Herbivory and leaf expansion of Cyathea phalerata Mart (Cyatheaceae) in subtropical Atlantic Forest, southern Brazil / Herbivoria e expansão foliar de Cyathea phalerata Mart. (Cyatheaceae) em Floresta Atlântica subtropical no sul do Brasil

Herbivory is an interaction with great impact on plant communities since relationships between herbivores and plants are fundamental to the distribution and abundance of species over time and space. The aim of this study was to monitor the rate of leaf expansion in the tree fern Cyathea phalerata and evaluate the damage caused by herbivores to leaves of different ages and whether such damage is related to temperature and precipitation. The study was performed in a subtropical Atlantic Forest fragment located in the municipality of Caraá, in the northeast hillside of Rio Grande do Sul state, in southern Brazil. We monitored 24 mature individuals of C. phalerata with croziers in a population of approximately 50 plants. Leaf expansion rate, percentage of damaged leaves and leaf blade consumption rate by herbivory were calculated. Monthly means for temperature and accumulated rainfall were calculated from daily data. Croziers of C. phalerata were found to expand rapidly during the first and second months after emergence (3.98 cm day-¹; 2.91 cm day-¹, respectively). Damage caused by herbivory was observed in all of the monitored leaves, but none of the plants experienced complete defoliation. The highest percentage (57%) of damaged leaves was recorded at 60 days of monitoring, and also the highest monthly consumption rate of the blade (6.04%) occurred with young, newly-expanded leaves, while this rate remained between 1.50 and 2.21% for mature leaves. Rates of monthly leaf consumption and damaged leaves showed positive and strong relationship with each other and with temperature. The rapid leaf expansion observed for C. phalerata can be considered a phenological strategy to reduce damage to young leaves by shortening the developmental period and accelerating the increase of defenses in mature leaves.

A herbivoria é uma interação de grande impacto sobre as comunidades de plantas, uma vez que as relações entre herbívoros e plantas são fundamentais para a distribuição e abundância das espécies ao longo do tempo e do espaço. O objetivo deste estudo foi monitorar a taxa de expansão foliar da samambaia arborescente Cyathea phalerata e avaliar o dano causado por herbívoros a folhas de diferentes idades, assim como verificar se este dano está relacionado à temperatura e à precipitação. O estudo foi realizado em um fragmento de Floresta Atlântica subtropical localizado no município de Caraá, na encosta nordeste do estado do Rio Grande do Sul, no sul do Brasil. Nós monitoramos 24 indivíduos maduros de C. phalerata com báculos em uma população de aproximadamente 50 plantas. A taxa de expansão foliar, a porcentagem de folhas danificadas e a taxa de consumo da lâmina foliar por herbivoria foram calculados. Médias mensais de temperatura e precipitação acumulada foram calculadas a partir de dados diários. Báculos de C. phalerata expandiram rapidamente durante o primeiro e o segundo mês após emergência (3,98 cm dia-¹; 2,91 cm dia-¹, respectivamente). O dano causado por herbivoria foi observado em todas as folhas monitoradas, mas nenhuma das plantas sofreu desfolhação completa. A maior porcentagem (57%) de folhas danificadas foi registrada aos 60 dias de monitoramento, e também a maior taxa de consumo mensal (6,04%) ocorreu em folhas jovens, recém expandidas, enquanto esta taxa permaneceu entre 1,50 e 2,21% em folhas maduras. As taxas mensais de consumo da lâmina foliar e de folhas danificadas mostraram relação positiva e forte entre si e com a temperatura. A rápida expansão foliar observada em C. phalerata pode ser considerada uma estratégia fenológica para reduzir o dano a folhas jovens, abreviando o período de desenvolvimento e acelerando o aumento das defesas em folhas maduras.

Genomes tell tales of spores versus seeds.

Genomes from ferns and a cycad reveal deep roots of plant reproduction.

DNA barcoding ferns in an unexplored tropical montane cloud forest area of southeast Oaxaca, Mexico.

DNA barcoding can be useful for species identification and phylogenetic analysis, but its effectivity has not been verified in most neotropical cloud forest plants. We tested three plastid barcodes, rbcLa, matK, and trnH-psbA, in selected pteridophytes, a well-represented group in these forests, from a little-explored area in Oaxaca, Mexico, applying the CBOL criteria for barcoding. We used BLASTn, genetic distance, and monophyly tree-based analyses employing neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference methods. Universal primers for rbcLa and trnH-psbA were successfully amplified and bi-directionally sequenced, but matK could not be amplified for most species. rbcLa showed the highest species discrimination in BLASTn (66.67%). trnH-psbA exhibited higher significant interspecific divergence values than rbcL and rbcLa + trnH-psbA (two-sample sign test, P value < 2.2e-16). Using NJ and ML phylogenetic trees, monophyletic species were successfully resolved (100%), differing only in support values and displaying full agreement with the most recent fern classification. ML trees showed the highest mean support value (80.95%). trnH-psbA was the only barcode that could detect the Elaphoglossoideae subfamily. Species discrimination did not increase using rbcLa + trnH-psbA. rbcLa is useful for fern barcoding, trnH-psbA is most helpful for phylogenetic analyses, and matK may not work as a universal barcoding marker.

De Novo Sporophyte Transcriptome Assembly and Functional Annotation in the Endangered Fern Species Vandenboschia speciosa (Willd.) G. Kunkel.

We sequenced the sporophyte transcriptome of Killarney fern (Vandenboschia speciosa (Willd.) G. Kunkel). In addition to being a rare endangered Macaronesian-European endemism, this species has a huge genome (10.52 Gb) as well as particular biological features and extreme ecological requirements. These characteristics, together with the systematic position of ferns among vascular plants, make it of high interest for evolutionary, conservation and functional genomics studies. The transcriptome was constructed de novo and contained 36,430 transcripts, of which 17,706 had valid BLAST hits. A total of 19,539 transcripts showed at least one of the 7362 GO terms assigned to the transcriptome, whereas 6547 transcripts showed at least one of the 1359 KEGG assigned terms. A prospective analysis of functional annotation results provided relevant insights on genes involved in important functions such as growth and development as well as physiological adaptations. In this context, a catalogue of genes involved in the genetic control of plant development, during the vegetative to reproductive transition, in stress response as well as genes coding for transcription factors is given. Altogether, this study provides a first step towards understanding the gene expression of a significant fern species and the in silico functional and comparative analyses reported here provide important data and insights for further comparative evolutionary studies in ferns and land plants in general.

A taxonomic and molecular survey of the pteridophytes of the Nectandra Cloud Forest Reserve, Costa Rica.

Floristic surveys are crucial to the conservation of biodiversity, but the vast majority of such surveys are limited to listing species names, and few take into account the evolutionary history of species. Here, we combine classical taxonomic and molecular phylogenetic (DNA barcoding) approaches to catalog the biodiversity of pteridophytes (ferns and lycophytes) of the Nectandra Cloud Forest Reserve, Costa Rica. Surveys were carried out over three field seasons (2008, 2011, and 2013), resulting in 176 species representing 69 genera and 22 families of pteridophytes. Our literature survey of protected areas in Costa Rica shows that Nectandra has an exceptionally diverse pteridophyte flora for its size. Plastid rbcL was selected as a DNA barcode marker and obtained for >95% of pteridophyte taxa at this site. Combined molecular and morphological analyses revealed two previously undescribed taxa that appear to be of hybrid origin. The utility of rbcL for species identification was assessed by calculating minimum interspecific distances and found to have a failure rate of 18%. Finally we compared the distribution of minimum interspecific rbcL distances with two other areas that have been the focus of pteridophyte molecular surveys: Japan and Tahiti. The comparison shows that Nectandra is more similar to Japan than Tahiti, which may reflect the biogeographic history of these floras.

Simple and Divided Leaves in Ferns: Exploring the Genetic Basis for Leaf Morphology Differences in the Genus Elaphoglossum (Dryopteridaceae).

Despite the implications leaves have for life, their origin and development remain debated. Analyses across ferns and seed plants are fundamental to address the conservation or independent origins of megaphyllous leaf developmental mechanisms. Class I KNOX expression studies have been used to understand leaf development and, in ferns, have only been conducted in species with divided leaves. We performed expression analyses of the Class I KNOX and Histone H4 genes throughout the development of leaf primordia in two simple-leaved and one divided-leaved fern taxa. We found Class I KNOX are expressed (1) throughout young and early developing leaves of simple and divided-leaved ferns, (2) later into leaf development of divided-leaved species compared to simple-leaved species, and (3) at the leaf primordium apex and margins. H4 expression is similar in young leaf primordia of simple and divided leaves. Persistent Class I KNOX expression at the margins of divided leaf primordia compared with simple leaf primordia indicates that temporal and spatial patterns of Class I KNOX expression correlate with different fern leaf morphologies. However, our results also indicate that Class I KNOX expression alone is not sufficient to promote divided leaf development in ferns. Class I KNOX patterns of expression in fern leaves support the conservation of an independently recruited developmental mechanism for leaf dissection in megaphylls, the shoot-like nature of fern leaves compared with seed plant leaves, and the critical role marginal meristems play in fern leaf development.

Symplasmic Isolation Contributes to Somatic Embryo Induction and Development in the Tree Fern Cyathea delgadii Sternb.

In this report, we describe studies on symplasmic communication and cellular rearrangement during direct somatic embryogenesis (SE) in the tree fern Cyathea delgadii. We analyzed changes in the symplasmic transport of low-molecular-weight fluorochromes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) and fluorescein (delivered to cells as fluorescein diacetate, FDA), within stipe explants and somatic embryos originating from single epidermal cells and developing during 16-d long culture. Induction of SE is preceded by a restriction in fluorochrome distribution between certain explant cells. Microscopic analysis showed a series of cellular changes like a decrease in vacuole size, increase in vacuole numbers, and increased density of cytoplasm and deposition of electron-dense material in cell walls that may be related with embryogenic transition. In somatic embryos, the limited symplasmic communication between cells was observed first in linear tri-cellular embryos. Further development of the fern embryo was associated with the formation of symplasmic domains corresponding to the four segments of the plant body. Using symplasmic tracers, we provided evidence that the changes in plasmodesmata permeability are corelated with somatic-to-embryogenic transition and somatic embryo development.

Weedy ferns (Polypodiopsida) in Argentina: diversity, distribution and impact on human activities and ecosystems.

Currently, in Argentina 368 species of true ferns (i.e. Polypodiopsida class) are distributed throughout the country, however, only four of them have been mentioned until now as weeds and ruderal species. The goal of this work was to generate an update of weedy ferns from Argentina, including morphology, distribution, and type of weed according to their impact on natural habitats and/or human activities. All Argentinian fern species were analyzed based on references, herbarium specimens, and field trips. As a result of our study 25 species were recorded from Argentina and classified as segetal, ecological, or aquatic weeds, and ruderal and/or toxic species. Current taxonomic identity, diagnostic characters, origins, habitats, geographical distribution, common names, and impact and potential risks were indicated by species. In addition, we provide a dichotomous key to species, presence of these species in southern South American countries, as well as and photographs in natural habitat. This work represents the first review on native and exotic ferns from Argentina that cause an impact on human activities or disturbe native habitats. The results provide information for the development of weed management tools and priority areas to implement them.

Physical space interacts with clonal fragmentation and nutrient availability to affect the growth of Salvinia natans.

Physical space, clonal fragmentation and nutrient availability can each affect the growth of clonal plants, but their interactive effect has been little studied. We grew un-fragmented (connected) and fragmented (disconnected) ramet pairs of the floating, clonal plant Salvinia natans in cylindrical containers with different diameters and heights (volumes) filled with solutions of two nutrient levels (high vs. low). To simulate competition environments that are commonly confronted by S. natans, we also added two ramets of another floating plants Spirodela polyrrhiza in each container. Biomass (total biomass, floating biomass and submerged biomass) and number of ramets of S. salvinia were higher in the containers with a larger diameter. Compared to the low nutrient level, the high nutrient level increased number of ramets, and altered submerged to floating mass ratio of S. salvinia. The impacts of physical space on floating mass and number of ramets were stronger under the high than under the low nutrient level. Clonal fragmentation positively affected biomass in the containers with a smaller volume (a smaller height and diameter), but had little impact in the containers with a larger volume (a larger height or diameter). Our results suggest that physical space can interact with nutrients and clonal fragmentation to affect the performance of S. salvinia under competition.

Fluctuations in cell cycle, morphology and metabolism of Anemia phyllitidis gametophytes are the most important hallmarks of GA3-induced antheridiogenesis.

The research object concerns partially explained mechanisms of plant hormone participation in male sex determination in plants, among them in A. phyllitidis gametophytes during GA3-induced antheridiogenesis. To provide an explanation of the mechanisms of fluorescence and white-light microscopy, cytophotometric, autoradiographic and spectrophotometic methods were used to study cell cycle, the number of nucleoli, the amount of DiOC6-stained IMN/ER, in which endoplasmic reticulum (ER) constitutes the main part, and its distribution as well as the amounts of proteins and chlorophylls and activities of acidic (Ac) and basic (Ba) phosphatases (Phases). It was revealed that antheridiogenesis was accompanied by cell cycle arrest at S-phase, changes of the number of nucleoli with simultaneous changes of the amount of IMN/ER and its distribution as well as fluctuations of protein amounts and of activities of acidic (Ac) and basic (Ba) phosphatases (Phases). The results indicated that initiation of antheridiogenesis in A. phyllitidis gametophytes by GA3 was related to the elevation of GAs/ANs in the culture media, during its induction phase, and the elevation of IMN/ER and GAs/ANs amounts, during expression phase of this process.

Key innovations in transition from homospory to heterospory.

Heterospory (i.e. dimorphic spores) is a long-lasting topic discussed in plant biology. It is observed in many of ferns, fern allies, and seed plants. The rise of heterospory and the mechanisms underlying its success in plant evolution are not clearly elucidated. In this short communication, an attempt is made to shed some light on these two questions.

Variability of fatty acid profiles in ferns: Relation to fern taxonomy and seasonal development.

Ferns are known to contain long chain polyunsaturated fatty acids which may provide health benefits. The objective of this study was to investigate ferns of Pacific temperate regions (Far East of Russia and New Zealand) as sources of valuable fatty acids: arachidonic (20:4n-6) and eicosapentaenoic (20:5n-3). Fatty acids were analyzed in fronds of 23 fern species from 12 families. Major fatty acids include: 18:3n-3 (6-68% of total fatty acids), 16:0 (6-33%), 18:2n-6 (5-46%), 18:1n-9 (1-60%), 20:4n-6 (1-16%). Polyunsaturated fatty acids of fern fronds belong to the omega-6 (16:2n-6, 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6) and omega-3 (16:3n-3, 18:3n-3, 18:4n-3, 20:3n-3, 20:4n-3, 20:5n-3) families. For the first time, Δ5-unsaturated polymethylene-interrupted fatty acids were reported for ferns: sciadonic (5,11,14-20:3) and juniperonic (5,11,14,17-20:4) acids (up to 1.9% and 0.4%, respectively). Fatty acid profiles in fern fronds were unrelated to fern taxonomy, but affected by spore presence: fronds with sporangia/spores contained more 18:1n-9 and/or 18:2n-6. The absolute content of 20:4n-6 was found to be relatively constant for a species in different seasons. 20:5n-3 was a minor fatty acid (traces-5%) which accumulates during the vegetation period. Young fronds of the New Zealand ferns Phymatosorus pustulatus and Pteridium esculentum were enriched in 20:4n-6, while aged fronds of Cyathea dealbata had the highest level of 20:5n-3. The mature fronds of the Far Eastern ferns Phegopteris connectilis, Dryopteris expansa, and Athyrium sinense were also enriched in 20:5n-3.

Separate and combined effects of glyphosate and copper on growth and antioxidative enzymes in Salvinia natans (L.) All.

The coexistence of glyphosate and copper is widely found in bodies of water and terrestrial ecosystems due to widespread application of herbicides and heavy metal. However, their joint ecotoxicological risks in aquatic environments remain unknown. The experiment investigated the individual and combined effects of glyphosate and copper on the growth and physiological response in Salvinia natans (L.) All. The results showed that their joint toxicity is related to concentration. Antagonistic effects were induced when plants were exposed to low concentrations of glyphosate and copper (≤1 + 0.2 mg l-1). Synergistic effects were elicited at higher doses (≥5 + 1 mg l-1). In addition, increased hydrogen peroxide levels indicated the occurrence of oxidative stress at individual or combined exposures. To cope with oxidative stress, S. natans can activate the antioxidant defense systems, including increased superoxide dismutase and changes in peroxidase, ascorbate peroxidase and catalase. High concentrations of combined pollution exceed the oxidative defense capabilities of plants, and therefore, malondialdehyde content increased significantly. Our results indicated that the ecotoxicity of glyphosate or copper may be exacerbated in aquatic environments and caused obvious damage to S. natans.

Can fern spores develop hydration memory in response to priming?

Fern spores and seeds initiate germination with fast water uptake, followed by a stationary phase with no appreciable water uptake and biochemical and metabolic processes that precede germination. After that, seed, germination is avoided by dehydration, as part of the priming treatments. After dehydration, seeds maintain their metabolic advances (hydration memory). As a result, rehydrated seeds germinate rapidly. We hypothesized that, as seeds, fern spores may be capable of developing hydration memory. To assess priming, spores of six fern species were exposed to: four or eight days of hydration in water (hydro-priming) or in a soil matrix (matrix-priming); or 1 month of hydration in the soil of the collection site (natural-priming). At the end of the treatments, the spores were dehydrated in the dark and germinated under laboratory conditions. Germination was evaluated using lag-time, germination rate and germination percentage. Priming treatments shortened lag time and/or increased germination rate or germination percentage in relation to the controls. Matrix-priming (8 days) reduced the spore germination percentage in three species. Our results provide evidence that fern spores possess a hydration memory that probably evolved in the soil bank and suggests that hydration-dehydration cycles within the natural soil might provide advantages for successful germination.

Meristem development and activity in gametophytes of the model fern, Ceratopteris richardii.

Ceratopteris richardii is a model fern species widely used to analyze various developmental processes and their regulation in gametophytes. The form of mature C. richardii gametophytes depends on the activity of the marginal meristem, but knowledge on meristem formation and structure is limited. Therefore, we analyzed cellular events accompanying the development of gametophytes using cell lineage and proliferation analyses to explain the establishment and functioning of the marginal meristem. We show that: i) gametophytes are devoid of the apical initial cell or the apical cell-based meristem in the early developmental stages; ii) the cells that are predestined to form the marginal meristem divide according to a stable pattern; iii) only one transient initial cell is present in the marginal meristem, and the selection of a new functioning initial cell is related to a stable sequence of its divisions. Our results contribute to a better understanding of the developmental events underlying gametophyte growth and marginal meristem functioning in Ceratopteris. The principles, which were established in this study and enabled the identification of functioning initial cells, can be applied to analyze genetic and/or physiological mechanism(s) governing meristem maintenance in vascular plants, both in developmental and evolutionary contexts.

Morfogénesis de la fase sexual de los helechos epífitos Microgramma mortoniana y Pleopeltis macrocarpa (Polypodiaceae) de la Reserva Natural Punta Lara, Buenos Aires, Argentina / Sexual morphogenesis phase of the epiphytic ferns Microgramma mortoniana and Pleopeltis macrocarpa (Polypodiaceae) from Punta Lara Natural Reserve, Buenos Aires, Argentina

Resumen Pleopeltis macrocarpa y Microgramma mortoniana son dos helechos epífitos de la familia Polypodiaceae hallados con baja frecuencia en la Reserva Natural Punta Lara (Buenos Aires, Argentina). El objetivo de este trabajo fue estudiar la morfogénesis de gametófitos epífitos para fortalecer poblaciones vulnerables y contribuir a su conservación. Se recolectaron hojas fértiles. Las esporas se sembraron en placas de Petri con medio de cultivo Dyer. Las esporas se caracterizaron por ser monoletes, elipsoidales, amarillentas y con ornamentación verrucosa. La germinación fue del tipo Vittaria y el desarrollo del gametofito es del tipo Drynaria. El esporofito de P. macrocarpa surgió a los 500 días y el de M. mortoniana a los 120. El patrón de germinación, el desarrollo del gametofito, el glóbulo lipídico en la célula protálica y los pelos unicelulares capitados en el margen podrían considerarse en la sistemática del grupo. La demora en la formación de esporofitos a través de la reproducción sexual, permite inferir que el éxito de su propagación estaría sujeto a la reproducción vegetativa.

Abstract The Punta Lara Natural Reserve is located on the riverside of the La Plata River in the province of Buenos Aires, Argentina. It is the Southernmost relict in the world of subtropical riparian forest. The epiphytic ferns studied in this work belong to the Polypodiaceae family: Microgramma mortoniana and Pleopeltis macrocarpa. Plant communities are subject to high levels of anthropization and introduction of exotic species. The goals of this work are to provide information on the morphogenesis of epiphytic gametophytes and to extend knowledge of their life cycles, contributing to their conservation. Sowing was carried out in Dyer medium. In both species the spores are monolete, ellipsoidal, yellowish and with verrucate sculpture. The equatorial diameter is 60-61 μm, the polar diameter is 39-42 μm. The germination is the Vittaria type; in M. mortoniana occurs at 20 days, while in P. macrocarpa occurs at six days. The filaments are uniseriate of 3-6 cells in length. The gametophyte development is Drynaria type. The cordated form is given after 40 days. In M. mortoniana, buds originated after 40 days. In P. macrocarpa, after 120 days, clathrate trichomes scale-like appear mainly on the margins of the gametophyte. The gametangia are typical of leptosporangiate ferns. The sporophyte of M. mortoniana emerged after 120 days and that of P. macrocarpa arose after 500 days, its blades are simple, spatulate and unicellular and multicellular branching hairs were observed. The germination pattern, gametophyte development, the presence of a lipid globule in the prothalic cell and the formation of unicellular capitated hairs are relevant characters that could be considered for systematic group. The delay in the formation of sporophytes through sexual reproduction, allows us to infer that the success of their establishment in situ would be given by the vegetative reproduction through creeping rhizomes and buds of gametophytes. Rev. Biol. Trop. 66(3): 1078-1089. Epub 2018 September 01.

Germination fitness of two temperate epiphytic ferns shifts under increasing temperatures and forest fragmentation.

Ferns are an important component of ecosystems around the world. Studies of the impacts that global changes may have on ferns are scarce, yet emerging studies indicate that some species may be particularly sensitive to climate change. The lack of research in this subject is much more aggravated in the case of epiphytes, and especially those that live under temperate climates. A mathematical model was developed for two temperate epiphytic ferns in order to predict potential impacts on spore germination kinetics, in response to different scenarios of global change, coming from increasing temperature and forest fragmentation. Our results show that an increasing temperature will have a negative impact over the populations of these temperate epiphytic ferns. Under unfragmented forests the germination percentage was comparatively less influenced than in fragmented patches. This study highlight that, in the long term, populations of the studied epiphytic temperate ferns may decline due to climate change. Overall, epiphytic fern communities will suffer changes in diversity, richness and dominance. Our study draws attention to the role of ferns in epiphytic communities of temperate forests, emphasizing the importance of considering these plants in any conservation strategy, specifically forest conservation. From a methodological point of view, the model we propose could be easily used to dynamically monitor the status of ecosystems, allowing the quick prediction of possible future scenarios, which is a crucial issue in biodiversity conservation decision-making.

Are rates of species diversification and body size evolution coupled in the ferns?

PREMISE OF THE STUDY: Understanding the relationship between phenotypic evolution and lineage diversification is a central goal of evolutionary biology. To extend our understanding of the role morphological evolution plays in the diversification of plants, we examined the relationship between leaf size evolution and lineage diversification across ferns. METHODS: We tested for an association between body size evolution and lineage diversification using a comparative phylogenetic approach that combined a time-calibrated phylogeny and leaf size data set for 2654 fern species. Rates of leaf size change and lineage diversification were estimated using BAMM, and rate correlations were performed for rates obtained for all families and individual species. Rates and patterns of rate-rate correlation were also analyzed separately for terrestrial and epiphytic taxa. KEY RESULTS: We find no significant correlation between rates of leaf area change and lineage diversification, nor was there a difference in this pattern when growth habit is considered. Our results are consistent with the findings of an earlier study that reported decoupled rates of body size evolution and diversification in the Polypodiaceae, but conflict with a recent study that reported a positive correlation between body size evolution and lineage diversification rates in the tree fern family Cyatheaceae. CONCLUSIONS: Our findings indicate that lineage diversification in ferns is largely decoupled from shifts in body size, in contrast to several other groups of organisms. Speciation in ferns appears to be primarily driven by hybridization and isolation along elevational gradients, rather than adaptive radiations featuring prominent morphological restructuring. The exceptional diversity of leaf morphologies in ferns appears to reflect a combination of ecophysiological constraints and adaptations that are not key innovations.

Azolla along a phosphorus gradient: biphasic growth response linked to diazotroph traits and phosphorus-induced iron chlorosis.

Azolla spp., a water fern often used for phytoremediation, is a strong phosphorus (P) accumulator due to its high growth rate and N2 fixing symbionts (diazotrophs). It is known that plant growth is stimulated by P, but the nature of the interactive response of both symbionts along a P gradient, and related changes in growth-limiting factors, are unclear. We determined growth, and N and P sequestration rates of Azolla filiculoides in N-free water at different P concentrations. The growth response appeared to be biphasic and highest at levels ≥10 P µmol l-1. Diazotrophic N sequestration increased upon P addition, and rates were three times higher at high P than at low P. At 10 µmol P l-1, N sequestration rates reached its maximum and A. filiculoides growth became saturated. Due to luxury consumption, P sequestration rates increased until 50 µmol P l-1. At higher P concentrations (≥50 µmol l-1), however, chlorosis occurred that seems to be caused by iron- (Fe-), and not by N-deficiency. We demonstrate that traits of the complete symbiosis in relation to P and Fe availability determine plant performance, stressing the role of nutrient stoichiometry. The results are discussed regarding Azolla's potential use in a bio-based economy.