Coordination of pinna, petiole, and root anatomical traits in 24 tropical-subtropical fern species.

Ferns are primitive vascular plants with diverse morphologies and structures. Plant anatomical traits and their linkages can reflect adaptation to the environment; however, these remain are still poorly understood in ferns. The main objective of this study was to explore whether there was structural coordination among and within organs in fern species. We measured 16 hydraulically related anatomical traits of pinnae, petioles, and roots of 24 representative fern species from the tropical and subtropical forest understory and analyzed trait correlation networks. In addition, we examined phylogenetic signals for the anatomical traits and analyzed co-evolutionary relationships. These results indicated that stomatal density and all petiole anatomical traits exhibited significant phylogenetic signals. Evolutionary correlations were observed between the tracheid diameter and wall thickness of the petiole and between the water transport capacity of the petiole and stomatal density. Conversely, anatomical traits of roots (e.g., root diameter) showed no phylogenetic signals and were not significantly correlated with those of the pinnae and petioles, indicating a lack of structural coordination between the below- and above-ground organs. Unlike angiosperms, vein density is unrelated to stomatal density or pinna thickness in ferns. As root diameter decreased, the cortex-to-stele diameter ratio decreased significantly (enhanced water absorption) in angiosperms but remained unchanged in ferns. These differences lead to different responses of ferns to climate change and improve our knowledge of the water adaptation strategies of ferns.

Conserved autophagy and diverse cell wall composition: unifying features of vascular tissues in evolutionarily distinct plants.

BACKGROUND AND AIMS: The formation of multifunctional vascular tissues represents a significant advancement in plant evolution. Differentiation of conductive cells is specific, involving two main pathways, namely protoplast clearance and cell wall modification. In xylogenesis, autophagy is a crucial process for complete protoplast elimination in tracheary elements, whose cell wall also undergoes strong changes. Knowledge pertaining to living sieve elements, which lose most of their protoplast during phloemogenesis, remains limited. We hypothesized that autophagy plays a crucial role, not only in complete cytoplasmic clearance in xylem but also in partial degradation in phloem. Cell wall elaborations of mature sieve elements are not so extensive. These analyses performed on evolutionarily diverse model species potentially make it possible to understand phloemogenesis to an equal extent to xylogenesis. METHODS: We investigated the distribution of ATG8 protein, which is an autophagy marker, and cell wall components in the roots of ferns, gymnosperms and angiosperms (monocots, dicot herbaceous plants and trees). Furthermore, we conducted a bioinformatic analysis of complete data on ATG8 isoforms for Ceratopteris richardii. KEY RESULTS: The presence of ATG8 protein was confirmed in both tracheary elements and sieve elements; however, the composition of cell wall components varied considerably among vascular tissues in the selected plants. Arabinogalactan proteins and ß-1,4-galactan were detected in the roots of all studied species, suggesting their potential importance in phloem formation or function. In contrast, no evolutionary pattern was observed for xyloglucan, arabinan or homogalacturonan. CONCLUSIONS: Our findings indicate that the involvement of autophagy in plants is universal during the development of tracheary elements that are dead at maturity and sieve elements that remain alive. Given the conserved nature of autophagy and its function in protoplast degradation for uninterrupted flow, autophagy might have played a vital role in the development of increasingly complex biological organizations, including the formation of vascular tissues. However, different cell wall compositions of xylem and phloem in different species might indicate diverse functionality and potential for substance transport, which is crucial in plant evolution.

Mechanical advantage makes stomatal opening speed a function of evaporative demand.

Stomatal opening in the light, observed in nearly all vascular land plants, is essential for providing access to atmospheric CO2 for photosynthesis. The speed of stomatal opening in the light is critical for maximizing carbon gain in environments in which light intensity changes, yet we have little understanding of how other environmental signals, particularly evaporative demand driven by vapor pressure deficit (VPD) influences the kinetics of this response. In angiosperms, and some fern species from the family Marsileaceae, a mechanical interaction between the guard cells and the epidermal cells determines the aperture of the pore. Here, we examine whether this mechanical interaction influences the speed of stomatal opening in the light. To test this, we investigated the speed of stomatal opening in response to light across a range of VPDs in seven plant species spanning the evolutionary diversity of guard cell and epidermal cell mechanical interactions. We found that stomatal opening speed is a function of evaporative demand in angiosperm species and Marsilea, which have guard cell and epidermal cell mechanical interactions. Stomatal opening speeds did not change across a range of VPD in species of gymnosperm and fern, which do not have guard cell mechanical interactions with the epidermis. We find that guard cell and epidermal cell mechanical interactions may play a key role in regulating stomatal responsiveness to light. These results provide valuable insight into the adaptive relevance of mechanical advantage.

Changes in endogenous abscisic acid and stomata of the resurrection fern, Pleopeltis polypodioides, in response to de- and rehydration.

PREMISE: While angiosperms respond uniformly to abscisic acid (ABA) by stomatal closure, the response of ferns to ABA is ambiguous. We evaluated the effect of endogenous ABA, hydrogen peroxide (H2 O2 ), nitric oxide (NO), and Ca2+ , low and high light intensities, and blue light (BL) on stomatal opening of Pleopeltis polypodioides. METHODS: Endogenous ABA was quantified using gas chromatography-mass spectrometry; microscopy results and stomatal responses to light and chemical treatments were analyzed with Image J. RESULTS: The ABA content increases during initial dehydration, peaks at 15 h and then decreases to one fourth of the ABA content of hydrated fronds. Following rehydration, ABA content increases within 24 h to the level of hydrated tissue. The stomatal aperture opens under BL and remains open even in the presence of ABA. Closure was strongly affected by BL, NO, and Ca2+ , regardless of ABA, H2 O2 effect was weak. CONCLUSIONS: The decrease in the ABA content during extended dehydration and insensitivity of the stomata to ABA suggests that the drought tolerance mechanism of Pleopeltis polypodioides is independent of ABA.

Functional traits and trait coordination change over the life of a leaf in a tropical fern species.

PREMISE: Plant ecological strategies are often defined by the integration of underlying traits related to resource acquisition, allocation, and growth. Correlations between key traits across diverse plants suggest that variation in plant ecological strategies is largely driven by a fast-slow continuum of plant economics. However, trait correlations may not be constant through the life of a leaf, and it is still poorly understood how trait function varies over time in long-lived leaves. METHODS: Here, we compared trait correlations related to resource acquisition and allocation across three different mature frond age cohorts in a tropical fern species, Saccoloma inaequale. RESULTS: Fronds exhibited high initial investments of nitrogen and carbon, but with declining return in photosynthetic capacity after the first year. In the youngest fronds, we found water-use efficiency to be significantly lower than in the oldest mature fronds due to increased transpiration rates. Our data suggest that middle-aged fronds are more efficient relative to younger, less water-use efficient fronds and that older fronds exhibit greater nitrogen investments without higher photosynthetic return. In addition, several trait correlations expected under the leaf economics spectrum (LES) do not hold within this species, and some trait correlations only appear in fronds of a specific developmental age. CONCLUSIONS: These findings contextualize the relationship between traits and leaf developmental age with those predicted to underlie plant ecological strategy and the LES and are among the first pieces of evidence for when relative physiological trait efficiency is maximized in a tropical fern species.

Light, rather than circadian rhythm, regulates gas exchange in ferns and lycophytes.

Circadian regulation plays a vital role in optimizing plant responses to the environment. However, while circadian regulation has been extensively studied in angiosperms, very little is known for lycophytes and ferns, leaving a gap in our understanding of the evolution of circadian rhythms across the plant kingdom. Here, we investigated circadian regulation in gas exchange through stomatal conductance and photosynthetic efficiency in a phylogenetically broad panel of 21 species of lycophytes and ferns over a 46 h period under constant light and a selected few under more natural conditions with day-night cycles. No rhythm was detected under constant light for either lycophytes or ferns, except for two semi-aquatic species of the family Marsileaceae (Marsilea azorica and Regnellidium diphyllum), which showed rhythms in stomatal conductance. Furthermore, these results indicated the presence of a light-driven stomatal control for ferns and lycophytes, with a possible passive fine-tuning through leaf water status adjustments. These findings support previous evidence for the fundamentally different regulation of gas exchange in lycophytes and ferns compared to angiosperms, and they suggest the presence of alternative stomatal regulations in Marsileaceae, an aquatic family already well known for numerous other distinctive physiological traits. Overall, our study provides evidence for heterogeneous circadian regulation across plant lineages, highlighting the importance of broad taxonomic scope in comparative plant physiology studies.

Different nitrogen acquirement and utilization strategies might determine the ecological competition between ferns and angiosperms.

BACKGROUND AND AIMS: The abundance or decline of fern populations in response to environmental change has been found to be largely dependent on specific physiological properties that distinguish ferns from angiosperms. Many studies have focused on water use efficiency and stomatal behaviours, but the effects of nutrition acquirement and utilization strategies on niche competition between ferns and flowering plants are rarely reported. METHODS: We collected 34 ferns and 42 angiosperms from the Botanic Garden of Hokkaido University for nitrogen (N), sulphur (S), NO3- and SO42- analysis. We then used a hydroponic system to compare the different N and S utilization strategies between ferns and angiosperms under N deficiency conditions. KEY RESULTS: Ferns had a significantly higher NO3--N concentration and NO3--N/N ratio than angiosperms, although the total N concentration in ferns was remarkably lower than that in the angiosperms. Meanwhile, a positive correlation between N and S was found, indicating that nutrient concentration is involved in assimilation. Pteris cretica, a fern species subjected to further study, maintained a slow growth rate and lower N requirement in response to low N stress, while both the biomass and N concentration in wheat (Triticum aestivum) responded quickly to N deficiency conditions. CONCLUSIONS: The different nutritional strategies employed by ferns and angiosperms depended mainly on the effects of phylogenetic and evolutionary diversity. Ferns tend to adopt an opportunistic strategy of limiting growth rate to reduce N demand and store more pooled nitrate, whereas angiosperms probably utilize N nutrition to ensure as much development as possible under low N stress. Identifying the effects of mineral nutrition on the evolutionary results of ecological competition between plant species remains a challenge.

The relationship between chlorophyllous spores and mycorrhizal associations in ferns: evidence from an evolutionary approach.

PREMISE: Approximately 14% of all fern species have physiologically active chlorophyllous spores that are much more short-lived than the more common and dormant achlorophyllous spores. Most chlorophyllous-spored species (70%) are epiphytes and account for almost 37% of all epiphytic ferns. Chlorophyllous-spored ferns are also overrepresented among fern species in habitats with waterlogged soils, of which nearly 60% have chlorophyllous spores. Ferns in these disparate habitat types also have a low incidence of mycorrhizal associations. We therefore hypothesized that autotrophic chlorophyllous spores represent an adaptation of ferns to habitats with scarce mycorrhizal associations. METHODS: We evaluated the coevolution of chlorophyllous spores and mycorrhizal associations in ferns and their relation to habitat type using phylogenetic comparative methods. RESULTS: Although we did not find support for the coevolution of spore type and mycorrhizal associations, we did find that chlorophyllous spores and the absence of mycorrhizal associations have coevolved with epiphytic and waterlogged habitats. Transition rates to epiphytic and waterlogged habitats were significantly higher in species with chlorophyllous spores compared to achlorophyllous lineages. CONCLUSIONS: Spore type and mycorrhizal associations appear to play important roles in the radiation of ferns into different habitat types. Future work should focus on clarifying the functional significance of these associations.

Fern fronds that move like pine cones: humidity-driven motion of fertile leaflets governs the timing of spore dispersal in a widespread fern species.

BACKGROUND AND AIMS: The sensitive fern, Onoclea sensibilis, is a widespread species in eastern North America and has an atypical timing of spore dispersal among temperate ferns. During early summer, this dimorphic species produces heavily modified spore-bearing fronds with leaflets tightly enveloping their sporangia and spores. These fronds senesce and persist above ground as dead mature structures until the following early spring when the leaflets finally open and spores are dispersed. While this timing of spore dispersal has been observed for over 120 years, the structural mechanisms underpinning this phenology have remained elusive. METHODS: Based on field observations, growth chamber manipulations and scanning electron microscopy, the mechanisms underlying this distinctive timing of spore dispersal in the sensitive fern were investigated. KEY RESULTS: I show that fertile leaflets of the sensitive fern move in direct response to changes in humidity, exhibiting structural and functional parallels with multicellular hygromorphic structures in seed plants, such as pine cones. These parallels include differences in cellulose microfibril orientation in cells on the abaxial and adaxial sides of the leaflet. The dynamics of this hygroscopic movement concomitant with regular abscission zones along the pinnules and coordinated senescence lead to the specific timing of early spring spore dispersal in the sensitive fern. CONCLUSIONS: While hygroscopic movement is common in seed-free plants, it mostly occurs in small structures that are either one or a few cells in size, such as the leptosporangium. Given its multicellular structure and integration across many cells and tissues, the movement and construction of the sensitive fern pinnules are more similar to structures in seed plants. The evolution of this complex trait in the sensitive fern efficiently regulates the timing of spore release, leading to early spring dispersal. This phenology likely gives gametophytes and subsequent sporophytes an advantage with early germination and growth.

Metabolism-mediated mechanisms underpin the differential stomatal speediness regulation among ferns and angiosperms.

Recent results suggest that metabolism-mediated stomatal closure mechanisms are important to regulate differentially the stomatal speediness between ferns and angiosperms. However, evidence directly linking mesophyll metabolism and the slower stomatal conductance (gs ) in ferns is missing. Here, we investigated the effect of exogenous application of abscisic acid (ABA), sucrose and mannitol on stomatal kinetics and carried out a metabolic fingerprinting analysis of ferns and angiosperms leaves harvested throughout a diel course. Fern stomata did not respond to ABA in the time period analysed. No differences in the relative decrease in gs was observed between ferns and the angiosperm following provision of sucrose or mannitol. However, ferns have slower gs responses to these compounds than angiosperms. Metabolomics analysis highlights that ferns have a higher accumulation of secondary rather than primary metabolites throughout the diel course, with the opposite being observed in angiosperms. Our results indicate that metabolism-mediated stomatal closure mechanisms underpin the differential stomatal speediness regulation among ferns and angiosperms, in which the slower stomatal closure in ferns is associated with the lack of ABA-responsiveness, to a reduced capacity to respond to mesophyll-derived sucrose and to a higher carbon allocation toward secondary metabolism, which likely modulates both photosynthesis-gs and growth-stress tolerance trade-offs.

Leaf water relations in epiphytic ferns are driven by drought avoidance rather than tolerance mechanisms.

Opportunistic diversification has allowed ferns to radiate into epiphytic niches in angiosperm dominated landscapes. However, our understanding of how ecophysiological function allowed establishment in the canopy and the potential transitionary role of the hemi-epiphytic life form remain unclear. Here, we surveyed 39 fern species in Costa Rican tropical forests to explore epiphytic trait divergence in a phylogenetic context. We examined leaf responses to water deficits in terrestrial, hemi-epiphytic and epiphytic ferns and related these findings to functional traits that regulate leaf water status. Epiphytic ferns had reduced xylem area (-63%), shorter stipe lengths (-56%), thicker laminae (+41%) and reduced stomatal density (-46%) compared to terrestrial ferns. Epiphytic ferns exhibited similar turgor loss points, higher osmotic potential at saturation and lower tissue capacitance after turgor loss than terrestrial ferns. Overall, hemi-epiphytic ferns exhibited traits that share characteristics of both terrestrial and epiphytic species. Our findings clearly demonstrate the prevalence of water conservatism in both epiphytic and hemi-epiphytic ferns, via selection for anatomical and structural traits that avoid leaf water stress. Even with likely evolutionarily constrained physiological function, adaptations for drought avoidance have allowed epiphytic ferns to successfully endure the stresses of the canopy habitat.

Stomatal morphology and physiology explain varied sensitivity to abscisic acid across vascular plant lineages.

Abscisic acid (ABA) can induce rapid stomatal closure in seed plants, but the action of this hormone on the stomata of fern and lycophyte species remains equivocal. Here, ABA-induced stomatal closure, signaling components, guard cell K+ and Ca2+ fluxes, vacuolar and actin cytoskeleton dynamics, and the permeability coefficient of guard cell protoplasts (Pf) were analyzed in species spanning the diversity of vascular land plants including 11 seed plants, 6 ferns, and 1 lycophyte. We found that all 11 seed plants exhibited ABA-induced stomatal closure, but the fern and lycophyte species did not. ABA-induced hydrogen peroxide elevation was observed in all species, but the signaling pathway downstream of nitric oxide production, including ion channel activation, was only observed in seed plants. In the angiosperm faba bean (Vicia faba), ABA application caused large vacuolar compartments to disaggregate, actin filaments to disintegrate into short fragments and Pf to increase. None of these changes was observed in the guard cells of the fern Matteuccia struthiopteris and lycophyte Selaginella moellendorffii treated with ABA, but a hypertonic osmotic solution did induce stomatal closure in fern and the lycophyte. Our results suggest that there is a major difference in the regulation of stomata between the fern and lycophyte plants and the seed plants. Importantly, these findings have uncovered the physiological and biophysical mechanisms that may have been responsible for the evolution of a stomatal response to ABA in the earliest seed plants.

Large-Scale Green Liver System for Sustainable Purification of Aquacultural Wastewater: Construction and Case Study in a Semiarid Area of Brazil (Itacuruba, Pernambuco) Using the Naturally Occurring Cyanotoxin Microcystin as Efficiency Indicator.

The aquaculture industry in Brazil has grown immensely resulting in the production of inefficiently discarded wastewater, which causes adverse effects on the aquatic ecosystem. The efficient treatment of aquaculture wastewater is vital in reaching a sustainable and ecological way of fish farming. Bioremediation in the form of the Green Liver System employing macrophytes was considered as wastewater treatment for a tilapia farm, COOPVALE, in Itacuruba, Brazil, based on previously demonstrated success. A large-scale system was constructed, and the macrophytes Azolla caroliniana, Egeria densa, Myriophyllum aquaticum, and Eichhornia crassipes were selected for phytoremediation. As cyanobacterial blooms persisted in the eutrophic wastewater, two microcystin congeners (MC-LR and -RR) were used as indicator contaminants for system efficiency and monitored by liquid-chromatography-tandem-mass-spectrometry. Two trial studies were conducted to decide on the final macrophyte selection and layout of the Green Liver System. In the first trial, 58% MC-LR and 66% MC-RR were removed and up to 32% MC-LR and 100% MC-RR were removed in the second trial. Additional risks that were overcome included animals grazing on the macrophytes and tilapia were spilling over from the hatchery. The implementation of the Green Liver System significantly contributed to the bioremediation of contaminants from the fish farm.

Pteridophytes in phytoremediation.

Soil contamination by heavy metals and metalloids is a serious problem which needs to be addressed. There are several methods for removal of contaminants, but they are costly, while the method of phytoremediation is eco-friendly and cost-effective. Pteridophytes have been found to remediate heavy metal-contaminated soil. Pteridophytes are non-flowering plant that reproduces by spores. Pteris vittata has been reported as the first fern plant to hyperaccumulate arsenic. The Pteris species belongs to the order Pteridales. Other ferns that are known phytoremediators are, for example, Nephrolepis cordifolia and Hypolepis muelleri (identified as phytostabilisers of Cu, Pb, Zn and Ni); similarly Pteris umbrosa and Pteris cretica accumulate arsenic in leaves. So, pteridophytes have a number of species that accumulate contaminants. Many of them have been identified, while various other are being explored. The present review article describes the phytoremediation potential of pteridophytes plants and suggests as a potential asset for phytoremediation programs.

On the evolutionary significance of horizontal gene transfers in plants.

Horizontal gene transfer (HGT) has long been seen as a crucial process in the evolution of prokaryotic species, but until recently it was thought to have little, if any, effect on the evolution of eukaryotic life forms. Detecting and describing HGT events in eukaryotes is difficult, making this phenomenon at times controversial. However, modern advances in genomics and bioinformatics have radically altered our view of HGT in eukaryotes, especially in plants. It now appears that HGT to and from plant lineages is more common than previously suspected. Importantly, the transfer of functional nuclear genes with adaptive significance has been reported in numerous taxa. Here we review several recent studies that have found evidence of the horizontal transfer of nuclear genes, and argue that HGT has undoubtedly had profound impacts on plant evolution as a whole.

Are stomata in ferns and allies sluggish? Stomatal responses to CO2 , humidity and light and their scaling with size and density.

Fast stomatal reactions enable plants to successfully cope with a constantly changing environment yet there is an ongoing debate on the stomatal regulation mechanisms in basal plant groups. We measured stomatal morphological parameters in 29 fern and allied species from temperate to tropical biomes and two outgroup angiosperm species. Stomatal dynamic responses to environmental drivers were measured in 16 ferns and the two angiosperms using a gas-exchange system. Principal components analyses were used to further reveal the structure-function relationships in stomata. We show a > 10-fold variation for stomatal opening delays and 20-fold variation for stomatal closing delays in ferns. Across species, stomatal responses to vapor pressure deficit (VPD) were the fastest, while light and [CO2 ] responses were slower. In most cases the outgroup species' reaction speeds to changes in environmental variables were similar to those of ferns. Correlations between stomatal response rate and size were apparent for stomatal opening in light and low [CO2 ] while not evident for closing reactions and changes in VPD. No correlations between stomatal density and response speed were observed. Together, this study demonstrates different mechanisms controlling stomatal reactions in ferns at different environmental stimuli, which should be considered in future studies relating stomatal morphology and function.

Epiphyte associations and canopy soil volume: nutrient capital and factors influencing soil retention in the canopy.

Canopy soil (CS) volume reflect epiphyte community maturity, but little is known about the factors that retain CS or species succession within it. Humus fern species (e.g. Phlebodium areolatum) appear capable of retaining CS. In ten Quercus spp. we sampled 987 epiphyte mats to examine the role of the common epiphyte species and crown traits determining CS volume, in order to infer successional stages and identify pioneer and late successional species. Branch traits (height, diameter and slope), CS volume and cover of the epiphyte species were determined for each mat. Nutrient content was determined in CS random samples of 12 epiphyte associations and sizes (one sample from each size quintile). A total of 60% of the mats lack CS. Cover of P. areolatum was the main variable explaining CS volume, and this species was present in 46.8% of those with CS. Epiphyte composition was highly variable, but pioneer (species appearing in monospecific mats, without CS) and late successional species could be identified. Canopy soil nutrient content was similar among the associations of epiphytes. Magnesium, Ca and pH decreased with CS volume, while P and N increased. Phlebodium areolatum is associated with high CS volumes and could act as a key species in its retention. Monospecific mats of pioneer species lack CS or have low volumes, while CS is much higher in mats with late successional species, but the mechanisms of CS formation and nutrient retention in response to interactions between epiphyte species remain to be tested.

A left-handed fern twiner in a Permian swamp forest.

The twining habit is a climbing strategy that helps slender plants grow upward by using circumnutation around other plants. In geological history, climbing may have already been present in the first Middle Devonian forests, as indicated by possible climbers among aneurophytalean progymnosperms [1] and lycopsids [2]. By the late Carboniferous, climbing was both more common and diverse - preserved in swamp forests with modes of attachment ranging from aerial roots to appendages modified into hooks and tendrils on the leaves [3]. However, all of these diagnoses of a climbing habit are based upon either indirect morphological characteristics of the purported climber or on direct physical contact with a host plant, but without direct preservation of twining [3,4]. Permineralized epiphytes have been preserved in the Carboniferous [5], but the interpretation of scars purported to have been caused by twiners that have been found on trunk compressions of potential host-plants has been questioned [5] (see Supplemental Information). Direct preservation of a climber engaged in true twining around a host has only been documented in the Miocene Shanwang Formation of Eastern China, albeit with the identity of the twiner difficult to establish and likely to be a self-twiner [6]. Here, we report a climbing fern engaged in left-handed twining around a seed plant from the early Permian Wuda Tuff fossil Lagerstätte of Inner Mongolia, China [7]. Moreover, the host plant is likely to also be a climber based on its overall form. Such a climber-climbing-a-climber phenomenon signals the potential ecological complexity of late Paleozoic forests.

Virtual issue: Ecology and evolution of pteridophytes in the era of molecular genetics.

The past quarter-century has witnessed a revolution in our understanding of the phylogenetics, systematics, and ecology of pteridophytes (ferns and lycophytes), particularly due to the rapid accumulation of plastid sequence data and a renewed interest in the ecology of the sexual phase of the life cycle. We here compile 19 papers recently published in the Journal of Plant Research dealing with the biology of pteridophytes, grouped into six categories: (1) breeding systems, (2) species complexes and polyploidization, (3) fossil taxa, (4) gametophyte ecology, (5) systematics, (6) biodiversity. We hope this collection of papers will be of value to researchers interested in this fascinating group of plants.