by transposon-mediated events.     

Pollinator shifts and the loss of style polymorphism in Narcissus papyraceus (Amaryllidaceae)

Darwin proposed that the driving force for the evolution of style polymorphisms is the promotion of cross‐pollination between style morphs, through accurate placement of pollen on the pollinator’s body. This hypothesis has received much attention, but the effect of different pollinators in the fitness of morphs remains poorly understood. Narcissus papyraceus is a style dimorphic species (long ‐L‐ and short ‐S‐ styled) with isoplethic (1 : 1) and L‐monomorphic populations, mainly visited by long‐tongued (LT) nocturnal and short‐tongued (ST) diurnal pollinators, respectively. We studied natural female fertility of morphs, and assessed the role of diurnal and nocturnal pollinators. We also quantified female fertility of the morphs in experimental populations with different morph ratio, exposed to predominately long‐ or short‐tongued pollinators. We found that with LT pollinators, both morphs were successfully pollinated in all morph ratio conditions, suggesting that these insects could be involved in maintenance of the polymorphism, although other factors may also play a role. However, with ST pollinators, S‐plants displayed less fertility than L‐plants, and mating among L‐plants was favoured, implying that the polymorphism is lost. These results underscore the role of pollinators on variations in style polymorphism.     

Seed cone and ovule ontogeny in Metasequoia, Sequoia and Sequoiadendron (Taxodiaceae–Coniferales)

TAKASO, T. & TOMUNSON, P. B., 1992. Seed cone and ovule ontogeny in Metasequoia, Sequoia and Sequoiadendron (Taxodiaceae–Coniferales). Structural features of seed cones, up to the initiation of ovules, are developed in the three genera in the summer and late fall prior to pollination in the following spring, when cones renew their further development. Bracts are initiated in a decussate manner in Metasequoia but spirally in Sequoia and Sequoiadendron. No ovuliferous scale is initiated, at most there is a shallow mound of tissue on the adaxial surface of the bract from which the ovules are developed. Metasequoia produces a single series of up to eight ovules in acropetal (centripetal order), Sequoia and Sequoiadendron produce a double series of six to nine ovules, also in acropetal order, since a second series of ovules appear distal to and alternate with the first series. Common features that unite the genera are the somewhat peltate configuration of the cone scales due to late intercalary expansion, the derivation of the vascular supply to the bract-ovule complex from a single bundle and the usual inversion of the seed during late development, to which can be added developmental features. The absence of tooth-like structures, present in some other Taxodiaceae, is discussed in relation to cone evolution in the family being determined by changes in developmental timing. Emphasis is made on the way in which features of morphogenesis, determine cone organization in the three genera independent of an interpretation that relies solely on hypothetical ancestral forms.     

Breeding systems in Cyrtanthus (Amaryllidaceae): variation in self‐sterility and potential for ovule discounting

• Breeding systems of plants determine their reliance on pollinators and ability to produce seeds following self‐pollination. Self‐sterility, where ovules that are penetrated by self‐pollen tubes that do not develop into seeds, is usually considered to represent either a system of late‐acting self‐incompatibility or strong early inbreeding depression. Importantly, it can lead to impaired female function through ovule or seed discounting when stigmas receive mixtures of self and cross pollen, unless cross pollen is able to reach the ovary ahead of self pollen (‘prepotency’). Self‐sterility associated with ovule penetration by self‐pollen tubes appears to be widespread among the Amaryllidaceae.
• We tested for self‐sterility in three Cyrtanthus species – C. contractus, C. ventricosus and C. mackenii – by means of controlled hand‐pollination experiments. To determine the growth rates and frequency of ovule penetration by self‐ versus cross‐pollen tubes, we used fluorescence microscopy to examine flowers of C. contractus harvested 24, 48 and 72 h after pollination, in conjunction with a novel method of processing these images digitally. To test the potential for ovule discounting (loss of cross‐fertilisation opportunities when ovules are disabled by self‐pollination), we pollinated flowers of C. contractus and C. mackenii with mixtures of self‐ and cross pollen.
• We recorded full self‐sterility for C. contractus and C. ventricosus, and partial self‐sterility for C. mackenii. In C. contractus, we found no differences in the growth rates of self‐ and cross‐pollen tubes, nor in the proportions of ovules penetrated by self‐ and cross‐pollen tubes. In this species, seed set was depressed (relative to cross‐pollinated controls) when flowers received a mixture of self and cross pollen, but this was not the case for C. mackenii.
• These results reveal variation in breeding systems among Cyrtanthus species and highlight the potential for gender conflict in self‐sterile species in which ovules are penetrated and disabled by pollen tubes from self pollen.

     

Callus induction, somatic embryogenesis and organogenesis in Narcissus confusus: correlation between the state of differentiation and the content of galanthamine and related alkaloids

In vitro cultures from two strains of Narcissus confusus (Amaryllidaceae) initiated from mature seeds were screened for their ability to produce alkaloids. Protocols for callus induction, somatic embryogenesis and organogenesis were established. The alkaloid contents were determined by HPLC. Undifferentiated calli produced small amounts of galanthamine, which increased with the degree of tissue differentiation. Scanning electron micrographs of the cultures at different stages of development were made. Embryogenic friable calli were maintained in culture for more than 2 years, retaining a high regeneration capability. All regenerated plants showed normal morphological characteristics. Received: 20 August 1997 / Revision received: 20 December 1997 / Accepted: 1 February 1998