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.

Testing the Distraction Hypothesis: Do extrafloral nectaries reduce ant-pollinator conflict?

Ant guards protect plants from herbivores, but can also hinder pollination by damaging reproductive structures and/or repelling pollinators. Natural selection should favour the evolution of plant traits that deter ants from visiting flowers during anthesis, without waiving their defensive services. The Distraction Hypothesis posits that rewarding ants with extrafloral nectar could reduce their visitation of flowers, reducing ant-pollinator conflict while retaining protection of other structures.We characterised the proportion of flowers occupied by ants and the number of ants per flower in a Mexican ant-plant, Turnera velutina. We clogged extrafloral nectaries on field plants and observed the effects on patrolling ants, pollinators and ants inside flowers, and quantified the effects on plant fitness. Based on the Distraction Hypothesis, we predicted that preventing extrafloral nectar secretion should result in fewer ants active at extrafloral nectaries, more ants inside flowers and a higher proportion of flowers occupied by ants, leading to ant-pollinator conflict, with reduced pollinator visitation and reduced plant fitness.Overall ant activity inside flowers was low. Preventing extrafloral nectar secretion through clogging reduced the number of ants patrolling extrafloral nectaries, significantly increased the proportion of flowers occupied by ants from 6.1% to 9.7%, and reduced plant reproductive output through a 12% increase in the probability of fruit abortion. No change in the numbers of ants or pollinators inside flowers was observed. This is the first support for the Distraction Hypothesis obtained under field conditions, showing ecological and plant fitness benefits of the distracting function of extrafloral nectar during anthesis. Synthesis. Our study provides the first field experimental support for the Distraction Hypothesis, suggesting that extrafloral nectaries located close to flowers may bribe ants away from reproductive structures during the crucial pollination period, reducing the probability of ant occupation of flowers, reducing ant-pollinator conflict and increasing plant reproductive success.

Ant-Pollinator Conflict Results in Pollinator Deterrence but no Nectar Trade-Offs.

Direct and indirect negative interactions between ant guards and pollinators on ant-plants are expected for two reasons. First, aggressive ants may deter pollinators directly. Second, pollinators benefit from plant investment in reproduction whilst ants benefit from plant investment in indirect defense, and resource allocation trade-offs between these functions could lead to indirect conflict. We explored the potential for ant-pollinator conflict in a Mexican myrmecophile, Turnera velutina, which rewards ants with extrafloral nectar and pollinators with floral nectar. We characterized the daily timing of ant and pollinator activity on the plant and used experiments to test for direct and indirect conflict between these two groups of mutualists. We tested for direct conflict by quantifying pollinator responses to flowers containing dead specimens of aggressive ant species, relative to unoccupied control flowers. We assessed indirect conflict by testing for the existence of a trade-off in sugar allocation between ant and pollinator rewards, evidenced by an increase in floral nectar secretion when extrafloral nectar secretion was prevented. Secretion of floral and extrafloral nectar, activity of ants and pollinators, and pollen deposition all overlapped in daily time and peaked within the first 2 h after flowers opened. We found evidence of direct conflict, in that presence of ants inside the flowers altered pollinator behavior and reduced visit duration, although visit frequency was unchanged. We found no evidence for indirect conflict, with no significant difference in the volume or sugar content of floral nectar between control plants and those in which extrafloral nectar secretion was prevented. The presence of ants in flowers alters pollinator behavior in ways that are likely to affect pollination dynamics, though there is no apparent trade-off between plant investment in nectar rewards for pollinators and ant guards. Further studies are required to quantify the effect of the natural abundance of ants in flowers on pollinator behavior, and any associated impacts on plant reproductive success.

Pollination biology of the hexaploid self-compatible species Turnera velutina (Passifloraceae).

The evolution of monomorphisms from heterostylous ancestors has been related to the presence of homostyly and the loss of self-incompatibility, allowing the occurrence of selfing, which could be advantageous under pollinator limitation. However, flowers of some monomorphic species show herkogamy, attraction and rewarding traits that presumably favour cross-pollination and/or a mixed mating system. This study evaluated the contributions of pollinators, breeding system and floral traits to the reproduction of Turnera velutina, a herkogamous monomorphic species. Floral visitors and frequency of visits were recorded, controlled hand cross-pollinations were conducted under greenhouse and natural conditions, and individual variation in floral traits was characterised to determine their contribution to seed production. Apis mellifera was the most frequent floral visitor. Flowers presented approach herkogamy, high variation in nectar features, and a positive correlation of floral length with nectar volume and sugar concentration. Seed production did not differ between manual self- and cross-pollinations, controls or open cross-pollinations, but autonomous self-pollination produced, on average, 82.74% fewer seeds than the other forms, irrespective of the level of herkogamy. Differences in seed production among autonomous self-pollination and other treatments showed that T. velutina flowers depend on insect pollination for reproduction, and that approach herkogamy drastically reduced seed production in the absence of pollen vectors. The lack of differences in seed production from manual cross- and self-pollinations suggests the possible presence of a mixed mating system in the studied population. Overall, this species was possibly derived from a distylous ancestor but appears fully capable of outcrossing despite being monomorphic.

Plant defence as a complex and changing phenotype throughout ontogeny.

BACKGROUND AND AIMS: Ontogenetic changes in anti-herbivore defences are common and result from variation in resource availability and herbivore damage throughout plant development. However, little is known about the simultaneous changes of multiple defences across the entire development of plants, and how such changes affect plant damage in the field. The aim of this study was to assess if changes in the major types of plant resistance and tolerance can explain natural herbivore damage throughout plant ontogeny. METHODS: An assessment was made of how six defensive traits, including physical, chemical and biotic resistance, simultaneously change across the major transitions of plant development, from seedlings to reproductive stages of Turnera velutina growing in the greenhouse. In addition, an experiment was performed to assess how plant tolerance to artificial damage to leaves changed throughout ontogeny. Finally, leaf damage by herbivores was evaluated in a natural population. KEY RESULTS: The observed ontogenetic trajectories of all defences were significantly different, sometimes showing opposite directions of change. Whereas trichome density, leaf toughness, extrafloral nectary abundance and nectar production increased, hydrogen cyanide and compensatory responses decreased throughout plant development, from seedlings to reproductive plants. Only water content was higher at the intermediate juvenile ontogenetic stages. Surveys in a natural population over 3 years showed that herbivores consumed more tissue from juvenile plants than from younger seedlings or older reproductive plants. This is consistent with the fact that juvenile plants were the least defended stage. CONCLUSIONS: The results suggest that defensive trajectories are a mixed result of predictions by the Optimal Defence Theory and the Growth-Differentiation Balance Hypothesis. The study emphasizes the importance of incorporating multiple defences and plant ontogeny into further studies for a more comprehensive understanding of plant defence evolution.

Understanding ontogenetic trajectories of indirect defence: ecological and anatomical constraints in the production of extrafloral nectaries.

BACKGROUND AND AIMS: Early ontogenetic stages of myrmecophytic plants are infrequently associated with ants, probably due to constraints on the production of rewards. This study reports for the first time the anatomical and histological limitations constraining the production of extrafloral nectar in young plants, and the implications that the absence of protective ants imposes for plants early during their ontogeny are discussed. METHODS: Juvenile, pre-reproductive and reproductive plants of Turnera velutina were selected in a natural population and their extrafloral nectaries (EFNs) per leaf were quantified. The anatomical and morphological changes in EFNs during plant ontogeny were studied using scanning electron and light microscopy. Extrafloral nectar volume and sugar concentration were determined as well as the number of patrolling ants. KEY RESULTS: Juvenile plants were unable to secrete or contain nectar. Pre-reproductive plants secreted and contained nectar drops, but the highest production was achieved at the reproductive stage when the gland is fully cup-shaped and the secretory epidermis duplicates. No ants were observed in juvenile plants, and reproductive individuals received greater ant patrolling than pre-reproductive individuals. The issue of the mechanism of extrafloral nectar release in T. velutina was solved given that we found an anatomical, transcuticular pore that forms a channel-like structure and allows nectar to flow outward from the gland. CONCLUSIONS: Juvenile stages had no ant protection against herbivores probably due to resource limitation but also due to anatomical constraints. The results are consistent with the growth-differentiation balance hypothesis. As plants age, they increase in size and have larger nutrient-acquiring, photosynthetic and storage capacity, so they are able to invest in defence via specialized organs, such as EFNs. Hence, the more vulnerable juvenile stage should rely on other defensive strategies to reduce the negative impacts of herbivory.