Palynological analysis of Dennstaedtiaceae taxa from the Paranaense Phytogeografic Province that produce monolete spores and its systematic implications (I): Blotiella lindeniana, Histiopteris incisa and Paesia glandulosa

ABSTRACT The genera of Dennstaedtiaceae have sporophytes with very different morphological characteristics between each other, and this feature has made difficult the systematic circumscription of the family. This reason makes necessary the study of new characters that allow a better understanding of the relations within the group. The spore morphology and wall structure of Blotiella lindeniana, Histiopteris incisa and Paesia glandulosa from the Paranaense Phytogeographic Province were studied using light microscope, and scanning and transmission electron microscope. The exospore has two layers and, according to the species, the exospore surface bears pila, echinae, verrucae, bacula and tubercles. The perispore has two or three layers and its surface is psilate, baculate or rugulate. The variability found in the sculpture of the spores and their stratification and ultrastructure of perispore reflects the morphological differences observed in the sporophyte of the species studied. Additionally, while the stratification and ultrastructure of the exospore is shared by the Dennstaedtiaceae species, their ornamentation could be a character to distinguish species into the clade "hypolepidoide". The finding of spores with similar characteristics in phylogenetically unrelated families allows us to suggest that palynological features do not have an evolutionary value to establish relationships between groups above the genus level.

Esporogénesis, esporodermo y ornamentaciónde esporas maduras en Lycopodiaceae / Sporogenesis, sporoderm and mature spore ornamentation in Lycopodiaceae

Studies on reproductive aspects, spore morphology and ultrastructure of Lycopodiaceae are not very common in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. In order to complete existing information, adding new and broader contributions on these topics, a comparative analysis of the sporogenesis ultrastructure, with emphasis on cytological aspects of the sporocyte coat development, tapetum, monoplastidic and polyplastidic meiosis, sporoderm ontogeny and ornamentation of the mature spores, was carried out in 43 taxa of eight genera of the Lycopodiaceae: Austrolycopodium, Diphasium, Diphasiastrum, Huperzia (including Phlegmariurus), Lycopodium, Lycopodiella, Palhinhaea and Pseudolycopodiella growing in the Andes of Colombia and the Neotropics. For this study, the transmission electron microscopy (TEM) samples were collected in Cauca and Valle del Cauca Departments, while most of the spores for scanning electron microscopy (SEM) analysis were obtained from herbarium samples. We followed standard preparation procedures for spore observation by TEM and SEM. Results showed that the sporocyte coat is largely composed by primary wall components; the sporocyte develop much of their metabolic activity in the production of their coat, which is retained until the spores release; protective functions for the diploid cells undergoing meiosis is postulated here for this layer. The abundance of dictyosomes in the sporocyte cytoplasm was related to the formation and development of the sporocyte coat. Besides microtubule activity, the membrane of sporocyte folds, associated with electrodense material, and would early determine the final patterns of spore ornamentation. Monoplastidic condition is common in Lycopodium s.l., whereas polyplastidic condition was observed in species of Huperzia and Lycopodiella s. l.. In monoplastidic species, the tapetum presents abundant multivesicular bodies, while in polyplastidic species, the secretory activity of the tapetum is less intense. Sporoderm development is centripetal, exospore is the first formed layer, then the endospore and, if present, perispore is the final deposited layer. Adult spores of the Lycopodiaceae showed two patterns of ornamentation: negative or caviform (foveolate spores) and positive or muriform ornamentation, the latter with two subtypes (rugate and reticulate spores). The spores of Huperzia are characteristically foveolate, the rugate spores were found in a few species of Huperzia and in all of the Lycopodiella s. l. taxa studied, while Lycopodium s.l. spores bear reticulate ornamentation. Numerous ornamentation traits are diagnostic at the specific level. The types of ornamentation found do not support the recent extreme fragmentation of the family in several genera, but could match, a priori, with the idea of three subfamilies. The findings of sporogenesis, extremely similar in all taxa studied, point more to consider fewer genera, more comprehensive, than the recent, markedsplitting of the family. Rev. Biol. Trop. 62 (3): 1161-1195. Epub 2014 September 01.

Estudios sobre aspectos reproductivos, morfología y ultraestructura de las esporas de Lycopodiaceae no son abundantes en la literatura científica y constituyen información esencial para apoyar las relaciones taxonómicas y sistemáticas en el grupo. Con el fin de completar la información existente, añadiendo contribuciones nuevas y más amplias sobre estos temas, se realizó un análisis comparado de la ultraestructura de la esporogénesis, con énfasis en aspectos citológicos que tienen que ver con la formación de la cubierta de los esporocitos, el tapete, las meiosis monoplastidial y poliplastidial, la ontogenia del esporodermo y la ornamentación de las esporas maduras en 43 táxones de ocho géneros de Lycopodiaceae: Austrolycopodium, Diphasium, Diphasiastrum, Huperzia (incluyendo Phlegmariurus), Lycopodium, Lycopodiella, Palhinhaea y Pseudolycopodiella que crecen en los Andes de Colombia y el Neotrópico. Para estudios con microscopía electrónica de trasmisión (MET) las muestras se recolectaron en los departamentos de Cauca y Valle del Cauca, mientras que la mayoría de las muestras para microscopía electrónica de barrido (MEB) provienen de material herborizado de colecciones. Para la observación de las muestras con MET y MEB se utilizaron protocolos estándar para el procesamiento de esporas. La cubierta de los esporocitos está formada por pared primaria; los esporocitos invierten gran parte de su actividad metabólica en la producción de esa cubierta, que es mantenida hasta la liberación de las esporas y tiene funciones de protección de las células que harán meiosis. La abundancia de dictiosomas en los esporocitos se relacionó con la formación y desarrollo de la cubierta. Además de la actividad de los microtúbulos, la presencia de sinuosidades y plegamientos asociados con material electro denso en la membrana de los esporocitos determinarían tempranamente los patrones de ornamentación de las esporas. La condición monoplastidial es común en Lycopodium s.l.y la poliplastidial se observó en Huperzia y Lycopodiella s. l. En especies monoplastidiales el tapete presenta abundantes cuerpos plurivesiculares, en las poliplastidiales la actividad secretora del tapete es menos intensa. El desarrollo del esporodermo es centrípeto, el exosporio se forma primero, seguido del endosporio y el perisporio, si está presente, se deposita de último. En las esporas adultas de Lycopodiaceae se encontraron dos patrones de ornamentación: negativo o caviforme (esporas foveoladas) y positivo o muriforme (esporas rugadas y reticuladas). Las esporas foveoladas son características de Huperzia; las rugadas de unas pocas especies de Huperzia y las especies de Lycopodiella s. l., mientras que las reticulada son típicas de Lycopodium s. l.. Numerosos caracteres de la ornamentación resultan diagnósticos en el nivel específico. Los tipos principales no apoyan la extrema fragmentación reciente de la familia en varios géneros, aunque podría coincidir, a priori, con la idea de tres subfamilias. Los hallazgos de la esporogénesis, extremadamente similar en todos los táxones estudiados, apuntan más a la unificación de los géneros en la familia que a su segregación.

Esporogénesis y esporas de Equisetum bogotense (Equisetaceae) de las áreas montañosas de Colombia / Sporogenesis and spores of Equisetum bogotense (Equisetaceae) from mountain areas of Colombia

Studies on some reproductive traits in Equisetum species are scarce and valuable to understand species distribution. Therefore, a detailed study of the sporogenesis process and spore development in E. bogotense is presented, with an analysis of the main events during meiosis, maturation of spores, spore wall ultrastructure, orbicules and elaters. Specimens were collected from 500 to 4 500m in Cauca, Colombia. Strobili at different maturation stages were fixed, dehydrated, embedded in resin, and ultra-microtome obtained sections were stained with Toluidine blue. Observations were made with optical microscopy with differential interference contrast illumination technique (DIC), transmission and scanning electron microscopy (TEM and SEM). Ultrathin sections (70-80μm) for TEM observations were stained with uranyl acetate and lead citrate; while samples for SEM observations, were fixed, dehydrated in 2.2-dimethoxypropane and dried at critical point as in standard methods. Strobili have numerous mature sporangiophores, each one with a peltate structure, the scutellum, bearing five-six sessile sporangia attached to the axis of strobilus by the manubrium. Immature sporocytes (spore mother cells) are tightly packed within the young sporangia. The sporocytes quickly undergo meiosis, by passing the stage of archesporium and give origin to tetrads of spores. The tapetum loses histological integrity during early stages of sporogenesis, intrudes as a plasmodial mass into the cavity of the sporangium, partially surrounding premeiotic sporocytes, and then, tetrads and adult spores. The tapetum disintegrates towards the end of the sporogenesis, leaving spores free within the sporangial cavity. Spores present several cytological changes that allow them to achieve greater size and increase the number of plastids, before reaching the adult stage. Sporoderm includes three layers external to the cytoplasmic membrane of the spore cell, and they are pseudoendospore, exospore and perispore. Viewed with SEM, the exospore is smooth to rugulate, with micro perforations, while the perispore is muriform, rugate, with narrow, delicate, discontinuous, randomly distributed folds delimiting incomplete, irregular areolae, externally covered by of different size, densely distributed orbicules. These orbicules are also found all over the external face and margins of the elaters, while the internal face is smooth and lack orbicules. Viewed with TEM, the exospore is a thick layer of fine granular material, while perispore is a thinner layer of dense, separate orbicules. The elaters are composed by two layers of fibrillar material: an inner layer with longitudinally oriented fibrils and an outer, thicker and less dense layer with fibrils transversely fibrils and abundant, external orbicules. It is suggested that the processes of ontogeny and characters of the sporoderm are relatively constant in Equisetum; however, sporogenesis in E. bogotense is synchronous and this condition has been observed so far only in E. giganteum, a tropical genus also found in Colombia.

Los estudios sobre aspectos reproductivos son escasos en Equisetum. Por eso, hemos realizado un análisis detallado del proceso de esporogénesis, desarrollo de las esporas, ultraestructura de procesos que tienen lugar durante la meiosis, formación de la pared esporal, orbículas y eláteres de E. bogotense, en especímenes procedentes del Cauca, Colombia. Los estudios se efectuaron mediante microscopía fotónica, electrónica de transmisión (TEM) y de barrido (SEM). Los estróbilos llevan numerosos esporangióforos maduros, cada uno con un escutelo peltado, unido al eje del estróbilo por el manubrio y portador de 5-6 esporangios sésiles. Los esporocitos experimentan meiosis dando origen a tétradas de esporas. El tapete pierde la integridad histológica en las primeras etapas de esporogénesis y rodea los esporocitos premeióticos, posteriormente a las tétradas y finalmente las esporas inmaduras, que experimentan cambios citológicos y de tamaño antes de alcanzar la etapa adulta. El esporodermo de las esporas adultas de E. bogotense consiste de seudoendosporio, exosporio y perisporio. Vistos con MEB, el exosporio de las esporas adultas es liso a rugulado con microperforaciones y el perisporio es muriforme, rugado, con pliegues delicados, estrechos, discontinuos, que se distribuyen al azar y delimitan aréolas incompletas. Externamente el perisporio está cubierto por orbículas, que se forman también en la cara externa y los márgenes de los eláteres. Vistos con TEM, el exosporio es una capa de material granular fino y el perisporio, una capa mucho más delgada con orbículas discretas. Los eláteres están formados por dos capas de naturaleza fibrilar, orientadas longitudinalmente y transversalmente. La esporogénesis en E. bogotense es sincrónica, similar a la de E. giganteum, otra especie de distribución tropical que también crece en Colombia.

Morfología de esporas y sinangios en especies neotropicales del helecho Marattia (Marattiaceae)

Spores morphology and synangia in neotropical fern species of Marattia (Marattiaceae). The Marattiaceae are represented by a small family of four to six genera that bear esporogenous structures of two types: sorus with free eusporangia in Angiopteris and Archangiopteris, and indurated synangium in Christensenia, Danaea and Marattia. Marattia is a pantropical genus of about eight to ten species in the paleotropic and seven to eight species in the neotropic. In order to describe the spores and sinangia morphology, this study analyzed the shape of the receptacles, and the position of the synangia, and evaluated the spores with SEM, of seven neotropical species of the genus Marattia: M. alata, M. cicutifolia, M. excavata, M. interposita, M. laevis, M. laxa y M. weinmanniifolia from several collections. The receptacles were fully developed in M. cicutifolia and M. laevis, and scarcely overelevated in the rest of the species. The synangium was ellipsoidal and had intramarginal to supramedial position in the laminae. The spores of Marattia were elliptic. Among the taxa, only monolete spores were found, with no trilete, aborted or deformed spores. The laesura was linear and reached about two of the total length of the spore. The perispore appears as a continuous thin layer deposited on the exospore according to its ornamentation in M. cicutifolia and M. laevis. It is smooth in M. alata, rugate in M. excavata and pustulate-rugate in two species: M. interposita and M. laxa. The exospore is echinate in M. cicutifolia and M. laevis and pustulate in the other species. In M. weinmannifolia spores produced by the same sinangium may have different ornamentation types. We concluded that, while the presence of ellipsoidal and superficial synangia and monolete spores aperture were generic traits, the micro and macro-ornamentation types of the perispores and exospores vary at specific level. Besides, macro-ornamentation can be bulliform (pustulate), a combination of bulliform and muriform types (pustulate-rugate), muriform (rugate-retate) and stelliform (echinate); finally, granular micro-ornamentation can be seen frecuently in perispores. Rev. Biol. Trop. 59 (4): 1833-1844. Epub 2011 December 01.

La familia Marattiaceae, que incluye de cuatro a seis géneros, presenta estructuras esporógenas de dos tipos: sinangios en Christensenia, Danaea y Marattia; y soros con eusporangios libres en Angiopteris y Archangiopteris. Marattia es un género pantropical con unas ocho-diez especies en el paleotrópico y siete-ocho en el neotrópico. Mediante MEB se estudiaron las esporas de las siete especies neotropicales de Marattia: M. alata, M. cicutifolia, M.excavata, M. interposita, M. laevis, M. laxa y M. weinmanniifolia, basado en la forma de los receptáculos y la posición de los sinangios en material proveniente de distintas colecciones. Los receptáculos son hiperdesarrollados en M. cicutifolia y M. laevis, y apenas sobreelevados en el resto de las especies. Los sinangios son elipsoidales y la posición en la lámina es intramarginal a medial. Las esporas son elípticas, siempre monoletes y no se encuentran esporas triletes, abortadas o deformadas. La lesura tiene una longitud aproximadamente igual a la mitad del diámetro de la espora. El perisporio aparece como una capa delgada continua que se deposita siguiendo los procesos del exosporio, en M. cicutifolia y M. laevis. Es liso en M. alata, rugado en M. excavata y pustulado-rugado en dos especies: M. interposita y M. laxa. El exosporio es equinado en M. cicutifolia y M. laevis, y pustulado en las otras especies. En M. weinmanniifolia, las esporas producidas por el mismo sinangio tienen diferentes tipos de ornamentación. La presencia de sinangios elipsoidales superficiales y de esporas monoletes, son rasgos genéricos; mientras que los tipos de micro-ornamentación y macroornamentación en exosporios y perisporios son caracteres diagnósticos específicos. La macro-ornamentación puede ser buliforme (pustulada), una combinación de buliforme y muriforme (pustulada-rugada), muriforme (rugada-retiada) y esteliforme (equinada); es frecuente la presencia de micro-ornamentación granular en el perisporio.

Morphology and ultrastructure of megaspores and microspores of Isoetes sehnemii Fuchs (Lycophyta)

The morphology and wall ultrastructure of megaspores and microspores of Isoetes sehnemii that grows in Brazil were analyzed as part of the study of the Isoetaceae present in Southern South America. The observations were performed with light, scanning and transmission electron microscopes. The megaspores are trilete, 350-450μm in equatorial diameter. The surface is reticulate. In section, the sporoderm is 100μm thick including the ornamentation. The wall is composed of a siliceous perispore, which consists of short fused flatten, elements forming a three-dimensional mesh. The exospore has two zones of different structure. The endospore is fibrillar. The microspores are monolete, 21-27μm in equatorial diameter. The sporoderm is composed of a sporopollinic rugulate perispore. A space between the paraexospore and the exospore is evident. The exospore is compact. The endospore is fibrillar. The ultrastructural analysis akes hoologies evident concerning structure and organization of the layers belo the perispore in both spore types. A possible similarity and stability in the ultrustructure of the present spores and fossils could be also inferred. In addition, there would be a correlation among the plant habitat, the spore ornamentation and the geographic distribution.

A morfologia e a ultraestrutura da parede de megasporos e microspores de Isoetes sehnemii que crescem no Brasil foram analisados como parte do estudo de Isoetaceae presente no sul da América do Sul. As observações foram realizadas com microscopias de luz e eletrônicas de transmissão e varredura. Os megasporos são triletes com 350-450μm de diâmetro equatorial. A superfície é reticulada. Em secção o esporoderma possui 100μm de espessura incluindo ornamentação. A parede é composta de um perisporo silicoso que consiste de elementos fusionados curtos e achatados formando uma rede tridimensional. O exosporo tem duas zonas com diferentes estruturas. O endosporo é fibrilar. Os microsporos são monoletes, 21-27μm de diâmetro equatorial. A esporoderme é composta por um perisporo esporopolínico rugulado. Um espaço entre o para-exosporo e o exosporo é evidente. O exosporo é compacto. O endosporo é fibrilar. A análise ultraestrutural torna evidente homologias relativas a estrutura e organização das camadas abaixo do perisporo em ambos os tipos de esporos. Uma possível similaridade e estabilidade na ultraestrutura do presente esporo e fósseis pode também ser inferida. Além disso, haveria uma correlação entre o habitat da planta, a ornamentação do esporo e a distribuição geográfica.

Palynological analysis of Sphaeropteris gardneri (Cyatheaceae, Pteridophyta)

The spore morphology and wall ultrastructure of Sphaeropteris gardneri (Hook.) R.M. Tryon from Brazil were analyzed with LM, SEM and TEM. The spores are trilete with an ornamentation formed of short low ridges with spines in their margins. The exospore is 2.5μm thick, two- layered in section and single or branched channels are present. The perispore is 1.2μm thick and two-layered. The inner layer has three strata: the inner stratum is formed of a network of branched and fused threads, the middle stratum has threads with a radial orientation and in the outer stratum thin, dark fibres are immersed in a less dense contrasted matrix. The outer layer of the perispore is the one that forms the echinate-ridges and is constituted of threads arranged in a compact way. Globules of different sizes are observed on the surface. The differences found in the perispore ornamentation and ultrastructure in Alsophila, which was previously studied, and those of Sphaeropteris, show a tendency to wall complexity.

A morfologia dos esporos e a ultraestrutura da parede de Sphaeropteris gardneri (Hook.) R.M. Tryon, Brasil, foram analisadas com MO, MEV e MET. Os esporos são trilete com uma ornamentação formada por cristas curtas e baixas e com espinhos em suas margens. O exosporo possui 2,5μm de espessura, duas camadas em secção e estão presentes canais simples ou ramificados. A camada interna possui três estratos: o estrato interno é formado por uma rede de filamentos ramificados e fusionados, o estrato médio tem fios com uma orientação radial e no estrato externo fino, fibras escuras estão imersas em uma matrix menos densa. A outra camada do perisporo é a que forma as cristas equinatas e é constituída de filamentos dispostos em um arranjo compacto. Glóbulos de diferentes tamanhos são observados na superfície. As diferenças encontradas na ornamentação do perisporo e na ultraestrutura do Alsophila estudado previamente e aqueles de Sphaeropteris mostram uma tendência à complexidade da parede.

Gametófitos y esporófitos jóvenes de cuatro especies de helechos del género Pteris (Pteridaceae) naturalizadas en América

Gametophytes and young sporophytes of four species of the fern genus Pteris (Pteridaceae) naturalized in the American continent. The pantropical fern genus Pteris L. has about 250 species of which 60 occur in the American continent. We studied the morphogenesis of the gametophyte, and the morphology of the young sporophytes of four species: P. cretica, P. ensiformis, P. multifida and P.vittata, together with a palynological analysis that includes the ability of spores to germinate. Gametophytes were obtained trough in vitro culture techniques with agar-gellified Knudson medium. Young sporophytes were placed in earth-sand (3:1) sterile substrate. We used light and SEM microscopy. Triletes spores predominate, but monolete, tetralete, and other types of apertura are often found. The viability of spores is not affected by the variation, so the term spore polymorphism is applied to the condition occurring among these species. Spore polymorphism is similar in P. cretica and P. multifida. Germination occurs following the Vittaria type, 3-7 days after the sowing. Filamentous, 3-5 celled gametophytes were found in P. cretica, P. multifida and P. vittata, and 7-9 celled in P. ensiformis. Development of gametophytes takes place following Adiantum type and eratopteris type. The symmetry of the laminae differ in gametophytes, those of P. ensiformis and P. multifida are similar and differ from the other two species, P. cretica and P. vittata. Gametophytes of P. ensiformis, P. multifida and P. vittata are bisexual and protandric, while male gametophytes were found in P. cretica. Antheridia correspond to the common leptosporangiate type; they are cylindric in P. vittata and ovoid in the other three species. Archegonia necks have 4 rows of 4 cells each. The sporophytes complete their development 3 months after sowing, and have indument close to the adult plants. P. cretica shows obligated apogamy. Rev. Biol. Trop. 58 (1): 89-102. Epub 2010 March 01.

El género pantropical Pteris L. tiene 250 especies de la cuales 60 están en el continente Americano. Se estudió la morfogénesis de los gametófitos, y la morfología de los esporófitos jóvenes de cuatro especies: P. cretica, P. ensiformis, P. multifida y P.vittata, junto con un análisis palinológico que incluye la capacidad de las esporas de germinar. Los gametófitos se obtuvieron mediante técnicas de cultivo in vitro. Los esporófitos jóvenes se trasladaron a sustrato estéril de tierra y arena (3:1). Se usó el microscopio de luz y el de barrido (SEM). Se encontraron esporas con diferentes tipos de aperturas. La germinación ocurre entre 3-7 días y corresponde al tipo Vittaria. Se encontraron gametófitos filamentosos formados por 3-5 células en P. cretica, P. multifida y P. vittata y por 7-9 células en P. ensiformis. El desarrollo gametofítico ocurre de dos formas: tipo Adiantum y tipo Ceratopteris. Los gametófitos de P. ensiformis, P. multifida y P. vittata son monoicos y protándricos. P. cretica desarrolla gametófitos anteridiados. Los anteridios corresponden al tipo común de los helechos leptosporangiados, son cilíndricos en P. vittata y ovoides en las otras tres especies. Los cuellos de los arquegonios tienen 4 hileras con 4 células cada una. Los esporófitos se desarrollan después de los 3 meses de su siembra y su indumento es semejante a las plantas adultas. P. cretica presenta apogamia obligada.

Characterisation and identification of broad spectrum antibiotic producing Streptomyces hygroscopicus D 1.5.

A streptomycete strain D1.5 capable of producing broad spectrum antiobiotic was isolated from soil. The morphological, cultural, physiological and biochemical characters were studied, compared to known species and identified as Streptmoyces hygroscopicus. Antibiotic activity of the strain was tested against both Gram positive and negative bacteria as well as fungi. It exhibited complete resistance to beta-lactum antibiotics.

Descripción de la espora y de los estadios previos del Mixosporidio Kudora Rosenbuschi, parásito muscular de Merluccio Hubbsi: ciclo de vida / Description of the spore and previous stages in the mixosporidian Kudoa Rosenbuschi, muscle parasite of Merluccius Hubbsi: life cycle

Se describen al microscopio óptico y electrónico la espora y los estadios previos del mixosporidio Kudoa Rosenbuschi, parásito muscular de Merluccius Hubbsi. La espora madura está formada por tres tipos celulares: esporoplasma, células valvares y células capsulares. Se trata de un parásito de ciclo directo, donde todos los estadios ocurren dentro de las células musculares. Se esboza así mismo el ciclo de vida para este protozoo