Pollinator-mediated selection on Krameria oil flowers: A flower-pollinator fit adaptation to an atypical oil-collecting behaviour?

BACKGROUND AND AIMS: Spatial variation in plant-pollinator interactions is a key driver of floral trait diversification. A so far overlooked qualitative aspect of this variation is the behavioural component on flowers that relates to the pollinator fit. We tested the hypothesis that variation in pollinator behaviour influences the geographical pattern of phenotypic selection across the distribution range of the oil-producing Krameria grandiflora (Krameriaceae). This variation mainly involves the presence or absence of flag petal grasping, which is only performed by representatives of Centris (Centridini, Apidae), an oil-collecting bee group highly associated with Krameriaceae pollination. METHODS: We quantified variation in floral traits and fitness and estimated pollinator-mediated selection in five populations at a large geographical scale comprising the entire species range. In each population, we sampled individual pollen arrival and germination as a fitness measure, indicating pollination success and pollination performance, which was then relativized and regressed on standardized flower-pollinator fit (flag-stigma distance), advertisement (sepal length) and reward (oil volume) traits. This generated mean-scaled selection gradients used to calculate geographical selection dispersion. KEY RESULTS: Unexpectedly, stronger selection was detected on the flower-pollinator fit trait in populations highly associated to the absence of the flag petal grasping. Geographical variation in selection was mainly attributed to differential selection on the flag-stigma distance generating a selection mosaic. This may involve influences of a spatial variation in pollinator behaviour as well as composition and morphology. CONCLUSIONS: Our results show the adaptive significance of the specialized "flag" petals of Krameria in the absence of the grasping behaviour and highlight the contribution of geographical variation in pollinator behaviour on flowers in driving selection mosaics, with implications for floral evolution, adaptation to pollinator fit and phenotypic diversity in specialized systems.

Phenotypic selection patterns in a hybrid zone between two Calceolaria species with contrasting pollinators: insights from field surveys and fitness assessments.

Hybrid zones provide natural experimental settings to test hypotheses about species divergence. We concentrated on a hybrid swarm in which oil-collecting bees and flower-pecking birds act as pollinators of two Calceolaria species. We asked whether both pollinators contributed to flower divergence by differentially promoting prezygotic fitness at the phenotypic extremes that represent parentals. We studied pollinator-mediated selection on phenotypic traits critical in plant-pollinator mechanical interaction, namely plant height, reward-to-stigma distance, and flower shape. We utilised the quantity and quality of pollen deposited as fitness measures and distinguished between the contribution of the two pollinator types. Results showed uni- and bivariate disruptive selection for most traits through pollen grains deposited by both pollinators. Bird-mediated fitness favoured low plants with a long reward-to-stigma distance and a straight corolla, while bee-mediated fitness favoured tall plants with a short reward-to-stigma distance and curved corolla. In addition, stabilising selection at one end of the phenotypic range showed a bird-mediated reproductive asymmetry within the swarm. The disruptive pattern was countered, albeit weakly, by hybrids receiving higher-quality pollen on the stigmas. Results suggest that pollinator-mediated selection promotes divergence of integrated flower phenotypes mechanically adjusted either to bees or birds underscoring the importance of pollinator specialisation in diversification.

Centre-periphery approaches based on geography, ecology and historical climate stability: what explains the variation in morphological traits of Bulnesia sarmientoi?

BACKGROUND AND AIMS: The centre-periphery hypothesis posits that higher species performance is expected in geographic and ecological centres rather than in peripheral populations. However, this is not the commonly found pattern; therefore, alternative approaches, including the historical dimension of species geographical ranges, should be explored. Morphological functional traits are fundamental determinants of species performance, commonly related to environmental stability and productivity. We tested whether or not historical processes may have shaped variations in tree and leaf traits of the Chaco tree Bulnesia sarmientoi. METHODS: Morphological variation patterns were analysed from three centre-periphery approaches: geographical, ecological and historical. Tree (stem and canopy) and leaf (leaf size and specific leaf area) traits were measured in 24 populations across the species range. A principal component analysis was performed on morphological traits to obtain synthetic variables. Linear mixed-effects models were used to test which of the implemented centre-periphery approaches significantly explained trait spatial patterns. KEY RESULTS: The patterns retrieved from the three centre-periphery approaches were not concordant. The historical approach revealed that trees were shorter in centre populations than in the periphery. Significant differences in leaf traits were observed between the geographical centre and the periphery, mainly due to low specific leaf area values towards the geographical centre. We did not find any pattern associated with the ecological centre-periphery approach. CONCLUSIONS: The decoupled response between leaf and tree traits suggests that these sets of traits respond differently to processes occurring at different times. The geographical and historical approaches showed centres with extreme environments in relation to their respective peripheries, but the historical centre has also been a climatically stable area since the Last Glacial Maximum. The historical approach allowed for the recovery of historical processes underlying variation in tree traits, highlighting that centre-periphery delimitations should be based on a multi-approach framework.

The role of ontogenetic allometry and nonallometric flower shape variation in species-level adaptive diversification - Calceolaria polyrhiza (Calceolariaceae) as a case study.

Organism shape changes predictably during ontogeny, resulting in specific patterns of ontogenetic allometry. In several plant and animal lineages, among-species variation in the shape of mature organisms mirrors variation along their growth trajectories. Hence, ontogenetic allometry is an important bias in evolution. This bias should be stronger at reduced evolutionary time scales, in which among-trait correlations had less time to evolve. Nevertheless, it was shown that adaptation of organism shape frequently involved departures from the ancestral ontogenetic allometry. Moreover, only a moderate fraction of shape variation is correlated with size during ontogeny. Hence, nonallometric variation in shape (NAVSh) is likely to contribute to adaptation, even at reduced evolutionary time scales. We explored the contributions of allometric variation in shape (AVSh), NAVSh, and size variation to adaptive evolution in the angiosperm species Calceolaria polyrhiza. This strongly relies on oil-collecting bees for pollination and experienced transitions in the size of pollinators during the last 2 Ma. Using geometric morphometrics, we described corolla morphology in several populations across its distribution range. Variation in corolla shape was decomposed into an allometric and a nonallometric component, and corolla size was estimated. We then looked for the correlation between these aspects of morphology and the pollinator. Our results suggest that adaptation to pollinators with different sizes relied on NAVSh, which resulted from shifts in the allometric slope and from shape changes that occurred early in flower development. We conclude that NAVSh can contribute to adaptation in flowering plants, even at the species-level.

Daily fluctuations in pollination effectiveness explain higher efficiency of native over exotic bees in Lepechinia floribunda (Lamiaceae).

BACKGROUND: Despite Stebbins' principle of the most efficient pollinator being proposed decades ago, the most important pollinators are still mainly identified using the frequency of visits to flowers. This shortcoming results in a gap between the characterization of the flower visitors of a plant species and a reliable estimation of the plant fitness consequences of the mutualistic interaction. The performance of a mutualistic visitor depends on its abundance, behaviour, effectiveness (pollen removal and deposition per unit time) and efficiency (seed set per unit time) conditioned by the temporal matching between pollinator activity and temporal patterns of maturation of the sexual functions of flowers. Although there have been recent attempts to provide a conceptual and methodological framework to characterize pollinators' performance, few have combined all key elements of visitors and plants to provide an accurate estimation of pollinators' performance under natural conditions. METHODS: We complement information on the flower biology and mating system of the sub-shrub Lepechinia floribunda (Lamiaceae) to provide a daily quantitative estimation of performance (effectiveness and efficiency) of the more abundant pollinators, i.e. native bumble-bees (Bombus spp.) and leafcutter bees (Megachile sp.), and the exotic honey-bee (Apis mellifera). KEY RESULTS: Unlike honey-bees or leafcutter bees, native bumble-bees matched the daily pattern of nectar production and stigma receptivity, and showed higher effectiveness and efficiency. Despite the overabundance of honey-bees, visits occurred mainly when stigmas were not receptive, thus reducing the honey-bees' overall performance. CONCLUSIONS: Bumble-bees appear to be the most important pollinators and potential historical mediators of reproductive trait evolution in L. floribunda. Because the production of seeds by bumble-bees involved fewer pollen grains for plants and less investment in floral display than honey-bees, contemporary and expected changes in pollinator abundance may affect future L. floribunda floral evolution. If bumble-bees were to be further displaced by anthropogenic disturbance or by competition with honey-bees, their lower efficiency will select for a larger floral display increasing reproductive costs. This scenario may also impose selection to reduce dichogamy to match honey-bee foraging activity.

Travelling to the south: Phylogeographic spatial diffusion model in Monttea aphylla (Plantaginaceae), an endemic plant of the Monte Desert.

Effects of Pleistocene climatic oscillations on plant phylogeographic patterns are relatively well studied in forest, savanna and grassland biomes, but such impacts remain less explored on desert regions of the world, especially in South America. Here, we performed a phylogeographical study of Monttea aphylla, an endemic species of the Monte Desert, to understand the evolutionary history of vegetation communities inhabiting the South American Arid Diagonal. We obtained sequences of three chloroplast (trnS-trnfM, trnH-psbA and trnQ-rps16) and one nuclear (ITS) intergenic spacers from 272 individuals of 34 localities throughout the range of the species. Population genetic and Bayesian coalescent analyses were performed to infer genealogical relationships among haplotypes, population genetic structure, and demographic history of the study species. Timing of demographic events was inferred using Bayesian Skyline Plot and the spatio-temporal patterns of lineage diversification was reconstructed using Bayesian relaxed diffusion models. Palaeo-distribution models (PDM) were performed through three different timescales to validate phylogeographical patterns. Twenty-five and 22 haplotypes were identified in the cpDNA and nDNA data, respectively. that clustered into two main genealogical lineages following a latitudinal pattern, the northern and the southern Monte (south of 35° S). The northern Monte showed two lineages of high genetic structure, and more relative stable demography than the southern Monte that retrieved three groups with little phylogenetic structure and a strong signal of demographic expansion that would have started during the Last Interglacial period (ca. 120 Ka). The PDM and diffusion models analyses agreed in the southeast direction of the range expansion. Differential effect of climatic oscillations across the Monte phytogeographic province was observed in Monttea aphylla lineages. In northern Monte, greater genetic structure and more relative stable demography resulted from a more stable climate than in the southern Monte. Pleistocene glaciations drastically decreased the species area in the southern Monte, which expanded in a southeastern direction to the new available areas during the interglacial periods.

Genetic diversity patterns of arbuscular mycorrhizal fungi associated with the mycoheterotroph Arachnitis uniflora Phil. (Corsiaceae).

Background and Aims: Arachnitis uniflora is a mycoheterotrophic plant that exploits arbuscular mycorrhizal fungi of neighbouring plants. We tested A. uniflora 's specificity towards fungi across its large latitudinal range, as well as the role of historical events and current environmental, geographical and altitudinal variables on fungal genetic diversity. Methods: Arachnitis uniflora mycorrhizas were sampled at 25 sites. Fungal phylogenetic relationships were reconstructed, genetic diversity was calculated and the main divergent lineages were dated. Phylogeographical analysis was performed with the main fungal clade. Fungal diversity correlations with environmental factors were investigated. Key Results: Glomeraceae fungi dominated, with a main clade that likely originated in the Upper Cretaceous and diversified in the Miocene. Two other arbuscular mycorrhizal fungal families not previously known to be targeted by A. uniflora were detected rarely and appear to be facultative associations. High genetic diversity, found in Bolivia and both northern and southern Patagonia, was correlated with temperature, rainfall and soil features. Conclusions: Fungal genetic diversity and its distribution can be explained by the ancient evolutionary history of the target fungi and by micro-scale environmental conditions with a geographical mosaic pattern.

The buck in the milkweed: evidence of male-male interference among pollinaria on pollinators.

Direct physical confrontation among conspecifics for access to mates is a form of sexual selection well known among animals, but not thought to take place in plants. Consequently, no structures are known that can be considered as weapons that evolved under such confrontation. Pollinaria of milkweeds may physically compete for access to attachment points on the pollinators' body, and occasionally pollinaria may link onto pre-existing pollinaria on a pollinator resulting in concatenation. We hypothesized that concatenation may result in interference between proximal and distal pollinaria, and that features of nonconcatenating pollinaria might be attributed to prevention of concatenation. We tested this by analyzing pollen donation efficiency, experimental manipulation of the phenotype and the phylogenetic patterns of co-occurrence of traits. It is shown that concatenation was able to diminish the reproductive performance of proximal pollinaria, that horns on pollinaria prevented concatenation, and that horn acquisition was correlated with a loss of concatenation. The experimental removal of horns in species that did not concatenate caused reversion to concatenation. The present work could be the first evidence of male physical struggles and of the acquisition of weapons related to these struggles, that are analogous to those known in animals.

Geographical differentiation in floral traits across the distribution range of the Patagonian oil-secreting Calceolaria polyrhiza: do pollinators matter?

BACKGROUND AND AIMS: The underlying evolutionary processes of pollinator-driven floral diversification are still poorly understood. According to the Grant-Stebbins model speciation begins with adaptive local differentiation in the response to spatial heterogeneity in pollinators. Although this crucial process links the micro- and macroevolution of floral adaptation, it has received little attention. In this study geographical phenotypic variation was investigated in Patagonian Calceolaria polyrhiza and its pollinators, two oil-collecting bee species that differ in body size and geographical distribution. METHODS: Patterns of phenotypic variation were examined together with their relationships with pollinators and abiotic factors. Six floral and seven vegetative traits were measured in 45 populations distributed across the entire species range. Climatic and edaphic parameters were determined for 25 selected sites, 2-16 bees per site of the most frequent pollinator species were captured, and a critical flower-bee mechanical fitting trait involved in effective pollination was measured. Geographical patterns of phenotypic and environmental variation were examined using uni- and multivariate analyses. Decoupled geographical variation between corolla area and floral traits related to the mechanical fit of pollinators was explored using a Mantel test. KEY RESULTS: The body length of pollinators and the floral traits related to mechanical fit were strongly correlated with each other. Geographical variation of the mechanical-fit-related traits was decoupled from variation in corolla size; the latter had a geographical pattern consistent with that of the vegetative traits and was mainly affected by climatic gradients. CONCLUSIONS: The results are consistent with pollinators playing a key role in shaping floral phenotype at a geographical scale and promoting the differentiation of two floral ecotypes. The relationship between the critical floral-fit-related trait and bee length remained significant even in models that included various environmental variables and an allometric predictor (corolla area). The abiotic environment also has an important role, mainly affecting floral size. Decoupled geographical variation between floral mechanical-fit-related traits and floral size would represent a strategy to maintain plant-pollinator phenotypic matching in this environmentally heterogeneous area.

Extreme variation in floral characters and its consequences for pollinator attraction among populations of an Andean cactus.

BACKGROUND AND AIMS: A South American cactus species, Echinopsis ancistrophora (Cactaceae), with dramatic among-population variation in floral traits is presented. METHODS: Eleven populations of E. ancistrophora were studied in their habitats in northern Argentina, and comparisons were made of relevant floral traits such as depth, stigma position, nectar volume and sugar concentration, and anthesis time. Diurnal and nocturnal pollinator assemblages were evaluated for populations with different floral trait combinations. KEY RESULTS: Remarkable geographical variations in floral traits were recorded among the 11 populations throughout the distribution range of E. ancistrophora, with flower lengths ranging from 4.5 to 24.1 cm. Other floral traits associated with pollinator attraction also varied in a population-specific manner, in concert with floral depth. Populations with the shortest flowers showed morning anthesis and those with the longest flowers opened at dusk, whereas those with flowers of intermediate length opened at unusual times (2300-0600 h). Nectar production varied non-linearly with floral length; it was absent to low (population means up to 15 microL) in short- to intermediate-length flowers, but was high (population means up to 170 microL) in the longest tubed flowers. Evidence from light-trapping of moths, pollen carriage on their bodies and moth scale deposition on stigmas suggests that sphingid pollination is prevalent only in the four populations with the longest flowers, in which floral morphological traits and nectar volumes match the classic expectations for the hawkmoth pollination syndrome. All other populations, with flowers 4.5-15 cm long, were pollinated exclusively by solitary bees. CONCLUSIONS: The results suggest incipient differentiation at the population level and local adaptation to either bee or hawkmoth (potentially plus bee) pollination.

New insights into the phylogenetic relationships, character evolution, and phytogeographic patterns of Calceolaria (Calceolariaceae).

Biogeographical patterns and diversification processes in Andean and Patagonian flora are not yet well understood. Calceolaria is a highly diversified genus of these areas, representing one of the most specialized plant-pollinator systems because flowers produce nonvolatile oils, a very unusual floral reward. Phylogenetic analyses with molecular (ITS and matK) and morphological characters from 103 Calceolaria species were conducted to examine relationships, to understand biogeographic patterns, and to detect evolutionary patterns of floral and ecological characters. Total evidence analysis retrieved three major clades, which strongly correspond to the three previously recognized subgenera, although only subgenus Rosula was retrieved as a monophyletic group. A single historical event explains the expansion from the southern to central Andes, while different parallel evolutionary lines show a northward expansion from the central to northern Andes across the Huancabamba Deflection, an important geographical barrier in northern Peru. Polyploidy, acquisition of elaiophores, and a nototribic pollination mechanism are key aspects of the evolutionary history of Calceolaria. Pollination interactions were more frequently established with Centris than with Chalepogenus oil-collecting bee species. The repeated loss of the oil gland and shifts to pollen as the only reward suggest an evolutionary tendency from highly to moderately specialized pollination systems.

'Prepackaged symbioses': propagules on roots of the myco-heterotrophic plant Arachnitis uniflora.

Arachnitis uniflora, a myco-heterotrophic plant species, has fleshy tuberous roots colonized by the arbuscular mycorrhizal fungal genus Glomus (Phylum Glomeromycota). These roots produce apical and lateral propagules, both reported here for the first time. The objective of the study was to characterize the ontogeny and structure of the propagules, and to determine their function. Scanning electron microscopy, laser scanning confocal microscopy and light microscopy were used to study the ontogeny and structure of the propagules. Propagules developed either from cortical parenchyma cells or from cells immediately beneath the root cap; they developed a shoot meristem and cells in the basal region which were colonized by various fungal structures including hyphae and vesicles. These propagules may detach from the roots, establishing new plants.

The southernmost myco-heterotrophic plant, Arachnitis uniflora: root morphology and anatomy.

Root morphology and anatomy of the myco-heterotrophic Arachnitis uniflora (Corsiaceae) were studied in relation to their association with a Glomus species (Glomeromycota). The mycorrhizal features were studied in three distinctive stages of development: (i) shoot and flower restricted to a small, underground bud; (ii) shoot and flower bud up to 1.5 cm; and (iii) shoot and flower already withered. The hyphae penetrate through and between the epidermal and exodermal cells; the exodermis and outer cortical cells become colonized in an inter- and intracellular manner, with some coils being formed in these layers. The fungi colonize the middle cortex, where intracellular vesicles in bundles are abundant. Arbuscules are formed profusely at very early stages of development, while in older stages they almost disappear and abundant vesicles are formed. Except for some details, the pattern of root colonization corresponds to a Paris-type. Presence of storage substances (starch and oil) also was recorded. Starch is produced and stored within root cells, mainly in the outer and inner root cortex. In senescent stages, plant and fungal tissues collapse.

Epiparasitic plants specialized on arbuscular mycorrhizal fungi.

Over 400 non-photosynthetic species from 10 families of vascular plants obtain their carbon from fungi and are thus defined as myco-heterotrophs. Many of these plants are epiparasitic on green plants from which they obtain carbon by 'cheating' shared mycorrhizal fungi. Epiparasitic plants examined to date depend on ectomycorrhizal fungi for carbon transfer and exhibit exceptional specificity for these fungi, but for most myco-heterotrophs neither the identity of the fungi nor the sources of their carbon are known. Because many myco-heterotrophs grow in forests dominated by plants associated with arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), we proposed that epiparasitism would occur also between plants linked by AMF. On a global scale AMF form the most widespread mycorrhizae, thus the ability of plants to cheat this symbiosis would be highly significant. We analysed mycorrhizae from three populations of Arachnitis uniflora (Corsiaceae, Monocotyledonae), five Voyria species and one Voyriella species (Gentianaceae, Dicotyledonae), and neighbouring green plants. Here we show that non-photosynthetic plants associate with AMF and can display the characteristic specificity of epiparasites. This suggests that AMF mediate significant inter-plant carbon transfer in nature.