Anatomical, histochemical, and developmental approaches reveal the long-term functioning of the floral nectary in Tocoyena formosa (Rubiaceae).

Tocoyena formosa has a persistent floral nectary that continues producing nectar throughout flower and fruit development. This plant also presents an intriguing non-anthetic nectary derived from early-developing floral buds with premature abscised corolla. In this study, we characterize the structure, morphological changes, and functioning of T. formosa floral nectary at different developmental stages. We subdivided the nectary into four categories based on the floral and fruit development stage at which nectar production started: (i) non-anthetic nectary; (ii) anthetic nectary, which follows the regular floral development; (iii) pericarpial nectary, derived from pollinated flowers following fruit development; and (iv) post-anthetic nectary that results from non-pollinated flowers after anthesis. The nectary has a uniseriate epidermis with stomata, nectariferous parenchyma, and vascular bundles, with a predominating phloem at the periphery. The non-anthetic nectary presents immature tissues that release the exudate. The nectary progressively becomes more rigid as the flower and fruit develop. The main nectary changes during flower and fruit development comprised the thickening of the cuticle and epidermal cell walls, formation of cuticular epithelium, and an increase in the abundance of calcium oxalate crystals and phenolic cells near the vascular bundles. Projections of the outer periclinal walls toward the cuticle in the post-anthetic nectary suggest nectar reabsorption. The anatomical changes of the nectary allow it to function for an extended period throughout floral and fruit development. Hence, T. formosa nectary is a bivalent secretory structure that plays a crucial role in the reproductive and defensive interactions of this plant species.

Root deformation affects mineral nutrition but not leaf gas exchange and growth of Genipa americana seedlings during the recovery phase after soil flooding / A deformação de raízes afeta a nutrição mineral mas não as trocas gasosas foliares e o crescimento de mudas de Genipa americana durante a fase de recuperação após o alagamento do solo

Root deformation (RD) caused by errors in the pricking out process are irreversible and very difficult to detect in container-grown seedlings at the time of planting in the field. The objective of this study was to evaluate the effects of RD on leaf gas exchange, growth, biomass allocation and mineral nutrition of G. americana seedlings during the recovery phase after soil flooding. Four-months-old seedlings, with and without RD, were flooded for 42 days and their recovery was evaluated 28 days after soil drainage. There were no significant interactions between RD and soil flooding for all leaf gas exchange, growth and mineral nutrition after soil drainage, with the exception of leaf P concentrations. In plants with no RD, the P concentration in leaves of non-flooded plants was significantly higher than that of plants with RD. Soil flooding and RD did not influence leaf or root N concentrations or whole-plant N content. RD increased the K concentration in the roots, but not in the leaves. Changes in the nutrient concentrations in leaves and roots indicate that RD may affect physiological performance of seedlings after planting in the field.

A deformação da raiz (RD) causada por erros no processo de repicagem é irreversível e difícil de detectar em mudas produzidas em embalagens no momento do plantio no campo. O objetivo deste estudo foi avaliar os efeitos do RD nas trocas gasosas foliares, crescimento, alocação de biomassa e nutrição mineral de mudas de G. americana na fase de recuperação após o alagamento do solo. Mudas com quatro meses de idade, com e sem RD, foram alagadas por 42 dias e a sua recuperação foi avaliada 28 dias após a drenagem do solo. Não houve interação significativa entre RD e alagamento do solo nas trocas gasosas foliares, crescimento e nutrição mineral após a drenagem, com exceção das concentrações de P foliar. Em plantas sem RD, a concentração de P nas folhas de plantas não alagadas foi significativamente maior que a das plantas com RD. O alagamento do solo e a RD não influenciaram as concentrações de N nas folhas e raízes, e no conteúdo de N na planta inteira. A RD aumentou a concentração de K nas raízes, mas não nas folhas. Alterações nas concentrações de nutrientes nas folhas e raízes indicam que a RD pode afetar o desempenho fisiológico das mudas após o plantio no campo.

Tradeoffs in the evolution of plant farming by ants.

Diverse forms of cultivation have evolved across the tree of life. Efficient farming requires that the farmer deciphers and actively promotes conditions that increase crop yield. For plant cultivation, this can include evaluating tradeoffs among light, nutrients, and protection against herbivores. It is not understood if, or how, nonhuman farmers evaluate local conditions to increase payoffs. Here, we address this question using an obligate farming mutualism between the ant Philidris nagasau and epiphytic plants in the genus Squamellaria that are cultivated for their nesting sites and floral rewards. We focused on the ants' active fertilization of their crops and their protection against herbivory. We found that ants benefited from cultivating plants in full sun, receiving 7.5-fold more floral food rewards compared to shade-cultivated plants. The higher reward levels correlated with higher levels of crop protection provided by the ants. However, while high-light planting yielded the greatest immediate food rewards, sun-grown crops contained less nitrogen compared to shade-grown crops. This was due to lower nitrogen input from ants feeding on floral rewards instead of insect protein gained from predation. Despite this tradeoff, farming ants optimize crop yield by selectively planting their crops in full sun. Ancestral state reconstructions across this ant-plant clade show that a full-sun farming strategy has existed for millions of years, suggesting that nonhuman farmers have evolved the means to evaluate and balance conflicting crop needs to their own benefit.

Seed dispersal by proboscis monkeys: the case of Nauclea spp.

Frugivorous vertebrates such as primates are important dispersal agents in tropical forests, although the role of folivorous colobines is generally not considered. However, recent studies reported seed dispersal by endo- and epizoochory in colobine primates, including the proboscis monkey (Nasalis larvatus), suggesting that the role colobines play in seed dispersal might have been underestimated. In the Lower Kinabatangan Floodplain in Sabah, Malaysian Borneo, we investigated whether seeds were still able to germinate after being ingested by proboscis monkeys. Faecal samples (n = 201) from proboscis monkeys were collected between 2015 and 2017. Intact seeds belonging to eight plant species were found in 77% of the faecal samples. Nauclea spp. were the most abundant plant species, accounting for 98% of all intact seeds. This study is the first to conduct germination trials on seeds defecated by proboscis monkeys. Higher germination success was recorded in ingested Nauclea spp. seeds than in control seeds, from both ripe and unripe Nauclea orientalis fruits (P < 0.001). Therefore, we suggest that proboscis monkeys play a role in seed dispersal by enhancing the germination success of defecated seeds for at least some plant species. Similar to other colobines, although proboscis monkeys may provide a lower contribution to seed dispersal (low seed diversity over short distances) than other sympatric frugivores, this study emphasises that proboscis monkeys do contribute to the dispersal of intact seeds, such as Nauclea spp., in potentially suitable riverine habitats.

Changes in cambial activity are related to precipitation patterns in four tropical hardwood species grown in Indonesia.

PREMISE: Cambial activity in some tropical trees varies intra-annually, with the formation of xylem rings. Identification of the climatic factors that regulate cambial activity is important for understanding the growth of such species. We analyzed the relationship between climatic factors and cambial activity in four tropical hardwoods, Acacia mangium, Tectona grandis, Eucalyptus urophylla, and Neolamarckia cadamba in Yogyakarta, Java Island, Indonesia, which has a rainy season (November-June) and a dry season (July-October). METHODS: Small blocks containing phloem, cambium, and xylem were collected from main stems in January 2014, October 2015 and October 2016, and examined with light microscopy for cambial cell division, fusiform cambial cells, and expanding xylem cells as evidence of cambial activity. RESULTS: During the rainy season, when precipitation was high, cambium was active. By contrast, during the dry season in 2015, when there was no precipitation, cambium was dormant. However, in October 2016, during the so-called dry season, cambium was active, cell division was conspicuous, and a new xylem ring formation was initiated. The difference in cambial activity appeared to be related to an unusual pattern of precipitation during the typically dry months, from July to October, in 2016. CONCLUSIONS: Our results indicate that low or absent precipitation for 3 to 4 months induces cessation of cambial activity and temporal periodicity of wood formation in the four species studied. By contrast, in the event of continuing precipitation, cambial activity in the same trees may continue throughout the year. The frequency pattern of precipitation appears to be an important determinant of wood formation in tropical trees.

Relative effectiveness of insects versus hummingbirds as pollinators of Rubiaceae plants across elevation in Dominica, Caribbean.

Most angiosperms rely on animal pollination for reproduction, but the dependence on specific pollinator groups varies greatly between species and localities. Notably, such dependence may be influenced by both floral traits and environmental conditions. Despite its importance, their joint contribution has rarely been studied at the assemblage level. At two elevations on the Caribbean island of Dominica, we measured the floral traits and the relative contributions of insects versus hummingbirds as pollinators of plants in the Rubiaceae family. Pollinator importance was measured as visitation rate (VR) and single visit pollen deposition (SVD), which were combined to assess overall pollinator effectiveness (PE). In the wet and cool Dominican highland, we found that hummingbirds were relatively more frequent and effective pollinators than insects, whereas insects and hummingbirds were equally frequent and effective pollinators at the warmer and less rainy midelevation. Furthermore, floral traits correlated independently of environment with the relative importance of pollinators, hummingbirds being more important in plant species having flowers with long and wide corollas producing higher volumes of dilute nectar. Our findings show that both environmental conditions and floral traits influence whether insects or hummingbirds are the most important pollinators of plants in the Rubiaceae family, highlighting the complexity of plant-pollinator systems.

Drying and reduction in sensitivity to desiccation of seeds of Alibertia edulis: the influence of fruit ripening stage.

The intense environmental degradation in tropical regions suitable for agriculture has decreased native forest populations of plants with important fruits and medicinal properties. Alibertia edulis is a native tree from the Brazilian Cerrado. Knowledge about the effects of drying and storage on the physiological behavior of its seeds may aid in its sustainable exploitation and conservation. The goal of the present study was to determine which fruit ripening stage results in A. edulis seeds with higher tolerance to desiccation, and to investigate the effectiveness of polyethylene glycol (PEG) induced osmotic stress in combination with abscisic acid (ABA) in increasing seed desiccation tolerance during storage. Seeds were dried in activated silica gel (fast) or under ambient conditions (slow). Seeds originating from mid-ripe or fully ripe fruits exhibited better physiological performance than those obtained from green fruits. Slow drying resulted in seeds with high physiological potential. Seeds may be stored for up to 180 days without losing viability when treated with -0.73 MPa PEG without ABA.

Diversity of epicotyl dormancy among tropical montane forest species in Sri Lanka.

Fruiting season of many Sri Lankan tropical montane species is not synchronised and may not occur when conditions are favourable for seedling establishment. We hypothesised that species with different fruiting seasons have different seed dormancy mechanisms to synchronise timing of germination with a favourable season for establishment. Using six species with different fruiting seasons, we tested this hypothesis. Germination and imbibition of intact and manually scarified seeds were studied. Effect of GA3 on germination was examined. Embryo length:seed length (E:S) ratio of freshly matured seeds and of those with a split seed coat was determined. Time taken for radicle and plumule emergence and morphological changes of the embryos were recorded. The radicle emerged from Ardisia missionis, Bheza nitidissima and Gaetnera walkeri seeds within 30 days, whereas it took >30 days in other species. Embryos grew in seeds of B. nitidissima and G. walkeri prior to radicle emergence but not in Microtropis wallichiana, Nothapodytes nimmoniana and Symplocos cochinchinensis. A considerable delay was observed between radicle and plumule emergence in all six species. Warm stratification and/or GA3 promoted germination of all species. All the tested species have epicotyl dormancy. Seeds of B. nitidissima and G. walkeri have non-deep simple morphophysiological epicotyl dormancy, and the other four species have non-deep physiological epicotyl dormancy. Differences in radicle and epicotyl dormancy promote synchronisation of germination to a favourable time for seedling development. Therefore, information on dormancy-breaking and germination requirements of both radicle and epicotyl are needed to determine the kind of dormancy of a particular species.

Colleters in Rubiaceae from forest and savanna: the link between secretion and environment.

This study aims to investigate colleters' secretory function, on cellular level, in Rubiaceae species from contrasting environments looking to explore the association between secretion and environment. We collected samples from eight species of Rubiaceae growing in forest and savanna having standard-type colleters with diverse histochemistry (hydrophilic, lipophilic and mixed secretions) and processed for both conventional and cytochemical study under transmission electron microscopy (TEM). The standard colleters, although similar in morphology and anatomy, exhibited marked differences on cellular level, especially in the abundance and topology of Golgi bodies, endoplasmic reticulum and plastids when comparing forest and savanna species. These differences were clearly aligned with the chemical nature of the secretions they produce, with predominance of hydrophilic secretions in forest species and lipophilic or mixed secretions in savanna species. The combination of methods in electron microscopy revealed the sites of synthesis and intracellular compartmentation of substances, the mechanisms of their secretion from the protoplast and confirmed the involvement of the outer walls of the epithelial cells in the elimination of exudates to the gland surface. Our study suggests a potential environment-associated plasticity of the secretory cells of standard-type colleters in modulating their secretory function performance.

Obligate plant farming by a specialized ant.

Many epiphytic plants have associated with ants to gain nutrients. Here, we report a novel type of ant-plant symbiosis in Fiji where one ant species actively and exclusively plants the seeds and fertilizes the seedlings of six species of Squamellaria (Rubiaceae). Comparison with related facultative ant plants suggests that such farming plays a key role in mutualism stability by mitigating the critical re-establishment step.

Does intraspecific behavioural variation of pollinator species influence pollination? A quantitative study with hummingbirds and a Neotropical shrub.

Floral visitors differ in their efficacy as pollinators, and the impact of different pollinator species on pollen flow and plant reproduction has been frequently evaluated. In contrast, the impact of intraspecific behavioural changes on their efficacy as pollinators has seldom been quantified. We studied a self-incompatible shrub Palicourea rigida (Rubiaceae) and its hummingbird pollinators, which adjust their behaviour according to floral resource availability. Fluorescence microscopy was used to access pollen tube growth and incompatibility reaction in pistils after a single visit of territorial or intruder hummingbirds in two populations. To characterise the plant populations and possible differences in resource availability between areas we used a three-term quadrat variance method to detect clusters of floral resources. Within-species variation in foraging behaviour, but not species identity, affected pollinator efficacy. Effectively, hummingbirds intruding into territories deposited more compatible pollen grains on P. rigida stigmas than territory holders in both study areas. Additionally, territory holders deposited more incompatible than compatible pollen grains. Our results imply that intraspecific foraging behaviour variation has consequences for pollination success. Quantifying such variation and addressing the implications of intraspecific variability contribute to a better understanding of the dynamics and consequences of plant-pollinator interactions.

Neighbourhood structure and light availability influence the variations in plant design of shrubs in two cloud forests of different successional status.

BACKGROUND AND AIMS: Plant design refers to the construction of the plant body or its constituent parts in terms of form and function. Although neighbourhood structure is recognized as a factor that limits plant survival and species coexistence, its relative importance in plant design is not well understood. We conducted field research to analyse how the surrounding environment of neighbourhood structure and related effects on light availability are associated with changes in plant design in two understorey plants (Palicourea padifolia and Psychotria elata) within two successional stages of a cloud forest in Costa Rica. METHODS: Features of plant neighbourhood physical structure and light availability, estimated using hemispherical photographs, were used as variables that reflect the surrounding environment. Measures of plant biomechanics, allometry, branching and plant slenderness were used as functional plant attributes that reflect plant design. We propose a framework using a partial least squares path model and used it to test this association. KEY RESULTS: The multidimensional response of plant design of these species suggests that decreases in the height-based factor of safety and increases in mechanical load and developmental stability are influenced by increases in maximum height of neighbours and a distance-dependence interference index more than neighbourhood plant density or neighbour aggregation. Changes in plant branching and slenderness are associated positively with light availability and negatively with canopy cover. CONCLUSIONS: Although it has been proposed that plant design varies according to plant density and light availability, we found that neighbour size and distance-dependence interference are associated with changes in biomechanics, allometry and branching, and they must be considered as key factors that contribute to the adaptation and coexistence of these plants in this highly diverse forest community.

Ontogenesis secretion and senescence of Tocoyena bullata (Vell.) Mart. (Rubiacaeae) colleters.

Colleters are secretory structure present on many families including Rubiaceae. Particular characteristics have been described about colleters secretory cells, however senescence process are still under debate. Tocoyena bullata (Vell.) Mart. (Rubiaceae) shoot apex were collected at Jardim Botânico do Rio de Janeiro, RJ/Brazil. Stipules were separated and fragments were fixed in 2.5% glutaraldehyde and 4.0% formaldehyde in 0.05 m sodium cacodylate buffer, pH 7.2, post fixed in 1.0% osmium tetroxide in the same buffer, dehydrated in acetone, critical-point-drying, sputtered coated and observed. For light microscopy fragments were fixed and dehydrated, infiltrated with historesin and stained with 1% toluidine blue. For transmission electron microscopy, the samples were infiltrated with Epoxi resin. Colleters are present on stipule adaxial surface. On the beginning of development, these structures are recognized as small projections. Later on, colleters differentiated and secrete by cuticle rupture. The colleters senescence occurs in a concomitant and indissoluble way of programmed cell death. Ultrastructural analyses during the process strongly suggest the senescence is based on a non-autolitic programmed cell death. T. bullata colleters, present at stipule abaxial surface are cylindrical secretory structures. Colleters secretory cells originated as stipule projections; differentiate; secrete and senesce by programmed cell death. The secretion and the cell dead occurs in a concomitantly and indissoluble way.

Partner choice through concealed floral sugar rewards evolved with the specialization of ant-plant mutualisms.

Obligate mutualisms require filtering mechanisms to prevent their exploitation by opportunists, but ecological contexts and traits facilitating the evolution of such mechanisms are largely unknown. We investigated the evolution of filtering mechanisms in an epiphytic ant-plant symbiotic system in Fiji involving Rubiaceae and dolichoderine ants, using field experiments, metabolomics, X-ray micro-computed tomography (micro-CT) scanning and phylogenetics. We discovered a novel plant reward consisting of sugary sap concealed in post-anthetic flowers only accessible to Philidris nagasau workers that bite through the thick epidermis. In five of the six species of Rubiaceae obligately inhabited by this ant, the nectar glands functioned for 10 d after a flower's sexual function was over. Sugar metabolomics and field experiments showed that ant foraging tracks sucrose levels, which only drop at the onset of fruit development. Ontogenetic analyses of our focal species and their relatives revealed a 25-fold increase in nectary size and delayed fruit development in the ant-rewarding species, and Bayesian analyses of several traits showed the correlated evolution of sugar rewards and symbiosis specialization. Concealed floral nectar forestalls exploitation by opportunists (generalist ants) and stabilizes these obligate mutualisms. Our study pinpoints the importance of partner choice mechanisms in transitions from facultative to obligate mutualisms.

A new strategy for controlling invasive weeds: selecting valuable native plants to defeat them.

To explore replacement control of the invasive weed Ipomoea cairica, we studied the competitive effects of two valuable natives, Pueraria lobata and Paederia scandens, on growth and photosynthetic characteristics of I. cairica, in pot and field experiments. When I. cairica was planted in pots with P. lobata or P. scandens, its total biomass decreased by 68.7% and 45.8%, and its stem length by 33.3% and 34.1%, respectively. The two natives depressed growth of the weed by their strong effects on its photosynthetic characteristics, including suppression of leaf biomass and the abundance of the CO2-fixing enzyme RUBISCO. The field experiment demonstrated that sowing seeds of P. lobata or P. scandens in plots where the weed had been largely cleared produced 11.8-fold or 2.5-fold as much leaf biomass of the two natives, respectively, as the weed. Replacement control by valuable native species is potentially a feasible and sustainable means of suppressing I. cairica.

Reciprocal herkogamy promotes disassortative mating in a distylous species with intramorph compatibility.

Mating patterns in heterostylous species with intramorph compatibility have the potential to deviate from symmetrical disassortative mating owing to ecological and reproductive factors influencing pollen dispersal. Here, we investigate potential and realized patterns of mating in distylous Luculia pinceana (Rubiaceae), a species with intramorph compatibility. Our analysis provides an opportunity to test Darwin's hypothesis that reciprocal herkogamy promotes disassortative pollen transfer. We combined measurements of sex-organ reciprocity and pollen production to predict potential pollen transfer and mating patterns in a population from SW China. Marker-based paternity analysis was then used to estimate realized patterns of disassortative and assortative mating at the individual and floral morph levels. Both potential and realized mating patterns indicated a significant component of disassortative mating, satisfying theoretical conditions for the maintenance of floral dimorphism. Levels of assortative mating (37.7%) were significantly lower than disassortative mating (62.3%), but numerous offspring resulting from intramorph mating were detected in the majority of maternal seed families in both floral morphs. Our results provide empirical support for Darwin's cross-promotion hypothesis on the function of reciprocal herkogamy, but indicate that in most heterostylous species strong diallelic incompatibility may be a general requirement for complete disassortative mating.

Occurrence of stomatal patchiness and its spatial scale in leaves from various sizes of trees distributed in a South-east Asian tropical rainforest in Peninsular Malaysia.

In this study, we demonstrated the occurrence of stomatal patchiness and its spatial scale in leaves from various sizes of trees grown in a lowland dipterocarp forest in Peninsular Malaysia. To evaluate the patterns of stomatal behavior, we used three techniques simultaneously to analyze heterobaric or homobaric leaves from five tree species ranging from 0.6 to 31 m in height: (i) diurnal changes in chlorophyll fluorescence imaging, (ii) observation and simulation of leaf gas-exchange rates and (iii) a pressure-infiltration method. Measurements were performed in situ with 1000 or 500 µmol m(-2) s(-1) photosynthetic photon flux density. Diurnal patterns in the spatial distribution of photosynthetic electron transport rate (J) mapped from chlorophyll fluorescence images, a comparison of observed and simulated leaf gas-exchange rates, and the spatial distribution of stomatal apertures obtained from the acid-fuchsin-infiltrated area showed that patchy stomatal closure coupled with severe midday depression of photosynthesis occurred in Neobalanocarpus heimii (King) Ashton, a higher canopy tree with heterobaric leaves due to the higher leaf temperature and vapor pressure deficit. However, subcanopy or understory trees showed uniform stomatal behavior throughout the day, although they also have heterobaric leaves. These results suggest that the occurrence of stomatal patchiness is determined by tree size and/or environmental conditions. The analysis of spatial scale by chlorophyll fluorescence imaging showed that several adjacent anatomical patches (lamina areas bounded by bundle-sheath extensions within the lamina) may co-operate for the distributed patterns of J and stomatal apertures.

Vivipary in Ophiorrhiza mungos L. - a rare phenomenon in angiosperms.

Vivipary, the precocious germination of seeds within the parent plant, is a specialised feature of evolutionary and biological importance that ensures survival of a plant. Reports on vivipary in angiosperms are rare, accounting for <0.1% of flowering plants. Here, we report a remarkable case of occurrence of vivipary in Ophiorrhiza mungos. A study was conducted to collect information on the morphology of the capsules that support vivipary, environmental factors that induce vivipary, survival mode and the survival of viviparous seedlings. The hydroscopic movement of the cup-shaped capsules of O. mungos was found to help in viviparous germination during the rainy season. Of the total seeds in a capsule, 70% showed viviparous germination. The seedlings remaining inside the capsule attain a height of 0.98 ± 0.4 cm and reach the ground when the capsule falls. On the ground, seedlings obtain easy anchorage to the substratum since they have already germinated. Vivipary appears to be an adaptation of O. mungos to the rainy season for ensuring viable offspring. This suggests that vivipary in this species might be artificially induced by continuous spraying with water to rescue seeds in all seasons for use in large-scale propagation to meet increasing market demand and conservation of this valuable anticancer medicinal herb.

Reproductive isolation between sympatric sister species, Mussaenda kwangtungensis and M. pubescens var. alba.

Reproductive isolation defines the biological species concept and plays a key role in the formation and maintenance of species. The relative contributions of different isolating stages has been suggested to be closely associated with phylogenetic relatedness. Few studies have focused on the relative contributions of pre- versus post-zygotic mechanisms, and even fewer have been conducted under strict phylogenetic frameworks. Pre- and post-zygotic reproductive isolation stages have been investigated in the sister species Mussaenda kwangtungensis and M. pubescens var. alba. The two species have partly overlapping distribution ranges and flowering times, while the principal pollinators differed strikingly for them, demonstrating strong pre-zygotic isolations. Natural hybrids were detected by simple sequence repeat markers and their maternal parents were identified based on chloroplast gene sequences. Five out of 81 individuals were suggested to be hybrids that fall into the categories F2, BC1, and BC2 by the NewHybrids analysis. Interspecific crossings resulted in significantly reduced fruit set and seed germination rates. Phylogenetic analysis revealed short Kimura-2-parameter distance between M. kwangtungensis and M. pubescens var. alba. These findings strongly supported the hypothesis that for species with a closer phylogenetic relationship, pre-zygotic isolation plays an important part in limiting gene exchange in sympatric areas.