THE ROLE OF INBREEDING DEPRESSION AND MATING SYSTEM IN THE EVOLUTION OF HETEROSTYLY

We investigated the role of morph‐based differences in the expression of inbreeding depression in loss of the mid‐styled morph from populations of tristylous Oxalis alpina. The extent of self‐compatibility (SC) of reproductive morphs, the degree of self‐fertilization, and the magnitude of inbreeding depression were investigated in three populations of O. alpina differing in their tristylous incompatibility relationships. All three populations exhibited significant inbreeding depression. In two populations with highly modified tristylous incompatibility, manifested as increased reciprocal compatibility between short‐ and long‐styled morphs, substantial SC and self‐fertilization of mid‐styled morphs were detected, and expected to result in expression of inbreeding depression in the progeny of mid‐styled morphs in the natural populations. In contrast, significant self‐fertility of the mid‐styled morph was absent from the population with typical tristylous incompatibility, and no self‐fertilization could be detected. Although self‐fertilization and expression of inbreeding depression should result in selection against the mid‐styled morph in the later stages of the transition from tristyly to distyly, in O. alpina selection against the mid‐styled morph in the early phases of the evolution of distyly is likely due to genic selection against mid‐alleles associated with modified tristylous incompatibility, rather than expression of inbreeding depression.     

The evolution of distyly from tristyly in populations of Oxalis alpina (Oxalidaceae) in the Sky Islands of the Sonoran Desert

The evolution of distyly from tristyly was investigated in populations of Oxalis alpina at high elevations throughout the Sky Islands of the Sonoran Desert. Incompatibility systems in tristylous populations, where self-incompatible short-, mid-, and long-styled morphs occur in populations, vary from those typical of tristylous species in which each morph is equally capable of fertilizing ovules of the other two morphs, to breeding systems in which incompatibility relationships are asymmetric. In these populations, selection against the allele controlling expression of the mid-styled morph is likely. The degree of modification of incompatibility in the short- and long-styled morphs in 10 populations was strongly associated with fewer mid-styled morphs, supporting models predicting the effect of these modifications of incompatibility on frequency of the mid-styled morph. Self-compatibility of the mid-styled morph may be important for maintaining the frequency of this morph, depending on the level of self-pollination, self-fertilization, and the extent of inbreeding depression. Modifications of incompatibility in tristylous populations and the distribution of distylous populations of O. alpina in the Sky Island region have similar geographic components, indicating the potential importance of historical factors in the evolution of distyly from tristyly.     

VARIATION IN MALE AND FEMALE REPRODUCTIVE SUCCESS AMONG FLORAL MORPHS IN THE TRISTYLOUS PLANT LYTHRUM SALICARIA (LYTHRACEAE)

The purpose of this study was to assess variation in male and female reproductive success among the three morphs of the tristylous plant, Lythrum salicaria. Fluorescent dyes were used as pollen analogs to determine whether morphs differ in their abilities to donate and receive pollen, and actual and potential seed set was measured with a hand pollination experiment. Dye transfer among morphs was highly asymmetric, with more frequent transfer from the short-styled morph to the long- and mid-styled morphs. This suggests that shorts are performing better at pollen donation and longs and mids are performing better at pollen receipt. All flowers on 95 plants were hand pollinated to test whether female reproductive success is more pollen-limited in the short-styled morph than in other morphs. Hand-pollinated short-styled plants had significantly higher total seed mass and more seeds per capsule than short controls, whereas hand pollination failed to increase seed set in long and mid morphs. As predicted, short-styled morphs showed significant pollen limitation, whereas seed set in long- and midstyled morphs was not pollen-limited. Thus, in Lythrum salicaria asymmetrical pollen flow generates morph-specific differences in male and female fitness.     

Fecundity and fitness in cross-compatible pollinations of tristylous North American Lythrum salicaria populations

As heterostyly and self incompatibility are linked in tristylous L. salicaria, all cross-compatible pollinations – those between anthers and styles of the same level – should produce viable seed. The rigor of this theory was tested using seed set and germination of cross-compatible pollinations in 18 naturalized L. salicaria populations in Minnesota/Wisconsin (n=342 genotypes; n=86 shorts, n=127 mids, and n=129 longs). Seed set for short-styled genotypes ranged from zero to 135 (36+38); mids–0–156, (53+39), and longs–0–151 (59+39). Mean seed set per capsule was not significantly different for mids and longs, but both were significantly greater than that of shorts (F=14, P<0.0001). Zero seed set frequently occurred in most compatible crosses, in contrast with theoretical expectations. The high percentage of populations deviating from normality must be due to incompatibility. An incompatibility system independent of heterostyly could cause this, where failed crosses result from matches of incompatibility specificities in pollen and pistil. This independence is questionable, however, given the non-significant difference in failed outcrosses for pooled χ² comparing within and between populations (χ²=0.395, P>0.5). A sporophytic incompatibility model is proposed with a minimum of three specificities. Zero seed set in compatible crosses is due to the addition of alleles from L. alatum, a distylous species that forms introgressive hybrids with L. salicaria. Reduced fecundity could increase the deficiency of shorts, and significantly greater seed germination of shorts could explain the continued presence of short-styled individuals in L. salicaria populations. Received: 3 February 2000 / Accepted: 25 March 2000     

Pollinator visitation in populations of tristylous Eichhornia paniculata in northeastern Brazil

Summary The frequencies of floral morphs in populations of tristylous Eichhornia paniculata often deviate from the theoretical expectation of equality. This variation is associated with the breakdown of tristyly and the evolution of self-fertilization. Differences in morph frequencies could result from selection pressures due to variable levels of insect visitation to populations and contrasting foraging behavior among the floral morphs. We estimated pollinator densities in 16 populations and quantified visitation sequences to morphs in five populations of E. paniculata in northeastern Brazil. Foraging behavior among floral morphs was measured as the frequency of visits to morphs relative to their frequency in the population (preference) and number of flights between inflorescences of the same versus different morphs (constancy). Pollinator density (number/m²/minute) was not correlated with population size, plant density or morph diversity. Pollinator densities varied most among populations of less than 200 plants. Whether pollinators discriminated among the morphs, depended on whether they primarily collected nectar or pollen. In four populations, nectar-feeding bees (Ancyloscelis and Florilegus spp.) and butterflies showed no consistent preference or constancy among the morphs. In contrast, pollen-collecting bees (Trigona sp.) visited a lower proportion of longstyled inflorescences than expected and tended to visit more mid-and short-styled inflorescences in succession, once they were encountered. Pollinator constancy for morphs did not result from differences in inflorescence production or spatial patchiness among the morphs. Although non-random pollinator visitation to morphs in heterostylous populations could potentially affect mating and hence morph frequencies, the observed visitation patterns in this study do not provide evidence that pollinators play a major role in influencing floral morph frequencies.     

Stylar polymorphism, reciprocity and incompatibility systems in Nymphoides montana (Menyanthaceae) endemic to southeastern Australia

Heterostyly is a stylar polymorphism that has been shaped by the evolution of floral characters adapted for efficient pollen transfer. Different types of stylar polymorphism are described in which the discrete characterization of the exact polymorphic type (e.g., distyly vs. stigma-height dimorphism) requires detailed floral measurements (e.g., sex-organ reciprocity). In clonal and aquatic Nymphoides montana, although the presence of two floral morphs that contain styles of two lengths has been previously reported, no studies have quantitatively estimated the level of reciprocity and/or described the stylar condition. Morphological variations and incompatibility relationships were explored between the two morphs in three southeastern Australian populations. In this study, one population is characterized as stigma-height dimorphism (i.e., two morphs with discrete variation in stigma height but little variation in anther height), whereas the other two populations are typical distylous (i.e., two morphs for reciprocal stigma and anther height). Nymphoides montana is dimorphic in a wide range of ancillary characters, including corolla size, stigma size, shape and papillae morphology, and pollen size, number and exine sculpture. Following glasshouse pollinations, full incompatibility systems were observed in the distylous populations, whereas the stigma-height dimorphic population showed between-morph variation in the extent of incompatibility. Despite the variation in sex-organ reciprocity and incompatibility, other lines of evidence appear to assure the maintenance of the stylar polymorphism in the N. montana study populations. All populations are nearly isoplethic (i.e., both morphs in equal frequencies), which is indicative of balanced polymorphisms that appear to be maintained by legitimate pollen transfer between the morphs.     

POLLEN-PISTIL INTERACTIONS IN TRISTYLOUS PONTEDERIA SAGITTATA (PONTEDERIACEAE). I. FLORAL HETEROMORPHISM AND STRUCTURAL FEATURES OF THE POLLEN TUBE PATHWAY

Heteromorphic characters and structural features of the pollen tube pathway are described in tristylous Pontederia sagittata to assess their influence on the pollination process and in mediating self-incompatibility behavior. Heteromorphic characters that distinguish the floral morphs include style length, stigma depth, stigmatic papillae length, stylar parenchyma cell length, area of the stylar canal, stamen height, anther size, and pollen size. Unlike several distylous species that have been investigated, the exine of pollen in P. sagittata was not strongly differentiated among the pollen types, and no differences in stigma cytochemistry were apparent. Features common to the floral morphs were a wet stigma, a hollow trilobed stylar canal separating into two sterile and one fertile canal, and a single anatropous ovule with a highly elaborated integumentary obturator. The similarity in structural features of the pollen tube pathway of P. sagittata to those found in monocotyledonous taxa with gametophytic self-incompatibility suggests that phylogenetic constraints may have influenced the evolution of trimorphic incompatibility in the Pontederiaceae.     

Reproductive consequences of interactions between clonal growth and sexual reproduction in Nymphoides peltata: a distylous aquatic plant

Distyly is a sexual polymorphism in which plant populations contain two floral morphs differing in morphology and physiology. The dimorphism serves to promote animal-mediated cross-pollination between the floral morphs. Clonal propagation can interfere with the functioning of distyly by compromising intermorph pollinations, resulting in reduced fertility. Here, we investigate the relations between clonal growth and sexual reproduction in the aquatic macrophyte Nymphoides peltata (Menyanthaceae). Surveys of morph representation in 30 populations from five regions of China revealed that most populations exhibited strongly biased morph ratios and 30% contained a single floral morph. Experimental pollinations indicate that N. peltata possesses a strong dimorphic incompatibility system preventing self and intramorph fertilizations. An experiment involving the manipulation of morph ratios in an experimental population and an investigation in a natural population with strong morph substructure both provided evidence that compatible pollen dilution limits fertility. Despite constraints on the functioning of distyly in N. peltata we found no evidence for evolutionary changes to the heterostylous syndrome, as reported in Nymphoides, including populations of N. peltata in other parts of its geographical range.     

Pollen and stigma size changes during the transition from tristyly to distyly in Oxalis alpina (Oxalidaceae)

• Pollen and stigma size have the potential to influence male fitness of hermaphroditic plants, particularly in species presenting floral polymorphisms characterised by marked differences in these traits among floral morphs. In this study, we take advantage of the evolutionary transition from tristyly to distyly experienced by Oxalis alpina (Oxalidaceae), and examined whether modifications in the ancillary traits (pollen and stigma size) respond to allometric changes in other floral traits. Also, we tested whether these modifications are in accordance with what would be expected under the hypothesis that novel competitive scenarios (as in distylous‐derived reproductive system) exert morph‐ and whorl‐specific selective pressures to match the available stigmas.
• We measure pollen and stigma size in five populations of O. alpina representing the tristyly–distyly transition.
• A general reduction in pollen and stigma size occurred along the tristyly–distyly transition, and pollen size from the two anther levels within each morph converged to a similar size that was characterised by whorl‐specific changes (increases or decreases) in pollen size of different anthers in each floral type.
• Overall, results from this study show that the evolution of distyly in this species is characterised not only by changes in sexual organ position and flower size, but also by morph‐specific changes in pollen and stigma size. This evidence supports the importance of selection on pollen and stigma size, which increase fitness of remaining morphs following the evolution of distyly, and raises questions to explore on the functional value of pollen size in heterostylous systems under pollen competition.

     

Correlated evolution of floral morphology and mating-type frequencies in a sexually polymorphic plant

In sexually polymorphic species, reproductive morphology governs mating patterns and the character of negative frequency-dependent selection. If local environmental conditions cause sexual morphs to differ between populations, then frequency-dependent selection should create corresponding geographic variation in morph frequencies. We investigate this relation with a model of morph-ratio evolution and analysis of geographic variation in the heterostylous plant Narcissus triandrus. Unlike other tristylous species, N. triandrus possesses both imperfect reciprocity among morphs in sex-organ position and a self-incompatibility system that permits outcrossing within and between morphs. We sampled 137 populations throughout the Iberian Peninsula for floral-morph ratios, and measured floral morphology in 31 populations. Morph ratios exhibited three atypical features: (1) predominance of the long-styled (L) morph; (2) absence of the mid-styled (M) morph from 17.5% of populations; and (3) a negative relation between the frequencies of the L and M morphs among populations. Morph ratios varied geographically, with decreasing frequency of the M morph from the southeast to the northwest of the species' range. Much of this variation accompanied allometric change in the positions of sex organs, especially the mid-level organs, with the M morph declining in frequency and ultimately being lost in large-flowered populations. Using multivariate multiple regression, we demonstrate that variation in floral morphology among populations predicts this geographic variation in morph frequencies. Our theoretical analysis illustrates that patterns of pollen transfer governed by imperfect sex-organ reciprocity can select for unequal equilibrium morph ratios like those observed for N. triandrus. We interpret the L-biased morph ratios and the unusual morphology of N. triandrus as a consequence of its atypical intramorph compatibility system.     

THE DEVELOPMENTAL BASIS OF TRISTYLY IN EICHHORNIA PANICULATA (PONTEDERIACEAE)

Eichhornia paniculata is a tristylous, self-compatible, emergent aquatic. A given plant produces flowers with either long, mid or short styles and two levels of stamens equal in length to the styles not found in that flower. Flowers of each morph have two whorls of three tepals, six stamens and three fused carpels. The six stamens differentiate into two sets of three stamens each. A relatively short set, having either short- or mid-level stamens, occurs on the upper side of the flower, while a relatively long set, having either mid- or long-level stamens, occurs on the lower side. Stamen level depends on differences among stamens in filament length and position of insertion on the floral tube. Floral parts arise in whorls of three, but the two stamen whorls do not form the two sets of stamens found in each mature flower. Instead, stamens from both whorls make up a given set. Floral differences among morphs are not present at flower origin or floral organ initiation. Morphological differences arise first among stamen sets. The two sets within a flower differ prior to meiosis in the size, number, and timing of comparable developmental events in the sporogenous cells. After these initial differences arise, anther size diverges. In later developmental stages differences in filament and floral tube length, cell size, and cell number, as well as differences in the length, cell size, and cell number of styles, develop among morphs. This sequence of developmental events suggests that the genes controlling development in different morphs do not control flower and floral organ initiation but are first morphologically visible in sporogenous cell differentiation.     

Heterostyly in the Lamiaceae: The case ofSalvia brandegeei

Heterostyly rarely occurs in families with strongly zygomorphic flowers. For this reason Darwin (1877) doubted whether heterostyly would occur in the Lamiaceae and recent reviews have not reported the floral polymorphism in this family. Here we describe distyly in a rare species ofSalvia restricted to bluffs and seaward canyons on Santa Rosa Island (Santa Barbara Co., California) and northwestern Baja California (Mexico).Salvia brandegeei is morphologically distylous with populations composed of equal frequencies of long-and short-styled morphs differing reciprocally in stigma and anther position. Controlled hand pollinations demonstrated no significant differences in the seed set of self, intramorph or intermorph pollinations. Unlike most heterostylous species investigated,S. brandegeei does not possess diallelic incompatibility or ancillary polymorphisms of pollen and stigmas. We propose that the evolution of distyly inS. brandegeei may have been associated with an ecological shift to a new environment in which protandry failed to prevent increased levels of geitonogamy. Heterostyly was then selected because it increased the proficiency of cross-pollination. The origin of distyly in self-compatibleS. brandegeei is consistent with Lloyd and Webb's theoretical model for the evolution of distyly.     

Breeding system and floral morphometry of distylous Psychotria L. species in the Atlantic rain forest, SE Brazil

General patterns of floral morphology and incompatibility mechanisms have been described for many distylous plants. The absence of these patterns in typically distylous groups, as observed especially in tropical environments, is interpreted as atypical distyly, or as a new reproductive strategy derived from it. Data are presented here on the morphological and compatibility relations between floral morphs of four Psychotria dimorphic species in the Atlantic rain forest in SE Brazil: Psychotria jasminoides, P. birotula, P. mapourioides, and P. pubigera. When significant differences were found, floral parts were larger in thrum flowers. Results of controlled crosses showed that most incompatible pollen tubes were arrested in the stigma, and only in a low proportion in the upper parts of the style. We conclude that, at the study site, the majority and most important morphological and mating features of typical distyly seem to be conserved in P. jasminoides and P. mapourioides, which presented reciprocal herkogamy, self and intramorph incompatibility, and a balanced morph ratio in the population. Typical distyly in P. birotula is supported by floral morphology, pollen tube data and morph ratio and, in P. pubigera, only by floral morphology and pollen tube data.     

Variation in expression of trimorphic incompatibility in Pontederia cordata L. (Pontederiaceae)

Summary Pontederia cordata L. (Pontederiaceae), a perennial diploid, possesses the rare genetic polymorphism tristyly. A controlled pollination programme was conducted over a three year period, under glasshouse conditions, on 36 clones of P. cordata var. cordata to examine the nature of the self-incompatibility system. The three major findings of the pollination study were: (1) the three floral morphs display different levels of self-incompatibility, (2) pollen from the two anther levels within a flower exhibits different compatibility behaviour in self-pollinations, (3) considerable individual genetic variation in the expression of self-incompatibility is evident among clones within floral morphs. Similar results were also obtained from a smaller study on 15 clones of P. cordata var. lancifolia conducted over a 6 month period. In common with other Pontederia species the mid-styled morph (M) of P. cordata produces large amounts of seed when self-pollinated with pollen from long-level anthers. A developmental model is proposed to explain the high level of self-compatibility of the M morph in Pontederia species. Self-pollination of segregating progenies from M and S morphs of known incompatibility status demonstrated that the expression of incompatibility is closely associated with style length. It is suggested that overall differences in incompatibility behaviour among the floral morphs may be due to the pleiotropic effects of major genes controlling sub-characters of the tristylous syndrome, rather than linked modifier genes. However, the variable expression of trimorphic incompatibility within floral morphs suggests that this variation may be polygenic in origin.     

Distyly and incompatibility in Psychotria homalosperma (Rubiaceae), an endemic plant of the oceanic Bonin (Ogasawara) Islands

Distyly rarely occurs on oceanic islands. Indeed, this form of polymorphism is absent on the Hawaiian Islands and in New Zealand. However, Psychotria homalosperma (Rubiaceae), which is endemic to the oceanic Bonin Islands of Japan, was presumed to maintain a distylous breeding system and floral morphology. We investigated the floral and reproductive characteristics of this species and examined incompatibility in self- and intramorph pollination. Psychotria homalosperma is morphologically and functionally distylous, based on findings with the examined populations, but its stigma and anther heights are not exactly reciprocal between the two morphs. This is the second example of distyly among the flora from the Bonin Islands, the other being the endemic P. boninensis. Both of these distylous species contrast with the Hawaiian Psychotria species, which have already become dioecious. The flowers of the long (L)-styled (pin) morph of the Bonin Islands plants are completely self- and intramorph incompatible, while the flowers of the short (S)-styled (thrum) morphs are partially intramorph compatible. The S-styled morph was more abundant in the examined populations from three islands of the archipelago, probably due to the existence of partial S–S intramorph compatibility.     

Genetic control of floral morph in tristylous Pickerelweed (Pontederia cordata L.)

Pickerelweed (Pontederia cordata L.) is a diploid (2n = 2x = 16) tristylous aquatic perennial. Populations usually contain 3 floral morphs that differ reciprocally in style length and anther height (referred to as the long-, mid-, and short-styled morphs, hereafter L-, M-, and S-morphs). The floral polymorphism promotes disassortative mating among the 3 floral morphs and is maintained in populations by negative frequency-dependent selection. The objective of this study was to determine the number of loci, number of alleles, and gene action controlling floral morph in pickerelweed. Three parental lines (one each of the L-, M-, and S-morph) were used to create S1 and F1 populations. F2 populations were produced through self-pollination of F1 plants. Progeny ratios of S1, F1, and F2 generations revealed that tristyly is controlled by 2 diallelic loci (S and M) with dominant gene action. The S locus is epistatic to the M locus, with the S-morph produced by plants with the dominant S allele (genotype S _ _ _). Plants with recessive alleles at the S locus were either L-morph (ssmm) or M-morph (ssM_). The results of this experiment demonstrate that the inheritance of tristyly in pickerelweed is the same as previously reported for several tristylous species in the Lythraceae and Oxalidaceae.     

CYTOGEOGRAPHIC EVIDENCE FOR THE EVOLUTION OF DISTYLY FROM TRISTYLY IN THE NORTH AMERICAN SPECIES OF OXALIS SECTION IONOXALIS

A survey of haploid chromosome numbers of 18 North American taxa of Oxalis section Ionoxalis was initiated to determine the relationship between ploidal level, geographic distribution, and the occurrence of tristyly and distyly. Although chromosome numbers in the section are variable, the majority of tristylous populations are diploid. Among the distylous taxa a greater diversity of ploidal levels exists, with the higher chromosome numbers predominating. In section Ionoxalis the majority of the tristylous taxa are geographically restricted endemics of southern Mexico, while the distylous taxa have more extensive distributions ranging to the north. The association of diploidy and geographic endemism in the majority of the tristylous taxa suggests that these species are relictual. A few widespread tristylous taxa are polyploid, and often somewhat weedy. The probable derivation of widespread polyploid species from the restricted diploid endemics of southern Mexico appears to have been accompanied by the evolution of distyly from tristyly. The frequent association of polyploidy and distyly in section Ionoxalis has apparently resulted from the concurrence of two evolutionary trends: increase in ploidal level and the derivation of distyly from tristyly.     

STIGMATIC POLLEN LOADS IN POPULATIONS OF PONTEDERIA CORDATA FROM THE SOUTHERN U.S.

Stigmatic pollen loads were examined in four tristylous populations of Pontederia cordata from the southern U.S. to evaluate Darwin's hypothesis that floral trimorphism promotes legitimate pollination. In each population, morph frequency, pollen production, the composition of stigmatic pollen loads and seed production were estimated. Goodness-of-fit tests and a measure of the efficiency of legitimate pollination were employed to evaluate pollen load patterns. Results from the four populations demonstrate that the long-styled (L) morph usually experiences legitimate pollination, whereas the mid-styled (M) and short-styled (S) morphs often display random pollination. However, at Taylor Road (Louisiana) all three floral morphs exhibited statistically significant levels of legitimate pollination. The size of legitimate pollen loads suggests that in populations of P. cordata tristyly may have only a minor influence on the fecundity of morphs. Two hypotheses are proposed to explain the maintenance of floral trimorphism in contemporary populations of Pontederia spp. The first assumes the polymorphism is selectively neutral; the second suggests that it contributes towards the male component of fitness by increasing the distance, and hence the number of genets, that pollen can be transported to by pollinators. In clonal species, such as Pontederia cordata, where geitonogamous pollinations are promoted by large floral displays, floral mechanisms that increase pollen carryover may have selective value.     

Floral trimorphism and monomorphism in continental and island populations of Eichhornia paniculata (Spreng.) Solms. (Pontederiaceae)

Eichhornia paniculata (Spreng.) Solms. (Pontederiaceae) is a short-lived perennial or annual of marshes, seasonal pools and ditches of lowland tropical South America, primarily NE Brazil, and the Caribbean islands of Cuba and Jamaica. Comparisons, made under uniform glasshouse conditions, of populations originating from seed collected in the two regions revealed striking differences in their floral biology and breeding systems. The majority of populations sampled in NE Brazil are tristylous. Floral trimorphism is associated with pollen trimorphism and minor differences in anther size and pollen production among the three stamen levels. Unlike the majority of heterostylous plants the floral morphs of E. paniculata are highly self-fertile. Populations sampled on the island of Jamaica are composed exclusively of self-pollinating, semi-homostylous, mid-styled forms. Flowers from these populations are smaller and less showy, with reduced pollen heteromorphism and significantly fewer pollen grains and ovules per flower, in comparison with trimorphic populations from Brazil. Individual genotypes from Jamaican populations display considerable developmental instability in floral expression, particularly with respect to filament elongation of the lower stamen level. It is proposed that self-pollinating populations of E. paniculata are evolutionarily derived from tristylous ancestors and that the shift in breeding system is favoured at low density, following population bottlenecks, where pollinator service is unreliable.     

Impact of mate availability, population size, and spatial aggregation of morphs on sexual reproduction in a distylous, aquatic plant

In distylous, self-incompatible plants, clonal propagation, unbalanced floral morph frequencies, and reduced population size can interfere with the functioning of distyly by compromising legitimate intermorph pollinations, resulting in reduced reproductive output. Here, we examined the mating system and the impact of mate availability, population size, and spatial aggregation of morphs on reproductive output in the distylous, clonal, aquatic plant Hottonia palustris. Controlled pollinations under greenhouse conditions detected no spontaneous selfing without the action of a pollen vector (autonomous autogamy) and demonstrated very low fruit and seed development after self-pollination. Intermorph (legitimate) crossings resulted in high reproductive output in both floral morphs (long- and short-styled individuals), whereas intramorph (illegitimate) crossings decreased fruit and seed development by more than 50%, indicating that the species has partial intramorph-incompatibility. In natural populations, small population size and increasing deviation of floral morph frequencies negatively affected reproductive outcome. Individuals of the majority morph type developed significantly fewer fruit and seeds than individuals of the minority morph type. This rapid decline in fecundity was symmetrical, indicating that regardless of which morph was in the majority, the same patterns of negative frequency-dependent mating occurred. Increasing spatial isolation between compatible morphs significantly reduced fruit and seed set in both morphs similarly. This study provides clear indications of frequency- and context-dependent mating in natural populations of a distylous plant species.