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.

Ontogenia de los esporangios, formación y citoquímica de esporas en licopodios (Lycopodiaceae) colombianos / Ontogeny of the sporangium, spore formation and cytochemistry in Colombian Lycopodials (Lycopodiaceae)

Studies on reproductive aspects of Lycopodiaceae are not very abundant in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. Here we present a detailed study of the ontogeny of sporangia and sporogenesis, and the chemical determination of several compounds generated during spore formation. The analyses were performed in 14 taxa of six genera of the family, Diphasiastrum, Diphasium, Huperzia (a genus which is treated here including Phlegmariurus), Lycopodiella, Lycopodium and Palhinhaea. Specimens were collected in three departments from the Colombian Andes between 1 454-3 677m altitude. Ontogeny was studied in small, 1cm long pieces of strobili and axis, which were fixed in glutaraldehyde or FAA, dehydrated in alcohol, embedded in LR White, sectioned in 0.2-0.5μm and stained with toluidine blue (TBO), a metachromatic dye that allows to detect both sporopollenin and lignin or its precursors, during these processes. For other studies, paraplast plus-embedded sections (3-5μm) were stained with safranin-fast green and alcian blue-hematoxylin. Chemical tests were also conducted in sections of fresh sporangia at different stages of maturity using alcian blue (mucopolysaccharides), Lugol solution (starch), Sudan III (lipids), phloroglucinol (lignin) and orcein (chromosomes). Sections were observed with photonic microscope equipped with differential interference contrast (DIC) and fluorescence microscopy (for spore and sporangium walls unstained). Strobili and sporangia were dehydrated with 2.2 dimethoxypropane, critical point dried and coated with gold for scanning electron microscopy (SEM). Our results indicated that the ontogeny of sporangia and sporogenesis were very similar to the previously observed in Huperzia brevifolia. Cutinisation occurs in early stages of development of sporangium cell walls, but in their final stages walls become lignified. As for the sporoderm development, the exospore is the first layer formed, composed by sporopollenin. The endospore deposits as a thin inner layer composed of cellulose, pectin and carboxylated polysaccharides. The perispore, if present, deposits at last. Mucopolysaccharides were found on the sporocyte coat and its abundance in sporangial cavity persists up to the immature tetrads stage, and then disappears. The lipids were abundant in the sporocytes, tetrads and spores, representing the main source of energy of the latter. In contrast, starch is not detected in the spores, but is abundant in premeiotic sporocytes and immature tetrads, developmental stages of high cellular metabolic activity. Intrinsic fluorescence corroborates the presence of lignin and cutin in the sporangium wall, while the sporopollenin is restricted to the exospore. The transfusion cells and the perispore are not always present. However, the processes of ontogeny and sporogenesis are extremely similar throughout the taxa studied, suggesting that they represent conservative family traits, nonspecific or generic.

Los estudios sobre aspectos reproductivos no son muy abundantes en la literatura científica sobre los taxones de Lycopodiaceae y constituyen información esencial para apoyar la taxonomía y relaciones sistemáticas en el grupo. Por lo tanto, se presenta aquí un análisis detallado de la ontogenia de los esporangios y esporogénesis, así como determinaciones químicas de varios compuestos generados durante la formación de las esporas. Los análisis se llevaron a cabo en 14 taxones de seis géneros de la familia: Diphasiastrum, Diphasium, Huperzia (un género que se trata aquí, incluyendo Phlegmariurus), Lycopodiella, Lycopodium y Palhinhaea. Las muestras fueron recolectadas en tres departamentos de los Andes de Colombia entre 1 454-3 677m de altitud. La ontogenia se estudió en trozos de estróbilos y ejes, de 1cm de largo, que se fijaron en glutaraldehido o FAA, se deshidrataron en alcohol, se incluyeron en LR White, se seccionaron en cortes de 0.2-0.5μm y se colorearon con azul de toluidina (TBO), un colorante metacromático que permite detectar tanto esporopolenina como lignina o sus precursores. Para estudios adicionales, secciones de 3-5μm de material incluido en paraplast plus se colorearon con safranina-verde rápido y azul alciánhematoxilina. Las pruebas químicas se llevaron a cabo en secciones de esporangios sin fijar en diferentes etapas de madurez utilizando azul alcián (mucopolisacáridos), solución de Lugol (almidón), Sudán III (lípidos), fluoroglucinol (lignina) y orceína (cromosomas). Las observaciones se efectuaron con microscopio fotónico equipado con contraste diferencial de interferencia (DIC) y microscopía de fluorescencia (para esporas y pared de los esporangios sin colorear). Para observaciones con microscopía electrónica de barrido (MEB), los estróbilos y esporangios se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Los resultados indican que la ontogenia de los esporangios y esporogénesis es muy similar a la observada previamente en Huperzia brevifolia. En las primeras etapas de desarrollo, las paredes celulares de la epidermis del esporangio se cutinizan y en las finales se lignifican. En el desarrollo del esporodermo, la primera capa que se forma es el exosporio, compuesto por esporopolenina. El endosporio es una capa interna delgada compuesta de celulosa, pectina y polisacáridos carboxilados. El perisporio, si está presente, es la última capa que se deposita. Los mucopolisacáridos se encontraron en la cubierta del esporocito, son abundantes en la cavidad esporangial hasta la etapa de tétradas inmaduras y luego desaparecen. Los lípidos son abundantes en esporocitos, tétradas y esporas, y representan la principal fuente de energía de estas. En contraste, el almidón no se detecta en las esporas pero es abundante en esporocitos premeióticos y tétradas inmaduras, ambos con gran actividad metabólica. La fluorescencia intrínseca corrobora la presencia de lignina y cutina en la pared del esporangio, mientras que la esporopolenina se limita al exosporio. Las células de transfusión y el perisporio no siempre están presentes. Sin embargo, los procesos de la ontogenia y esporogénesis son extremadamente similares en todos los taxones estudiados, lo que sugiere que representan rasgos típicos de familia, no específicos ni genéricos.

[Ontogeny of strobili, sporangia development and sporogenesis in Equisetum giganteum (Equisetaceae) from the Colombian Andes]. / Ontogenia de los estróbilos, desarrollo de los esporangios y esporogénesis de Equisetum giganteum (Equisetaceae) en los Andes de Colombia.

Studies on the ontogeny of the strobilus, sporangium and reproductive biology of this group of ferns are scarce. Here we describe the ontogeny of the strobilus and sporangia, and the process of sporogenesis using specimens of E. giganteum from Colombia collected along the Rio Frio, Distrito de Sevilla, Piedecuesta, Santander, at 2200m altitude. The strobili in different stages of development were fixed, dehydrated, embedded in paraffin, sectioned using a rotatory microtome and stained with the safranin O and fast green technique. Observations were made using differential interference contrast microscopy (DIC) or Nomarski microscopy, an optical microscopy illumination technique that enhances the contrast in unstained, transparent. Strobili arise and begin to develop in the apical meristems of the main axis and lateral branches, with no significant differences in the ontogeny of strobili of one or other axis. Successive processes of cell division and differentiation lead to the growth of the strobilus and the formation of sporangiophores. These are formed by the scutellum, the manubrium or pedicel-like, basal part of the sporangiophore, and initial cells of sporangium, which differentiate to form the sporangium wall, the sporocytes and the tapetum. There is not formation of a characteristic arquesporium, as sporocytes quickly undergo meiosis originating tetrads of spores. The tapetum retains its histological integrity, but subsequently the cell walls break down and form a plasmodium that invades the sporangial cavity, partially surrounding the tetrads, and then the spores. Towards the end of the sporogenesis the tapetum disintegrates leaving spores with elaters free within the sporangial cavity. Two layers finally form the sporangium wall: the sporangium wall itself, with thickened, lignified cell walls and an underlying pyknotic layer. The mature spores are chlorofilous, morphologically similar and have exospore, a thin perispore and two elaters. This study of the ontogeny of the spore-producing structures and spores is the first contribution of this type for a tropical species of the genus. Fluorescence microscopy indicates that elaters and the wall of the sporangium are autofluorescent, while other structures induced fluorescence emitted by the fluorescent dye safranin O. The results were also discussed in relation to what is known so far for other species of Equisetum, suggesting that ontogenetic processes and structure of characters sporoderm are relatively constant in Equisetum, which implies important diagnostic value in the taxonomy of the group.