Evolutionary consequences of ecological factors: pollinator reliability predicts mating‐system traits of a perennial plant

The reproductive‐assurance hypothesis predicts that mating‐system traits will evolve towards increased autonomous self‐pollination in plant populations experiencing unreliable pollinator service. We tested this long‐standing hypothesis by assessing geographic covariation among pollinator reliability, outcrossing rates, heterozygosity and relevant floral traits across populations of Dalechampia scandens in Costa Rica. Mean outcrossing rates ranged from 0.16 to 0.49 across four populations, and covaried with the average rates of pollen arrival on stigmas, a measure of pollinator reliability. Across populations, genetically based differences in herkogamy (anther–stigma distance) were associated with variation in stigmatic pollen loads, outcrossing rates and heterozygosity. These observations are consistent with the hypothesis that, when pollinators are unreliable, floral traits promoting autonomous selfing evolve as a mechanism of reproductive assurance. Extensive covariation between floral traits and mating system among closely related populations further suggests that floral traits influencing mating systems track variation in adaptive optima generated by variation in pollinator reliability.     

MULTILEVEL ANALYSIS OF MORPHOMETRIC DATA FROM NATURAL PLANT POPULATIONS: INSIGHTS INTO ONTOGENETIC,GENETIC, AND SELECTIVE CORRELATIONS IN DALECHAMPIA SCANDENS

I examined patterns of covariation of three morphometric blossom characters [gland area (GA), gland–stigma distance (GSD), and bract length (BL)] within genets, among genets, and among populations of the tropical vine, Dalechampia scandens (Euphorbiaceae). Covariance between BL and GA was evenly distributed among the three levels. This observation, coupled with developmental information, indicates that the two characters change size similarly during development, that there is probably genetic covariance between them (apparently caused by pleiotropy), and that the genetic covariance may have constrained (at least proximally) the course of population differentiation with respect to these characters. Most covariance between GSD and GA occurred at the among-population level. This observation, coupled with developmental information, indicates that there is negligible ontogenetic covariance and that within populations there is probably little or no genetic covariance between the two characters. Among-population covariance has probably been caused by natural selection operating in a correlated fashion on characters that functionally interact in pollination.     

Genetic constraints predict evolutionary divergence in Dalechampia blossoms

If genetic constraints are important, then rates and direction of evolution should be related to trait evolvability. Here we use recently developed measures of evolvability to test the genetic constraint hypothesis with quantitative genetic data on floral morphology from the Neotropical vine Dalechampia scandens (Euphorbiaceae). These measures were compared against rates of evolution and patterns of divergence among 24 populations in two species in the D. scandens species complex. We found clear evidence for genetic constraints, particularly among traits that were tightly phenotypically integrated. This relationship between evolvability and evolutionary divergence is puzzling, because the estimated evolvabilities seem too large to constitute real constraints. We suggest that this paradox can be explained by a combination of weak stabilizing selection around moving adaptive optima and small realized evolvabilities relative to the observed additive genetic variance.     

Quantitative studies of the mating system in two sympatric species of Ipomoea (Convolvulaceae)

The rates of outcrossing in sympatric populations of Ipomoea purpurea and I, hederacea were estimated (using electrophoretic markers) to be 70% and 7% respectively. The difference in outcrossing rate is not apparently due to differences in pollinator service received by the species, but is associated with differences in anther-stigma distance. In I. purpurea stigmas are generally exserted and there is much genetic variation for anther-stigma distance. Variation in this character has a significant effect on the ease with which selfpollination occurs. In contrast there is no variation for the character in the I. hederacea population, the anthers being invariably held at the same level as the stigma, an arrangement promoting self-pollination.     

REPRODUCTIVE ASSURANCE MECHANISMS IN THREE TAXA OF THE MIMULUS GUTTATUS COMPLEX (SCROPHULARIACEAE)

The evolution of inbreeding in plants has often been attributed to selection for the ability to set seed in the absence of mates or pollinators. Mechanisms of reproductive assurance in five populations of mixed mating Mimulus guttatus, three populations of inbreeding M. platycalyx, and two populations of inbreeding M. nasutus were examined in a pollinator-free greenhouse. Reproductive assurance was manifested in all populations by autofertility, vegetative reproduction, or both. The inbreeding taxa had significantly greater levels of autofertility and less vegetative reproduction. Three modes of autofertility were identified: 1) due to corolla abscission only, occurring in three M. guttatus populations; 2) due to both corolla abscission and direct anther-stigma contact by curling of the lower stigmatic lobe into the anthers, occurring in two M. guttatus populations; and 3) direct stigma-anther contact by stigma curling alone prior to corolla abscission, found in each M. platycalyx and M. nasutus population. Stigma-anther distance and its interaction with stigma curling contributed to differences in autofertility among populations. Significant levels of intrapopulation quantitative genetic variation were found for seven of ten traits examined; average levels were similar between inbreeding and mixed mating populations. Genetic variation within populations for autofertility per se was not detected, but significant levels controlling stigma-anther distance were found in two M. guttatus populations. These results show that evolution of inbreeding by natural selection for reproductive assurance is possible in Mimulus, and illustrate the complex changes in floral dynamics and morphology it may involve.     

Morphometric analysis of floral variation in the Pyrola picta species complex (Ericaceae): interpretation and implications for ecological and phylogenetic differentiation

The Pyrola picta species complex of western North America comprises four species (P. picta, P. dentata, P. aphylla and P. crypta) that grow sympatrically in some parts of their collective ranges, have remarkably similar flowers and share pollinators. These species do not exhibit the genetic signatures typical of random or heterospecific mating, but instead show genetic divergence patterns indicating that they maintain surprising levels of reproductive isolation. To better understand how species boundaries are maintained, the current study uses statistical ordination analyses to determine whether species isolation across shared geographical ranges might be achieved through subtle differences in floral characters among species. The possible contribution of differences in flowering phenology (e.g. temporal reproductive isolation) to reproductive isolation was also evaluated for the small subset of populations in which two or more species occur in direct sympatry. Among species in the P. picta complex, there are both phylogenetic and geographical trends in some floral characteristics, whereas other characters do not covary with either geography or species identity. In several sympatric populations, differences in flowering phenology among species suggest that timing plays a major role in non‐random (i.e. mainly conspecific) mating. The conclusions of this study are that reproductive isolation in the P. picta species complex is reinforced by differences in the timing of floral maturation and the morphologies of androecium and floral display characters. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 00 , 000–000.     

GENETIC AND ENVIRONMENTAL VARIATION IN FLORAL TRAITS AFFECTING OUTCROSSING RATE IN CLARKIA TEMBLORIENSIS (ONAGRACEAE)

Clarkia tembloriensis exhibits a wide range of variation among its natural populations in outcrossing rate and in separation of male and female function in space (anther-stigma separation or herkogamy) and in time (protandry). Here we show that outcrossing rate is highly correlated with protandry and anther-stigma separation. Both genetic and environmental variation contribute to inter- and intrapopulation variation in protandry and anther-stigma separation. Interpopulation differentiation for protandry and anther-stigma separation was found to be polygenic. Genetic variation for protandry and anther-stigma separation within populations was demonstrated by a significant among-family variance in two populations with contrasting breeding systems. Environmental effects on the expression of mating system traits were manifested in two ways. First, significant variation among lathhouse benches suggests that small-scale environmental heterogeneity may affect the development of floral traits. Second, protandry was shortened under hot summer conditions. Hence, hotter and drier habitats, typical of the more self-pollinating populations of C. tembloriensis, can promote self-pollination purely through environmental effects.     

Comparing Variational Properties of Homologous Floral and Vegetative Characters in Dalechampia scandens: Testing the Berg Hypothesis

The Berg hypothesis posits that, in plants with specialized pollination systems, floral characters should evolve to become integrated with each other and decoupled from vegetative characters. We test this hypothesis by comparing serially homologous and morphologically similar characters in leaves and involucral bracts in the Neotropical vine Dalechampia scandens, which has a specialized pollination system based on resin-collecting bees. The involucral bracts serve a number of specialized floral functions, including signaling and protection, that may put them under stronger selection for precision than the less specialized leaves. The homology and morphological similarity of the leaves and bracts allow us to make a sharper test of Berg’s hypothesis than is possible in most other systems. We found support for the hypothesis in that the bracts had lower coefficients of variation than the leaves for comparable traits. Also in support of the hypothesis, we found essentially zero phenotypic correlations between bracts and leaves at the same time that we found moderate correlations between different leaves and between different bracts. In contradiction to the hypothesis, however, we did not find higher correlations among traits within bracts than within leaves, and we found no evidence of bracts being more developmentally stable than the leaves.     

Morphological variability in rupicolous species of the Acianthera prolifera complex (Orchidaceae) occurring in southeastern Brazil

We carried out multivariate morphometric analysis of 23 floral characters in seven populations of a complex of four species of Acianthera (Orchidaceae) occurring in Brazilian campo rupestre (rocky field) vegetation (A.?hamosa, A.?limae, A.?modestissima, and A.?prolifera) that flower synchronously and are partially intercompatible, and one putative hybrid population between A.?limae and A.?prolifera. We also carried out cluster analysis involving these eight populations plus 21 populations of a previously published study belonging to another species complex of Acianthera occurring in campo rupestre, including 12 floral characters in the analysis. Allopatric species pollinated by the same group of Diptera showed higher floral similarity among themselves than to a sympatric species pollinated by another group of Diptera. Such patterns indicate the existence of floral convergence in allopatric species and/or radiation in sympatric species. The analysis also indicated that there is more floral similarity between species of different complexes but that share the same group of pollinators. Large overlap was observed between A.?limae and the putative sympatric hybrids, indicating the occurrence of later generations of hybrids and/or individuals of A.?limae with introgression. The results do not support A.?hamosa and A.?modestissima as distinct species. These taxa are geographically isolated, occurring in different environments, are recognized only by vegetative characters that show high phenotypic plasticity, and share the same pollinators, being interfertile.     

Causes of covariation of phenotypic traits among populations

Morphological and life-history traits often vary among populations of a species. Traits generally do not vary independently, but show patterns of covariation that can arise from genetic and environmental influences on phenotype. Covariance of traits may arise at an among-population level when genetically influenced traits diverge among populations in a correlated manner. Genetic correlations caused by pleiotropy and/or gene linkage can cause traits to evolve together, but among-population covariance can also arise among traits that are not genetically correlated. For example, “selective covariance” can arise when natural selection directly causes correlated change in a suite of traits. Similarly, mutation, migration, and drift may also sometimes cause correlated genetic changes among populations. Because covariation of traits among populations can arise by several different processes, the evolution of suites of traits must be interpreted with great caution. We discuss the sources of among-population covariance and illustrate one approach to identifying the sources' using data on floral traits of Dalechampia scandens (Euphorbiaceae).     

VARIATION IN POLLEN FLOW WITHIN AND AMONG POPULATIONS OF IPOMOPSIS AGGREGATA

Pollen dispersal is a major component of gene flow in plant populations. It can influence microevolution within and among populations as well as the evolution of floral characters that affect dispersal. Most previous studies have relied on point estimates to characterize dispersal distances, even though there is likely to be substantial intrapopulational and interpopulational variation. We measured variation in pollen dispersal for the hummingbird-pollinated herb Ipomopsis aggregata (Polemoniaceae), using powdered fluorescent dyes to estimate pollen movement. Analysis of 5–6 natural populations in each of three years indicated that mean and mean squared distances of pollen dispersal, measured over the reproductive lifespan of individual plants, varied more than threefold among populations and years. Dispersal distances also shifted over the season within a given population. Unlike the variation among populations, these seasonal changes were associated in part with changes in flower density. The mean distance of pollen dispersal from an individual plant was unrelated to the date of first flowering, but did reflect two floral characters. Plants with higher variance in stamen length across flowers delivered pollen farther on average, as predicted by computer simulations of pollen carryover. Plants with lower mean stamen lengths also delivered pollen farther. Such effects of plant characters on pollen dispersal are a critical prerequisite for dispersal to evolve in response to its effects on fitness.     

Mismatch in the distribution of floral ecotypes and pollinators: insights into the evolution of sexually deceptive orchids

Plants are predicted to show floral adaptation to geographic variation in the most effective pollinator, potentially leading to reproductive isolation and genetic divergence. Many sexually deceptive orchids attract just a single pollinator species, limiting opportunities to experimentally investigate pollinator switching. Here, we investigate Drakaea concolor, which attracts two pollinator species. Using pollinator choice tests, we detected two morphologically similar ecotypes within D. concolor. The common ecotype only attracted Zaspilothynnus gilesi, whereas the rare ecotype also attracted an undescribed species of Pogonothynnus. The rare ecotype occurred at populations nested within the distribution of the common ecotype, with no evidence of ecotypes occurring sympatrically. Surveying for pollinators at over 100 sites revealed that ecotype identity was not correlated with wasp availability, with most orchid populations only attracting the rare Z. gilesi. Using microsatellite markers, genetic differentiation among populations was very low (G_ST = 0.011) regardless of ecotype, suggestive of frequent gene flow. Taken together, these results may indicate that the ability to attract Pogonothynnus has evolved recently, but this ecotype is yet to spread. The nested distribution of ecotypes, rather than the more typical formation of ecotypes in allopatry, illustrates that in sexually deceptive orchids, pollinator switching could occur throughout a species' range, resulting from multiple potentially suitable but unexploited pollinators occurring in sympatry. This unusual case of sympatric pollinators highlights D. concolor as a promising study system for further understanding the process of pollinator switching from ecological, chemical and genetic perspectives.     

POLLINATION OF TWO SYMPATRIC SPECIES OF DALECHAMPIA (EUPHORBIACEAE) IN SURINAME BY MALE EUGLOSSINE BEES

Dalechampia brownsbergensis and D. fragrans co-occur in Suriname, and both are pollinated by fragrance-collecting male euglossine bees. Dalechampia brownsbergensis appears to bloom year-round and is pollinated by relatively few species of bees, including Euglossa tridentata and E. gaianii. In contrast, D. fragrans appears to bloom from late October through early December and is visited and pollinated by at least 13 species of euglossines. Field observations of pollination indicated that the two species did not share pollinators. However, when the flowers of D. fragrans were “transplanted” into a population of D. brownsbergensis, the main pollinator of D. brownsbergensis also visited the flowers of D. fragrans. The pollinators of D. fragrans, however, did not visit the flowers of D. brownsbergensis. Partial sharing of pollinators may have only a small negative impact on the two sympatric plant species at this site because they flower simultaneously only part of the year, and they are often spatially separated from one another.     

Genetic diversity in the tetraploid sand dune endemic Deschampsia mackenzieana and its widespread iploid progenitor D. Cespitosa (Poaceae)

Electrophoretic variation was examined in 14 populations of tetraploid Deschampsia mackenzieana, an endemic of the Athabasca sand dunes in northern Saskatchewan, Canada, and 20 populations of its geographically widespread diploid progenitor, D. cespitosa. Three of the D. cespitosa populations were sympatric with the endemic on the Athabasca sand dunes. Populations of the endemic were found to have fewer alleles per locus (1.22 vs. 1.52), fewer alleles per polymorphic locus (2.17 vs. 2.70), lower percent polymorphic loci (18.9 vs. 30.5), and lower heterozygosity (0.062 vs. 0.119) than progenitor populations. Species level genetic diversity parameters also indicated that D. mackenzieana was genetically depauperate relative to its progenitor D. cespitosa. Deschampsia mackenzieana had no novel alleles but did share one allele with sympatric progenitor populations that did not occur in populations of D. cespitosa from other habitats. Although both species were found to partition most of their genetic diversity within populations, D. mackenzieana did have more of its limited genetic diversity partitioned among populations than D. cespitosa. The close genetic relationship between D. mackenzieana and sympatric populations of D. cespitosa may suggest the endemic tetraploid evolved from the sympatric diploid gene pool in the Athabasca sand dune region. The low levels of genetic diversity in D. mackenzieana suggest a restricted origin with limited gene flow from the progenitor since speciation.     

Floral traits and mating systems in sister species of <Emphasis Type="Italic">Nicotiana</Emphasis>: interpopulational variability and sympatry effects

Mating systems of Angiosperms are important determinants of population genetic structure and evolutionary potential. Nicotiana longiflora and N. plumbaginifolia are self-compatible, sister species, with contrasting floral morphology and can be found in allopatry and sympatry in North Argentina. In two sympatric and 10 nearby allopatric populations we studied their natural interpopulational variability and sympatry effects on corolla length and anther–stigma distance. We also estimated seed set by selfing and via pollinators using pollination treatments. Both corolla length and anther–stigma distance varied significantly among N. longiflora, but not among N. plumbaginifolia populations. We did not detect an effect of sympatry in either species for any of the floral traits studied. Pollination treatments suggest that N. longiflora is mainly an outcrosser, although selfing occurs at some extent. Seed set attributed to pollinators was significantly higher in N. longiflora whereas most seeds in N. plumbaginifolia were sired through self-pollination. In N. plumbaginifolia, selfing seems to assure reproduction in sympatric populations, where floral visitors have a strong preference for N. longiflora. Corolla length was significantly negatively correlated with an increase in the percentage of self-seeds estimated by pollination treatments. Within N. longiflora, anther–stigma distance showed a positive correlation with selfing.     

Novel microsatellite markers for Dalechampia scandens (Euphorbiaceae) and closely related taxa: application to studying a species complex

We developed novel microsatellite markers for D alechampia scandens L. (Euphorbiaceae). The target plants belong to a distinct, but undescribed, species in the D . scandens species complex, characterized by small resin‐producing glands. In total, 110 alleles over 36 novel markers were identified across 39 individuals from three populations. The number of alleles varied from one to seven, with an average of 3.06 ± 0.26 alleles per locus. The developed markers, along with previously developed ones for a large‐glanded D . scandens species, were tested for amplification in 11 additional species of the genus D alechampia. Four markers did not produce any detectable allele in 37 individuals from two populations of the large‐glanded species. Average expected heterozygosity across all small‐ and large‐glanded specific loci was 0.36 and 0.15, for the small and large glanded populations, respectively. Cross‐species amplification showed that 89% of all markers were successfully amplified in at least one of the 11 other D alechampia species. These microsatellite markers may be useful for detecting undescribed species in the D . scandens species complex, and can be used for comparative analyses of genetic structure, mating system and phylogeography of other D alechampia species.     

Comparative intra‐ and interspecific sexual organ reciprocity in four distylous Primula species in the Himalaya‐Hengduan Mountains

• Distyly is a mechanism promoting cross‐pollination within a balanced polymorphism. Numerous studies show that the degree of inter‐morph sexual organ reciprocity (SOR) within species relates to its pollen‐mediated gene flow. Similarly, a lower interspecific SOR should promote interspecific isolation when congeners are sympatric, co‐blooming and share pollinators. In this comparative study, we address the significance of SOR at both intra‐ and interspecific levels.
• Seventeen allopatric and eight sympatric populations representing four Primula species (P. anisodora, P. beesiana, P. bulleyana and P. poissonii) native to the Himalaya‐Hengduan Mountains were measured for eight floral traits in both long‐ and short‐styled morphs. GLMM and spatial overlap methods were used to compare intra‐ and interspecific SOR.
• While floral morphology differed among four Primula species, SOR within species was generally higher than between species, but in species pairs P. poissonii/P. anisodora and P. beesiana/P. bulleyana, the SOR was high at both intra‐ and interspecific levels. We did not detect a significant variation in intraspecific SOR or interspecific SOR when comparing allopatric versus sympatric populations for all species studied.
• As intraspecific SOR increased, disassortative mating may be promoted. As interspecific SOR decreased, interspecific isolation between co‐flowering species pairs also may increase. Hybridisation between congeners occurred when interspecific SOR increased in sympatric populations, as confirmed in two species pairs, P. poissonii/P. anisodora and P. beesiana/P. bulleyana.

     

Comparative floral and vegetative differentiation between two European Aquilegia taxa along a narrow contact zone

As a first step in determining the identity and relative importance of the evolutionary forces promoting the speciation process in two closely related European taxa of Aquilegia, we investigated the levels of morphological variation in floral and vegetative characters over the narrow region where their ranges enter into contact, and evaluate the relative importance of both types of traits in their differentiation. A total of 12 floral and ten vegetative characters were measured on 375 plants belonging to seven A. vulgaris populations and six A. pyrenaica subsp. cazorlensis populations located in southeastern Spain. Floral and vegetative morphological differentiation occur between taxa and among populations within taxa, but only vegetative characters (particularly plant height and leaf petiolule length) contribute significantly to the discrimination between taxa. Differentiation among populations within taxa is mostly explained by variation in floral traits. Consequently, morphological divergence between the two taxa cannot be interpreted as an extension of among-population differences occurring within taxa. Multivariate vegetative, but not floral, similarity between populations could be predicted from geographical distance. Moreover, the key role of certain vegetative traits in the differentiation of A. vulgaris and A. p. cazorlensis could possibly be attributable to the contrasting habitat requirements and stress tolerance strategies of the two taxa. These preliminary findings seem to disagree with the currently established view of the radiation process in the genus Aquilegia in North America, where the differentiation of floral traits seems to have played a more important role.     

Effect of floral organ sizes on female reproductive success in<Emphasis Type="Italic">Erythronium japonicum</Emphasis> (Liliaceae)

We examined the effects of floral organ size on female reproductive success in self-incompatibleErythronium japonicum. We measured tepal size and anther-stigma separation and investigated the relationship between these measurements and fruiting and seeding success. We found that tepal length was positively correlated with fruiting success and the number of seeds per fruit This suggests that pollinator attraction is affected by tepal length in f.japonicum and that the number of pollinator visits affects female reproductive success. Anther-stigma separation was the most variable floral trait measured and was not correlated with either fruiting or seeding success in this species, suggesting that the proportion of outcross pollen deposited on stigmas by pollinators does not increase with anther-stigma separation. This is inconsistent with a previous report onEtyyhtonium. grandiflorum. Pollinator size might explain this interspecific difference in the effect of anther-stigma separation on female reproductive success.     

BREEDING SYSTEMS IN ASCLEPIAS INCARNATA L., A. SYRIACA L., AND A. VERTICILLATA L.

A series of experimental pollinations involving three sympatric species provided strong evidence for physiological and/or genetic barriers to hybridization in Asclepias. Pollen tubes were observed to penetrate the style in some interspecific crosses of all species pairs. Aniline blue fluorescence microscopy also demonstrated pollen tube penetration of foreign ovules following pollinations between A. incarnata and A. verticillata. However, none of the 279 total pollinations attempted between species yielded mature seed, indicating the presence of late pre-fertilization or early post-fertilization incompatibility. Intraspecific pollinations in greenhouse and field populations revealed greater crossability between populations than within populations of a single species. Self-pollinations of A. verticillata were unsuccessful, while 29% and 4% of those of A. incarnata and A. syriaca, respectively, yielded mature follicles. It is suggested that the potential for autogamy, combined with floral mechanisms requiring pollination by insects, insure the advantages of both the genetic variability promoted by outcrossing and the reproductive assurance of uniparental reproduction. Strong reproductive barriers between species reduce the change of intergradation where species occur sympatrically.