Resumen Antecedentes: La embriología de las estructuras reproductivas masculinas en Fouquieriaceae ha sido poco estudiada. La ontogenia de la pared de la antera, la microsporogénesis, la microgametogénesis y la morfología del polen fueron descritas en detalle para Fouquieria fasciculata. Preguntas: ¿Cómo se llevan a cabo los procesos de microsporogénesis y microgametogénesis en Fouquieria fasciculata? ¿Cómo es la morfología del grano de polen maduro? Especie de estudio: Fouquieria fasciculata (Fouquieriaceae). Sitio de estudio y fecha: Cañón del Río Estórax cerca de El Plátano, Querétaro, México. Métodos: Botones florales y flores fueron recolectadas, procesadas y analizadas con microscopía de luz y microscopía electrónica de barrido. Resultados: El desarrollo de la pared de la antera es de tipo Dicotiledóneo. La pared de la antera consiste en una epidermis con cutícula gruesa, un endotecio con engrosamientos fibrosos en forma de U, una capa media y un tapete secretor con células uni o binucleadas. Se caracterizaron dos tipos de orbículas: orbículas con forma de dona y orbículas agregadas con apariencia ruminada. La microsporogénesis es sucesiva, las tétradas de microsporas son tetraédricas. Los granos de polen son prolados, tricolporados con exina reticulada-heterobrocada y son dispersados en etapa bicelular. Conclusiones: Esta investigación es el primer estudio embriológico detallado de las estructuras reproductivas masculinas en Fouquieriaceae. Características novedosas fueron descritas en Fouquieria fasciculata, tales como la diferenciación de un tapete secretor y la presencia de orbículas, descritas por primera vez y contribuyen a caracterizar la embriología de Fouquieriaceae.

Abstract Background: Embryology of the male reproductive structures in Fouquieriaceae has been poorly studied. The ontogeny of the anther wall, microsporogenesis, microgametogenesis, and pollen morphology were described in detail for Fouquieria fasciculata. Questions: How microsporogenesis and microgametogenesis are carried out in Fouquieria fasciculata? How is the morphology of mature pollen grain? Study species: Fouquieria fasciculata (Fouquieriaceae). Study site and date: Río Estórax Canyon, near the El Plátano, Querétaro, Mexico. Methods: Floral buds and flowers at different stages of development were collected and processed for further analysis with light and scanning electron microscopy. Results: The development of the anther wall is of Dicotyledonous type. The anther wall consists of a single-layered epidermis covered by a thick cuticle, an endothecium with U-shaped fibrous thickenings, one middle layer and a secretory tapetum with uni or binucleate cell. Two orbicules types were characterized: doughnut-shape orbicules and aggregated orbicules with ruminate appearance. Microsporogenesis is successive, the microspore tetrads are tetrahedral. Pollen grains are prolate, tricolporate with reticulate-heterobrochate exine, and are shed in the two-celled stage. Conclusions: This research is the first comprehensive study of male structures ontogeny of Fouquieriaceae. Remarkable features were described in Fouquieria fasciculata, such as the differentiation of a secretory tapetum and the presence of orbicules, which are described for the first time and contribute to characterize the embryology of Fouquieriaceae.

Abstract Pérez, Y. J.; A. M. González & M. B. Angulo. 2023. Male gametophyte development and cytogenetics of natural diploid and polyploid species of the South American genus Lessingianthus (Asteraceae, Vernonieae). Darwiniana, nueva serie 11(1): 71-91. In view of the scarce cytoembryological knowledge of Lessingianthus and in order to understand the contribution of polyploidy to the evolution of the genus we perform a developmental survey of anthers with the main objective of study if the different ploidy level affect the anther and pollen development in Lessingianthus species and infer the male fertility of diploid and polyploid species. Our results showed that diploid and polyploid species have normal anther development, thus polyploidy did not affect the anther wall development. The following male embryological traits are conserved among the species: tetrasporangiate anthers, four-layered anther wall of dicotyledonous development, with uniseriate persistent epidermis, invasive tapetum, tetrahedral or isobilateral tetrads, and 3-celled pollen grains. Diploid and polyploid species had high male fertility: both presented relatively high meiotic regularity and therefore formed normal viable spores independently of the ploidy level. Lessingianthus argenteus, the only species with high irregular behavior, might be a neopolyploid species. The eight studied species of the genus presented different percentages of meiotic irregularity at the different phases observed. In diploid specimens the irregularities were concentrated in anaphase of meiosis I, whereas in polyploid specimens the irregularities were dispersed through the phases of meiosis II. The main abnormal behavior identified, and therefore the main mechanisms of unreduced microspore formation, were chromosomes out of plate, bridges without fragments and asynchrony of meiosis I and II. These features broadened the male cytoembryological knowledge of the genus, confirming the suggestion that embryological characters related to male gametophyte development are conserved among the studied species. The results contribute to understand the mechanisms of polyploid formation which is an important process during the evolution of the genus.

Resumen Pérez. Y. J.; A. M. González & M. B. Angulo. 2023. Desarrollo del gametofito masculino y citogenética de especies naturales diploides y poliploides del género sudamericano Lessingianthus (Asteraceae, Vernonieae). Darwiniana, nueva serie 11(1): 71-91. En vista del escaso conocimiento citoembriológico de Lessingianthus y con el fin de comprender la contribución de la poliploidía a la evolución del género, realizamos un estudio de desarrollo de las anteras con el principal objetivo de estudiar si los diferentes niveles de ploidía afectan el desarrollo de las anteras y el polen en las especies de Lessingianthus, e inferir la fertilidad masculina de las especies diploides y poliploides. Nuestros resultados mostraron que las especies poliploides tienen desarrollo normal de las anteras, por lo tanto la poliploidía no afecta el desarrollo de la pared de las anteras. Los siguientes caracteres embriológicos masculinos están conservados entre las especies: anteras tetrasporangiadas, pared de la antera de cuatro capas de desarrollo dicotiledóneo, epidermis uniseriada persistente, tapete invasivo, tétradas tetraédricas o isobilaterales, y granos de polen tricelulares. Tanto las especies diploides como las poliploides tienen una alta fertilidad masculina: ambas presentan una regularidad meiótica relativamente alta y, por tanto, forman esporas viables normales independientemente del nivel de ploidía. Lessingianthus argenteus, la única especie con un comportamiento irregular elevado, podría ser una especie neopoliploide. Las ocho especies estudiadas del género presentaron diferentes porcentajes de irregularidad meiótica en las distintas fases observadas. En los ejemplares de las especies diploides las irregularidades se concentraron en la anafase de la meiosis I, mientras que en los ejemplares de las especies poliploides las irregularidades se dispersaron por las fases de la meiosis II. Los principales comportamientos anormales identificados, y por tanto los principales mecanismos de formación de microsporas no reducidas, fueron cromosomas fuera de placa, puentes sin fragmentos y asincronía de las meiosis I y II. Estos caracteres amplían el conocimiento citoembriológico masculino del género, a la vez que confirman que los caracteres embriológicos relacionados al desarrollo del gametofito masculino son conservados entre las especies estudiadas. Los resultados contribuyen a la comprensión y determinación de los mecanismos de formación de poliploides, un proceso importante durante la evolución del género.

Abstract The objective of this work was to investigate the causes of sterility in a new set of triploid banana (Musa spp.) cultivars and to assess the chances of obtaining some progenies by manual cross-pollination. The developmental stages of female gametophyte were histologically recorded in ovules of four distinct triploid banana cultivars. Samples were taken on the day of flower opening and pollination and three days later. Morphologically mature embryo sacs were very rare in the two clones of the 'Cavendish' subgroup and in 'Prata Anã'. In 'Mysore', it occurred at a variable and low frequency. In 'Terrinha', it was extremely common. In the last two cases, the embryo sac maturation process was clearly continuing between the sampling days of flower opening and three days later. No pollen tube was positively identified in pollinated flowers within the integuments of any ovule of the older explants. Different proportions of mature embryo sacs are observed among the studied triploid genotypes of banana. The presence of mature embryo sacs is not an indication of functional capacity. Other causes of infertility in cultivated bananas can be due to some event immediately before or after fertilization, for instance, pollination tube growth failure or seed abortion. Musa spp. spp crosspollination. crosspollination cross pollination. cross-pollination later Cavendish 'Cavendish Prata Anã. Anã . Anã' Mysore, Mysore , 'Mysore' frequency Terrinha, Terrinha 'Terrinha' common cases explants capacity fertilization instance abortion 'Mysore 'Terrinha

Resumo O objetivo deste estudo foi investigar as causas da esterilidade em um novo conjunto de cultivares triploides de banana (Musa spp.) e avaliar as possibilidades de obter progênies por meio de polinização manual. Os estados de desenvolvimento do gametófito feminino foram histologicamente observados em óvulos de quatro cultivares de banana triploide. As amostras foram colhidas no dia da abertura das flores e da polinização e três dias depois. Os sacos embrionários morfologicamente maduros mostraram-se raros em dois clones do subgrupo 'Cavendish' e em 'Prata Anã'. Em 'Mysore', ocorreu a uma frequência variável e baixa. Em 'Terrinha', foi extremamente comum. Nos dois últimos casos, o processo de maturação dos sacos embrionários claramente continuava entre o dia da amostragem na floração e os três dias depois. Nenhum tubo polínico foi identificado em flores polinizadas nos integumentos de qualquer óvulo nos explantes mais velhos. Diferentes proporções de sacos embrionários são observadas entre os genótipos de bananas triploides. A presença de sacos embrionários não indica capacidade funcional. Outras causas de infertilidade em bananas cultivadas podem ser em razão de algum evento imediatamente antes ou depois da fertilização, como ausência de cariogamia ou aborto das sementes. Musa spp. spp manual triploide mostraramse mostraram se Cavendish 'Cavendish Prata Anã. Anã . Anã' Mysore, Mysore , 'Mysore' baixa Terrinha, Terrinha 'Terrinha' comum casos velhos funcional fertilização sementes 'Mysore 'Terrinha

Abstract Cyathea corcovadensis and Cyathea phalerata are tree ferns native to Brazil, endangered in the state of Rio Grande do Sul. Spore germination and gametophyte development in media with different nutrient formulations and activated charcoal were evaluated, aiming to develop a process for obtaining plants of the two species. Spores were sown in four semi-solid culture media: Meyer, Dyer, MS with 50% and MS with 25% of the original macronutrient concentration. For each medium, 10 replicates were carried out (flasks with 5 mg of spores/30 mL of medium), with and without 1% activated charcoal, respectively. Spore germination and gametophytic development (laminar and cordate stages) were quantified at 30, 60 and 90 days of culture. Cyathea corcovadensis and C. phalerata germinated and developed gametophytes in all media. For both species, the highest percentages of germination and cordate gametophytes (more advanced development stage) were recorded in Meyer medium without activated charcoal, which has higher concentrations of macronutrients and no micronutrients compared to the other evaluated media. We recommend cultivating the plants in Meyer medium for greater gametophytic development and subsequent sporophyte obtention, as a biotechnological tool for C. corcovadensis and C. phalerata conservation and for environmental restoration and enrichment using these tree ferns. Brazil Sul species semisolid semi solid Dyer 50 25 concentration 1 flasks spores30 spores 30 spores/3 , medium) respectively laminar stages 6 9 C more stage obtention 2 spores3 3 spores/

Resumo Cyathea corcovadensis e Cyathea phalerata são samambaias arborescentes nativas do Brasil, ameaçadas de extinção no estado do Rio Grande do Sul. A germinação de esporos e o desenvolvimento de gametófitos em meios com diferentes formulações de nutrientes e carvão ativado foram avaliados, visando ao desenvolvimento de um processo para obtenção de plantas das duas espécies. Esporos foram semeados em quatro meios de cultura semi-sólidos: Meyer, Dyer, MS com 50% e MS com 25% da concentração original dos macronutrientes. Para cada meio, foram realizadas 10 repetições (frascos com 5 mg de esporos/30 mL de meio), respectivamente com e sem 1% de carvão ativado. A germinação dos esporos e o desenvolvimento gametofítico (estádios laminar e cordiforme) foram quantificados aos 30, 60 e 90 dias de cultivo. Cyathea corcovadensis e C. phalerata germinaram e desenvolveram gametófitos em todos os meios. Para ambas as espécies, as maiores porcentagens de germinação e de gametófitos cordiformes (estádio de desenvolvimento mais avançado) foram registradas no meio Meyer sem carvão ativado, que se caracteriza por maiores concentrações de macronutrientes e ausência de micronutrientes em comparação com os demais meios avaliados. Recomendamos cultivar as plantas em meio Meyer para maior desenvolvimento gametofítico e subsequente obtenção de esporófitos, como uma ferramenta biotecnológica para a conservação de C. corcovadensis e C. phalerata e restauração e enriquecimento ambiental usando estas samambaias arborescentes. Brasil Sul avaliados espécies semisólidos semi sólidos semi-sólidos Dyer 50 25 1 frascos esporos30 30 esporos/3 , meio) estádios cordiforme 6 9 cultivo C estádio avançado esporófitos 2 esporos3 3 esporos/

Abstract In vitro spore culture and gametophyte characteristics may contribute understanding the life cycle in different fern taxa and also to systematic and taxonomic studies. The family Woodsiaceae contains two genera: Physematium and Woodsia. Physematium montevidensis is the only species recognized for South America, and up to now, no information has been recorded on the gametophytic phase. The aim of this work is to study the development pattern, the morphology and sexual expression of the gametophyte of Physematium montevidensis to provide information about its reproductive biology. The germination pattern was of the Vittaria-type and the gametophyte development of the Aspidium-type. Gametangia appeared 30-40 days after spore germination. Both, cordiform bisexual and spatulate-elongated male gametophytes developed in the cultures. The sporophytes emerged near 3 months after spore sowing. The gametophyte development of Physematium montevidensis and its morphological features are broadly similar to those registered in other taxa of Woodsiaceae. studies genera Woodsia America now phase biology Vittariatype Vittaria type Aspidiumtype. Aspidiumtype Aspidium type. Aspidium-type 3040 30 40 30-4 Both spatulateelongated spatulate elongated cultures sowing 304 4 30-

Resumo O cultivo de esporos in vitro e as características dos gametófitos podem contribuir para a compreensão do ciclo de vida em diferentes táxons de samambaias e também para estudos sistemáticos e taxonômicos. Woodsiaceae contém dois gêneros, Physematium e Woodsia. Physematium montevidensis é a única espécie registrada para a América do Sul. Até o momento, a fase gametofítica desta espécie é desconhecida.. O objetivo deste trabalho é estudar o padrão de desenvolvimento, a morfologia e a expressão sexual do gametófito de Physematium montevidensis para fornecer informações sobre sua biologia reprodutiva. O padrão de germinação foi do tipo-Vittaria e o desenvolvimento do gametófito do tipo- Aspidium. Os gametângios surgiram 30-40 dias após a germinação dos esporos. Ambos, gametófito bisexual-cordiforme e masculino espatulado-alongado se desenvolveram nas culturas. Os esporófitos emergiram em aproximadamente três meses após a semeadura dos esporos. O desenvolvimento do gametófito de Physematium montevidensis e suas características morfológicas são amplamente semelhantes aos registrados em outros táxons de Woodsiaceae. taxonômicos gêneros Woodsia Sul momento desconhecida desconhecida. reprodutiva tipoVittaria tipo Vittaria Aspidium 3040 30 40 30-4 Ambos bisexualcordiforme bisexual cordiforme espatuladoalongado espatulado alongado culturas 304 3 4 30-

Abstract Pteris congesta an endemic and endangered species was investigated to identify morphological differences between its sterile and fertile leaves. Our findings have unveiled, leaf dimorphism in both morphology and growth patterns between fertile and sterile leaves. The sterile leaves petioles exhibited equivalent growth to the laminae, achieving complete leaf expansion within a month. In contrast, the petioles of fertile leaves elongated more than twice the size of the leaf blades that remain closed for the initial 30 days. Spore germination was carried out in both sterile and non-sterile soils, and juvenile sporophytes were cultivated to allow for morphological comparisons with those collected from the wild. One month after sowing the spores germinated in both soils resulting in a uniseriate germ filament Vittaria-type, and differentiated into laminar gametophytes, forming a Ceratopteris-type prothallus. The first leaf of the sporophyte emerged two months after sowing, and its development was completed one month later. Based on our studies, we propose that seedlings cultivated from spores germinated in soil can be used for the reintroduction of endangered fern species. unveiled laminae contrast 3 days nonsterile non wild Vittariatype, Vittariatype Vittaria type, type Vittaria-type gametophytes Ceratopteristype Ceratopteris prothallus later studies

Resumo Pteris congesta, uma espécie endêmica e ameaçada de extinção, foi investigada para identificar diferenças morfológicas entre suas folhas estéreis e férteis. Revelamos que há dimorfismo foliar tanto morfológico como também nos padrões de crescimento entre as folhas férteis e estéreis. Os pecíolos das folhas estéreis crescem de forma equivalente às lâminas e alcançam a expansão foliar completa em um mês. Em contraste, as folhas férteis alongam mais que o dobro do tamanho das lâminas foliares e permanecem fechadas nos primeiros 30 dias. A germinação de esporos foi realizada em solos estéreis e não estéreis, e esporófitos juvenis foram cultivados para permitir comparações morfológicas com aqueles coletados na natureza. Um mês após a semeadura, os esporos germinaram em ambos os solos resultando em filamentos germinativos unisseriados do tipo Vittaria que se diferenciaram em gametófitos laminares, formando um prótalo do tipo Ceratopteris. A primeira folha do esporófito emergiu dois meses após a semeadura, e seu desenvolvimento foi concluído um mês depois. Com base em nossos estudos, propomos que mudas cultivadas a partir de esporos germinados no solo possam ser utilizadas para a reintrodução de espécies de samambaias ameaçadas de extinção. congesta extinção contraste 3 dias natureza semeadura laminares Ceratopteris depois estudos

Resumen El desarrollo de los prótalos y de los esporófitos jóvenes de Asplenium blepharophorum Bertol. se describen y se ilustran. Los ejemplares fueron recolectados en un bosque mesófilo de montaña en San Antonio, 1 km al norte de Amecameca, Municipio de Amecameca, Estado de México. Las esporas fueron sembradas en frascos de vidrio de 125 ml con cerámica (barro molido), piedra de río, tela de mosquitero y tres soportes de cultivo: maquique, musgo y tierra de hoja, todo previamente esterilizado y cerrado con plástico y alambre de cobre. El ciclo fue isospórico con germinación tipo Vittaria y desarrollo del prótalo tipo Aspidium. Los gametófitos fueron unisexuados masculinos y femeninos; en maquique se formaron solo femeninos. Se observó dimorfismo sexual, los prótalos masculinos cordados con cauda y los femeninos cordados con gran cantidad de rizoides y cojinete, los gametangios se apreciaron inmaduros. Los esporófítos apogámicos se obtuvieron en maquique a partir de los 351 días, en musgo a los 356 y en tierra de hoja a los 473 días. Se discute la posibilidad de que sea una entidad híbrida o poliploide debido a que se desarrollaron esporófitos apogámicos en los tres sustratos y la mayoría de los esporangios tenían 32 esporas.

Abstract Prothallus development and the young sporophytes of Asplenium blepharophorum Bertol., are described and illustrated. The studied material was collected in a cloud forest in San Antonio, 1 km north of Amecameca municipality, State of México. The spores were sown in a glass vessel of 125 ml prepared in the following manner: the glasses were layered with ceramic, river stone, mosquito net and the three natural substrates: maquique, moss and plant litter, all previously sterilized and enclosed in a plastic bag tied with copper wire. The cycle was isosporeus with Vittaria type germination and prothallial development corresponding to the Aspidium type. The gametophytes were male and female; in maquique, only gametophyte female. The prothallus with sexual dimorphism, the male chordate with cauda and the female chordate with numerous rhizoids and pads, the gametangia did not mature. Young sporophytes were apogamous obtained around 351 days after sowing in maquique, 356 in moss and the plant litter 473. The possibility of it being a hybrid or polyploid entity is discussed because apogamic sporophytes developed in the three substrates and most of the sporangia had 32 spores.

Abstract Background: Fouquieriaceae is a small family that consists of a single genus, Fouquieria, with eleven species occurring mostly in arid and semiarid regions of Mexico and the southwestern United States. The development of the ovule and the female gametophyte has been poorly characterized in species of the family; thus, it is proposed to carry out a detailed study on the development of the ovule, megasporogenesis and megagametogenesis in Fouquieria fasciculata. Questions: How are the developmental patterns of the ovule and the female gametophyte in Fouquieria fasciculata? Study species: Fouquieria fasciculata (Willd. ex Roem. & Schult.) Nash (Fouquieriaceae). Methods: Embryology, morphology and histochemical tests of floral buds and flowers at pre-anthesis of Fouquieria fasciculata were examined using light microscopy and scanning electron microscopy. Results: The ovules of Fouquieria fasciculata are anatropous, bitegmic and tenuinucellate, and the micropyle is formed by the inner integument. The innermost layer cells of the inner integument form an integumentary tapetum or endothelium. The cells of integumentary tapetum are characterized by very conspicuous wall thickenings on the inner tangential side, and are radially elongated surrounding the nucellus. The pattern of development of the female gametophyte is monosporic Polygonum-type, and the formation of a lateral haustorium was observed. Conclusions: This investigation describes in detail the development of the ovule and the female gametophyte of Fouquieria fasciculata. Our results show interesting features, such as the differentiation of an integumentary tapetum, a monosporic Polygonum-type pattern of development and the formation of lateral haustorial arm in the female gametophyte.

Resumen Antecedentes: Fouquieriaceae consiste únicamente del género Fouquieria, con once especies que se distribuyen principalmente en las regiones áridas y semiáridas de México y el suroeste de los Estados Unidos. El desarrollo del óvulo y del gametofito femenino ha sido pobremente caracterizado en especies de la familia. Por lo tanto, investigaciones detalladas sobre la embriología, morfología e histoquímica contribuirán al conocimiento de la biología reproductiva de la familia Fouquieriaceae. Preguntas: ¿Cómo es el patrón de desarrollo del óvulo y del gametófito femenino en Fouquieria fasciculata? Especies de estudio: Fouquieria fasciculata (Willd. ex Roem. & Schult.) Nash (Fouquieriaceae). Métodos: Se investigó la morfología y la embriología de botones florales y de flores en preantesis de Fouquieria fasciculata utilizando microscopía de luz y electrónica de barrido. Resultados: Los óvulos de Fouquieria fasciculata son anátropos, bitégmicos y tenuinucelados, el tegumento interno forma el micrópilo. La capa más interna del tegumento interno se diferencia en un tapete tegumentario o endotelio. Las células del tapete tegumentario presentan evidentes engrosamientos en las paredes internas tangenciales y están radialmente alargadas rodeando la nucela. El patrón de desarrollo del gametofito femenino es monospórico de tipo Polygonum y se forma un haustorio lateral. Conclusiones: Esta investigación describe detalladamente el desarrollo del óvulo y del gametofito femenino en Fouquieria fasciculata. Los resultados muestran características relevantes, como la diferenciación de un tapete tegumentario a partir del tegumento interno y el desarrollo de un haustorio lateral en el gametofito femenino.

Resumo Lessingianthus plantaginoides (Vernonieae, Asteraceae) é um pequeno arbusto com tetraploidia natural que habita as formações rochosas da América do Sul. A população estudada ocorre e se estende por uma região de uma reserva privada com elevada biodiversidade local e endemismos de animais e vegetais. Com o intuito de fornecer conhecimentos sobre a citologia desta espécie tetraploide, realizamos um estudo ontogenético dos gametófitos masculinos e femininos de L. plantaginoides; fizemos uma análise meiótica detalhada e avaliamos a fertilidade desta espécie; documentamos e demos destaque às implicações taxonômicas dos seus aspectos reprodutivos. Lessingianthus plantaginoides apresentou os seguintes traços dos gametófitos masculinos e femininos: desenvolvimento da parede da antera é do tipo dicotiledôneo, tétrades tetraédricas, grãos de pólen maduros tricelular; desenvolvimento do megásporo calazal, saco embrionário monospórico e desenvolvimento do megagametófito tipo Polygonum. O comportamento meiótico é regular, os esporos são tétrades de igual tamanho e os grãos de pólen são altamente tingíveis. Lessingianthus plantaginoides é um autotetraplóide com alta diplodização que se reproduz sexualmente e tem uma elevada regularidade meiótica; o que é aparentemente responsável pelo seu potencial de colonização. Ao que tudo indica a especiação poliplóide desempenha um papel significativo no estabelecimento e diversificação do gênero.

Abstract Lessingianthus plantaginoides (Vernonieae, Asteraceae) is a small natural tetraploid shrub that inhabits rocky highlands from South America. The population studied inhabits and covers an extensive region of a private reserve with high local biodiversity and animal and plant endemisms. With the purpose of providing insights into the cyto-embryology of this tetraploid species, the aims of this study were: to perform an ontogenetic study of the male and female gametophytes of L. plantaginoides; to carry out detailed meiotic analysis and evaluate the fertility of this species; to document and provide highlights on taxonomic implications of their reproductive aspects. Lessingianthus plantaginoides presented the following male and female gametophyte traits: dicotyledonous type of anther wall development, tetrahedral tetrads, 3-celled mature pollen grains; development of the chalazal megaspore, monosporic embryo sac and Polygonum type of megagametophyte development. The meiotic behavior was regular, the spores were tetrads of equal size and the pollen grains were highly stainable. Lessingianthus plantaginoides is a highly diplodized autotetraploid that reproduces sexually and has high meiotic regularity; which is apparently responsible for its colonization potential. It now seems certain that polyploid speciation plays a significant role in the establishment and diversification of the genus.

Resumo O uso de técnicas de micropropagação é crucial para a conservação de espécies de musgos ameaçadas e para o reestabelecimento da natureza. Este estudo teve como objetivo estabelecer culturas in vitro de fragmentos de gametófitos de dez espécies de musgos do Cerrado. Após a desinfestação com álcool e solução de hipoclorito de sódio, explantes de musgo foram cultivados em meio Knop. As espécies Bryum argenteum, B. coronatum, Isopterygium tenerifolium, Leucobryum crispum, Pogonatum pensilvanicum e Vitalia cuspidifera foram sucessivamente estabelecidas com taxa de eficiência variando de 1 a 31,2%. No entanto, não foram obtidas culturas assépticas para as espécies Barbula indica, Bryum densifolium, Fissidens flaccidus e Sphagnum platyphylloides. Contudo, alguns explantes contaminados apresentaram desenvolvimento. Assim, todas as dez espécies foram submetidas as técnicas de resgate para estabelecer culturas em condições assépticas, a partir de explantes parcialmente contaminados (estabelecimento indireto). Consequentemente, o estabelecimento indireto resultou no aumento das chances de estabelecimento de culturas in vitro para a maioria das espécies. Portanto, o estabelecimento indireto como nova metodologia de cultivo in vitro foi bastante viável para a propagação das espécies de musgos listadas nesta pesquisa. Este fato é especialmente importante para fins de conservação, principalmente para espécies cujo material é sensível ou escasso.

Abstract The use of micropropagation techniques is crucial for the conservation of endangered moss species and their reestablishment in nature. This study aimed to establish in vitro cultures of gametophyte fragments of ten species of Cerrado mosses. After disinfestation with alcohol and commercial bleach, moss explants were grown in Petri dishes containing Knop medium. The species Bryum argenteum, B. coronatum, Isopterygium tenerifolium, Leucobryum crispum, Pogonatum pensilvanicum, and Vitalia cuspidifera were successively established with efficiency rate ranging from 1 to 31.2%. However, no aseptic cultures were obtained for the species Barbula indica, Bryum densifolium, Fissidens flaccidus, and Sphagnum platyphylloides. Even though, a few contaminated explants of these species were able to develop and grow. Thus, all ten species were submitted to rescue techniques to establish cultures in aseptic conditions, from partially contaminated explants (indirect establishment). Consequently, the indirect establishment resulted in higher percentages of explant development, which enhanced the establishment of in vitro cultures for most of the species tested. This fact is especially important for conservation purposes, mainly for species whose material is sensitive or scarce. Therefore, indirect establishment as a new in vitro culture methodology was a viable form of propagating the bryophyte species listed in this research. This fact is essential for conservation purpose, especially for species whose material is sensitive or scarcer.

Abstract Background: Although there have been studies on the sexual phase of some species of Thelypteridaceae, none have included germination types and prothallial development. Question: Is there variation in the type of germination and prothallial development in the gametophytes of four species in the family Thelypteridaceae? Study species: Four species of the family Thelypteridaceae: Meniscium falcatum, Goniopteris mollis, Goniopteris liebmannii, and Goniopteris minor of the states of Chiapas, Oaxaca, and Tabasco. Methods: Spores were collected, sown in Petri dishes with Thompson culture medium, stored in transparent plastic bags, and placed in a plant growth chamber. The various stages of gametophyte development were fixed in FAA with 0.8 % sucrose, dehydrated in gradual alcohols, critical point dried, mounted on stubs, and coated with gold. The photomicrographs were obtained using a scanning electron microscope (SEM) Model Jeol JSM5310-LV. Results: The spores are monolete and perinate. Germination is of the Vittaria type, and prothallial development of the Aspidium type. Adult gametophytes are cordiform-spatulate, with numerous glandular, simple and single-celled hairs, antheridia and archegonia are of the leptosporangiate type. The first sporophyte leaf is bi or trilobate, with free dichotomous venation, with capitate-glandular hairs, simple, unicellular and acicular hairs, and hypostomatic polycytic stomata. Conclusions: The differences observed among the studied species are: spore size, perispore ornamentation, germination and emergence timing of the rhizoids, type and size of hairs, and development time of gametangia and sporophytes.

Resumen Antecedentes: Existen trabajos sobre la fase sexual para algunas especies de Thelypteridaceae, pero no hacen mención sobre tipos de germinación y desarrollo protálico. Pregunta: ¿Existe variación en el tipo de germinación y desarrollo protálico en los gametofitos de cuatro especies de la familia Thelypteridaceae? Especies de estudio: Cuatro especies de la familia Thelypteridaceae: Meniscium falcatum, Goniopteris mollis, Goniopteris liebmannii y Goniopteris minor, de los estados de Chiapas, Oaxaca y Tabasco. Métodos: Se recolectaron esporas, se sembraron en cajas Petri con medio de cultivo Thompson, guardándose en bolsas de plástico transparentes y se colocaron en una cámara de crecimiento de plantas. Las fases de desarrollo de los gametofitos se fijaron en FAA con sacarosa al 0.8 %, se deshidrataron en alcoholes graduales, se desecaron en punto crítico, se montaron en porta-muestras y se recubrieron con oro. Las microfotografías se obtuvieron usando un microscopio electrónico de barrido (MEB) Modelo Jeol JSM5310-LV. Resultados: Las esporas son monoletes y perinadas. La germinación es de tipo Vittaria, y el desarrollo protálico de tipo Aspidium. Los gametofitos adultos son cordiforme-espatulados, con pelos-capitados glandulares, simples y unicelulares. Los anteridios y arquegonios son del tipo leptosporangiados. La primera hoja del esporofito tiene forma lobada, bi o trilobada, con venación dicotómica abierta, con pelos-capitados glandulares, simples unicelulares y aciculares y estomas polocíticos hipostomáticos. Conclusiones: Las diferencias observadas entre las especies estudiadas son: tamaño de las esporas, ornamentación del perisporio, períodos de germinación y emergencia del rizoide, tipo y tamaño de los pelos, tiempo de desarrollo de los gametangios y esporofitos.

Resumen Introducción: Pteridium aquilinum es un helecho heliófilo ampliamente distribuido en México. Es una especie pionera que usualmente se encuentra en hábitats alterados y tiene gran relevancia ecológica por sus propiedades alelopáticas, su resistencia al fuego y a condiciones de sequía. Objetivo: Realizar una descripción integral de la forma y ornamentación de las esporas de P. aquilinum, así como de la morfogénesis del gametofito, incluyendo el tipo de germinación y el desarrollo protálico (resaltando su morfología). Además, describir la anatomía del esporofito joven desarrollado en condiciones in vitro. Metodología: Se utilizó microscopía electrónica de barrido y técnica histológica de parafina para la descripción de las fases del gametofito y esporofito. Resultados: Las esporas son globulares y el trilete tiene ornamentación granulada, mientras que su germinación fue de tipo Vittaria y el desarrollo protálico correspondió al tipo Adiantum. Los anteridios se observaron 13 días después de la siembra, mientras que los arquegonios surgieron al día 17. La primera hoja del esporofito apareció entre los días 60 y 70. Al cuarto mes, la fase esporofítica desarrollada in vitro mostró la anatomía típica de la especie a esta edad, dado que no presentó vitrificación. El análisis histológico del rizoma, la base del peciolo, el raquis de primer orden y la lámina mostraron los sistemas de tres tejidos bien diferenciados. Las modificaciones anatómicas observadas in vitro, como una dictiostela monocíclica y sólo una banda de esclerénquima en el rizoma, podrían atribuirse a la edad de los individuos. Por otra parte, los estomas están presentes en la superficie adaxial de la lámina, que corresponde al tipo anomocítico. Aunque estos estomas se formaron en condiciones in vitro, es importante resaltar que son completamente funcionales. Conclusiones: Nuestro trabajo describe por primera vez la morfo-anatomía de las fases gametofítica y esporofítica del ciclo de vida de P. aquilinum en condiciones in vitro. Nuestros resultados posiblemente permitan la exploración con mayor profundidad de las propiedades biológicas, fisiológicas y ecológicas de la especie.

Abstract Introduction: Pteridium aquilinum is a heliophilous fern widely distributed in Mexico. It is a pioneer species usually found within disturbed habitats with high ecological relevance because of its allelopathic properties, its resistance to fire and drought conditions. Objective: Provide an integrative description regarding P. aquilinum spores shape and ornamentation, and also on gametophyte morphogenesis, including the type of germination and the prothallic development (highlighting its morphology). Furthermore, the anatomy of young sporophyte grown under in vitro conditions was described. Methods: We used scanning electron microscopy and histological paraffin technique to describe the gametophytic and sporophytic phases. Results: The evaluation of the spores showed the presence of globular morphology and triletes with granulated ornamentation; while their germination was Vittaria-type, the prothallic development corresponded to the Adiantum-type. The antheridia were observed 13 days following to the sowing, whereas archegonia arose at day 17. The first leaf of the sporophyte appeared between days 60 and 70. At fourth month, the in vitro grown sporophyte showed the typical anatomy of the individuals of the species at this age, since it did not exhibit vitrification. The histological analysis of rhizome, the petiole base, first order rachis and lamina showed the three tissue systems well differentiated. The anatomical modifications observed in vitro, such as a non-polycyclic dictyostele and just one band sclerenchyma in the rhizome, may be attributed to the individuals age. Moreover, the stomata are present on the adaxial surface of lamina, corresponding to the anomocytic type. Although these stomata were developed under in vitro conditions, it is important to highlight that they are completely functional. Conclusions: Our work describes for the first time the morpho-anatomy of both the gametophytic and the sporophytic phases in the life cycle of P. aquilinum under in vitro conditions. Our results indicate that this method will allow deeper exploration of biological, physiological and ecological properties of the species.

ABSTRACT Objective: to extract and determine the antiviral activity of compounds from Chondracanthus chamissoi and Chlorella peruviana against DENV-2 in Vero-76 cells. Materials and methods: carrageenan from Chondracanthus chamissoi and soluble carbohydrates from Chlorella peruviana were extracted, and toxicity tests in VERO-76 cells and antiviral activity were determined. Results: carrageenan from Chondracanthus chamissoi sporophyte and gametophyte phases were obtained, the compound was identified as k-carrageenan using infrared light. Nine sugars (ribose, xylose, arabinose, fructose, mannose, galactose, sucrose, maltose, and lactose) were identified using chromatography in the soluble carbohydrate mix from Chondracanthus chamissoi, and four sugars (glucose, sucrose, maltose, and lactose) from Chlorella peruviana were identified. Compounds from Chondracanthus chamissoi and Chlorella peruviana soluble carbohydrate solutions did not show any cytotoxic effect, carbohydrates from the raw extract from Chlorella peruviana did have this effect. All fractions from the raw extract from the gametophyte phase from Chondracanthus chamissoi were positive in the plate reduction test at 1:5 dilution. K-carrageenan from Chondracanthus chamissoi in both phases, as well as the crude extracts of soluble carbohydrates from Chondracanthus chamissoi and Chlorella peruviana as well as the soluble carbohydrate solution from Chlorella peruviana inhibited growth of dengue virus, but that was not the case with the carbohydrates of the raw extract from Chlorella peruviana. Conclusions: compounds obtained from Chondracanthus chamissoi and Chlorella peruviana show antiviral activity against DENV-2, so it is necessary to continue studies aiming to determine the antiviral potential of these fractioned and purified compounds.

RESUMEN Objetivo: extraer y evaluar la actividad antiviral de los compuestos de Chondracanthus chamissoi y Chlorella peruviana contra DENV-2 en células Vero-76. Materiales y métodos: se extrajeron el carragenano de Chondracanthus chamissoi, los carbohidratos solubles de Chondracanthus chamissoi y Chlorella peruviana y se realizó la prueba de toxicidad en células VERO-76 y la evaluación de la actividad antiviral. Resultados: se obtuvieron carragenanos de la fase de esporofito y gametofito de Chondracanthus chamissoi, los mismos que fueron identificados, mediante infrarrojo, como k-carragenano. Por cromatografía se identificaron nueve azúcares (ribosa, xilosa, arabinosa, fructuosa, manosa, galactosa, sucrosa, maltosa y lactosa) en la muestra de carbohidratos solubles de Chondracanthus chamissoi fase gametofito y cuatro azucares (glucosa, sucrosa, maltosa y lactosa) en la de Chlorella peruviana. Los compuestos de Chondracanthus chamissoi y la solución de carbohidratos solubles de Chlorella peruviana no presentaron efecto citotóxico; los carbohidratos del extracto crudo de Chlorella peruviana sí los tuvieron. Todas las fracciones del extracto crudo de Chondracanthus chamissoi fase gametofítica fueron positivas por la prueba de reducción del número de placas(50) a la dilución 1:5. El k-carragenano de Chondracanthus chamissoi en ambas fases y los extractos crudos de carbohidratos solubles de Chondracanthus chamissoi, Chlorella peruviana y la solución de carbohidratos solubles de Chlorella peruviana inhibieron el crecimiento del virus dengue, pero no los carbohidratos del extracto crudo de la Chlorella peruviana. Conclusiones: los compuestos obtenidos de Chondracanthus chamissoi y Chlorella peruviana presentan actividad antiviral contra DENV-2 por lo cual es necesario continuar los estudios del potencial antiviral de estos compuestos fraccionados y purificados.

Abstract The aim of this paper is to investigate the ultrastructural events that occur during pollen grains development, with emphasis in pollen grain wall and tapetum ontogeny in Ziziphus jujuba, Z. mucronata, Paliurus spina-christi (Paliureae) and Gouania ulmifolia (Gouanieae). Anthers at different developmental stages were processed according to classic techniques for transmission electron microscopy. Differences in the number of endothecium layers and in the number of tapetal cell nuclei were found. Tapetal cells present an anastomosing tubular network and large vesicles with fibrillar content in the cytoplasm. Pollen grain development and ontogeny of pollen grain wall are similar in the four species. The number of endothecium layers, the number of nuclei of the tapetal cells and tapetal cells ultrastructure of the four species support the phylogenetic relationships previously published for the Rhamnaceae family. Tapetal vesicles with fibrillar or polysaccharide content seem to be an exclusive characteristic of the tribes Paliureae and Gouanieae. Some ultrastructural characters of the pollen grain wall development are common to other species of Rhamnaceae, such as the primexine matrix present at the microspore mother cell stage, the aperture entirely built up during the tetrad stage, the thick and fibrillar intine, and the granular infractectum.

Resumen Efectos del hongo Pestalotiopsis maculans (Ascomycota: Amphisphaeriales) en el desarrollo gametofítico del helecho Lygodium venustum (Lygodiaceae). Lygodium Sw. es el único género de la familia Lygodiaceae, con alrededor de 25 a 40 especies que habitan en su mayoría en regiones tropicales del mundo. En Argentina se desarrollan dos especies; uno de ellas, L. venustum, crece en el noreste del país. Algunas especies de Lygodium son invasivas, mientras que para algunos taxones se han reportado usos medicinales. Como parte de un proyecto para indagar sobre la biología reproductiva de los helechos nativos en Argentina, se encontró un hongo durante el cultivo in vitro de esporas de L. venustum. Asimismo, se observaron manchas marrones oscuras y pústulas eruptivas en hojas fértiles. Así, surgieron como objetivos de este trabajo: 1) recuperar e identificar el hongo presente en el esporofito y en los cultivos de esporas, 2) investigar el impacto del hongo en la generación gametofítica y 3) realizar experimentos con esporas no tratadas para demostrar que el hongo está asociado a las esporas recolectadas de esporofitos infestados. Se aplicaron técnicas fitopatológicas para aislar el hongo y luego lograr cultivos monospóricos. Las características morfo-biométricas de los conidios correspondieron a las de Pestalotiopsis maculans (Corda) Nag Raj [= Pestalotiopsis guepinii (Desm.) Steyaert] (Amphisphaeriales, Ascomycota). La re-inoculación de conidios en gametofitos sanos condujo a su necrosis y muerte. Los porcentajes de germinación de esporas fueron inicialmente más altos en los cultivos no esterilizados, lo que sugiere que el protocolo de desinfección pudo haber afectado la germinación de las esporas. Este es el primer reporte de Pestalotiopsis maculans sobre los esporofitos de Lygodium venustum. La información que surge de nuestro trabajo podría ser útil para el control biológico de otras especies de Lygodium, consideradas malezas en otras regiones del mundo.

Abstract Lygodium Sw. is the only genus of the family Lygodiaceae, with around 25 to 40 species inhabiting mostly tropical regions worldwide. In Argentina two species develop; one of them, L. venustum, grows in the Northeast of the country. Some species of Lygodium are invasive whereas medicinal uses have been reported for some taxa. As part of a project to ascertain on the reproductive biology of native ferns in Argentina, a fungus was found during in vitro spore culture of L. venustum. Dark brown spots and eruptive pustules were also observed in fertile leaves. Thus, emerged as objectives of this work: 1) to retrieve and identify the fungus from the sporophyte and from the spores cultures, 2) to inquire on the impact of the fungus on gametophyte generation, and 3) to perform experiments with untreated spores to demonstrate that the fungus is associated to the spores collected from infested sporophytes. Phytopathological techniques were applied in order to isolate the fungus and then to achieve monosporic cultures. The morpho-biometric features of the conidia corresponded with those of Pestalotiopsis maculans (Corda) Nag Raj [= Pestalotiopsis guepinii (Desm.) Steyaert] (Amphisphaeriales, Ascomycota). Re-inoculation of conidia on healthy gametophytes conducted to their necrosis and death. The percentages of spore germination were initially higher in unsterilized cultures, suggesting that disinfection protocol may have affected spore germination. This is the first report of Pestalotiopsis maculans on the sporophytes of Lygodium venustum. The information that emerges from our work could be useful for the biocontrol of other species of Lygodium, considered weeds in other regions of the world.