Thrum Stigmas Research Articles

Embryological background of low seed set in distylous common buckwheat (Fagopyrum esculentum Moench) with biased morph ratios, and biostimulant-induced improvement of it

The biased ratio (1 : 2.7–1 : 19) of long-styled Pin and short-styled Thrum flowers (anisoplethy) in common buckwheat (Fagopyrum esculentum) with low seed set (9.8–33.1%) is documented for the first time in two cultivars (Kora, Panda) and two strains (PA13, PA14). To establish the reasons for low grain yield we studied pollen, embryo sacs, embryos, counted stigmas with compatible pollen and with compatible pollen tubes, and recorded seed set under semi-controlled conditions with open access of pollinators. We also sought to improve seed yield via exogenous application of eight biostimulants at the beginning of flowering. Pin pollen supply to Thrum stigmas was low, due to the imbalance of flower morphs. This did not affect seed set or male success in either flower morph. The pollen of Pin or Thrum was highly viable (97.9–99.9%) in all studied cultivars and strains, germinating well on compatible stigmas. The female success of both flower types was much lower; 49–59% of the ovules exhibited signs of degeneration (whole flower buds, ovules only) or abortion (mature embryo sacs, proembryos, embryos); the highest share of mature embryo sac abortions resulted from degeneration of synergids or the whole egg apparatus. Three biostimulants (Gibberellic acid, putrescine, Asahi SL) in PA13 and six (1-Naphthaleneacetic acid, Gibberellic acid, TYTANIT, putrescine, 6-Benzylaminopurine, Asahi SL) in PA14 decreased embryo abortions (4–12 fold) and increased seed set (0.4–2.4 times), but seed set was still low and never exceeded 33% (the highest value of the untreated with biostimulants plants). Biostimulant treatments were most effective on PA14 strain increasing seed set in 7 out of 8 treatments. These were Gibberellic acid, putrescine and Asahi SL improving seed set of two among four analysed genotypes.

Structural differences of the stigmatic papillae in distylousLinum hirsutumand monostylousLinum tenuifolium

The stigmatic receptive surface of distylous Linum hirsutum and monostylous L. tenuifolium were investigated by using a light and a scanning microscope and cytochemical differences were identified. The papillate stigma is linear in both morphs of L. hirsutum and is capitate in L. tenuifolium . The stigma surface of the pin (long-styled) and thrum (short-styled) morphs in L. hirsutum demonstrates distinct differences. In the thrum morph, a cap like zone consisting of a pectic substance is present between the cellulose-pectin layer and the cuticle at the apical region but the papillae in the pin morph lack a cap zone. In the thrum morph, the cuticle seems to be discontinuous at some regions and is disrupted with thinner patches. In the pin morph the cuticle is continuous at the surface of papillae. The stigma of the pin morph is of the dry type, the thrum stigma of the wet type. In the homomorphic L. tenuifolium lack a cap zone, the cuticle is continuous. Therefore, it seems to be the dry type of stigma.

Three‐dimensional reciprocity of floral morphs in wild flax (Linum suffruticosum): a new twist on heterostyly

Here, we studied the floral morphology and pollination of the distylous plant Linum suffruticosum (Linaceae) in southern Spain. We observed a previously unreported form of distyly that involved twisting and bending of styles and stamens during floral development to achieve three-dimensional reciprocity of anthers and stigmas in the long-styled (pin) and short-styled (thrum) morphs. This developmental pattern causes pin pollen to be placed on the underside of pollinating Usia flies (Bombyliidae), and thrum pollen to be placed on the top of the thorax and abdomen. The pin stigmas contact the flies on the dorsum, apparently picking up predominantly thrum pollen, and the thrum stigmas contact the flies on the ventral surface, apparently picking up predominantly pin pollen. This form of heterostyly would appear on morphological grounds to be far more efficient in dispersing pollen between compatible morphs than the typical pin-thrum system. If so, this plant fits Darwin's prediction of efficient pollen flow between heterostylous morphs more closely than anything Darwin himself reported. Molecular phylogenetic analyses indicate that this form of heterostyly evolved in a lineage that already had typical heterostyly. The analyses also indicate that there have been several independent origins of heterostyly in Linum and at least one reversal to stylar monomorphism.

Stigma morphology in distylous and non-heterostylous species ofVillarsia (Menyanthaceae)

Stigma morphology was examined with the SEM in 14 of the 16 species ofVillarsia. In nine of the ten distylous species studied, stigmas of the floral morphs were strongly dimorphic in length, shape, configuration of the receptive surface, and in the size and density of their papillae. Thrum stigmas ofVillarsia, in contrast to those of most other distylous species, are not simply smaller versions of the conspecific pin stigmas, but generally exhibit an array of morph-specific characters. Thrum stigma lobes may be broader than those of pins, they may have undulate margins, lobes subdivided into secondary lobes, papillae more extensively distributed than in pins, and various combinations of these traits occur. The traits that distinguish thrum from pin stigmas achieve an increase in the receptive area and may enhance more efficient pollen capture by the shorter and less accessible thrum stigmas. The morphogenesis of the stigma shape dimorphism appears to involve processes more complex than inhibition of elongation in thrum styles. InVillarsia, the stigma dimorphisms are species-specific. No correlations were found between morphologies of the stigma and the different breeding systems in distylous species. Stigmas of the four non-heterostylous species examined resemble the thrum stigma type found in most distylousVillarsia species.

The control of incompatibility in distylous Pulmonaria affinis Jordan (Boraginaceae)

In P. affinis, pin pollen is shorter on average than thrum pollen. Pins have more coronal hooks on stigmatic papillae than thrums, but gaps between papillae are relatively smaller for thrums. Pin stigmas receive more pollen than thrum stigmas. Thrum stigmas receive more (dissortive) pin pollen, but pin stigmas are assortively pollinated. Pollen only germinates when trapped below papilla coronas. On thrum stigmas, most trapped pollen is pin. Pollen germination is better on thrum stigmas than pin stigmas, and thrum stigmas show a close relationship between numbers of legitimate pollen grains, numbers of germinating grains, and numbers of pollen tubes in the style. There is no inhibition of illegitimate pollen germination. Illegitimate pollen tubes are inhibited in the style. Incompatibility operates by a combination of dissortive pollination, dissortive pollen trapping, and stylar pollen tube inhibition. All heteromorphic features differing between pins and thrums are implicated in the inhibition of within-morph fertilization in thrums.

Fine Structure and Cytochemistry of the Stigma Surface and Incompatibility in some Distylous Linum Species

The receptive surface of the stigma in distylous Linum grandiflorum and L. pubescens was studied by electron microscopy and cytochemical techniques. In both floral morphs a proteinaceous-lipoidal coating is present on the papilla surface. In thrum stigmas the cuticle is highly irregular and pitted at the papilla tip. The cuticle is dislodged and torn at anthesis and an osmiophilic secretion product is released within a pectinaceous matrix. The secretion product stains for proteins and lipids and contributes to adhesion of pollen. In the larger pin papillae the cuticle is wavy, continuous, thicker than in thrum papillae and adjoins the cell wall. In both species the surface of the two types of pollen grains is coated with lipids and protein. A similar behaviour of the male gametophyte is observed in incompatible pollinations of L. pubescens, L. mucronatum and L. grandiflorum. In intramorph thrum pollinations pollen tubes are arrested within the stigma. In intramorph pin pollinations the majority of pollen grains fail to adhere to the stigma. Low permeability to water in pin papillae, as determined by the neutral red test, may be a factor preventing imbibition of the few adhering grains. Tubes of the few germinated grains are inhibited inside the stigma. On the part of the stigma, the difference in the major site of inhibition in the two intramorph incompatible combinations may be accounted for by the dissimilar properties of the papillae, i.e. the occurrence of wet thrum stigmas and dry pin stigmas. Functionally, the unusual association of sporophytic incompatibility with wet thrum stigmas is attributed to retention of the secretory material on individual papillae.

Differential localization of enzymes in the stigmatic exudates ofPrimula obconica

The stigmatic dimorphisms in the distylous speciesPrimula obconica, which has been dealt with in an earlier paper, has been described further. An ultrastructural localization of various enzymes in the exudates is hereby revealed, and evidence is given for intermorph differences. Esterases are confined to the lipid phase of the exudates, including the lipid globuli in the papillae walls of both morphs, but they are not found in the pellicle of the dry thrum stigma. However, this pellicle exhibits acid phosphatase activity, as does the lumen of the blisters in the viscous exudate on pin stigmas. The blisters are presumed to hold the watery phase of the pin secretion. From the present findings and from previous results with LM cytochemical methods it is suggested that the sites of peroxidase activity resemble those of acid phophatases.

The dry and wet stigmas ofPrimula obconica: Ultrastructural and cytochemical dimorphisms

The stigmatic papillae of the distylous speciesPrimula obconica are studied by means of cytochemical, light and electron microscopic techniques. The papillae on thrum stigmas are smaller than those on pin stigmas. At the bud stage, secretory vesicles are not a conspicious part of the cytoplasm, although certain signs of secretory activity are present. The young papillae bear the thin, superficial pellicle typical to dry stigmas. Small vesicles are numerous in mature papillae of both morphs, and seem to originate from the ER. A layer of closely packed, osmiophilic globuli is present in the outermost part of mature walls of pin papillae. At sites with cuticle disruption, the globuli seem to migrate outwards to be incorporated with the copious, blistery exudate. Due to this exudate the pin stigma is characterized as wet. Cytochemical tests suggest that the exudate contains mainly lipids, and different carbohydrates and protein are detected. It reacts positively in tests for peroxidase, acid phosphatase, and non-specific esterases. The thrum stigma remains dry at maturity, with a distinct pellicle also reacting positively to the enzyme tests. Only a few, scattered osmiophilic globuli, sized and situated as those in the pin papillae walls, are found in the thrum walls, and they do not form a proper layer.

The distyly in Primula elatior (L.) Hill (Primulaceae), with a study of flowering phenology and pollen flow

Flowers of Primula elatior exhibit all the dimorphic traits typical of distyly. The difference in pollen production between pin and thrum flowers is not thought to be caused by the pollen size dimorphism. The percentage of pin pollen which is probably viable exceeds that of thrum pollen. In the population studied the morph ratio is 1:1, and plants are randomly dispersed with regard to morph type. Bumblebees are the main pollinators, and analysis of the stigmatic pollen load in naturally |xjllinated flowers shows that pin stigmas receive many more pollen grains than thrum stigmas. Intramorph pollination is considerable in pins, while thrums are subject to some differential pollination. Emasculation experiments have shown that intraflower pollination in the thrum morph is negligible. Aging of pin flowers has a reducing effect on their total pollen loads, while in thrums there is no change in total pollen load after the first days of anthesis.

HETEROSTYLY AND INCOMPATIBILITY IN VILLARSIA CAPITATA (MENYANTHACEAE)

SummaryVillarsia capitata, a diploid annual of Western Australia, has distylous flowers. Results of a crossing program indicate that its distyly is associated with an incompatibility system. Intramorph pollinations produced 0.4‐0.8 seeds per pollination; intermorph pollinations produced 10.2 seeds per pollination in pins and 3.3 in thrums. About sixty percent of pin ovules and twenty percent of thrum ovules mature to seed following intermorph pollinations. These differences may result from the smaller size of thrum stigmas, from greater difficulty in transferring pollen to thrum stigmas in artificial pollinations, or from unknown causes. Villarsia capitata is a highly specialized member of the genus in its capitate, bracteate inflorescences and its annual habit, yet it retains a breeding system that is primitive in the Menyanthaceae.

Heterostyly inPrimula. 1. Fine-structural and cytochemical features of the stigma and style inPrimula vulgaris huds

The stigmas of the heterostylous genusPrimula are of the “dry” type without a free-flowing surface secretion. The papillae of the stigma surface cells of the two morphs, in pin (stigma exserted) and thrum (stamens exserted), bear a thin proteinaceous surface pellicle, overlying a discontinuous cuticle. The vacuoles of the papillate cells contain tannins, and tannin cells extend in files through the stigma heads and form a loose sheath surrounding the pollen-tube transmitting tract in the styles. The cells of the transmitting tissue in the stigma heads have a normal complement of organelles, and abundant ribosomal endoplasmic reticulum. The intercellular spaces contain an internal secretion which reacts cytochemically for both carbohydrate and protein. The transmitting tract in the styles forms a central core surrounded by several vascular strands. The cells are elongated, and the intercellular spaces here also have a carbohydrate-protein content. In a compatible pollination, thrum pollen tubes enter the stigma by penetrating the cuticle at the tip or on the flank of the pin papilla. Pin tubes on the thrum stigma enter between adjacent papillae, penetrating the thin cuticle at the base. The tubes grow through the transmitting tracts in the intercellular material.

Pollen-pistil interaction in Linum grandiflorum

A detailed investigation of the receptive surface of the stigma of a dimorphic taxon, Linum grandiflorum, was carried out using scanning electron microscopy, cytochemistry and polyacrylamide gel electrophoresis. The stigma surfaces of the pin and thrum morphs showed distinct differences. The stigma of the pin morph was of the dry type and the papillae were covered with a uniform cuticle-pellicle layer. The stigma of the thrum morph, on the other hand, resembled the wet type; the cuticle-pellicle layer was disrupted at places and a secretion product was released onto the surface of the stigma. Coomassie blue staining material was present on the surface of only the thrum stigma. Although esterases and acid phosphatases were present on the stigma of both the morphs, their activity was invariably higher on the thrum stigma. Polyacrylamide gel electrophoresis of stigma leachates also showed distinct differences in the protein profiles of the two morphs.

Pollen flow inPrimula veris (Primulaceae)

Observations on two natural and one artificial distylous populations ofPrimula veris in Britain revealed very high proportions of intramorph pollen on pin stigmas and moderate proportions of intermorph pollen on thrum stigmas. The average ratio of pin: thrum pollen production per flower was 2.4:1. Large amounts of pollen remained in dehisced anthers, perhaps reflecting low levels of pollinator activity. Population size and density had no clear effect on pollen flow patterns, which are asymmetrical for the two morphs.

Pollen flow in a population of Primula vulgaris Huds.

A British population of Primula vulgaris was visited four times between 1971 and 1976 for study of the nature and functioning of distyly. The stainability and production of pollen from pins exceeded those of thrums. About 20% of pin pollen and 48% of thrum pollen was removed from anthers by insect visitors. Pins and thrums were present in a 1:1 ratio in 1971 but pins outnumbered thrums in 1976. Thrums produced more seeds per flower than pins though the number of ovules produced by each was similar. Two methods for collecting stigmas from open flowers and analyzing them for pollen loans produced somewhat different results. For pin stigmas, the pollen load consisted of 2–23% thrum pollen; for thrum stigmas, the pollen load consisted of 0–71% pin pollen with most stigmas having less than 50% pin pollen. In general, intermorph pollen flow is less than would be expected if pollen flow were random. It is probable that most intramorph pollen on stigmas is a result of self- or geitonogamous pollination. The extensive literature concerning the natural pollinators of the primrose is reviewed. Although Darwin's hypothesis concerning the functional significance of distyly in promoting intermorph pollination was never quantified, the pollen flow patterns observed in P. vulgaris are unexpectedly deviant and are similar to those patterns observed in several unrelated heterostylous species in other plant families.

An unusual homostyle inHedyotis caerulea (Rubiaceae)

Hedyotis caerulea, a spring flowering herb widely distributed in eastern North America, has distylous flowers that differ by a number of morphological and physiological traits. The presence of a strong incompatibility system is indicated by the fact that intramorph crosses or self-pollinations produced little or no seed and intermorph crosses produced copious seed. An unusual homostyle was located that had most floral characters intermediate between pin and thrum flowers, although its pollen size was that of pin. The homostyle was only moderately self-compatible, and its pollen did not behave as pin pollen on thrum stigmas. It appears that, despite their intermediate position and morphology, the homostyle stigmas were fundamentally pin. Although the exceptional rarity of the homostyle suggests that it is an inadaptive failure, workers should watch for its further occurrence since additional study of it may provide insights into the genetic and physiological basis of heterostyly in theRubiaceae.

Pollen flow in distylous populations of Amsinckia (Boraginaceae)

Stigmatic pollen loads were analyzed from naturally pollinated pin and thrum form flowers of Amsinckia douglasiana and A. vernicosa var. furcata. Pin stigmas captured more total pollen than thrum stigmas. Pins experienced either net self-pollination or random pollination. Thrum stigmas experienced significant disassortative pollination. Comparing pollen loads from intact and emasculated thrum flowers of A. douglasiana indicated that self-pollination and geitonogamy were relatively unimportant in the pollination of the thrum form. The level of disassortative pollination of A. vernicosa var. furcata does not appear to be high enough to account for the level of disassortative mating observed by progeny testing, suggesting that this species may possess an incomplete stylar self-incompatibility system such as has been reported in A. grandiflora.

The Reproductive System of Amsinckia grandiflora, a Distylous Species

The flowers of Amsinckia grandiflora are distylous and the pollen is dimorphic in size. The single knozwn population of this annual species contained a 1: 1 representation of the two morphs during two seasons, but a 2: 1 pin: thrum ratio in a third season. The chief insect visitor to the flowers is Anthophora edwardsii, a bee that preferentially removes thrum pollen. Pin stigmas receive more pollen grains than thrum stigmas and also a greater proportion of illegitimate pollen than thrums. Seasonal fluctuations in the representation of the two morphs have no striking influence on pollen-flow patterns. Two sympatric, aggressive homostylous weeds of the same genus produce more seeds per flower than does A. grandiflora. It is possible that the greater reproductive success of these zweedy species has allowed them to displace A. grandiflora over much of the latter's former range. Amsinckia grandiflora Kleeberger (Boraginaceae) is an annual herb now restricted to a single, small population in San Joaquin County, California. The flowers are distylous, and the two floral forms differ conspicuously in the position of the anthers and the length of the style (Fig. 1-4). The floral morphology and highly restricted distribution of this species con- trast sharply with those of a number of other members of the genus, such as A. tessellata Gray and A. intermedia Fischer c Amsinckia is of particular interest because of the occurrence within it of distyly and an array of derived homostylous systems. In view of the con- trasts between the floral heteromorplhism and restricted distribution of A. grandiflora, a primitive species of the genus (Ray & Chisaki, 1957b), and those of its more successful homostylous congeners, I have studied the reproductive system of this species. This paper presents the results of this study and suggests that the present restricted distribution of the species is perhaps associated with its archaic and relatively inefficient reproductive system.

INCOMPATIBILITY AND THE POLLEN ECONOMY OF JEPSONIA PARRYI

Jepsonia parryi (Saxifragaceae) has heterostylous flowers and is strongly self‐incompatible. Pin flowers have long styles, large stigmas, short stamens, and numerous, small pollen grains with finely sculptured walls. Thrum flowers have short styles, small stigmas, long stamens, and fewer, larger pollen grains with coarsely sculptured walls. Pin plants and thrum plants occur in a 1:1 ratio in field populations. Although the insect pollinators of J. parryi transfer ample compatible pollen to pin and thrum stigmas to account for full seed production, much of the pollen deposited on stigmas is incompatible. Analysis of the pollen deposits on stigmas collected from field populations indicates that compatible “legitimate” pollination of pin and thrum flowers is essentially random and is not obviously aided by floral dimorphism. It is suggested that although heterostyly had a positive adaptive value in the past evolutionary history of Jepsonia it is no longer adaptive under the present pollination regime, although it is maintained because of its strong genetic fixity.

The Breeding System of Lithospermum caroliniense: Adaptation and Counteradaptation

The breeding system of the perennial herb Lithospermum caroliniense was analyzed in a population located near Zion, Lake County, Illinois. The species is both heterostylic and functionally cleistogamic, its populations containing plants which are solely chasmogamic, solely cleistogamic, or partially cleistogamic. Heterostyly is accompanied by pollen dimorphism so that levels of legitimate pollen flow may be accurately measured by examining stigmas and noting the size of the legitimate pollen loads. Legitimate pollen flow was measured at Zion, and it was found that 63% of the pin stigmas bore legitimate grains as compared with 42% of the thrum stigmas. The average numbers of such grains on all pin and thrum stigmas were 4.88 and 2.75, respectively, the range being one to 45 in the former and one to 43 in the latter. The distribution of legitimate loads on both stigmas was markedly leptokurtic. Average pollen loads per plant were related to the proximity of legitimate pollen donor. Illegitimate loads ranged...