Pollen-pistil Incompatibility Research Articles

Partial and asymmetrical reproductive isolation between two sympatric tropical shrub species: Cnidoscolus aconitifolius and C. souzae (Euphorbiaceae)

AbstractReproductive isolation is conferred by several barriers that occur at different stages of reproduction. Comprehensive reviews on the topic have identified that barriers occurring prior to zygote formation are often stronger than those that occur afterward. However, the overrepresentation of temperate perennial herbs in the current literature precludes any generalization of this pattern to plants that present other life forms and patterns of distribution. Here, we assessed reproductive isolation barriers and their absolute contribution to reproductive isolation and asymmetry in Cnidoscolus aconitifolius and C. souzae, two closely related tropical shrub species that co‐occur on the Yucatan peninsula. The reproductive barriers assessed were phenological mismatch, pollinator differentiation, pollen–pistil incompatibility (three pre‐zygotic barriers), fruit set failure, and seed unviability (post‐zygotic barriers). Reproductive isolation between the study species was found to be complete in the direction C. aconitifolius to C. souzae, but only partial in the opposite direction. One post‐zygotic barrier was the strongest example. Most barriers, particularly the pre‐zygotic examples, were asymmetrical and predicted the direction of heterospecific pollen flow and hybrid formation from C. souzae to C. aconitifolius. Both parental species, as well as the hybrids, were diploid and had a chromosome number 2n = 36. More studies with tropical woody perennials are required to fully determine whether this group of plants consistently shows stronger post‐zygotic barriers.

Pre- and post-pollination barriers between two exotic and five native Sagittaria species: Implications for species conservation

Anthropogenic introduction of species has resulted in a breakdown of geographical barriers and hybridization in previously allopatric species. Thus, examining hybridization proneness of exotic species contributes to revealing its potential threat. Moreover, reproductive barriers may be strengthened or weakened due to long-term geographical isolation for these newly sympatric species. However, few studies have evaluated multiple barriers between alien and native species. In this study, we quantified the importance of four pre-pollination barriers (phenological, floral traits, pollen production, and floral constancy) and four post-pollination barriers (pollen-pistil incompatibility, seed set, seed viability, and seedling survival) between two introduced and five native Sagittaria species. Results showed that introduced S. platyphylla was cross-compatible with two native species, whereas introduced S. montevidensis was incapable of hybridizing with any native species. Different barriers were asymmetric within species pairs and multiple barriers acted in concert to maintain species boundaries. Post-pollination barriers contributed more to total reproductive isolation in native species, whereas pre-pollination barriers played a stronger role in total reproductive isolation for two introduced species. Seed set was the only barrier that was positively correlated with genetic distance. Our results provide a perspective to better understand reproductive barriers for secondary contact species. We highlight the importance of monitoring hybridization events before human introduction and the possible conservation strategies to remove invasive species with hybridization proneness.

Genetic Diversity of Genes Controlling Unilateral Incompatibility in Japanese Cultivars of Chinese Cabbage.

In recent years, unilateral incompatibility (UI), which is an incompatibility system for recognizing and rejecting foreign pollen that operates in one direction, has been shown to be closely related to self-incompatibility (SI) in Brassica rapa. The stigma- and pollen-side recognition factors (SUI1 and PUI1, respectively) of this UI are similar to those of SI (stigma-side SRK and pollen-side SP11), indicating that SUI1 and PUI1 interact with each other and cause pollen-pistil incompatibility only when a specific genotype is pollinated. To clarify the genetic diversity of SUI1 and PUI1 in Japanese B. rapa, here we investigated the UI phenotype and the SUI1/PUI1 sequences in Japanese commercial varieties of Chinese cabbage. The present study showed that multiple copies of nonfunctional PUI1 were located within and in the vicinity of the UI locus region, and that the functional SUI1 was highly conserved in Chinese cabbage. In addition, we found a novel nonfunctional SUI1 allele with a dominant negative effect on the functional SUI1 allele in the heterozygote.

Intraspecific crossability and compatibility within Solanum aethiopicum

Understanding hybridization barriers is relevant for germplasm conservation and utilization. The prezygotic barriers to hybridization include floral morphological differences like pistil and stamen length, pollen characteristics and pollen-pistil interactions. This study sought to elucidate the reproductive biology of Solanum aethiopicum; its mating systems and compatibility barriers. Eight genotypes of Solanum aethiopicum were examined for differences in floral morphology, phenology and cross compatibility in a full diallel mating design, with assessment of fruit set, seed set and seed viability. In-vivo pollen tube growth was observed for failed crosses at 24, 48 and 72 h after pollination. All genotypes had heterostyly flowers, with predominantly small white petals. Incompatibility was observed in five out of 39 combinations. All selfed genotypes displayed compatibility implying the genotypes are self-compatible. Pollen–pistil incompatibility, which was exhibited in four out of the five failed cross combinations, occurred on the stigma, upper style and lower style, a phenomenon typical in Solanaceae. Solanum aethiopicum is self-compatible and majorly self-pollinating but has features that support cross-pollination.

Reproductive isolation between wild and domesticated chaya (Cnidoscolus aconitifolius) in sympatry.

Reproductive isolation is a necessary condition for plant domestication in their domestication centre where crops co-occur with their wild progenitors. However, the identification of reproductive barriers and their relative contribution to reproductive isolation have been overlooked in plants under domestication. We assessed pre- and post-pollination reproductive barriers and their relative contribution to reproductive isolation between wild and domesticated chaya (Cnidoscolus aconitifolius) in its domestication centre. We found that wild and domesticated chaya both exhibit a high degree of reproductive isolation. However, the reproductive isolation barriers exhibited some asymmetry: while pre-pollination barriers (differential pollen production and pollinator specificity) were only detected in wild plants, post-pollination barriers (pollen-pistil incompatibility and/or failure to set fruit) were observed in both wild and domesticated plants. We conclude that complete reproductive isolation has evolved in sympatry in co-occurring domesticated and wild chaya.

Pre-zygotic reproductive isolation between two synchronopatric Opuntia (Cactaceae) species in the Brazilian Chaco.

Opuntia (Cactaceae) is known for high rates of hybridization and ploidisation, resulting in the formation of new species. The occurrence of two sympatric and closely related species of Opuntia, O.elata and O.retrorsa, in Brazilian Chaco enabled us to test the hypothesis that pre-zygotic reproductive isolation mechanisms operate in both species. We monitored the flowering period, as well as floral biology, and compared the morphological variation of floral structures through measurements, performed intra- and interspecific cross-pollination tests, and recorded the guild of floral visitors and pollinators. Flowering was seasonal and highly synchronous. Floral biology exhibits similar strategies, and although floral morphology differs significantly in many of the compared structures, such morphological variation does not result in the selection of exclusive pollinators. Floral visitors and pollinators are oligolectic bees shared by both species. Opuntia elata and O.retrorsa are self-compatible. While interspecific cross-pollination (bidirectional) resulted in germination, the pollen tube did not penetrate the stigma. Opuntia elata and O.retrorsa are closely related; however, they are isolated and do not hybridise in Brazilian Chaco. We found that both have weak pre-pollination barriers, but that they are strongly isolated by pollen-pistil incompatibility, i.e. post-pollination barrier.

Cross-incompatibility and self-incompatibility: unrelated phenomena in wild and cultivated potatoes?

Knowledge of internal hybridization barriers is relevant for germplasm conservation and utilization. The two pre-zygotic barriers are pollen–pistil self-incompatibility (SI) and cross-incompatibility (CI). To ascertain whether SI and CI were phenotypically related phenomena in potatoes, extensive intra- and interspecific, both intra- and interploidy breeding relationships were established, without previous assumptions on the compatibility behavior of the studied germplasm. Pollen–pistil relationships were analyzed at the individual genotype/accession/family level. In two seasons, 828 intra- and interspecific genotypic combinations were performed, using accessions of the wild potatoes Solanum chacoense Bitter (2n = 2x = 24), S. gourlayi Hawkes (2n = 2x = 24; 2n = 4x = 48), and S. spegazzinii Bitter (2n = 2x = 24), full-sibling (hereinafter “full-sib”) families (2n = 2x = 24) within/between the latter two diploids, and S. tuberosum L. (2n = 4x = 48) cultivars. Pollen–pistil incompatibility occurred in the upper first third of the style (I1/3) in all selfed diploids. In both the intra- and interspecific combinations, the most frequent relationship was compatibility, followed by I1/3, but incompatibility also occurred in the stigma and the style (middle third and bottom third). We observed segregation for these relationships in full-sib families, and unilateral and bilateral incompatibility in reciprocal crosses between functional SI genotypes. Cross-incompatibility in potatoes is, apparently, controlled by genes independent of the S-locus or its S-haplotype recognition region (although molecular evidence is needed to confirm it), with segregation even within accessions.

Effect of humidity on intergeneric pollinations of iap (Inhibition of Alien Pollen) sorghum [Sorghum bicolor (L.) Moench

The production of interspecific or intergeneric progeny involving Sorghum spp. is greatly enhanced by the presence of the iap (Inhibition of Alien Pollen) allele in sorghum [Sorghum bicolor (L.) Moench]. Variation in the expressivity of the iap phenotype has been observed in previous studies, leading to the hypothesis that the humidity during pollination is affecting the degree of alien pollen adhesion and pollen tube growth. The objective of this study was to determine the optimum humidity for maximum maize pollen tube growth on sorghum florets homozygous for iap. Growth chambers were used to create environments of 45, 65, and 85 % humidity with reciprocal crosses made between the 65 and 85 % humidity chambers. Maize pollen was dusted onto the stigmas of Tx3361 (iapiap) and ATx623 (IapIap). Pollen adhesion and germination were higher for pollinations with Tx3361 at all humidity levels but the greatest adhesion and germination was observed at 45 % humidity for Tx3361. Under this humidity, pollen tube growth to the style and ovary was observed. The results indicate that lower humidity maximizes maize pollen adhesion and germination on the stigmas of Tx3361. Analysis of the differences in adhesion between Tx3361 and ATx623 indicate that Iap affects pollen adhesion at the stigma surface and may be controlling pollen–pistil incompatibility in interspecific crosses in sorghum.

Characterization of mode of reproduction in Commiphora wightii [(Arnot) Bhandari] reveals novel pollen–pistil interaction and occurrence of obligate sexual female plants

Guggul [Commiphora wightii (Arnot) Bhandari], a polygamous woody tree valued for its medicinal oleoresin gum rich in guggulsterone, is reported to reproduce via sporophytic apomixis. Details about its natural diversity, and mode and extent of sexual reproduction are, however, scanty. Therefore, a comprehensive investigation of guggul reproduction was made employing histology, controlled pollination, flow cytometry and RAPD analyses of progeny to assess the occurrence and extent of sexual reproduction. We report the discovery of obligate sexual female plants of guggul through these studies. Also, we document a unique pollen–pistil incompatibility that prevents all but one pollen tube growth into the style to effect fertilization. Consequently, obligate sexual female plants produced single-seeded fruit although each flower contains four ovules. In apomictic plants bearing more than one seed per fruit, at most only one seed was of sexual origin. Further, flow cytometric analysis conclusively demonstrated that endosperm development occurs either autonomously or following triple fusion. Autonomous endosperm development was invariably associated with endoreduplication, a unique feature of apomixis in guggul. Despite predominance of apomixis, a low frequency of sexual reproduction was found to persist in apomictic plants yielding new genetic variation. RAPD analysis clearly distinguished accessions and was useful in identifying sexual progenies. The implications of the novel pollen–pistil interaction on establishment and spread of apomixis in guggul are discussed. The study has not only revealed novel features of guggul reproduction but also opened new opportunities for molecular genetic analysis of sporophytic apomixis and breeding improvement of guggul.

EVALUATION OF THE REPRODUCTIVE PROCESS AS THE CAUSE FOR LOW FRUIT SET IN TWO JAPANESE PLUM CULTIVARS

The reproductive process has been evaluated to ascertain the causes of low fruit set in two Japanese plum cultivars, 'Sweet August' and 'Rubirosa'. In order to evaluate pollen germination, pollen-pistil incompatibility and ovule viability, different experiments were performed in orchard conditions. Pollen viability was determined by assessing the percentage of in vitro pollen germination. The self-(in)compatibility of both cultivars and their cross-compatibility with 'Champion' were examined by self- and cross-pollination experiments followed by observation of pollen tube growth under the microscope. Likewise, the S-RNase alleles of each cultivar were determined by PCR amplification of the S-RNase gene. Finally, ovule development was examined under the microscope. While 'Sweet August' behaved as self-incompatible, 'Rubirosa' appears to be self-compatible. The observation of pollen germination and pollen tube growth in the different crosses analysed showed that fruit set was not limited by the absence of compatible pollen. However, a high percentage of degenerated ovules were observed in flowers from both cultivars. This premature ovule degeneration is likely to be the cause of low fruit set in both cultivars.

A genetic approach to the species problem in wild potato

Wild potatoes are native to the Americas, where they present very wide geographical and ecological distribution. Most are diploid, obligate out-crossers due to a multiallelic gametophytic self-incompatibility (S) locus that prevents self-fertilisation and crossing between individuals carrying identical S-alleles. They have two alternative modes of reproduction: sexual (by seeds) and asexual (by stolons and tubers), which provide, respectively, for genetic flexibility in changing environments and high fitness of adapted genotypes under stable conditions. Since the early twentieth century, their taxonomic classification has been mostly based on morphological phenotypes (Taxonomic Species Concept). More recently, attempts have been made to establish phylogenetic relationships, applying molecular tools in samples of populations (accessions) with a previously assigned specific category. However, neither the reproductive biology and breeding relations among spontaneous populations nor the morphological and genetic variability expected in obligate allogamous populations are considered when the taxonomic species concept is applied. In nature, wild potato populations are isolated through external and internal hybridisation barriers; the latter, which are genetically determined, can be either pre-zygotic (pollen-pistil incompatibility) or post-zygotic (abortion of embryo, endosperm or both tissues, sterility, and hybrid weakness and breakdown in segregating generations). The internal barriers, however, can be incomplete, providing opportunities for hybridisation and introgression within and between populations and ploidy levels in areas of overlap. The widespread occurrence of spontaneous hybrids in nature was recognised in the mid-twentieth century. Using genetic approaches, results have been obtained that provide strong support to the assertion that populations are at different stages of genetic divergence and are not at the end of the evolutionary process, as presupposed by the Taxonomic Species Concept. Furthermore, since wild potatoes have uniparental and biparental overlapping generations, the Biological Species Concept - developed for sexually reproducing biparental organisms - cannot be applied to them. In this paper, morphological, genetic, molecular and taxonomic studies in wild potato are reviewed, considering the genetic consequences of their reproductive biology, in an attempt to shed light on the species problem, because of its relevance in germplasm conservation and breeding.

Lack of Fruit Set Caused by Ovule Degeneration in Japanese Plum

Some japanese plum (Prunus salicina) cultivars are particularly prone to erratic fruit set showing very low or even null fruit set for reasons that are not clear. To ascertain the causes of lack of fruit set in some of them, different factors intervening in the reproductive process have been evaluated using flowers of three cultivars, Angeleno, Rubirosa, and Sweet August, from commercial orchards with records of very low fruit set in previous seasons and compared with a producing cultivar, Simka. Different cultivars coincident at flowering with the cultivars studied were evaluated as adequate pollenizers in each orchard. To determine which factors that intervene in the reproductive process could be related to the lack of fruit set, microscopic observations of pollen germination, pollen tube growth, pollen–pistil incompatibility, and ovule development were analyzed in flowers of different pollination treatments. Results allowed dismissal of pollen viability, pollen transfer, and pollen–pistil incompatibility as the cause of lack fruit set. However, the observation of ovule development revealed a high incidence of premature ovule degeneration on final fruit set in the three low-producing cultivars. The lack of fruit set in orchards with no apparent adverse environmental conditions is traditionally studied by analyzing the pollination process and the pollen–pistil incompatibility relationships, but the stage of development of the ovules is not usually considered. The approach used in this work may prove valuable to other species and situations of lack of fruit set, which could help to identify the causes for premature ovule degeneration.

Pollen Tube Behavior in Different Mandarin Hybrids

Seedlessness is an important trait in the evaluation of commercial mandarin for fresh consumption. However, in the last decade, the presence of seeds in fruit of cultivars considered as seedless has become a problem in different citrus-growing areas because the commercial value is depreciated. Seeds have appeared concomitantly with the introduction of new cultivars that appear to be cross-compatible. To overcome this problem, different strategies have been explored, but a definitive answer is still elusive. The search for alternatives contrasts with how little is known about the basis of the problem: the pollen-pistil incompatibility reaction in mandarin, and the intercompatibility relationship between different cultivars. In this work, we characterized the pollen-pistil incompatibility in the two commonly grown mandarin cultivars Fortune and Nova, and evaluated the intercompatibility relationship between six cultivars with different genetic origins; these cultivars included Fortune (Citrus clementina Hort. ex Tan. × Citrus reticulata Blanco), Nova [(Citrus paradisi Macf. × C. reticulata) × C. clementina), Comune Clementine (C. clementina), Avana apireno (C. reticulata), Primosole (Citrus unshiu Marcov. × C. reticulata), and Simeto (C. unshiu × Citrus deliciosa Ten.). Following the controlled hand pollination in the field, we evaluated pollen tube performance after self- and cross-pollination, as well as intercultivar compatibility by observing pollen tube growth. The results show the self-incompatibility of these cultivars with the pollen tube having been arrested in the style, which explains their seedless condition when planted in solid blocks. The study of intercompatibility indicates a different pollen tube behavior depending on the genotype, as well as on the cross combinations. These results provide a basis to evaluate self- and intercompatibility in citrus, and the effect of close planting of some cultivars.

Reproductive biology and intergeneric breeding compatibility of ornamental Portulaca and Calandrinia (Portulacaceae)

Portulaca grandiflora Hook and P. umbraticola Kunth (Portulacaceae) are popular garden annuals, and have been bred for improved ornamental value. However, limited research has been published on hybridisation of Portulaca, with no reports on intergeneric hybridisation. Calandrinia balonensis Lindley and Calandrinia sp. nov. (not yet fully classified) are floriferous Australian Portulacaceae species, with potential as novel flowering pot plants, and are potential candidates for breeding with ornamental Portulaca. We studied the reproductive biology of these four species and breeding compatibility for reciprocal crosses of P. grandiflora × C. balonensis (2n = 18) and P. umbraticola × C. sp. nov. (2n = 24). All four species produced seeds for intraspecific outcrosses. P. grandiflora and C. sp. nov. are partially self-compatible whereas P. umbraticola and C. balonensis are highly self-incompatible. Autogamy was detected only for P. grandiflora. Reciprocal crosses of P. grandiflora × C. balonensis and P. umbraticola × C. sp. nov. with similar chromosome numbers did not produce seeds, primarily because of pollen–pistil incompatibility that prevents pollen-tube growth within the stigmata. Methods to overcome hybridisation barriers of these species combinations need to be established to create novel products for ornamental horticulture.

NATURAL SELECTION REINFORCES SPECIATION IN A RADIATION OF NEOTROPICAL RAINFOREST PLANTS

The importance of reinforcement, that is, natural selection that strengthens reproductive isolation between incipient species, remains controversial. We used two approaches to test for reinforcement in a species radiation of Neotropical gingers in the genus Costus. First, we conducted an intensive study of Costus pulverulentus and Costus scaber, two recently diverged species that co-occur and share hummingbird pollinators. The hummingbird pollinators transfer pollen between these Costus species, but hybrids are rarely found in nature. By performing pollinations between populations of C. pulverulentus and C. scaber from three sites across the species' geographic ranges, we find that pollen-pistil incompatibilities acting prior to fertilization have evolved only between locally sympatric populations, whereas geographically distant populations within the region of sympatry and allopatric populations remain fully interfertile. Second, we conducted a comparative study of isolating mechanisms across the genus. We find lower seed set due to pollen-pistil incompatibility between species pairs that co-occur and experience pollen transfer in nature compared to species pairs that are otherwise isolated, regardless of genetic distance. Taken together, these studies indicate that crossing barriers prevent potentially maladaptive hybridization and effectively reinforce the speciation process. Our results add to mounting evidence for reinforcement from animal studies and show that plant speciation may also involve complex mate recognition systems. Reinforcement may be particularly important in rapidly diverging lineages where ecological factors play a primary role in reproductive isolation, as may often be the case in tropical communities.

Microsporogenesis and crossing behavior of a tetraploid, interspecific inter-EBN hybrid potato

The commercial potato Solanum tuberosum L. autotetraploid, has many wild relatives with the ploidy range of 2–6×, which are suitable sources of traits interesting in potato breeding. However, not all of these species can be readily crossed with cultivated potato due to pre- and post-zygotic barriers. Post-zygotic barrier on Solanum spp. is attributed to their difference in endosperm balance number (EBN). According to the EBN successful crosses need to have a 2:1 ratio of maternal to paternal EBN in hybrid endosperm. Thus, only parents with the same EBN can be crossed. The cultivated potato is 4EBN, while wild allotetraploid relatives such as S. stoloniferum are 2EBN. In this study, meiosis and crossability of an exceptional tetraploid 3EBN hybrid obtained from our previous research by crossing S. stoloniferum × S. tuberosum with 2 and 4EBN testers were studied. Although the exact chromosome pairing analysis was not possible, the formation of several polyvalents in each meiocyte suggests that recombination could occur between different genomes. Meiosis had many irregularities such as unequal segregation as well as having laggard chromosome and micronuclei. Crosses involving the interspecific hybrid, as the male parent with 2 and 4EBN testers were not successful, some of which were apparently due to the pollen–pistil incompatibility. Interestingly controlled self-pollination resulted in viable seeds. Crosses of the interspecific hybrid as pistillate parent in the absence of pre-zygotic barrier in most case were successful. Results of the study confirmed the segregation of EBN value in the gametes of hybrid. It was expected that backcross progeny of this hybrid would be a good source for selecting aneutetraploids.

Combined Analysis of S-Alleles in European Pear by Pollinations and PCR-based S-Genotyping; Correlation between S-Phenotypes and S-RNase Genotypes

Pollen–pistil incompatibility in european pear (Pyrus communis L.) compromises adequate orchard pollination and fruit set and restricts cross-fertility between cultivars suitable as parents in breeding programs. Genetic control is simple, with a single locus expressed gametophytically in pollen controlling the rejection of the pollen tube in the style. Semicompatible pollination arises when only one allele of a pollen parent matches the pistil. Semicompatible test-crosses using partially S-genotyped european pear cultivars allowed the discrimination of 14 S-alleles (S1 to S14) at the phenotypic level and the assignment of 33 cultivars to 13 incompatibility groups. Partial genomic sequences of the S-RNase gene, spanning between the C1 and C5 conserved regions, were obtained for each new S-allele identified (S6 to S14). These sequences and those reported previously for the S1 to S5 RNases allowed a set of consensus primers amplifying all 14 S-RNase alleles to be designed. Allele-specific PCR allowed discrimination between those S-RNases giving amplification products of similar size with consensus primers. These two approaches provided a method for the molecular identification of all 14 S-alleles in european pear. With this methodology, we demonstrate that the S-RNase genotypes inferred from PCR exactly matches the S-phenotypes deduced from test-crosses. Comparison of the sequences obtained with those of S-RNases already published allowed us to relate S-alleles between studies. This will allow the prediction of cross-incompatibility among an even larger number of european pear cultivars.

Assessment of the Possibility of Direct Crossing Between Cultivated Potato and Two Wild Allotetraploid Relatives

The cultivated potato Solanum tuberosum L. is an autotetraploid with Endosperm Balance Number (EBN) of 4. There are some allotetraploid 2EBN wild species with desirable traits for potato improvement. However, it is very difficult to cross between these two groups due to differences in EBN. Eleven genotypes of autotetraploid cultivated potato including the six cultivars of subsp. Tuberosum (tbr) and five clones of subsp. Andigena (adg) were crossed with two wild allotetraploid species: S. acaule (acl) and S. stoloniferum (sto). For asses the pollen-pistil incompatibility, some ofpollinated flowers were fixed in Carnoy's solution and stained with aniline blue. Evaluation with fluorescent microscope revealed that pre-zygotic pollen-pistil incompatibility in these crosses is frequent. The pistils of both subsp. of cultivated potato were incompatible with pollens of acl but when sto used as staminate parent it was compatible with five cultivars of subsp. Tuberosum, but incompatible with most ofsubsp. Andigena (4 clones). From 739, 4x, 4EBN x 4x, 2EBN crosses we couldn't get any viable seed. In reciprocal crosses, allotetraploid species were pollinated with a mix of pollens from Tuberosum or Andigena clones. The crosses of sto x adg, acl x adg and acl x tbr were compatible but in the case of sto x tbr the situation was not clear. From these crosses 157 fruits were obtained but most of them contained only shrink seed and we find just one hybrid from the sto x tbr combination. The hybrid was tetraploid (2n = 4x = 48) and vigorous with profuse flowering and good fertility. When this hybrid was used as the pistillate parent it was compatible with both parental species.

Environmental effects on pollen–pistil compatibility between Phlox cuspidata and P. drummondii (Polemoniaceae): implications for hybridization dynamics

Postpollination mechanisms of reproductive isolation can critically influence the amount of gene flow between hybridizing species. While much evidence exists for genetically based pollen-pistil incompatibility, we show that environmental variation also influences the postpollination performance of heterospecific pollen in the annual Phlox hybrid system. Thus, the environmental segregation of species can influence hybridization dynamics. We found that P. cuspidata was restricted to soils of low Ca concentrations in the field and performed better under experimentally low Ca; P. drummondii was able to inhabit high-Ca soils and sometimes performed better in this environment. To determine whether soil Ca influenced pollen-pistil compatibility in a manner that alters pollen siring success, single-donor pollinations were performed in a completely factorial crossing design between species, maternal Ca environments, and paternal Ca environments. Maternal and paternal environments interacted in their effects on pollen-pistil compatibility for both inter- and intraspecific crosses, such that pollen performance was highest when mothers and fathers were grown in different soil Ca environments. These results suggest that when Phlox species predictably inhabit different environments, environmental heterogeneity can impede the processes of speciation and local adaptation by enhancing the performance of pollen dispersed across species and environments.

REPRODUCTIVE ISOLATION BETWEEN TWO CLOSELY RELATED HUMMINGBIRD POLLINATED NEOTROPICAL GINGERS

Empirical estimates of the relative importance of different barriers to gene flow between recently diverged species are important for understanding processes of speciation. I investigated the factors contributing to reproductive isolation between Costus pulverulentus and C. scaber (Costaceae), two closely related hummingbird-pollinated understory Neotropical herbs. I studied broad-scale geographic isolation, microhabitat isolation, flowering phenology, overlap in pollinator assemblages, floral constancy by pollinators, mechanical floral isolation, pollen-pistil interactions, seed set in interspecific crosses, and postzygotic isolation (hybrid seed germination, greenhouse survival to flowering, and pollen fertility). Aside from substantial geographic isolation, I found evidence for several factors contributing to reproductive isolation in the sympatric portion of their geographic ranges, but the identity and relative strength of these factors varied depending on the direction of potential gene flow. For C. pulverulentus as the maternal parent, mechanical floral isolation was the most important factor, acting as a complete block to interspecific pollen deposition. For C. scaber as the maternal parent, microhabitat isolation, pollinator assemblage, mechanical floral isolation, and postpollination pollen-pistil incompatibility were important. Overall, prezygotic barriers were found to be strong, resulting in 100% reproductive isolation for C. pulverulentus as the maternal parent and 99.0% reproductive isolation for C. scaber as the maternal parent. Some postzygotic isolation also was identified in the F1 generation, increasing total isolation for C. scaber to 99.4%. The results suggest that ecological factors, including habitat use and plant-pollinator interactions, contributed to speciation in this system and evolved before extensive intrinsic postzygotic isolation.