Unidirectional transitions in nectar gain and loss suggest food deception is a stable evolutionary strategy in Epidendrum (Orchidaceae): insights from anatomical and molecular evidence.

BACKGROUND: Nectar gain and loss are important flower transitions observed in angiosperms, and are particularly common in orchids. To understand such transitions, the availability of detailed anatomical data and species-level phylogenies are crucial. We investigated the evolution of food deception in Epidendrum, one of the largest orchid genera, using genus phylogeny to map transitions between nectar gain and loss among different clades. Associations between anatomical and histochemical changes and nectar gain and loss were examined using fresh material available from 27 species. The evolution of nectar presence/absence in Epidendrum species was investigated in a phylogenetic framework of 47 species, using one nuclear and five plastid DNA regions available from GenBank and sequenced in this study. RESULTS: The presence or absence of nectar was strongly associated with changes in the inner epidermal tissues of nectaries. Nectar-secreting species have unornamented epidermal tissue, in contrast to the unicellular trichomes found on the epidermis of food deceptive species. Bayesian tests confirmed that transitions occurred preferentially from nectar presence to nectar absence across the Epidendrum phylogeny. In addition, independent nectar loss events were found across the phylogeny, suggesting a lack of constraint for these transitions. CONCLUSIONS: Ornamented nectaries may play an important role in the deceptive pollination strategy by secreting volatile organic compounds and providing tactile stimuli to pollinators. The recurrent and apparently irreversible pattern of nectar loss in Epidendrum suggests that food deception may constitute an alternative evolutionarily stable strategy, as observed in other orchid groups.

Imaging of glyphosate uptake and identification of early microscopic markers in leaves of C3 and C4 glyphosate-resistant and -susceptible species.

Details of glyphosate uptake are not fully elucidated, and although this herbicide promotes important alterations in the plant phenotype few hours after its application (early responses), a detailed description of the presumable changes in plant anatomy is still poorly assessed by now. Due to glyphosate effects over leaf permeability, the use of an inert fluorescent tracer may allow the observation of the uptake event in situ. In addition, microscopic markers might put a light on the recognition of glyphosate-resistant (GR) and -susceptible (GS) species or varieties, which may vary in C3 and C4 species due to their putative distinct leaf anatomy. Here we aimed (i) to provide a new technique to track the route of glyphosate formulation towards leaf tissues using a fluorescent tracer, and (ii) to describe the early specific microscopic alterations in GR and GS -C3 or -C4 caused by the glyphosate formulation. Roundup Transorb® was applied in seedlings cultivated in a greenhouse and response alterations in leaf anatomy were described. Lucifer Yellow CH (LYCH) was applied over the same region where glyphosate formulation was previously applied to track the alterations in leaf permeability caused by this herbicide. LYCH successfully tracked the glyphosate formulation uptake, reaching the vascular bundles of GS species, and becoming retained in leaf tissues of GR species. All species exhibited a decrease in chlorophyll content at the site of glyphosate application regardless of their photosynthetic metabolism or susceptibility. GS species showed alterations in chloroplast morphology and activity of non-enzymatic antioxidants (carotenoids and flavonoids), in addition to symptoms indicating a process of accelerated cell senescence. A specific type of cell necrosis (hypersensitive response) was observed in GR-C4 species as a way to prevent the translocation of this herbicide, while GR-C3 species accumulated phenolic compounds inside the vacuole, probably sequestrating and inactivating the glyphosate action. This study provides a reliable tool to track glyphosate formulation uptake in situ and is the first attempt to the identification of early specific microscopic markers caused by glyphosate formulation.

Strong but permeable barriers to gene exchange between sister species of Epidendrum.

PREMISE OF THE STUDY: The investigation of reproductive barriers between sister species can provide insights into how new lineages arise, and how species integrity is maintained in the face of interspecific gene flow. Different pre- and postzygotic barriers can limit interspecific gene exchange in sympatric populations, and different sources of evidence are often required to investigate the role of multiple reproductive isolation (RI) mechanisms. METHODS: We tested the hypothesis of hybridization and potential introgression between Epidendrum secundum and Epidendrum xanthinum, two Neotropical food-deceptive orchid species, using nuclear and plastid microsatellites, experimental crosses, pollen tube growth observations, and genome size estimates. KEY RESULTS: A large number of hybrids between E. secundum and E. xanthinum were detected, suggesting weak premating barriers. The low fertility of hybrid plants and the absence of haplotype sharing between parental species indicated strong postmating barriers, reducing interspecific gene exchange and the development of advanced generation hybrids. Despite the strength of reproductive barriers, fertile seeds were produced in some backcrossing experiments, and the existence of interspecific gene exchange could not be excluded. CONCLUSIONS: Strong but permeable barriers were found between E. secundum and E. xanthinum. Indeed, haplotype sharing was not detected between parental species, suggesting that introgression is limited by a combination of genic incompatibilities, including negative cytonuclear interactions. Most taxonomic uncertainties in this group were potentially influenced by incomplete RI barriers between species, which mainly occurred sympatrically.

Floral colleters in Pleurothallidinae (Epidendroideae: Orchidaceae).

PREMISE OF THE STUDY: The term colleter is applied to trichomes or emergences positioned close to developing vegetative and floral meristems that secrete a sticky, mucilaginous, and/or lipophilic exudate. Several ecological functions are attributed to these glands, but none are exclusive to colleters. Patterns of morphology and distribution of colleters may be valuable for systematics and phylogeny, especially concerning problematic and large groups such as the subtribe Pleurothallidinae, and are also essential to understand the evolution of these glands in Orchidaceae as a whole. METHODS: We used scanning electron and light microscopy to examine the structure and occurrence of trichomes on bracts and sepals and in the invaginations of the external ovary wall (IEOW) in flowers in several developmental stages from species in seven genera. KEY RESULTS: The exudate was composed of polysaccharides, lipophilic, and phenolic compounds. Colleters were secretory only during the development of floral organs, except for the glands in the IEOW that were also active in flowers at anthesis. After the secretory phase, fungal hyphae were found penetrating senescent trichomes. CONCLUSIONS: Trichome-like colleters seem to be a widespread character in Epidendroideae, and digitiform colleters are possibly the common type in this subfamily. Mucilage from IEOW colleters may aid in the establishment of symbiotic fungi necessary for seed germination. The presence of colleters in the IEOW may be a case of homeoheterotopy, in which extrafloral nectaries that produce simple sugar-based secretions (as in other orchid species) have changed to glands that produce secretions with complex polysaccharides, as in Pleurothallidinae.

Evolution of connective glands reveals a new synapomorphy for Malpighiaceae and the hidden potential of staminal glands for Malpighiales systematics.

Connective glands are important morphological characters for the taxonomy of some genera of Malpighiaceae, with few recent studies having just elucidated these glands' anatomical and ecological functions. In order to test the systematic relevance of connective glands to the currently accepted phylogenetic informal clades of Malpighiaceae, we characterised the anatomy and/or histochemistry of two-thirds of Malpighiaceae genera and ten species from nine families of Malpighiales to test: 1. Do connective glands occur in the flowers of all informal clades of Malpighiaceae?; and 2. Are they taxonomically relevant to characterise those clades? We sampled 25 genera and 26 species of Malpighiaceae, processing their anthers using traditional anatomical methods and characterising their glands using light microscopy and SEM imaging. Selected species were subjected to histochemical tests, and an additional 21 genera and 33 species of Malpighiaceae and nine families (ten species) of Malpighiales were included in our sampling from the literature. Three anatomical characters were scored, coded and mapped using Maximum Likelihood methods onto the molecular phylogeny of Malpighiaceae. All sampled species of Malpighiaceae showed connective glands characterised as epidermal or trichomal elaiophores. Our character-mapping analyses recovered connective elaiophores as a new synapomorphy for Malpighiaceae. Different types of epidermal or trichomal elaiophores were recovered as homoplasies for the Christianella and Banisteriopsis clades and the genera Byrsonima, Camarea and Cottsia. Our analyses also recovered the glands' place of insertion in the stamen and the exudate type as potential new synapomorphies or homoplasies for the families of Malpighiales sampled. Our results propose the connective elaiophores as a new synapomorphy for Malpighiaceae and hypothesise the role that different staminal glands might play in the systematics of Malpighiales. Further comprehensive anatomical studies are still needed for the staminal glands of most families of this order to shed new light on the patterns recovered in our study.

Floral glands in myophilous and sapromyophilous species of Pleurothallidinae (Epidendroideae, Orchidaceae)-osmophores, nectaries, and a unique sticky gland.

Pleurothallidinae orchids have been the focus of many multidisciplinary studies due to their challenging systematics and taxonomy. The synapomorphies already recognized in the group are mostly related to floral characters, the last proposed being the occurrence of alkanes in the floral fragrance. The composition of the floral bouquet varied significantly among the studied species, leading us to hypothesize that the variations in volatiles emitted could be linked to the structure of osmophores, especially when comparing the myophilous and sapromyophilous pollination syndromes. Sepals and labellum at different developmental stages of seven Brazilian Pleurothallidinae species were examined using light, scanning, and transmission electron microscopy. Nectar reabsorption was assessed by Lucifer Yellow CH tracer and imaged under confocal microscopy. Nectaries were restricted to the labellum of the myophilous species, whereas osmophores occurred in the dorsal and/or lateral sepals, varying according to species. In the sapromyophilous species, floral nectaries were not detected and osmophores were restricted to the labellum. Osmophore structure was correlated with the volatiles emitted, being the trichome osmophores notably present on the sepals of both myophilous species that possess nectaries. For the first time, we demonstrated reabsorption of the released nectar in Pleurothallidinae and the occurrence of a unique gland named sticky-exudate glands, which occurred in the lateral sepals and labellum of Echinosepala aspasicensis, a sapromyophilous species, that released a heterogeneous exudate composed of polysaccharides and lipids. Similar glands have been reported in Bulbophyllum, highlighting the convergence between both groups.

Fluorescence emission spectra of target chloroplast metabolites (flavonoids, carotenoids, lipofuscins, pheophytins) as biomarkers of air pollutants and seasonal tropical climate.

Chloroplasts have luminescent metabolites-chlorophyll being the most known one-whose fluorescence emission may be a useful tool to assess the physiological status of the plant. Some antioxidants (flavonoids and carotenoids), and byproducts of membrane rupture (lipofuscins) and chlorophyll degradation (pheophytins), are chloroplasts' fluorescent metabolites directly involved in plant response to environmental stressors and pollutants and may act as a biomarker of stress. Here we hypothesized that climatic variations and air pollutants induce alterations in the emission profile of chloroplasts' fluorescent metabolites in Tillandsia usneoides (Bromeliaceae). To test this hypothesis, an active biomonitoring study was performed during 2 years in five polluted sites located at the Metropolitan Region of Campinas (São Paulo State, Brazil), aiming to identify target chloroplasts' fluorescent metabolites acting as biomarkers of environmental stress. In situ identification and quantification of the intensity of the fluorescence emission from target metabolites (flavonoids, carotenoids, lipofuscins, and pheophytins) were performed by the observation of fresh leaf sections under confocal laser scanning microscopy. Changes in the profile of fluorescence emission were correlated with local climate and air pollution data. The fluorescence emissions of flavonoids and carotenoids varied seasonally, with significant influence of rainfall and NO2. Our results expand the use of T. usneoides as a bioindicator by using alterations in the fluorescence emission profile of chloroplast metabolites. This application may be especially interesting for NO2 biomonitoring.

The passion fruit liana (Passiflora edulis Sims, Passifloraceae) is tolerant to ozone.

Passiflora edulis Sims is a liana species of high economic interest and is an interesting model plant for understanding ozone action on disturbed vegetation. In this work we hypothesized that P. edulis has adaptive responses to oxidative stress that enable it to tolerate ozone damage based on its capacity to grow under a diversity of environmental conditions and to dominate disturbed areas. We exposed seedlings to three levels of ozone in a Free-Air Controlled Exposure (FACE) system (22, 41 and 58 ppb h AOT40 and 13.52, 17.24 and 20.62 mmol m-2 POD0, over 97 days) for identifying its tolerance mechanisms. Anatomical (leaf blade structure and fluorescence emission of chloroplast metabolites), physiological (leaf gas exchange, growth rate and biomass production) and biochemical (pigments, total sugars, starch, enzymatic and non-enzymatic antioxidant metabolites, reactive oxygen species and lipid peroxidation derivatives) responses were assessed. Ozone caused decreased total number of leaves, hyperplasia and hypertrophy of the mesophyll cells, and accelerated leaf senescence. However, O3 did not affect carbohydrates content, net photosynthetic rate, or total biomass production, indicating that the carboxylation efficiency and associated physiological processes were not affected. In addition, P. edulis showed higher leaf contents of ascorbic acid, glutathione (as well high ratio between their reduced and total forms), carotenoids, and flavonoids located in the chloroplast outer envelope membrane. Our results indicate that P. edulis is an O3-tolerant species due to morphological acclimation responses and an effective antioxidant defense system represented by non-enzymatic antioxidants, which maintained the cellular redox balance under ozone.

Volatile organic compounds and nitric oxide as responses of a Brazilian tropical species to ozone: the emission profile of young and mature leaves.

The emission profile of volatile organic compounds (VOCs) and nitric oxide (NO) in young and mature leaves of Croton floribundus was assessed in plants exposed to filtered air (FA) and ozone-enriched filtered air (FA+O3). After the period of exposure, leaves were enclosed in polyethylene terephthalate bags and VOCs were collected in young and mature leaves. Both young and mature leaves constitutively emitted the same VOC, but the concentrations were higher in young leaves. O3 exposure induced the emission of sesquiterpenes (mainly ß-caryophyllene) known as antioxidant compounds that may scavenge O3. Young leaves were the highest emitters of sesquiterpenes. O3 induced a rapid accumulation of NO in different tissues and leaf developmental stages; this accumulation was marked in palisade and spongy parenchyma cells in young and mature leaves, respectively. O3 altered the levels of the signaling compound methyl salicylate (MeSA). Moreover, our data showed that NO together with VOC emissions, such as geranyl acetate, α-cadiene, trans-farnesol, cis-ß-farnesene, and MeSA, participate of plant defense mechanisms against the oxidative damage caused by O3.

Efficiency of biomonitoring methods applying tropical bioindicator plants for assessing the phytoxicity of the air pollutants in SE, Brazil.

In the tropical region, the greatest challenge of the biomonitoring approach is to establish linear relationships between biomarkers measured in plants and pollutant concentrations, since the bioindicator responses can be intensified or restricted by climatic variations. In southeastern Brazil, there are two regions affected by air pollution, where the Atlantic Forest remains and should be preserved. Consequently, both areas have been monitored by biomonitoring procedures using standardized and tropical plants. The industrial complex settled in Cubatão is one of the world's most famous examples of environmental pollution and degradation, with consequent decline of the Atlantic Forest. An oil refinery is among the most polluting industries in the Cubatão region. The other region is located in the Metropolitan Region of Campinas (MRC). The MRC has been affected by high levels of air pollutants originated from road traffic and is responsible for over 80% of CO, NOx, and hydrocarbon emissions and develops industrial activities that emit about 70% of the particulate matter present in the region. Both regions are distinguished by the climate, despite the fact that they are only about 130 km far from each other. Several studies carried out by our group in these regions aimed to establish the best native tree species and respective potential biomarkers for future assessment of pollution effects on tropical Forests. We present a critical review about the efficiency of native species compared to standardized bioindicator plants considering antioxidant defense system, nutrient accumulation, and microscopic aspects when exposed to atmospheric pollutants and climate.

Agricultural use of Samarco's spilled mud assessed by rice cultivation: A promising residue use?

Mining activity is one of the main responsible for accumulation of potentially toxic elements in the environment. These contaminants are absorbed by plants served as food that could be a risk to human health, such rice. Rice is a staple food with known accumulation of toxic elements. The recent collapse of a mining dam operated by Samarco Mining Company spilled around 50 million m3 of Fe-mining waste in the environment, including rivers and farming areas. In the present study, concentrations of As, Cd, Hg, Pb, Co, Zn, Mn, Cu, Fe, Al, Se, and Sr were determined in soils, roots and grains of rice plants cultivated in soil containing Samarco's residual mud (0, 16, 34 and 50%). Further, rice plant agronomic parameters (chlorophyll, carotenoids, grain yield, mass, height) were assessed. Rice cultivated at Samarco's residual mud produced grains with low levels of As, Cd and Pb. However, the excess of mud (50%) during the rice cultivation reduced roots' growth and grains yield. Chlorophyll (a and b) and carotenoids contents were significantly lower in all mud cultivations, mainly mud-50%. Our findings suggest that plant alterations induced by the mud were associated to the deficiency of nutrients and the physical properties of the mud. Soil fertilization by organic matter and top soil provided conditions for plant development. Therefore, considering the experimental conditions here used, we showed that is possible the use of the affected land for agriculture and reforestation after soil amendment.

Tillandsia usneoides: A successful alternative for biomonitoring changes in air quality due to a new highway in São Paulo, Brazil.

Tillandsia usneoides is an aerial epiphytic bromeliad that absorbs water and nutrients directly from the atmosphere by scales covering its surface. We expanded the use of this species as a broader biomonitor based on chemical and structural markers to detect changes in air quality. The usefulness of such comprehensive approach was tested during the construction and opening of a highway (SP-21) in São Paulo State, Brazil. The biomonitoring study was performed from 2009 to 2012, thus comprising the period during construction and after the highway inauguration. Metal accumulation and structural alterations were assessed, in addition to microscopy analyses to understand the metal chelation in plant tissues and to assess the causes of alterations in the number and shape of scale cells. Altogether, our analyses support the use of this species as a wide biomonitor of air quality in urbanized areas.

Synergism between ozone and light stress: structural responses of polyphenols in a woody Brazilian species.

Microscopic studies on isolated ozone (O3) effects or on those in synergy with light stress commonly report the induction of polyphenols that exhibit different aspects within the vacuole of photosynthesizing cells. It has been assumed that these different aspects are randomly spread in the symptomatic (injured) regions of the leaf blade. Interestingly, secretory ducts that constitutively produce polyphenols also exhibit these same variations in their vacuolar aspect, in a spatial sequence related to the destiny of these cells (e.g., programmed cell death (PCD) in lytic secretion processes). Here, we demonstrate that the deposition pattern of polyphenols prior to the establishment of the hypersensitive-like response, a type of PCD caused by O3, follows the same one observed in the epithelial cells of the constitutive lysigenous secretory ducts. Astronium graveolens, an early secondary Brazilian woody species, was selected based on its susceptibility to high light and presence of secretory ducts. The synergism effects were assessed by exposing plants to the high O3 concentrations at an urban site in São Paulo City. Confocal, widefield and light microscopies were used to examine polyphenols' occurrence and aspects. The spatial pattern of polyphenols distribution along the leaflets of plants submitted to the synergism condition, in which a dense vacuolar aspect is the target of a cell destined to death, was also observed in the constitutive secretory cells prior to lysis. This similar structural pattern may be a case of homology of process involving both the constitutive (secretory ducts) and the induced (photosynthesizing cells) defenses.

Tillandsia usneoides: a successful alternative for biomonitoring changes in air quality due to a new highway in São Paulo, Brazil.

Tillandsia usneoides is an aerial epiphytic bromeliad that absorbs water and nutrients directly from the atmosphere by scales covering its surface. We expanded the use of this species as a broader biomonitor based on chemical and structural markers to detect changes in air quality. The usefulness of such comprehensive approach was tested during the construction and opening of a highway (SP-21) in São Paulo State, Brazil. The biomonitoring study was performed from 2009 to 2012, thus comprising the period during construction and after the highway inauguration. Metal accumulation and structural alterations were assessed, in addition to microscopy analyses to understand the metal chelation in plant tissues and to assess the causes of alterations in the number and shape of scale cells. Altogether, our analyses support the use of this species as a wide biomonitor of air quality in urbanized areas.

Ozone-induced responses in Croton floribundus Spreng. (Euphorbiaceae): metabolic cross-talk between volatile organic compounds and calcium oxalate crystal formation.

Here, we proposed that volatile organic compounds (VOC), specifically methyl salicylate (MeSA), mediate the formation of calcium oxalate crystals (COC) in the defence against ozone (O3) oxidative damage. We performed experiments using Croton floribundus, a pioneer tree species that is tolerant to O3 and widely distributed in the Brazilian forest. This species constitutively produces COC. We exposed plants to a controlled fumigation experiment and assessed biochemical, physiological, and morphological parameters. O3 induced a significant increase in the concentrations of constitutive oxygenated compounds, MeSA and terpenoids as well as in COC number. Our analysis supported the hypothesis that ozone-induced VOC (mainly MeSA) regulate ROS formation in a way that promotes the opening of calcium channels and the subsequent formation of COC in a fast and stable manner to stop the consequences of the reactive oxygen species in the tissue, indeed immobilising the excess calcium (caused by acute exposition to O3) that can be dangerous to the plant. To test this hypothesis, we performed an independent experiment spraying MeSA over C. floribundus plants and observed an increase in the number of COC, indicating that this compound has a potential to directly induce their formation. Thus, the tolerance of C. floribundus to O3 oxidative stress could be a consequence of a higher capacity for the production of VOC and COC rather than the modulation of antioxidant balance. We also present some insights into constitutive morphological features that may be related to the tolerance that this species exhibits to O3.

A light in the shadow: the use of Lucifer Yellow technique to demonstrate nectar reabsorption.

BACKGROUND: Nectar reabsorption is a widely known phenomenon, related to the strategy of resource-recovery and also to maintain the nectar homeostasis at the nectary. The method currently performed to demonstrate nectar being reabsorbed involves the use of radioactive tracers applied to the nectary. Although this method works perfectly, it is complex and requires specific supplies and equipment. Therefore, here we propose an efficient method to obtain a visual demonstration of nectar reabsorption, adapting the use of Lucifer Yellow CH (LYCH), a fluorescent membrane-impermeable dye that can enter the vacuole by endocytosis. RESULTS: We applied a LYCH solution to the floral nectary (FN) of Cucurbita pepo L., which is a species known for its ability of nectar reabsorption, and to the extrafloral nectary (EFN) of Passiflora edulis Sims which does not reabsorb the secreted nectar. In all tests performed, we observed that LYCH stained the nectary tissues differentially according to the reabsorption ability of the nectary. The treated FN of C. pepo presented a concentrated fluorescence at the epidermis that decreased at the deeper nectary parenchyma, until reaching the vascular bundles, indicating nectar reabsorption in the flowers of the species. In contrast, treated EFN of P. edulis presented fluorescence only at the cuticle surface, indicating that nectar is not reabsorbed by that particular tissue. CONCLUSION: LYCH is an efficient marker to demonstrate nectar reabsorption.