Effects of anther-stigma position on cross-pollination efficiency in a hermaphroditic plant.

PREMISE: Evolution of cross-pollination efficiency depends on the genetic variation of flower traits, the pollen vector, and flower trait matching between pollen donors and recipients. Trait matching has been almost unexplored among nonheterostylous species, and we examined whether the match of anther length in pollen donors and stigma length in pollen recipients influences the efficiency of cross-pollination. To explore potential constraints for evolutionary response, we also quantified genetic variation and covariation among sepal length, petal length and width, stamen length, style length, and herkogamy. METHODS: We created 58 experimental arrays of Turnera velutina that varied in the extent of mismatch in the position of anthers and stigmas between single-flowered plants. Genetic variation and correlations among flower traits were estimated under greenhouse conditions. RESULTS: Style length, but not herkogamy, influenced the efficiency of cross-pollination. Plants with stamen length that matched the style length of other plants were more efficient pollen donors, whereas those with the style protruding above the stamens of other plants were more efficient pollen recipients. Significant broad-sense heritability (0.22 > hB 2 < 0.42) and moderate genetic correlations (0.33 > r < 0.85) among floral traits were detected. CONCLUSIONS: Our results demonstrated that anther-stigma mismatch between flowers contributed to variation in the efficiency of cross-pollination. The genetic correlations between stamen length and other floral traits suggests that any change in cross-pollination efficiency would be driven by changes in style rather than in stamen length.

Biomechanical properties of non-flight vibrations produced by bees.

Bees use thoracic vibrations produced by their indirect flight muscles for powering wingbeats in flight, but also during mating, pollination, defence and nest building. Previous work on non-flight vibrations has mostly focused on acoustic (airborne vibrations) and spectral properties (frequency domain). However, mechanical properties such as the vibration's acceleration amplitude are important in some behaviours, e.g. during buzz pollination, where higher amplitude vibrations remove more pollen from flowers. Bee vibrations have been studied in only a handful of species and we know very little about how they vary among species. In this study, we conducted the largest survey to date of the biomechanical properties of non-flight bee buzzes. We focused on defence buzzes as they can be induced experimentally and provide a common currency to compare among taxa. We analysed 15,000 buzzes produced by 306 individuals in 65 species and six families from Mexico, Scotland and Australia. We found a strong association between body size and the acceleration amplitude of bee buzzes. Comparison of genera that buzz-pollinate and those that do not suggests that buzz-pollinating bees produce vibrations with higher acceleration amplitude. We found no relationship between bee size and the fundamental frequency of defence buzzes. Although our results suggest that body size is a major determinant of the amplitude of non-flight vibrations, we also observed considerable variation in vibration properties among bees of equivalent size and even within individuals. Both morphology and behaviour thus affect the biomechanical properties of non-flight buzzes.

Climatic and soil characteristics account for the genetic structure of the invasive cactus moth Cactoblastis cactorum, in its native range in Argentina.

Background: Knowledge of the physical and environmental conditions that may limit the migration of invasive species is crucial to assess the potential for expansion outside their native ranges. The cactus moth, Cactoblastis cactorum, is native to South America (Argentina, Paraguay, Uruguay and Brazil) and has been introduced and invaded the Caribbean and southern United States, among other regions. In North America there is an ongoing process of range expansion threatening cacti biodiversity of the genus Opuntia and the commercial profits of domesticated Opuntia ficus-indica. Methods: To further understand what influences the distribution and genetic structure of this otherwise important threat to native and managed ecosystems, in the present study we combined ecological niche modeling and population genetic analyses to identify potential environmental barriers in the native region of Argentina. Samples were collected on the host with the wider distribution range, O. ficus-indica. Results: Significant genetic structure was detected using 10 nuclear microsatellites and 24 sampling sites. At least six genetic groups delimited by mountain ranges, salt flats and wetlands were mainly located to the west of the Dry Chaco ecoregion. Niche modeling supports that this region has high environmental suitability where the upper soil temperature and humidity, soil carbon content and precipitation were the main environmental factors that explain the presence of the moth. Environmental filters such as the upper soil layer may be critical for pupal survival and consequently for the establishment of populations in new habitats, whereas the presence of available hosts is a necessary conditions for insect survival, upper soil and climatic characteristics will determine the opportunities for a successful establishment.

The role of within-plant variation in nectar production: an experimental approach.

BACKGROUND AND AIMS: Nectar, a plant reward for pollinators, can be energetically expensive. Hence, a higher investment in nectar production can lead to reduced allocation to other vital functions and/or increased geitonogamous pollination. One possible strategy employed by plants to reduce these costs is to offer variable amounts of nectar among flowers within a plant, to manipulate pollinator behaviour. Using artificial flowers, we tested this hypothesis by examining how pollinator visitation responds to inter- and intra-plant variation in nectar production, assessing how these responses impact the energetic cost per visit. METHODS: We conducted a 2 × 2 factorial experiment using artificial flowers, with two levels of nectar investment (high and low sugar concentration) and two degrees of intra-plant variation in nectar concentration (coefficient of variation 0 and 20 %). The experimental plants were exposed to visits (number and type) from a captive Bombus impatiens colony, and we recorded the total visitation rate, distinguishing geitonogamous from exogamous visits. Additionally, we calculated two estimators of the energetic cost per visit and examined whether flowers with higher nectar concentrations (richer flowers) attracted more bumblebees. KEY RESULTS: Plants in the variable nectar production treatment (coefficient of variation 20 %) had a greater proportion of flowers visited by pollinators, with higher rates of total, geitonogamous and exogamous visitation, compared with plants with invariable nectar production. When assuming no nectar reabsorption, variable plants incurred a lower cost per visit compared with invariable plants. Moreover, highly rewarding flowers on variable plants had higher rates of pollination visits compared with flowers with few rewards. CONCLUSIONS: Intra-plant variation in nectar concentration can represent a mechanism for pollinator manipulation, enabling plants to decrease the energetic costs of the interaction while still ensuring consistent pollinator visitation. However, our findings did not provide support for the hypothesis that intra-plant variation in nectar concentration acts as a mechanism to avoid geitonogamy. Additionally, our results confirmed the hypothesis that increased visitation to variable plants is dependent on the presence of flowers with nectar concentration above the mean.

Geographic isolation and long-distance gene flow influence the genetic structure of the blue fan palm Brahea armata (Arecaceae).

The formation of the Baja California Peninsula (BCP) has impacted the microevolutionary dynamics of different species in ways that depend on biological traits such as dispersal capacity. Plants with relatively low levels of vagility have exhibited high genetic divergence between the BCP and Continental mainland. Brahea armata (Arecaceae) is a palm species inhabiting the northern part of the BCP and Sonora; its distribution occurs in isolated oases of vegetation. We aimed to evaluate the influence of the formation of the BCP on the genetic structure of B. armata using nuclear microsatellites and chloroplast markers (cpDNA) to compare patterns of genetic diversity and structure with previous published studies. Because gene flow through seeds is usually more limited compared to pollen flow, we expect to find stronger genetic structure at (cpDNA) than at nuclear markers. Moreover, larger genetic structure might also be explained by the smaller effective population size of cpDNA. We analyzed six microsatellite markers and two cpDNA regions. The main results indicated high levels of genetic differentiation among isolated populations located in the BCP, while low genetic differentiation was found between southern populations of the BCP and Sonora, suggesting long distance gene flow. In contrast, chloroplast markers indicated high levels of genetic structure between BCP and Sonora populations, suggesting asymmetrical gene flow between pollen (measured by nuclear microsatellites) and seed (cpDNA markers). This study provides valuable information on genetic diversity of B. armata that can be relevant for conservation and management; and develops microsatellites markers that can be transferred to other Brahea species.

Priming by Insects: Differential Effects of Sympatric and Allopatric Priming upon Plant Performance and Tolerance to Herbivory.

Plants have evolved multiple mechanisms to defend themselves from their multiple herbivores. Thus, being able to recognise among them and respond accordingly is fundamental for plant survival and reproduction. Defence priming prepares the plant to better or more rapidly respond to future damage; however, while it is considered an adaptive trait, to date, no studies have evaluated the extent and specificity of the priming recognition. To estimate the costs, benefits and specificity of priming, we used a highly specialist plant-insect system (Datura stramonium-Lema daturaphila) and performed a reciprocal transplant experiment with two populations where a priming stimulus (sympatric vs. allopatric) and a damage treatment (sympatric) were applied. We found no evidence of a fitness cost of priming, given that primed plants without damage showed no reduction in fitness. In contrast, our treatments affected the probability of bud abortion. That is, when damaged plants received no priming or the priming came from an allopatric insect, the likelihood of aborting the first bud was 1.9 times greater compared to plants being primed by their sympatric insect. We also found that damaged plants primed with an allopatric insect produced 14% fewer seeds compared to plants receiving a sympatric priming stimulus. Tolerance to herbivore damage was also the lowest when plants received the priming stimulus from an allopatric insect. Overall, these results suggest that, in our study system, plants recognise their local insect population reducing the negative effect of damage through a tolerance response.

Effects of herbivory and its timing on reproductive success of a tropical deciduous tree.

BACKGROUND AND AIMS: The implications of herbivory for plant reproduction have been widely studied; however, the relationship of defoliation and reproductive success is not linear, as there are many interacting factors that may influence reproductive responses to herbivore damage. In this study we aimed to disentangle how the timing of foliar damage impacts both male and female components of fitness, and to assess when it has greater impacts on plant reproductive success. METHODS: We measured herbivore damage and its effects on floral production, male and female floral attributes as well as fruit yield in three different phenological phases of Casearia nitida (Salicaceae) over the course of two consecutive years. Then we tested two models of multiple causal links among herbivory and reproductive success using piecewise structural equation models. KEY RESULTS: The effects of leaf damage differed between reproductive seasons and between male and female components of fitness. Moreover, the impact of herbivory extended beyond the year when it was exerted. The previous season's cumulated foliar damage had the largest impact on reproductive characters, in particular a negative effect on the numbers of inflorescences, flowers and pollen grains, indirectly affecting the numbers of infructescences and fruits, and a positive one on the amount of foliar damage during flowering. CONCLUSIONS: For perennial and proleptic species, the dynamics of resource acquisition and allocation patterns for reproduction promote and extend the effects of herbivore damage to longer periods than a single reproductive event and growing season, through the interactions among different components of female and male fitness.

Disentangling the effects of local resources, landscape heterogeneity and climatic seasonality on bee diversity and plant-pollinator networks in tropical highlands.

Land-use alteration and climate seasonality have profound effects on bee species diversity by influencing the availability of nesting and floral resources. Here, using twelve sites embedded in an agriculture-forest mosaic in the tropical highlands of Guatemala, we investigated the relative effects of climate seasonality and landscape heterogeneity on bee and floral-resource community structure and on their mutualistic network architecture. We found that climate seasonality affected bee diversity, which was higher in the wet season and associated positively with the availability of floral resources across both seasons. Bee community composition also differed between seasons and it was mainly driven by floral-resource richness and the proportion of agricultural, semi-natural and forest cover. In addition to the effects on bee diversity, climate seasonality also affected flower-bee visitation networks. We documented higher relative (null model corrected) nestedness in the dry season compared to the wet season. Niche partitioning as a result of competition for scarce resources in the dry season could be the process driving the differences in the network structure between seasons. Furthermore, relative nestedness was consistently smaller than zero, and relative modularity and specialization were consistently larger than zero in both seasons, suggesting the existence of isolated groups of interacting partners in all our flower-bee visitation networks. Our results highlight the effect of climatic seasonality and the importance of preserving local floral resources and natural heterogeneous habitats for the conservation of bee communities and their pollination services in tropical highlands.

Local dispersal pathways during the invasion of the cactus moth, Cactoblastis cactorum, within North America and the Caribbean.

Cactoblastis cactorum, a species of moth native to Argentina, feeds on several prickly pear cactus species (Opuntia) and has been successfully used as a biological control of invading Opuntia species in Australia, South Africa and native ruderal Opuntia species in some Caribbean islands. Since its introduction to the Caribbean its spread was uncontrolled, invading successfully Florida, Texas and Louisiana. Despite this long history of invasion, we are still far from understanding the factors determining the patterns of invasion of Cactoblastis in North America. Here, we explored three non-mutually exclusive explanations: a) a stepping stone model of colonization, b) long distance colonization due to hurricanes, and/or c) hitchhiking through previously reported commercial routes. Genetic diversity, genetic structure and the patterns of migration among populations were obtained by analyzing 10 nuclear microsatellite loci. Results revealed the presence of genetic structure among populations of C. cactorum in the invaded region and suggest that both marine commercial trade between the Caribbean islands and continental USA, as well as recurrent transport by hurricanes, explain the observed patterns of colonization. Provided that sanitary regulations avoiding human-mediated dispersal are enforced, hurricanes probably represent the most important agent of dispersal and future invasion to continental areas.

Ontogenetic changes in the targets of natural selection in three plant defenses.

The evolution of plant defenses has traditionally been studied at single plant ontogenetic stages, overlooking the fact that natural selection acts continuously on organisms along their development, and that the adaptive value of phenotypes can change along ontogeny. We exposed 20 replicated genotypes of Turnera velutina to field conditions to evaluate whether the targets of natural selection on different defenses and their adaptative value change across plant development. We found that low chemical defense was favored in seedlings, which seems to be explained by the assimilation efficiency and the ability of the specialist herbivore to sequester cyanogenic glycosides. Whereas trichome density was unfavored in juvenile plants, it increased relative plant fitness in reproductive plants. At this stage we also found a positive correlative gradient between cyanogenic potential and sugar content in extrafloral nectar. We visualize this complex multi-trait combination as an ontogenetic defensive strategy. The inclusion of whole-plant ontogeny as a key source of variation in plant defense revealed that the targets and intensity of selection change along the development of plants, indicating that the influence of natural selection cannot be inferred without the assessment of ontogenetic strategies in the expression of multiple defenses.

Natural selection acting on integrated phenotypes: covariance among functional leaf traits increases plant fitness.

Plant functional strategies are usually accomplished through the simultaneous expression of different traits, and hence their correlations should be promoted by natural selection. The adaptive value of correlations among leaf functional traits, however, has not been assessed in natural populations. We estimated intraspecific variation in leaf functional traits related to the primary metabolism and anti-herbivore defence in a population of Turnera velutina. We analysed whether natural selection favoured the expression of individual traits, particular combinations of traits or leaf phenotypic integration. Patterns of covariation among traits were related to water and nitrogen economy, and were similar among genotypes, but the magnitude of their phenotypic integration differed by 10-fold. Although families did not differ in the mean values of leaf functional traits, directional selection favoured low nitrogen content and low chemical defence, high content of chlorophyll, sugar in extrafloral nectar and trichome density. Families with higher phenotypic integration among leaf traits grew faster and produced more flowers. We suggest that the coordinated expression of leaf traits has an adaptive value, probably related to optimisation in the expression of traits related to water conservation and nitrogen acquisition.

Evolutionary transition between bee pollination and hummingbird pollination in Salvia: Comparing means, variances and covariances of corolla traits.

Covariation among traits can modify the evolutionary trajectory of complex structures. This process is thought to operate at a microevolutionary scale, but its long-term effects remain controversial because trait covariation can itself evolve. Flower morphology, and particularly floral trait (co)variation, has been envisioned as the product of pollinator-mediated selection. Available evidence suggests that major changes in pollinator assemblages may affect the joint expression of floral traits and their phenotypic integration. We expect species within a monophyletic lineage sharing the same pollinator type will show not only similarity in trait means but also similar phenotypic variance-covariance structures. Here, we tested this expectation using eighteen Salvia species pollinated either by bees or by hummingbirds. Our findings indicated a nonsignificant multivariate phylogenetic signal and a decoupling between means and variance-covariance phenotypic matrices of floral traits during the evolution to hummingbird pollination. Mean trait value analyses revealed significant differences between bee- and hummingbird-pollinated Salvia species although fewer differences were detected in the covariance structure between groups. Variance-covariance matrices were much more similar among bee- than hummingbird-pollinated species. This pattern is consistent with the expectation that, unlike hummingbirds, bees physically manipulate the flower, presumably exerting stronger selection pressures favouring morphological convergence among species. Overall, we conclude that the evolution of hummingbird pollination proceeded through different independent transitions. Thus, although the evolution of hummingbird pollination led to a new phenotypic optimum, the process involved the diversification of the covariance structure.

On the adaptive value of monomorphic versus dimorphic enantiostyly in Solanum rostratum.

Background and Aims: Enantiostyly is a reproductive system with heteromorphic flowers characterized by asymmetrical deflection of the style, either to the left or to the right of the floral axis. There are two types of enantiostyly. In monomorphic enantiostyly, plants produce the two types of flowers in the same individual. Dimorphic enantiostyly is restricted to only seven species and their populations consist of individuals producing either the right or the left flower type. It is hypothesized that the dimorphic form is derived from monomorphic ancestors because it functions as an outcrossing mechanism. We tested this latter hypothesis and investigated if monomorphic enantiostyly is resistant to invasion by individuals with dimorphic enantiostyly, because it functions as a reproductive assurance mechanism. Methods: To determine the conditions favouring the invasion of dimorphic enantiostyly, measurements of reproductive success and outcrossing rates in 15 natural flowering patches of Solanum rostratum were made. To test if monomorphic enantiostyly provides a reproductive assurance mechanism, experimental plants with either manually created dimorphic or natural monomorphic reproductive systems were exposed to two different pollination scenarios (flower density treatments), and reproductive success and outcrossing rates were measured. Key Results: Naturally flowering patches experienced severe pollination limitation, showed marked differences in reproductive success and had relatively high outcrossing rates. Plants in the experimental patches also showed pollination limitation and high outcrossing rates. Individuals with dimorphic enantiostyly expressed higher reproductive and outcrossing advantages under high-density conditions. These advantages disappeared in the low-density treatment, where the monomorphic form attained a higher reproductive success and no differences in outcrossing rates were detected. Conclusions: Monomorphic enantiostyly should be resistant to invasion of the dimorphic form because the prevalent ecological conditions favour the maintenance of geitonogamous individuals that are able to take advantage of ecological heterogeneity and generalized pollination limitation.

Different patterns of colonization of Oxalis alpina in the Sky Islands of the Sonoran desert via pollen and seed flow.

Historical factors such as climatic oscillations during the Pleistocene epoch have dramatically impacted species distributions. Studies of the patterns of genetic structure in angiosperm species using molecular markers with different modes of inheritance contribute to a better understanding of potential differences in colonization and patterns of gene flow via pollen and seeds. These markers may also provide insights into the evolution of reproductive systems in plants. Oxalis alpina is a tetraploid, herbaceous species inhabiting the Sky Island region of the southwestern United States and northern Mexico. Our main objective in this study was to analyze the influence of climatic oscillations on the genetic structure of O. alpina and the impact of these oscillations on the evolutionary transition from tristylous to distylous reproductive systems. We used microsatellite markers and compared our results to a previous study using chloroplast genetic markers. The phylogeographic structure inferred by both markers was different, suggesting that intrinsic characteristics including the pollination system and seed dispersal have influenced patterns of gene flow. Microsatellites exhibited low genetic structure, showed no significant association between geographic and genetic distances, and all individual genotypes were assigned to two main groups. In contrast, chloroplast markers exhibited a strong association between geographic and genetic distance, had higher levels of genetic differentiation, and were assigned to five groups. Both types of DNA markers showed evidence of a northward expansion as a consequence of climate warming occurring in the last 10,000 years. The data from both types of markers support the hypothesis for several independent transitions from tristyly to distyly.

Testing the hypothesis that biological modularity is shaped by adaptation: Xylem in the Bursera simaruba clade of tropical trees.

The study of modularity allows recognition of suites of character covariation that potentially diagnose units of evolutionary change. One prominent perspective predicts that natural selection should forge developmental units that maximize mutual functional independence. We examined the module-function relation using secondary xylem (wood) in a clade of tropical trees as a study system. Traditionally, the three main cell types in wood (vessels, fibers, and parenchyma) have respectively been associated with three functions (conduction, mechanical support, and storage). We collected samples from nine species of the simaruba clade of Bursera at fifteen sites and measured thirteen anatomical variables that have traditionally been regarded as reflecting the distinct functions of these cell types. If there are indeed (semi) independently evolving modules associated with functions, and cell types really are associated with these functions, then we should observe greater association between traits within cell types than between traits from different cell types. To map these associations, we calculated correlation coefficients among anatomical variables and identified modules using cluster and factor analysis. Our results were only partially congruent with expectations, with associations between characters of different cell types common. These results suggest causes of covariation, some involving selected function as predicted, but also highlighting the tradeoffs and shared developmental pathways limiting the evolutionary independence of some cell types in the secondary xylem. The evolution of diversity across the simaruba clade appears to have required only limited independence between parts.

A comparison of floral integration between selfing and outcrossing species: a meta-analysis.

BACKGROUND AND AIMS: Floral integration is thought to be an adaptation to promote cross-fertilization, and it is often assumed that it increases morphological matching between flowers and pollinators, increasing the efficiency of pollen transfer. However, the evidence for this role of floral integration is limited, and recent studies have suggested a possible positive association between floral integration and selfing. Although a number of explanations exist to account for this inconsistency, to date there has been no attempt to examine the existence of an association between floral integration and mating system. This study hypothesized that if pollinator-mediated pollen movement among plants (outcrossing) is the main factor promoting floral integration, species with a predominantly outcrossing mating system should present higher levels of floral integration than those with a predominantly selfing mating system. METHODS: A phylogenetically informed meta-analysis of published data was performed in order to evaluate whether mating system (outcrossing vs. selfing) accounts for the variation in floral integration among 64 species of flowering plants. Morphometric floral information was used to compare intra-floral integration among traits describing sexual organs (androecium and gynoecium) and those corresponding to the perianth (calix and corolla). KEY RESULTS: The analysis showed that outcrossing species have lower floral integration than selfing species. This pattern was caused by significantly higher integration of sexual traits than perianth traits, as integration of the latter group remained unchanged across mating categories. CONCLUSIONS: The results suggest that the evolution of selfing is associated with concomitant changes in intra-floral integration. Thus, floral integration of sexual traits should be considered as a critical component of the selfing syndrome.

The evolution of signal-reward correlations in bee- and hummingbird-pollinated species of Salvia.

Within-individual variation in floral advertising and reward traits is a feature experienced by pollinators that visit different flowers of the same plant. Pollinators can use advertising traits to gather information about the quality and amount of rewards, leading to the evolution of signal-reward correlations. As long as plants differ in the reliability of their signals and pollinators base their foraging decisions on this information, natural selection should act on within-individual correlations between signals and rewards. Because birds and bees differ in their cognitive capabilities, and use different floral traits as signals, we tested the occurrence of adaptive divergence of the within-individual signal-reward correlations among Salvia species that are pollinated either by bees or by hummingbirds. They are expected to use different floral advertising traits: frontal traits in the case of bees and side traits in the case of hummingbirds. We confirmed this expectation as bee- and hummingbird-pollinated species differed in which specific traits are predominantly associated with nectar reward at the within-individual level. Our findings highlight the adaptive value of within-individual variation and covariation patterns, commonly disregarded as 'environmental noise', and are consistent with the hypothesis that pollinator-mediated selection affects the correlation pattern among floral traits.

Genetic, morphological, geographical and ecological approaches reveal phylogenetic relationships in complex groups, an example of recently diverged pinyon pine species (Subsection Cembroides).

Elucidating phylogenetic relationships and species boundaries within complex taxonomic groups is challenging for intrinsic and extrinsic (i.e., technical) reasons. Mexican pinyon pines are a complex group whose phylogenetic relationships and species boundaries have been widely studied but poorly resolved, partly due to intrinsic ecological and evolutionary features such as low morphological and genetic differentiation caused by recent divergence, hybridization and introgression. Extrinsic factors such as limited sampling and difficulty in selecting informative molecular markers have also impeded progress. Some of the Mexican pinyon pines are of conservation concern but others may remain unprotected because the species boundaries have not been established. In this study we combined approaches to resolve the phylogenetic relationships in this complex group and to establish species boundaries in four recently diverged taxa: P. discolor, P. johannis, P. culminicola and P. cembroides. We performed phylogenetic analyses using the chloroplast markers matK and psbA-trnH as well as complete and partial chloroplast genomes of species of Subsection Cembroides. Additionally, we performed a phylogeographic analysis combining genetic data (18 chloroplast markers), morphological data and geographical data to define species boundaries in four recently diverged taxa. Ecological divergence was supported by differences in climate among localities for distinct genetic lineages. Whereas the phylogenetic analysis inferred with matK and psbA-trnH was unable to resolve the relationships in this complex group, we obtained a resolved phylogeny with the use of the chloroplast genomes. The resolved phylogeny was concordant with a haplotype network obtained using chloroplast markers. In species with potential for recent divergence, hybridization or introgression, nonhierarchical network-based approaches are probably more appropriate to protect against misclassification due to incomplete lineage sorting. The boundaries among genetic lineages were delimited by the inclusion of morphological, geographical and ecological data in the haplotype network. These multiple lines of evidence help to assign species boundaries in this complex group. P. johannis, P. discolor, P. culminicola and P. cembroides are different species based on their genetic, morphological and ecological niche differences. We suggest a reevaluation of the conservation status of these species considering the information generated in this study.

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.