Genome-wide data reveal cryptic diversity and hybridization in a group of tree ferns.

Discovery of cryptic diversity is essential to understanding both the process of speciation and the conservation of species. Determining species boundaries in fern lineages represents a major challenge due to lack of morphologically diagnostic characters and frequent hybridization. Genomic data has substantially enhanced our understanding of the speciation process, increased the resolution of species delimitation studies, and led to the discovery of cryptic diversity. Here, we employed restriction-site-associated DNA sequencing (RAD-seq) and integrated phylogenomic and population genomic analyses to investigate phylogenetic relationships and evolutionary history of 16 tree ferns with marginate scales (Cyatheaceae) from China and Vietnam. We conducted multiple species delimitation analyses using the multispecies coalescent (MSC) model and novel approaches based on genealogical divergence index (gdi) and isolation by distance (IBD). In addition, we inferred species trees using concatenation and several coalescent-based methods, and assessed hybridization patterns and rate of gene flow across the phylogeny. We obtained highly supported and generally congruent phylogenies inferred from concatenated and summary-coalescent methods, and the monophyly of all currently recognized species were strongly supported. Our results revealed substantial evidence of cryptic diversity in three widely distributed Gymnosphaera species, each of which was composite of two highly structure lineages that may correspond to cryptic species. We found that hybridization was fairly common between not only closely related species, but also distantly related species. Collectively, it appears that scaly tree ferns may contain cryptic diversity and hybridization has played an important role throughout the evolutionary history of this group.

Herbivory and leaf expansion of Cyathea phalerata Mart. (Cyatheaceae) in subtropical Atlantic Forest, southern Brazil

Abstract 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.

Resumo 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.

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

Abstract 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.

Resumo 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.

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.

Providing the missing links in fern life history: Insights from a phenological survey of the gametophyte stage.

Premise: The entire life cycle of ferns has been documented, yet their life histories are still poorly understood. In particular, the phenology of fern gametophytes remains largely unknown. To address this issue, we demonstrated a new ecological approach to explore the phenological link between spore release and gametophyte maturation within the life history of a tree fern species. Methods: We conducted a serial survey of Alsophila podophylla gametophyte abundance in the field, and recorded the time of its spore release. Every two months for one year, all terrestrial fern gametophytes in an unsampled subplot were collected and identified using tissue-direct PCR. Results: We found temporal differences in gametophyte abundances, with a sevenfold difference between the highest and lowest months. The number of spores released was linked to the gametophyte abundance two months later. The switch from gametophyte to juvenile sporophyte was found to be most correlated with precipitation. Discussion: The observed fluctuation in gametophyte abundance and population structure was likely associated with the phenology of spore release and environmental factors. Importantly, these findings provide the first evidence of phenological links between different developmental stages in a fern's life history.

Integrating tissue-direct PCR into genetic identification: An upgraded molecular ecology approach to survey fern gametophytes in the field.

Premise: The gametophytes of different fern species collected in the field can be difficult to distinguish because of their morphological similarities. Nonetheless, emerging molecular ecology techniques are starting to be used to tackle such limitations. Here, using case studies and a detailed protocol, we demonstrate a convenient methodology, tissue-direct PCR (TD-PCR), that foregoes a traditional DNA extraction and facilitates the identification of fern gametophytes, as well as enabling the elucidation of their natural distribution. Methods: Based on updated plastome information, we designed a universal primer set targeting the trnL-L-F region, which is effective across extant ferns. We used this primer set to perform TD-PCR on the case-studied populations of Taiwanese Lomariopsis gametophytes, using the generated sequences for their identification. In the case study concerning the microhabitat preference of Vaginularia junghuhnii, we designed and used a taxon-specific primer set. Results: Compared with approaches requiring DNA extraction, the use of TD-PCR with either universal or taxon-specific primers could save significant time, money, labor, and research materials in the genetic identification of fern gametophytes. Discussion: The use of modern genetic tools can aid in the identification of fern gametophytes. An updated TD-PCR strategy not only facilitates the DNA-based identification of gametophytes, but also promotes new avenues of research for investigating these plants in the field.

Enhancing In Vitro Production of the Tree Fern Cyathea delgadii and Modifying Secondary Metabolite Profiles by LED Lighting.

The tree ferns are an important component of tropical forests. In view of this, the enhancement of in vitro production of these plants is needed. Thus, the effect of different light-emitting diodes (LEDs) as well as control fluorescent lamps (Fl) and a 3-week-long period of darkness at the beginning of in vitro culture on micropropagation of the tree fern Cyathea delgadii Sternb. was analysed. Moreover, the photosynthetic pigment content and secondary metabolite profiles were estimated. The period of darkness contributed to a high production of somatic embryo-derived sporophytes and a low production of gametophytes. The formation of new sporophytes was stimulated by RBY (35% red, 15% blue, and 50% yellow) and B (100% blue) lights when the stipe explants or whole young sporophytes were used in the culture, respectively. The elongation of the roots and leaves was stimulated by RBfR light (35% red, 15% blue, and 50% far red), while root production increased under RBY light. The RB (70% red and 30% blue) and B lights stimulated the accumulation of chlorophyll better than Fl light. The most abundant metabolite found in the plant extracts was trans-5-O-caffeoylquinic acid (1.013 µg/mg of dry weight). The extract obtained from plants growing in a greenhouse had the best antioxidant activity.

First Multi-Organ Full-Length Transcriptome of Tree Fern Alsophila spinulosa Highlights the Stress-Resistant and Light-Adapted Genes.

Alsophila spinulosa, a relict tree fern, is a valuable plant for investigating environmental adaptations. Its genetic resources, however, are scarce. We used the PacBio and Illumina platforms to sequence the polyadenylated RNA of A. spinulosa root, rachis, and pinna, yielding 125,758, 89,107, and 89,332 unigenes, respectively. Combining the unigenes from three organs yielded a non-redundant reference transcriptome with 278,357 unigenes and N50 of 4141 bp, which were further reconstructed into 38,470 UniTransModels. According to functional annotation, pentatricopeptide repeat genes and retrotransposon-encoded polyprotein genes are the most abundant unigenes. Clean reads mapping to the full-length transcriptome is used to assess the expression of unigenes. The stress-induced ASR genes are highly expressed in all three organs, which is validated by qRT-PCR. The organ-specific upregulated genes are enriched for pathways involved in stress response, secondary metabolites, and photosynthesis. Genes for five types of photoreceptors, CRY signaling pathway, ABA biosynthesis and transduction pathway, and stomatal movement-related ion channel/transporter are profiled using the high-quality unigenes. The gene expression pattern coincides with the previously identified stomatal characteristics of fern. This study is the first multi-organ full-length transcriptome report of a tree fern species, the abundant genetic resources and comprehensive analysis of A. spinulosa, which provides the groundwork for future tree fern research.

Tree Fern Cyathea lepifera May Survive by Its Phytotoxic Property.

Cyatheaceae (tree ferns) appeared during the Jurassic period and some of the species still remain. Those species may have some morphological and/or physiological characteristics for survival. A tree fern was observed to suppress the growth of other ligneous plants in a tropical forest. It was assumed that the fern may release toxic substances into the forest floor, but those toxic substances have not yet been identified. Therefore, we investigated the phytotoxicity and phytotoxic substances of Cyathea lepifera (J. Sm. ex Hook.) Copel. An aqueous methanol extract of C. lepifera fronds inhibited the growth of roots and shoots of dicotyledonous garden cress (Lepidum sativum L.), lettuce (Lactuca sativa L.), and alfalfa (Medicago sativa L.), and monocotyledonous ryegrass (Lolium multiflorum Lam.), timothy (Phleum pratense L.), and barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.). The results suggest that C. lepifera fronds may have phytotoxicity and contain some phytotoxic substances. The extract was purified through several chromatographic steps during which inhibitory activity was monitored, and p-coumaric acid and (-)-3-hydroxy-ß-ionone were isolated. Those compounds showed phytotoxic activity and may contribute to the phytotoxic effects caused by the C. lepifera fronds. The fronds fall and accumulate on the forest floor through defoliation, and the compounds may be released into the forest soils through the decomposition process of the fronds. The phytotoxic activities of the compounds may be partly responsible for the fern's survival.

Colonización y estructura de la comunidad de hongos micorrízicos arbusculares en Alsophila firma (Cyatheaceae) en bosque mesófilo de montaña en Veracruz, México / Colonization and structure of arbuscular mycorrhizal fungi community in Alsophila firma (Cyatheales: Cyatheaceae) from a tropical montane cloud forest in Veracruz, México

Alsophila firma is a tree fern that is distributed mainly in tropical montane cloud forest (TMCF) and is considered as a threatened species. Arbuscular mycorrrhizal fungi (AMF) have been proposed as an alternative in rescue programs of endangered species. However, our knowledge about diversity of AMF and mycorrhizal status of the species of TMCF is limited. In Mexico TMCF shows different degrees of conservation because of fragmentation and land use change. In this study, we evaluated the level of colonization, richness and abundances of spores of AMF in three fragments with different conservation status: conserved (100 years), secondary vegetation (17 years) and disturbed. For this, soil samples and roots were collected from five individuals of A. firma per site, with at least 100m away from each other; a total of 100cm of roots were analysed per site. Root samples showed AMF and occasionally dark septate fungi (DSF) colonizations. For the overall study, 19 species of AMF were recorded: Gigaspora (7), Acaulospora (4), Glomus (4), Funneliformis (2), Sclerocystis (1) and Scutellospora (1). The dominant species in the three sites were Funneliformis geosporum and Acaulospora scrobiculata. The highest diversity (H’) and evenness (J’) (p<0.05) were found in the conserved site (H’=1.7, J’=0.66), when compared to the secondary vegetation (H’=1.5, J’=0.61), and the disturbed site (H’=0.74, J’=0.41). Statistical analysis showed that the AMF degree of colonization was significantly higher (p<0.05) in the conserved site; although, the disturbed site showed low richness and abundances of AMF, the degree of root colonization did not differ statistically (p<0.05) with the secondary vegetation site. Chao2 (Richness estimation model) showed that the number of analysed samples were sufficient to represent the structure of the AMF communities with values >90%. The present study confirmed that A. firma is a mycorrhizal species that exhibits high levels of colonization even in disturbed sites. We suggest that F. geosporum and A. scrobiculata may have the potential to inoculate the gametophyte and young sporophyte of A. firma, to support restoration programs, because of their abundances and high tolerance to disturbed sites.

Alsophila firma es un helecho arborescente que crece principalmente en bosque mesófilo de montaña (BMM) y se encuentra en la categoría de riesgo. Los hongos mico- rrízicos arbusculares (HMA) se han propuesto como una alternativa para programas de recuperación de especies nativas en peligro. Sin embargo, sabemos poco sobre la diversidad de HMA y del estatus micorrízico de las espe- cies vegetales del BMM. En México el BMM presenta diferentes grados de conservación debido a la fragmentación y el cambio de uso de suelo. Se evaluó el grado de colonización, riqueza y abundancia de esporas de los HMA presentes en fragmentos de BMM con diferente historia de manejo: conservado (100 años), en recuperación (17 años) y alterado. Se estudiaron muestras de suelo y raíces toma- das de 5 individuos de A. firma por cada sitio, con al menos 100m de distancia entre individuos. Se analizaron en total 100cm de raíz por cada sitio. Las muestras de raíz presentaron colonización principalmente de HMA y ocasionalmente por hongos septados oscuros (HSO). Se encontraron 19 especies: Gigaspora (7), Acaulospora (4), Glomus (4), Funneliformis (2), Sclerocystis (1) y Scutellospora (1). Las especies dominantes en los tres sitios fueron Funneliformis geosporum y Acaulospora scrobiculata. La mayor diversidad (H’) y equitatividad (J’) (p<0.05) se presentaron en el sitio conservado (H’=1.7; J’ 0.66) con respecto al sitio en recuperación (H’=1.5, J’ 0.61) y alterado (H’=0.74, J’=0.41). Los niveles de colonización micorrízica total de raíz fueron estadísticamente mayores (p<0.05) en el sitio conservado. Aunque el sitio alterado presenta una baja riqueza y abundancia de HMA la colonización micorrízica total de raíz no fue estadísticamente diferente (p<0.05) respecto al sitio en recuperación. El estimador Chao2 indica que las muestras analizadas reflejan la estructura general de la comunidad de HMA con valores superiores al 93%. El presente estudio confirma que A. firma es una especie micorrízica con altos niveles de colonización incluso en sitios alterados. Se sugiere que las especies F. geosporum y A. scrobiculata por su alta tolerancia a sitios alterados pueden tener potencial para inocular gametofitos y esporofitos de A. firma para incrementar la supervivencia en programas de restauración.

Germination and gametophyte development of Cyathea corcovadensis (Raddi) Domin (Cyatheaceae) from spores stored at low temperatures / Germinação e desenvolvimento gametofítico de Cyathea corcovadensis (Raddi) Domin (Cyatheaceae) com esporos armazenados em baixas temperaturas

The tree fern Cyathea corcovadensis (Raddi) Domin is an endangered species in the state of Rio Grande do Sul, Brazil. It currently occurs only in the northern segment of the coastal region. Spore storage would help in conservation programs since it maintains genetic variability and provides material for in vitro cultures. Current study evaluates the effect of low temperatures combined to different spore storage times on the germination and initial gametophyte development of C. corcovadensis. Spores were divided into two groups: spores of the first group were sowed immediately in Meyer culture medium with nystatin, at pH 4.0, while spores of the second group were stored at 7, -20 and -196°C during 60, 120, 180, and 365 days and then sowed in the same medium. Spore storage at 7 and -196°C for 365 days not only provided higher germination percentages than those reported for recently-collected spores but also stimulated gametophytic development. The latter was demonstrated by the higher percentages of laminar gametophytes in these treatments. The possibility of storing spores provides material for in vitro experiments, which is of special interest for C. corcovadensis due to its ornamental potential and conservation status.

A samambaia arborescente Cyathea corcovadensis (Raddi) Domin está em perigo de extinção no Estado do Rio Grande do Sul, Brasil, ocorrendo somente no norte da região costeira. O armazenamento de esporos pode auxiliar programas de conservação, uma vez que oferece a possibilidade de manutenção da variabilidade genética e fornece material para culturas in vitro. O objetivo deste estudo foi avaliar o efeito de temperaturas baixas combinadas com diferentes tempos de armazenamento de esporos sobre a germinação e o desenvolvimento inicial de gametófitos de C. corcovadensis. Os esporos foram divididos em dois grupos: os do primeiro grupo foram semeados imediatamente em meio de cultura Meyer com nistatina, com pH 4,0 e os do segundo grupo foram armazenados a 7, -20 e -196°C durante 60, 120, 180 e 365 dias e então semeados no meio supra mencionado. O armazenamento de esporos a 7 e -196°C durante 365 dias propiciou porcentagens de germinação superiores àquelas observadas para esporos recém coletados e também estimulou o desenvolvimento gametofítico, o que foi demonstrado pelos elevados valores de porcentagem de gametófitos laminares observados nestes tratamentos. A possibilidade de armazenar esporos oferece material para experimentos in vitro, o que é de especial interesse para C. corcovadensis, considerando seu potencial ornamental e estado de conservação.

In vitro propagation of Cyathea atrovirens (Cyatheaceae): spore storage and sterilization conditions / Propagación in vitro de Cyathea atrovirens (Cyatheaceae): almacenamiento de esporas y condiciones de esterilización

Cyathea atrovirens occurs in a wide range of habitats in Brazil, Paraguay, Uruguay and Argentina. In the Brazilian State of Rio Grande do Sul, this commonly found species is a target of intense exploitation, because of its ornamental characteristics. The in vitro cultura is an important tool for propagation which may contribute toward the reduction of extractivism. However, exogenous contamination of spores is an obstacle for the success of aseptic long-term cultures. This study evaluated the influence of different sterilization methods combined with storage conditions on the contamination of the in vitro cultures and the gametophytic development of C. atrovirens, in order to establish an efficient propagation protocol. Spores were obtained from plants collected in Novo Hamburgo, State of Rio Grande do Sul, Brazil. In the first experiment, spores stored at 7oC were surface sterilized with 0.5, 0.8 and 2% of sodium hypochlorite (NaClO) for 15 minutes and sown in Meyer’s culture medium. The cultures were maintained in a growth room at 26±1ºC for a 12-h photoperiod and photon flux density of 100μmol/m²/s provided by cool white fluorescent light. Contamination was assessed at 60 days, and gametophytic development was scored at 30, 60, 120 and 130 days of in vitro culture, analyzing 300 individuals for each treatment. There was no significant difference in culture contamination among the different sodium hypochlorite concentrations tested, and all treatments allowed for the development of cordiform gametophytes at 130 days of culture. In the second experiment, spores stored at 7 and -20°C were divided into two groups. Half of the spores were surface sterilized with 2% of NaClO for 15 minutes and the other half was not sterilized. All spores were sown in Meyer’s medium supplemented with one of the following antibiotics: nystatin, Micostatin® and actidione. The culture conditions and the procedures used for evaluating contamination and gametophytic development were the same described for the first experiment. No contamination was observed in spores stored at -20°C and treated with NaClO and actidione. In all treatments, cordiform gametophytes presenting antheridia were observed at 120 days. The percentages of these gametophytes increased from 120 to 130 days and no significant differences were observed among treatments. Archegonia were observed on cordiform gametophytes at 130 days. The findings provide data relevant to in vitro propagation procedures of this species, which may increase the availability of plants for ornamental purposes, therefore contributing to the reduction of the exploitation of endangered tree ferns species. Rev. Biol. Trop. 62 (1): 299-308. Epub 2014 March 01.

Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) se presenta en una amplia gama de hábitats en Brasil, Paraguay, Uruguay y Argentina. Debido a sus características ornamentales, la especie es objeto de intensa explotación. El cultivo in vitro es una herramienta importante para la propagación lo que puede contribuir a la reducción del impacto de las actividades extractivas. Sin embargo, la contaminación exógena de esporas es un obstáculo para el éxito de cultivos asépticos a largo plazo. Este estudio evaluó la influencia de diferentes métodos de esterilización en combinación con las condiciones de almacenamiento sobre la contaminación de los cultivos in vitro y el desarrollo gametofítico de C. atrovirens. En el primer experimento, las esporas almacenadas a 7°C se esterilizaron superficialmente con 0.5, 0.8 y 2% de hipoclorito de sodio (NaClO) durante 15 minutos y se sembraron en medio de cultivo de Meyer. Aunque no hubo diferencia en la contaminación de lós cultivos entre las concentraciones de hipoclorito de sodio de las diferentes pruebas, en el tratamiento con 2% NaClO se observó un mayor porcentaje de gametofitos cordiformes a los 130 días. En el segundo experimento, las esporas almacenadas a 7 y -20°C fueron divididas en dos grupos. La mitad de las esporas se esterilizaron con 2% de NaClO durante 15 minutos y la otra mitad no fue esterilizada. Todas las esporas se sembraron en medio de Meyer suplementado con uno de los siguientes antibióticos: nistatina, Micostatin® o actidiona. No se observó contaminación de las esporas almacenadas a -20°C y tratadas con NaClO y actidiona. En todos los tratamientos, se observaron gametofitos cordiformes con anteridios y arquegonios. Los resultados proporcionan datos relevantes para la propagación in vitro de C. atrovirens, que pueden aumentar la disponibilidad de las plantas para fines ornamentales, contribuyendo así a la reducción de la exploración de las especies de helechos arborescentes en peligro de extinción.

Ophiodiaporthe cyatheae gen. et sp. nov., a diaporthalean pathogen causing a devastating wilt disease of Cyathea lepifera in Taiwan.

The scaly tree fern, Cyathea lepifera, in Taiwan has been devastated by an ascomycetous pathogen in recent years. This fungus resembles species of Diaporthe, but unlike anamorphs of Diaporthe that produce two types of conidia, its anamorph produces one conidium type. It is described herein as Ophiodiaporthe cyatheae gen. et sp. nov. Through pathogenicity tests, O. cyatheae was demonstrated to be the causal agent of the C. lepifera wilt disease. Of interest, sporulating structures of O. cyatheae have not been found on C. lepifera plants but in culture thus far. The mating system is homothallic. Phylogenetic analyses based on combined sequences of nSSU-rDNA, nLSU-rDNA, EF1-α-1 and RPB2 placed O. cyatheae in Diaporthaceae. Combined sequences of EF1-α-2 and TUB indicated that O. cyatheae had its origin within Diaporthe.

Caudex growth and phenology of Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) in secondary forest, southern Brazil / Crescimento do cáudice e fenologia de Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) em floresta secundária, no sul do Brasil

The leaf production and senescence, formation and release of spores of Cyathea atrovirens (Langsd. & Fisch.) Domin were analysed based on the monthly monitoring of 50 plants growing in a secondary forest, in the municipality of Novo Hamburgo, in the state of Rio Grande do Sul, during the year 2004. The caudex height and number of mature and fertile leaves were recorded annually in 2004-09. In 2004, monthly production and senescence of leaves were concomitant, without total leaf abscission. Population synchrony at emergence (Z = 0.86) and leaf senescence (Z = 0.82) increased in spring but did not correlate with temperature and photoperiod. All individuals were fertile and the sporangia production and spore liberation presented higher and equal synchrony (Z = 0.84) respectively in spring and summer. Sporangia production was related with temperature and photoperiod, however taller plants did not produce more fertile leaves. Phenological events analysed were not influenced by precipitation, as expected for forests in non-seasonal climate. Over five years (2004-09), the annual mean caudex growth varied between 1.19 and 2.50 cm.year-1 and the plants appeared to have an ability to maintain a relatively stable amount of leaves throughout this period.

A produção e a senescência de folhas, a formação e a liberação de esporos de Cyathea atrovirens (Langsd. & Fisch.) Domin, durante o ano de 2004, foram analisadas a partir do monitoramento mensal de 50 plantas, crescendo em floresta secundária, no município de Novo Hamburgo, Estado do Rio Grande do Sul, Brasil. A altura do cáudice e o número de folhas foram mensurados anualmente de 2004-2009. Em 2004, a produção e a senescência mensal de folhas foram concomitantes, evitando a abscisão foliar total. A sincronia da população na renovação (Z = 0,86) e na senescência foliar (Z = 0,82) aumentou na primavera, porém esses eventos não se relacionaram com temperatura e fotoperíodo. Todos os indivíduos estavam férteis e a produção de esporângios e a liberação de esporos apresentaram uma sincronia maior e igual (Z = 0,84), respectivamente, na primavera e no verão. A produção de esporângios se relacionou com temperatura e fotoperíodo, porém plantas mais altas não produziram mais folhas férteis. Os eventos fenológicos analisados não foram influenciados pela precipitação, tal como esperado para florestas sob clima não sazonal. Durante cinco anos (2004-2009), a média anual do crescimento do cáudice variou de 1,19 a 2,50 cm.ano-1 e as plantas demonstraram capacidade de manter o número de folhas relativamente estável ao longo do período.

A natural shear-thickening water-soluble polymer from the fronds of the black tree fern, Cyathea medullaris: Influence of salt, pH and temperature.

A water-soluble polymer was extracted from the fronds of the black tree fern (Cyathea medullaris or "Mamaku" in Maori) and characterized under various physico-chemical conditions. The rheological properties (in the semi-dilute region) and particle size and charge (in the dilute region) were investigated under various salt (0-1M NaCl), pH (1-12) and temperature conditions (5-80°C), using rheometry and dynamic light scattering techniques. A 7% (w/w) Mamaku crude extract showed strong shear-thickening properties at high salinity levels (1M NaCl) and over the whole range of pH (1-12). However, the thickening properties disappeared above 50°C. Apparent viscosity remained constant over the pH range 3-9, although the particle size systematically decreased with increasing pH. Overall, Mamaku solutions showed very good salt and pH resistance and exhibited strong temperature dependency. Hydrophobic and electrostatic interactions are not likely to be the cause of the shear-thickening phenomenon observed. Other forces such as hydrogen bonding may play a dominant role on the formation of shear-induced associations.

Biodiversity and spatial distribution of epiphytic ferns on Alsophila setosa Kaulf. (Cyatheaceae) caudices in Rio Grande do Sul, Brazil / Biodiversidade e distribuição espacial de filicíneas epifíticas sobre cáudices de Alsophila setosa Kaulf. (Cyatheaceae) no Rio Grande do Sul, Brasil

The extractive exploitation of the tree fern Alsophila setosa Kaulf. alters forest formations and diminishes the availability of micro-habitat for epiphytes. A survey of epiphytic fern communities on A. setosa at 16 study sites in different forest formations in the State of Rio Grande do Sul, Brazil, documented the occurrence of 31 species representing 16 genera and six families. The greatest richness of species occurred in Polypodiaceae (39 percent) and in the genus Asplenium L. (22 percent). Habitual holoepiphyte was the predominant ecological category, representing 61 percent of the species. Similarity analysis demonstrated heterogeneity in the composition of the epiphytic ferns at the study sites and indicated that the vegetation type is not the main factor for floristic difference. The lowest total specific richness (5) was recorded for the seasonal deciduous forest site. The majority of the sites presented similar averages of phorophyte height and epiphyte richness per caudex. In 25 percent of the sites the height of the host plants presented significant correlation with specific richness. Considering that the majority of the epiphytes are habitual and that some of them occur exclusively or preferentially on tree ferns, the maintenance of these host plants in the vegetation is essential for the conservation of epiphytic species.

A exploração extrativista da samambaia arborescente Alsophila setosa Kaulf. altera as formações florestais e diminui a disponibilidade de microhabitats para epífitos. Um levantamento das comunidades de filicíneas epifíticas sobre A. setosa em 16 sítios de diferentes formações florestais, no Estado do Rio Grande do Sul, Brasil, documentou a ocorrência de 31 espécies representando 16 gêneros e seis famílias. A maior riqueza de espécies ocorreu em Polypodiaceae (39 por cento) e no gênero Asplenium L. (22 por cento). A categoria ecológica predominante foi a de holoepífito habitual, representando 61 por cento das espécies. A análise de similaridade evidenciou heterogeneidade na composição de filicíneas epifíticas dos sítios estudados e indicou que o tipo vegetacional não é o principal fator de diferenciação florística. A menor riqueza específica total (5) foi registrada em sítio de floresta estacional decidual. A maioria dos sítios apresentou médias similares de altura dos forófitos e de riqueza de epífitos por cáudice. Em 25 por cento dos sítios, a altura das plantas hospedeiras apresentou correlação significativa com riqueza específica. Considerando que a maioria dos epífitos é habitual e que alguns deles ocorrem exclusivamente ou preferencialmente sobre samambaias arborescentes, a manutenção dessas plantas hospedeiras, na vegetação, é essencial para a conservação de espécies epifíticas.

Composição e distribuição vertical de pteridófitas epifíticas sobre Dicksonia sellowiana Hook. (Dicksoniaceae), em floresta ombrófila mista no sul do Brasil / Composition and vertical distribution of epiphytic pteridophytes on Dicksonia sellowiana Hook. (Dicksoniaceae), in mixed ombrophylous forest in Southern Brazil

A extração ilegal de Dicksonia sellowiana Hook., uma espécie de samambaia arborescente, da Floresta Ombrófila Mista, no sul do Brasil, modifica a estrutura da vegetação e reduz a disponibilidade de microhabitats para espécies epifíticas. A composição e a distribuição vertical de pteridófitas epifíticas foram estudadas sobre D. sellowiana. O estudo foi desenvolvido em área de Floresta Ombrófila Mista, do Parque Municipal da Ronda, no Estado do Rio Grande do Sul, Brasil. Foram selecionados 164 forófitos e seus cáudices foram divididos em intervalos de 1 m, a partir do solo. Em cada intervalo, foi registrada a ocorrência de espécies de pteridófitas e foi calculada a freqüência por forófitos e por intervalos. Foram registradas 20 espécies, pertencentes a 13 gêneros e sete famílias, sendo que houve o predomínio de holoepífitos habituais. A maior riqueza específica foi encontrada em Aspleniaceae (6) e no gênero Asplenium L. (6). As espécies com maior freqüência relativa nos forófitos foram Trichomanes angustatum Carmich., Blechnum binervatum (Poir.) C.V. Morton & Lellinger e Vittaria lineata (L.) Sm.. Considerando uma sub-amostra de 60 forófitos, T. angustatum e B. binervatum apresentaram freqüência decrescente de ocorrência a partir do solo até 4 m de altura. A riqueza média foi maior nos três primeiros intervalos. A riqueza encontrada sobre os cáudices de D. sellowiana representa 67 por cento do total de pteridófitas epifíticas encontradas na área de Floresta Ombrófila Mista, no Parque Municipal da Ronda e demonstra a importância dessa planta hospedeira para a manutenção de espécies epifíticas, no ambiente florestal.

Dicksonia sellowiana Hook., a tree fern species, is illegally extracted from the Mixed Ombrophylous Forest of southern Brazil, what changes the vegetation structure and reduces the availability of micro-habitats for epiphytic species. The composition and the vertical distribution of epiphytic pteridophytes were studied on D. sellowiana. The study was carried out in a Mixed Ombrophylous Forest area, at the Parque Municipal da Ronda in the State of Rio Grande do Sul, Brazil. One hundred and sixty four phorophytes were selected and their caudices were divided at intervals of 1 m, from the ground. At each interval the occurrence of pteridophyte species was recorded, and the frequency by phorophyte and by interval was calculated. Twenty species were recorded, from 13 genera and seven families, and habitual holoephiphytes predominated. The largest specific richness was found in Aspleniaceae (6) and in the Asplenium L. genus (6). The species with the highest relative frequency on the phorophytes were Trichomanes angustatum Carmich., Blechnum binervatum (Poir.) C.V. Morton & Lellinger and Vittaria lineata (L.) Sm.. Considering a sub-sample of 60 phorophytes, T. angustatum and B. binervatum had decreasing frequency of occurrence from the ground up to 4 m. The average richness was higher in the first three intervals. The richness found over the caudices of D. sellowiana represented 67 percent of the total epiphytic pteridophytes found in Mixed Ombrophylous Forest area, at the Parque Municipal da Ronda and this shows the importance of this host plant for the maintenance of epiphytic species in the forest environment.

Cultivo de Oncidium baueri Lindley (Orchidaceae) em substratos a base de coco / Oncidium baueri Lindley (Orchidaceae) cultivation in coconut-based substrates

Entre os substratos utilizados no cultivo de orquídeas, a fibra de coco vem se destacando como promissor substituto do xaxim, material utilizado por muitos produtores e colecionadores de orquídeas, porém ameaçado de extinção. Objetivou-se, neste trabalho, foi avaliar a eficiência de substratos à base de coco, no cultivo da orquídea Oncidium baueri. As mudas foram cultivadas em vasos de polipropileno, permanecendo em viveiro com 50 por cento de luminosidade. Os tratamentos constituíram-se de xaxim desfibrado, coco desfibrado, coco em pó, coco desfibrado + coco em pó, coxim (coco em cubos), coxim + coco em pó e coxim + coco desfibrado. Após dezenove meses, foram avaliadas as variáveis: altura das plantas, diâmetro dos pseudobulbos, comprimento da maior raiz, massa seca das raízes, número de brotações e pH dos substratos. O delineamento experimental foi inteiramente casualizado, com sete tratamentos e dez repetições. Para todas as variáveis avaliadas, os substratos coco em pó e a mistura de coco desfibrado com coco em pó mostraram-se tão eficientes ao desenvolvimento das plantas quanto o xaxim desfibrado, podendo substituí-lo no cultivo de O. baueri.

Among the several substrates used in orchid growing, coconut fiber is being considered the most promising substitute for xaxim (tree fern fiber), material highly used by orchid growers and collectors, which has become endangered. The objective of this study was to evaluate the efficiency of coconut-based substrates in the cultivation of the Brazilian orchid Oncidium baueri. Seedlings were grown in polypropylene vases kept in a nursery with 50 percent of lighting. Treatments included: defibered tree fern (control), de-fibered coconut, coconut powder, de-fibered coconut + coconut powder, coxim (diced coconut.), coxim + coconut powder and coxim + de-fibered coconut. Plant height, pseudo bulbs diameter, number of buds, root dry matter weight, root length and pH substrate were evaluated 19 months later. The experimental design was entirely casualized, with 7 treatments and 10 replications. For all analyzed variables, the substrates powdered coconut and the mixture of defibered coconut with powdered coconut were as efficient to the plants development as the defibered tree fern and can substitute it in the O.baueri cultivation.