Microsporogénesis y ultraestructura de los granos de polen en la planta del cacao, Theobroma cacao (Malvaceae)

Introducción: No conocemos estudios sobre la microsporogénesis de la planta de cacao, y poco se sabe sobre la ultraestructura de sus granos de polen. Objetivo: Describir la microsporogénesis y ultraestructura de los granos de polen en T. cacao. Métodos: Procesamos más de 30 flores para cada etapa floral, teñidas con Safranina-Azul Alcian, PAS-Amidoblack y Lacmoid. Para la microscopía de transmisión procesamos las muestras en resina y las teñimos con azul de toluidina. Para microscopía electrónica de barrido, fijamos y deshidratamos en 2.2-dimetoxipropano, secamos hasta un punto crítico y recubrimos con oro. Resultados: Anteras diferenciadas por una masa celular en los extremos distales a los filamentos estaminales. Durante el desarrollo la pared de las anteras presenta varios estratos celulares y al madurar se reducen a la epidermis y al endotecio. Las células madre de microsporas se dividen por meiosis para formar tétradas. El tapete es secretor e intacto hasta que se liberan los granos, para luego degenerar. Los granos de polen son isopolares, esferoidales, pequeños, tricolpados. La ultraestructura presenta una esporodermis semitectada, con ornamentación reticulada, y un retículo heterobrochado con el muri sin ornamentación. La exina se deposita antes que la intina. Los orbículos son individuales, lisos y de tamaño variado. Hay abundante polenkit en el tectum y entre las columelas. La intina es delgada, pero se desarrolla ampliamente en las áreas del colpo, formando una intina interna compacta y una intina externa inusual con una apariencia columelada. Conclusión: La estructura y el desarrollo de las anteras siguen el patrón de las angiospermas. La microsporogénesis simultánea y la deposición centrípeta de la esporodermis se conocen de Malvaceae, pero los caracteres de la intina son nuevos para la familia.

Introduction: We know of no studies on the microsporogenesis of the cocoa plant, and little is known about the ultrastructure of its pollen grains. Objective: To describe microsporogenesis and ultrastructure of pollen grains in T. cacao. Methods: We processed over 30 flowers for each floral stage and stained with Safranin-Alcian Blue, PAS-Amidoblack and Lacmoid. For transmission microscopy we processed samples on resin and stained with toluidine blue. For scanning electron microscopy, we fixed and dehydrated in 2.2-dimethoxypropane, critically dried and coated with gold. Results: Anthers differentiated by a cellular mass at the ends distal to the staminal filaments. During development, the anther wall has several cellular layers reduced, at maturity, to the epidermis and endothecium. Microspore mother cells divide by meiosis to form tetrads. The tapetum is secretory and intact until the grains are released, to later degenerate. Pollen grains are isopolar, spheroidal, small, tricolpate. Ultrastructure has a semi-tectate sporodermis, with reticulate ornamentation, and heterobrochated reticulum with the muri without ornamentation. Exine is deposited before intine. The orbicles are individual, smooth, and varied in size. There is abundant pollenkitt on the tectum and between the columellae. The intine is thin, but develops widely in the colpus areas, forming a compact internal intine and an unusual external intine with a columellated appearance. Conclusion: Anther structure and development follows the angiosperm pattern. Simultaneous microsporogenesis and centripetal deposition of the sporodermis are known from Malvaceae, but intine characters are novel for the family.

Histology and structure of the testicles in three species of Atelopus frogs (Anura: Bufonidae) endemic to the Sierra Nevada de Santa Marta, Colombia / Histología y estructura de los testículos en tres especies de ranas Atelopus (Anura: Bufonidae) endémicas de la Sierra Nevada de Santa Marta, Colombia

Abstract Introduction: Testicular histology constitutes one of the least explored aspects in frogs of the genus Atelopus. This taxonomic group shows an alarming population decline; therefore, its reproductive biology is one of the greatest topics of interest for its conservation. Objective: To describe the testicular morphology and the spermatogenetic lineage cells in adult males of Atelopus laetissimus, Atelopus nahumae, and Atelopus carrikeri in the Sierra Nevada de Santa Marta, Colombia. Methods: During June - July 2017 and 2018, sampling was conducted in the localities of San Lorenzo and Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), to collect 15 adult males, 5 per species. Testes samples were fixed in Bouin to be processed by the standard paraffin-embedding technique. Histological sections (3 μm) were stained with Hematoxylin-eosin and Mallory-Heidenhain-Azan-Gomori's. For the description and photographic register of the germ cells, the photonic microscopy technique was used with the differential interference contrast system. Results: The testes are oval organs, compact, light yellow color, and with little vascularization. Externally, they are surrounded by a thin albuginea tunic constituted by regular dense connective tissue. Inside this layer, they are composed of numerous seminiferous tubules of hexagonal contour, in which germ cell cysts are distinguished at different stages of spermatogenesis (spermatogonia I and II, spermatocyte I and II, and early and late spermatids) and spermiogenesis (spermatozoa in fascicles and free spermatozoa). Separating the seminiferous structures is the interstitial tissue in which Leydig cells and blood vessels stand out. Additionally, in the cranial part of the testis, the Bidder's organ was found, formed by two distinguishable regions, the cortex and the medulla. In the cortex, there are previtellogénic oocytes of different sizes surrounded by a monolayer of flat follicular cells. For its part, the medullary region is the connective tissue that nourishes the oocytes and is constituted by blood capillaries. Conclusions: The gonads of the three species analyzed present a cystic cellular organization similar to other anurans, where all stages of spermatogenesis and spermiogenesis were identified, possibly indicating a continuous reproductive activity. Likewise, the Bidder's organ is reported for the first time in the three Atelopus species, which allows suggesting a possible sexual reversion in case of a population decrease of females as a reproductive strategy.

Resumen Introducción: La histología testicular constituye uno de los aspectos menos explorados en las ranas del género Atelopus. Este grupo taxonómico ostenta un declive poblacional alarmarte, es por ello, que su biología reproductiva resulta uno de los temas de mayor interés para su conservación. Objetivo: Describir la morfología testicular y las células del linaje espermatogénico en machos adultos de Atelopus laetissimus, Atelopus nahumae y Atelopus carrikeri en la Sierra Nevada de Santa Marta, Colombia. Métodos: Durante Junio - Julio de 2017 y 2018 se realizaron muestreos en las localidades de San Lorenzo y Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), para recolectar 15 machos adultos, 5 por especie. Las muestras de testículo se fijaron en Bouin para ser procesadas mediante la técnica estándar de inclusión en parafina. Las secciones histológicas (3 μm) se tiñeron con Hematoxilina-eosina y Mallory-Heidenhain-Azan-Gomori's. Para la descripción y registro fotográfico de las células germinales, se utilizó la técnica de microscopía fotónica con el sistema de contraste diferencial de interferencia. Resultados: Los testículos son órganos ovalados, compactos, de color amarillo claro y con poca vascularización. Externamente, están rodeados por una delgada túnica albugínea constituida por tejido conectivo denso regular. Al interior de esta capa se componen por numerosos túbulos seminíferos de contorno hexagonal, en los que se distinguen quistes de células germinativas en diferentes etapas de la espermatogénesis (espermatogonia I y II, espermatocito I y II y espermátidas tempranas y tardías) y espermiogénesis (espermatozoides en fascículos y espermatozoides libres). Separando las estructuras seminíferas se halla el tejido intersticial en el que se destacan las células de Leydig y los vasos sanguíneos. Adicionalmente, en la parte craneal del testículo se encontró el órgano de bidder formado por dos regiones diferenciables, la corteza y la medula. En la corteza se aprecian ovocitos previtelogénicos en diferente tamaño rodeados por una monocapa de células foliculares planas. Por su parte, la región medular es el tejido conectivo que nutre los ovocitos y está constituido por capilares sanguíneos. Conclusiones: Las gónadas de las tres especies analizadas presentan una organización celular quística de manera similar con otros anuros, donde se identificó todos los estadios de la espermatogénesis y espermiogénesis indicando posiblemente una actividad reproductiva continua. Así mismo, se reporta por primera vez el órgano de bidder en las tres especies de Atelopus, lo cual permite sugerir una posible reversión sexual en caso de una disminución poblacional de las hembras como una estrategia reproductiva.

Microsporogénesis y micromorfología del polen de la planta Alcea rosea (Malvaceae) / Microsporogenesis and micromorphology of pollen grains of the plant Alcea rosea (Malvaceae)

Resumen Introducción: Los estudios sobre microsporogénesis, micromorfología y estructura de los granos de polen en Malvaceae son escasos. Objetivos: Describir el proceso de microsporogénesis y aspectos micromorfológicos de los granos de polen en A. rosea. Métodos: Se procesaron más de 30 andróforos de acuerdo con los protocolos estándar para incrustar y seccionar en parafina. Las secciones obtenidas se tiñeron con Azul de Safranina-Alcian, las anteras inmaduras y no fijadas se tiñeron con Azul de anilina. Se procesaron secciones de resina adicionales de los andróforos y se tiñeron con azul de toluidina. Se observaron secciones ultrafinas con microscopía electrónica de transmisión (MET). Para la observación con microscopía electrónica de barrido (MEB), el material se fijó y deshidrató en 2,2 dimetoxipropano, luego se secó hasta un punto crítico y se recubrieron con oro. Resultados: las anteras se diferencian de una masa celular en los extremos distales de los filamentos del estambre. La pared de la antera madura presenta una capa externa de células epidérmicas y una capa interna, el endotecio. Las células madre de microesporas se dividen por mitosis y luego experimentan meiosis para formar tétradas. El tapete es inicialmente celular y forma una sola capa de células y luego pierde integridad celular al invadir el lóculo de microsporangio, formando un periplasmodio. Durante la formación de la esporodermis, primero se deposita la exina y luego la intina. Para el momento de la liberación de los granos de polen, el tapete se ha degenerado por completo. Los granos de polen son pantoporados, apolares, con simetría radial, esferoidales, con espinas, báculas, gránulos y microgránulos. El téctum está perforado con fovéoleas dispuestas homogéneamente en toda la superficie y con polenkit. La exina es ancha (5-6 µm) y consta de una endexina gruesa de 3.5 a 4 µm y una ektexina fina (0.6-0.7 µm). La ultraestructura muestra columelas claramente definidas formando el infratéctum. Se aprecian tricomas nectaríferos unicelulares glandulares capitados (TG) cubriendo toda la superficie de los filamentos de los estambres. Conclusiones: La estructura y desarrollo de las anteras sigue los patrones conocidos de las angiospermas. La microsporogénesis simultánea y el depósito centrípeto de la esporodermis se han descrito previamente para Malvaceae.

Abstract Introduction: Studies on microsporogenesis, micromorphology and structure of pollen grains in Malvaceae are scarce. Objectives: To describe the process of microsporogenesis and micromorphological aspects of pollen grains in A. rosea. Methods: Androphores were processed according to standard protocols for sectioning in paraffin. The obtained sections were stained with Safranin-Alcian blue, Aniline blue was used for immature and unfixed anthers and for resin sections of the androphores, Toluidine blue. Ultrathin sections were observed with transmission electron microscopy. For observation with scanning electron microscopy the material was fixed and dehydrated in 2.2 dimethoxypropane, dried to a critical point and coated with gold. Results: Anthers differentiate from a cell mass at the distal ends of the stamen filaments. The wall of the mature anther presents an outer layer of epidermal cells and an inner layer, the endothecium. Microspore mother cells divide by mitosis and then undergo meiosis to form tetrads. The tapetum is initially cellular and forms a single layer of cells and then loses cellular integrity by invading the microsporangium locule, forming a periplasmodia, by the time the pollen grains are released it degenerated. During sporodermis formation, exine is first deposited and then intine. Pollen grains are pantoporate, apolar, with radial symmetry, spheroidal, with spines, bacula, granules and microgranules. Tectum is perforated with foveolae arranged homogeneously over the whole surface and pollenkit is present. Exine is broad and consists of a thick 3.5 to 4 µm endexine and a thin ektexine (0.6-0.7 µm). The ultrastructure shows columellae forming the infratectum. Capitate glandular unicellular nectariferous trichomes covers the whole surface of the stamen filaments. Conclusions: The structure and development of the anthers follows the known patterns for angiosperms. Simultaneous microsporogenesis and centripetal deposit of the sporodermis have been previously described for Malvaceae.

Micromorfología y ultraestructura de las anteras y los granos de polen en diez genotipos élite de Theobroma cacao (Malvaceae) / Micromorphology and ultrastructure of anthers and pollen grains in ten elite genotypes of Theobroma cacao (Malvaceae)

Resumen Introducción: A pesar de que T. cacao es una especie importante a nivel mundial por la producción de cacao, es poco lo que se conoce sobre la micromorfología y estructura de las anteras y los granos de polen. Objetivos: Describir y analizar la estructura y micromorfología de las anteras y los granos de polen de 10 genotipos élite de esta importante especie tropical. Métodos: Se tomaron más de 30 anteras de flores en antesis de los 10 genotipos élite de T. cacao del banco de germoplasma ex situ del Centro de Investigaciones Suiza-Agrosavia (Rionegro, Santander-Colombia). El material se procesó de acuerdo con los protocolos estándar para embeber y seccionar en parafina. Las secciones obtenidas (3 μm) se tiñeron con azul de Safranina-Alcian para discriminar estructuras con paredes primarias y secundarias y polifenoles totales. Además, se usó la técnica PAS-Amidoblack para diferenciar entre polisacáridos estructurales y de reserva, así como proteínas. Para la determinación de esporopolenina y polifenoles se usó la tinción azul de toluidina y finalmente para descripciones adicionales se aplicó la tinción azul alcián-PAS-hematoxilina. Las observaciones se realizaron mediante microscopio fotónico y microscopio de epifluorescencia. Para la observación con microscopía electrónica de barrido (MEB), las anteras con los granos de polen se fijaron y deshidrataron en 2.2 dimetoxipropano, luego se desecaron hasta un punto crítico y finalmente se recubrieron con oro. Resultados: Las anteras son bitecas y están sostenidas por un largo filamento formado por un estrato epidérmico, tejido parenquimatoso y un haz vascular. La dehiscencia ocurre longitudinalmente a través del estomio. La pared de la antera madura está formada por una capa epidérmica monoestratificada, una capa de células endoteliales con engrosamientos fibrilares lignificados y se pueden apreciar restos celulares del tapete y abundantes orbículas recubriendo la cavidad de los microesporangios. Los tejidos epidérmicos y parenquimatosos de las anteras almacenan polifenoles. Las orbículas son generalmente esféricas, psiladas y exhiben las mismas reacciones de tinción y fluorescencia que la exina de los granos de polen. Los granos de polen son mónades, isopolares, pequeños (16-19 µm) con amb circular, esferoidales, tricolpados con colpos medianos o cortos (5-10 µm) con membrana ornamentada, semitectatos, reticulados, heterobrochados, las paredes del retículo ornamentadas o no, con microgránulos de diferente tamaño o escabrados. Los análisis estadísticos mostraron que existen diferencias significativas en el tamaño de los granos de polen (P ˂ 0.05). Se observa que los granos de polen más pequeños son los del genotipo TCS 19 (16.890 µm) y se diferencian del resto de genotipos, y entre estos no se observan diferencias significativas. Solo dos genotipos (SCC 19 y SCA 6) presentaron polenkit y solo uno tiene paredes perforadas (SCA 6). Conclusiones: La estructura y micromorfología de las anteras de T. cacao son similares a las descritas para otras Malvaceae. Así mismo, los granos de polen mostraron variaciones de tamaño, ornamentación de las paredes y del lumen del retículo y presencia de polenkit. Sin embargo, no se observó relación entre las variaciones de los caracteres micromorfológicos analizados en los granos de polen y los modelos de compatibilidad polínica reportados para estos genotipos.

Abstract Introduction: Despite the fact that T. cacao is an important species worldwide for cocoa production, little is known about the micromorphology and structure of anthers and pollen grains. Objectives: To describe and analyze the structure and micromorphology of the anthers and pollen grains of 10 elite genotypes of this important tropical species. Methods: More than 30 anthers of flowers in anthesis were taken of the 10 elite genotypes of T. cacao from the ex situ germplasm bank of the Suiza-Agrosavia Research Center (Rionegro, Santander-Colombia). The anthers with the pollen grains were fixated and processed according to the standard protocols for embedding and sectioning in paraffin. Sections obtained (3 μm thick) were stained with Safranin-Alcian blue to discriminate structures with primary and secondary walls and total polyphenols. Additionally, the samples were also stained with the PAS-Amidoblack technique was used to differentiate between structural and reserve polysaccharides as well as proteins. Toluidine blue staining was used for the determination of sporopollenin and polyphenols and finally Alcian blue-PAS-Hematoxylin staining was applied for additional descriptions. Observations were made using photonic microscopy and epifluorescence microscopy. For observation with scanning electron microscopy (SEM) the anthers with the pollen grains were fixed and dehydrated in 2.2 Dimethoxypropane, then desiccated to critical point and finally coated with gold. Results: The anthers are dithecal and supported by a long filament made up of an epidermal stratum, parenchymal tissue, and a vascular bundle. The dehiscence occurs longitudinally through the stomium. The anther wall is made up of a monostratified epidermal layer, followed by a layer of endothecial cells with lignified fibrillar thickenings, cellular remnants of tapetum and abundant orbicules can be seen covering the cavity of the microsporangia. The epidermal and parenchymal tissues of the anthers are abundant in polyphenols. Orbicules are generally spherical, psilated, and these exhibit the same staining and fluorescence reactions as exine from pollen grains. The pollen grains are monades, isopolar, small (16-19 µm) with circular amb, spheroidal, tricolpate with medium or short colpi (5-10 µm) with sculptured membrane, semitectate, reticulated, heterobrochate, sculptured or non- sculptured walls, with microgranules of different size or scabrate. The statistical analyzes showed that there are significant differences in the size of the pollen grains (P ˂ 0.05). It is observed that the smallest pollen grains are those of the TCS 19 genotype (16.890 µm) and are different from the other genotypes, and among these there are no significant differences. Only two genotypes (SCC 19 and SCA 6) showed pollenkit and only one has perforated walls (SCA 6). Conclusions: The structure and micromorphology of the anthers of T. cacao are similar to those described for other Malvaceae. Likewise, the pollen grains showed variations in size, ornamentation of the sporoderm and the lumen of the reticulum and the presence of pollenkitt. However, no relationship was observed between the micromorphological characters analyzed in the pollen grains and the pollen compatibility models reported for these genotypes.

Ontogenia de los esporangios y esporogénesis del helecho Phymatosorus scolopendria (Polypodiaceae)

Introducción: Las investigaciones sobre ontogenia de los soros, esporangios, paráfisis receptaculares y esporogénesis de los helechos leptosporangiados son escasas en la literatura científica. Objectivos: Describiry analizar la ontogenia de los soros, esporangios, paráfisis receptaculares y esporogénesis de Phymatosorus scolopendria. Métodos: Entre marzo y mayo 2017 (época lluviosa del año) se recolectaron frondas fértiles de P. scolopendria en el campus de la Universidad de Antioquia, Medellín-Colombia.Las frondas fértiles, en diferentes etapas del desarrollo se fijaron y procesaron de acuerdo a protocolos estándar para la inclusión y corte en parafina y resina. Las secciones de 0.5 µm obtenidas en resina se tiñeron con azul de Toluidina que tiñe diferencialmente paredes primarias y secundarias, resalta núcleos celulares, y esporopolenina y de manera secundaria tiñe polifenoles. Para descripciones detalladas, otros cortes se tiñeron con Safranina-azul de alciano que discrimina entre componentes de pared primaria, secundaria, núcleos, cutícula y polifenoles; Hematoxilina-azul de alciano para resaltar núcleos y paredes primarias y Fluoroglucinol ácido para detectar lignina. Las observaciones y registro fotográfico se efectuaron con microscopio fotónico. Para la observación y descripción con microscopía electrónica de barrido (MEB), los soros se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Resultados: Los soros son exindusiados, superficiales, vascularizados y de desarrollo mixto, se encuentran asociados a paráfisis receptaculares multicelulares uniseriadas. Durante el desarrollo del soro primero se diferencian las células epidérmicas receptaculares que darán origen a los esporangios y posteriormente las células que originarán a las paráfisis receptaculares. El esporangio es de tipo leptosporangio de pedicelos largos de una o dos filas de células. Los anillos de los esporangios muestran paredes secundarias con engrosamientos en forma de "U" ricos en lignina. La meiosis es simultánea y las tétradas de esporas se disponen de forma decusada o tetragonal. El tapete celular es inicialmente uniestratificado pero por una división mitótica de tipo periclinal, se torna biestratificado. Las células del estrato interno del tapete pierden la integridad estructural dando origen a un tapete plasmodial que invade los esporocitos en meiosis, el estrato externo persiste hasta la etapa de esporas maduras. En las diferentes etapas de desarrollo del esporodermo, primero se forma el exosporio, compuesto por esporopolenina, seguida del endosporio, conformado por celulosa, pectina y polisacáridos carboxilados y finalmente el perisporio. Los polifenoles fueron detectados, principalmente, en las vacuolas de las células de los esporangios, paráfisis y células receptaculares. Para el momento de la liberación de las esporas, tanto la capa externa del tapete celular como el plasmodial han degenerado por completo. En la cavidad esporangial se aprecian orbículas adyacentes a las esporas. Conclusiones: la ontogenia de los esporangios y esporogénesis de P. scolopendria es similar al descrito previamente para helechos leptosporangiados. Adicionalmente, se indica que las paráfisis receptaculares presentes en los soros de P. scolopendria tienen la función de protección de los esporangios durante las primeras etapas del desarrollo.

Introduction: Research about the ontogeny of sori, sporangia, receptacular paraphyses and sporogenesis of leptosporangiate ferns are scarce in the scientific literature. Objectives: To describe and analyze the ontogeny of sori, sporangia, receptacular paraphyses and sporogenesis of Phymatosorus scolopendria. Methods: Fertile fronds of P. scolopendria were collected in the campus of the Universidad de Antioquia, Medellín, Colombia, during the months March and May (annual rain season) of 2017. The fertile fronds of the samples at different developmental stages were fixed and processed according to the standard protocols for embedding and sectioning in paraffin and resin. Sections of 0.5 µm obtained in resin were stained with Toluidine blue, which differentially stains primary and secondary walls, highlights the cell nucleus and sporopolenin and secondarily stains polyphenols. For detailed descriptions, additional sections were processed with Safranin-Alcian blue, allowing the distinction of components of primary and secondary walls, nuclei, cuticle and polyphenols; Hematoxylin-Alcian blue to enhance nuclei and primary walls and Phloroglucinol-HCl for lignin. Observations and photographic records were done with a photonic microscope. For the observations and descriptions with scanning electron microscopy (SEM), the sori were dehydrated with 2,2-dimethoxypropane, critical point dried and coated with gold. Results: The sori are exindusiate, superficial, vascularized and have mixed development; they are associated with uniseriate and multicellular receptacle paraphyses. During the development of the sori, the epidermal cells of the receptacle that will form the sporangia are the first differentiated followed by those forming the receptacle paraphyses. The sporangium is leptosporangiate, with long stalks formed by one or two cell rows. The annulus of the sporangia displays secondary walls with U-shaped thickenings rich in lignin. The meiosis is simultaneous and the spore tetrads are arranged in a decussate or tetragonal shape. The cellular tapetum is initially unistratified but becomes bistratified after a periclinal division. The cells of the internal strata of the cellular tapetum loose structural integrity giving rise to a plasmodial tapetum that invades the meiotic sporocytes. During the sporoderm development, the sporopollenin-composed exospore is the first formed followed by the endospore, composed by cellulose, pectin and carboxylated polysaccharides; the process ends with the perispore. Polyphenols were mainly detected on vacuoles in cells of the sporangium, paraphysis and receptacle. When the time comes for the spore maturation, the remnants of cellular and the plasmodial tapeta have fully degenerated. Abundant orbicles are seen near the spores in the sporangial cavity. Conclusions: The ontogeny of the sporangia and sporogenesis of P. scolopendria are similar to the previously described for leptosporangiate ferns. Furthermore, in P. scolopendria, the receptacle paraphyses of the sori have a role protecting the sporangium during the early development stages.

Alteraciones histopatológicas causadas por la roya Puccinia nakanishikii (Pucciniales: Pucciniaceae) en plantas de Cymbopogon citratus (Poaceae)

Introducción: Los aspectos histopatológicos e histoquímicos relacionados con el ataque de royas en plantas, así como su relación con los diferentes estados espóricos, son escasos en la literatura científica. Objetivos: Describir y analizar los aspectos histopatológicos e histoquímicos en Cymbopogon citratus y su relación con los diferentes estados espóricos de la roya Puccinia nakanishikii. Métodos: Durante abril y agosto 2013 se recolectaron hojas sanas e infectadas con Puccinia nakanishikii en la escarpa noroccidental de la meseta de Bucaramanga-Colombia. Las muestras con lesiones en diferentes etapas del desarrollo se fijaron y procesaron de acuerdo a protocolos estándar para la inclusión y corte en parafina y resina. Las secciones obtenidas en parafina (5-7 µm) fueron teñidas con Safranina-azul de Alcian y azul de Alcian-Hematoxilina. En tanto que las secciones obtenidas en resina (0.5 µm) se tiñeron con azul de Toluidina. También se elaboraron secciones a mano alzada para análisis de autofluorescencia. Las observaciones y registro fotográfico se efectuaron con microscopio fotónico y microscopía de epifluorescencia. Para observaciones con microscopía electrónica de barrido (MEB), las muestras se fijaron en Glutaraldehído, se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Resultados: Las hojas son por lo general hipostomáticas, con células epidérmicas largas y cortas formando filas paralelas y con la presencia de tricomas unicelulares espinosos y microtricomas. La superficie abaxial está cubierta por una densa capa de ceras epicuticulares y la adaxial está formada por agrupaciones de células buliformes y células epidérmicas de contorno rectangular o cuadrado. En el mesófilo no hay diferenciación entre parénquima de empalizada y esponjoso y su anatomía refleja el metabolismo C4 presente en esta gramínea. Se observó la formación de urediosoros y teliosoros hipófilos. Las urediosporas son la fase de reinfección y estas tienen de 4-5 poros germinativos ecuatoriales y su pared es equinulada. Las teliosporas son de pared lisa y de pedicelo persistente. Las urediosporas forman tubos de geminación por lo general sobre la superficie abaxial de la hoja y se desarrollan en dirección de los estomas, por donde penetran al interior del mesófilo. No se observó la presencia de apresorios. La epidermis se desprende y levanta por del desarrollo de las urediosporas y las paráfisis capitadas, a medida que el urediosoro crece. Con el avance de la infección, los tejidos fotosintéticos se desorganizan, pierden la autofluorescencia de la clorofila y las células sufren necrosis. Posteriormente, los tejidos vasculares se fragmentan y colapsan. Para este momento, la infección se ha extendido sobre toda la lámina foliar llevando a la muerte de la hoja y defoliación de la planta. Durante etapas avanzadas de la infección en los urediosoros se observaron picnidios, probablemente del hiperparásito Sphaerellopsis, asociados estrechamente a los tejidos infectados por la roya. Conclusiones: Puccinia nakanishikii se desarrolla sobre las hojas de Cymbopogon citratus produciendo urediosoros y teliosoros. Las urediosporas son la fase de reinfección, y las teliosporas solo se observaron en etapas avanzadas de la infección. La epidermis y los tejidos fotosintéticos son severamente afectados por la necrosis celular. En etapas avanzadas de la infección los tejidos vasculares se ven afectados.

Introduction: Histopathological and histochemical aspects linked to the attack of fungal rusts to plants, as well as its relation with the different spore stages are topics rather scarce in the scientific literature. Objective: To describe and analyze the histopathological and histochemical aspects of Cymbopogon citratus and its relation with the different stages of the spores from the rust fungi Puccinia nakanishikii. Methods: During the months April and August 2013, leaves healthy and infected by Puccinia nakanishikii were collected in the Northwestern scarp of the Bucaramanga-Colombia plateau. The samples with injuries on diverse developmental stages were fixated and processed according to the standard protocols for embedding and sectioning in paraffin and resin. Sections obtained from paraffin (5-7 µm) were stained with Safranin-Alcian blue and Alcian blue-Hematoxylin. On the other hand, sections obtained from resin (0.5 µm) were stained with Toluidine blue. Further, freehand sections were obtained for an autofluorescence analysis. The observations and photographic record were done via photonic microscope and epifluorescence microscope. For the observations via scanning electron microscopy (SEM), the samples were fixated in Glutaraldehyde, dehydrated with 2,2 dimethoxypropane, then desiccated to critical point and finally coated with gold. Results: The leaves are generally hypostomatic, with long and short epidermic cell forming parallel rows and showing unicellular prickle trichomes and micro-trichomes. The abaxial surface is covered by epicuticular wax forming a dense layer. The adaxial epidermis is formed by groupings of bulliform cells and epidermal cells with rectangular or squared contour. In the mesophyll, there is no differentiation between palisade and spongy parenchyma, its anatomy reflects the C4 metabolism. The formation of uredosori and teliosori both hypophyllous was observed. Urediniospores are the reinfecting agents phase, they have 4-5 equatorial germ pores and echinulate wall. Teliospores have smooth wall and a persistent pedicel. The urediniospores form a germ tube, generally on the abaxial leaf surface, these tubes develop towards the stomata reaching the mesophyll interior. No appressorium were observed. The epidermis limiting the uredosorus detaches due the development and pressure that exert both the urediniospores and capitate paraphyses. As the infection progresses, autofluorescense of the chlorophyll is lost and the cells undergo necrotic processes. Afterwards, the phloem collapses and the xylem becomes slightly disorganized. At this moment, the infection is extended along the whole leaf blade, resulting in the leaf death and the plant defoliation. On advanced stages of the infection, the uredosori showed pycnidia, probably belonging to the hyperparasite Sphaerellopsis, these structures were closely associated to the rust infected tissues. Conclusions: Puccinia nakanishikii develops on the leaves of Cymbopogon citratus producing uredosori and teliosori. Urediniospores are the reinfective stage, teliospores were only observed at late stages of the infection. The epidermis and photosynthetic tissue are severely affected by cell necrosis. The vascular tissues are deeply affected on the advances stages of the infection.

Ontogenia e histoquímica de los esporangios y escamas receptaculares del helecho epífito Pleopeltis macrocarpa (Polypodiaceae)

Introducción: Las investigaciones sobre la ontogenia de los esporangios y más aún, de la estructura y función de las escamas receptaculares presentes en los soros de algunas especies de helechos, son escasos en la literatura científica. Objetivos: Describir y analizar la ontogenia de los esporangios y las escamas receptaculares de Pleopeltis macrocarpa. Metodología: Durante marzo y mayo de 2017 se recolectaron frondas fértiles de esta especie en los troncos de árboles en el vivero El Edén de las Flores en el municipio de Rionegro, Antioquia-Colombia. Las muestras se fijaron y procesaron de acuerdo a protocolos estándar para la inclusión y corte en parafina y resina. Las secciones obtenidas en resina (0.5 µm) se tiñeron con azul de Toluidina. Para descripciones adicionales sobre la anatomía e histoquímica se aplicaron reactivos específicos para determinar paredes primarias, secundarias, núcleos, lignina, polifenoles, polisacáridos, sustancias pécticas y celulosa. Las observaciones y registro fotográfico se efectuaron con microscopio fotónico y microscopía de epifluorescencia. Para observaciones con microscopía electrónica de barrido (MEB), los soros se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Resultados: Los soros son superficiales, vascularizados y de desarrollo mixto y están cubiertos por escamas receptaculares que se desprenden con la maduración de los esporangios. El esporangio de tipo leptosporangio tiene pedicelos largos de paredes primarias, anillos de los esporangios muestran paredes secundarias con engrosamientos en forma de "U" ricos en lignina. Las células epidérmicas de los receptáculos originan a los esporangios y las escamas receptaculares. Los eventos de división mitótica de estas dos estructuras son inicialmente similares, pero luego divergen para la diferenciación reproductiva y vegetativa de estos dos órganos. La meiosis es simultánea y las tétradas de esporas se disponen de forma decusada o tetragonal. El tapete celular es inicialmente uniestratificado pero por una división mitótica se torna biestratificado. Las células del estrato interno del tapete se rompen dando origen a un tapete plasmodial. En el desarrollo del esporodermo, primero se forma el exosporio, compuesto por esporopolenina, luego el endosporio compuesto de celulosa, pectina y polisacáridos carboxilados y finalmente el perisporio. Los resultados histoquímicos y de epifluorescencia indican que las paredes celulares tanto de los esporangios como las escamas receptaculares inmaduras son de naturaleza celulósica. Al madurar, estas estructuras, así como las células de la pared del esporangio mantienen esta composición. En tanto que las células epidérmicas de los escudos de las escamas receptaculares maduras se caracterizan por mostrar cutícula engrosada. Los polifenoles están presentes durante todas las etapas de desarrollo de los esporangios y escamas receptaculares. Los almidones son abundantes en etapas tempranas del desarrollo en las células del receptáculo y primordios de los esporangios. Conclusiones: La ontogenia de los esporangios de P. macrocarpa es similar al descrito para helechos leptosporangidos. Las escamas receptaculares son estructuras principalmente de protección, su morfología y composición de las paredes celulares evitan la desecación o perdida de humedad en los esporangios durante las etapas lábiles de su desarrollo. Estos resultados concuerdan con la función de protección atribuida a las escamas peltadas pluricelulares presentes en las estructuras vegetativas de algunas especies de helechos y angiospermas tolerantes a la sequía.

Introduction: The ontogeny of sporangia and furthermore the structure and function of the receptacle scales showed by the sori of some fern species are topics scarcely represented in the scientific literature. Objectives: To describe and analyze the ontogeny of sporangia and receptacle scales of Pleopeltis macrocarpa. Methods: During March and April of 2017, fertile fronds of P. macrocarpa were collected from tree stems located in the plant nursery "El Edén de las flores", municipality of Rionegro, Antioquia, Colombia. The samples were fixed and processed according to the standard protocols for embedding and sectioning in paraffin and resin. Sections obtained in resin (0.5 µm) were stained with Toluidine blue. The additional descriptions of the anatomy and histochemistry required specific reagents, applied for the determination of primary walls, secondary walls, nuclei, lignin, polyphenols, polysaccharides, pectic substances and cellulose. The observations and photographic records were performed by photonic and epifluorescence microscopy. For the scanning electron microscopy (SEM) technique, the sori were dehydrated with 2,2- Dimethoxypropane, dried to critical point and coated with gold. Results: The sori are superficial, vascularized and have mixed development, covered by receptacle scales that detach as the sporangia reaches maturity. The leptosporangiate type sporangium have long stalks of primary walls, the annulus of the sporangia shows secondary walls with "U" shaped thickenings rich in lignin. The epidermal cells of the receptacle originate the sporangia and receptacle scales. The mitotic division events of these two structures are initially similar, but then diverge for the reproductive and vegetative differentiation of these two organs. Meiosis is simultaneous and the spore tetrads are arranged in a decussate or tetragonal shape. The cellular tapetum is initially unstratified but becomes bistratified by mitotic division. The inner layer of the tapetum cells break originating a plasmodial tapetum. During the sporoderm development, the first structure formed is the exospore, composed of sporopolenin, followed by the endospore composed of cellulose, pectin and carboxilated polysaccharides, and finally the perispore. The histochemistry and epifluorescence results indicate that both the sporangia and immature receptacle scales have cell walls of cellulosic. These structures as well as those of the sporangium wall cells maintain its composition during maturation. Whereas, the epidermal wall cells of the shields from the mature receptacle scales are characterized by thickened cuticle. The polyphenols are present during all the development stages of the sporangia and receptacle scales. Starch is abundant in the early stages of development of the receptacle cells and sporangial primordia. Conclusions: The ontogeny of the sporangia of P. macrocarpa is similar to the described for leptosporangiate ferns. The receptacle scales are mainly protective structures, its morphology and cell wall composition prevent desiccation or humidity loss of the sporangia during the labile stages of development. These results agree with the protective function attributed to the peltated pluricellular scales present in the vegetative structures of drought tolerant species of ferns and angiosperms.

ANATOMÍA FOLIAR COMPARADA DE Gaiadendron punctatum Y Tripodanthus belmirensis (LORANTHACEAE) / Comparative Leaf Anatomy of Gaiadendron punctatum and Tripodanthus belmirensis (LORANTHACEAE)

RESUMEN Se describe la anatomía foliar de las especies Gaiadendron punctatum y Tripodanthus belmirensis, al objeto de estudiar posibles caracteres que permitan una identificación precisa de estos dos géneros de la familia Loranthaceae, de hábito arbustivo o arbóreo. Las muestras se procesaron y sometieron a tinción con técnicas clásicas para su observación al microscopio óptico. Ambas especies presentaron similitudes como una epidermis monoestratificada y estomas de tipo rubiáceo. Sin embargo, se observó una composición anatómica claramente diferenciada en aspectos como el mesófilo, forma y ubicación de las células epidérmicas y la presencia de acumulaciones de súber en G. punctatum o idioblastos abundantes en T. belmirensis. Se construyó una clave dicotómica para la determinación de especies con base en caracteres anatómicos de la hoja entre las especies del género Tripodanthus y G. punctatum, además se discute brevemente el uso de caracteres anatómicos en la determinación y soporte de entidades taxonómicas diferenciables dentro de la familia Loranthaceae.

ABSTRACT Leaf anatomy of Gaiadendron punctatum and Tripodanthus belmirensis species is described with the aim of exploring possible characters that allow a precise identification of these two genera, characterized by tree or shrub habit, belonging to Loranthaceae family. Samples were processed and stained with routine techniques for observation on optical microscope. Both species showed similarities, such as one-layered epidermis and rubiaceous type stomata. However, a different anatomic composition was observed in aspects such as: mesophyll, position and shape of epidermic cells and presence of suber accumulations in G. punctatum, or numerous idioblasts in T. belmirensis. A dichotomous key was constructed for species determination based on anatomical leaf characters, between the species of genus Tripodanthus and G. punctatum. Furthermore, the use of anatomical characters in determination and support of distinguishable taxonomical entities inside Loranthaceae is also briefly discussed.

Caracteres estructurales y ultraestructurales de la gametogénesis de Chara hydropitys (Charophyceae) / Structural and ultrastructural characters of the gametogenesis of Chara hydropitys (Charophyceae)

Resumen Los oosporangios y anteridios de Charophyceae son los órganos de reproducción sexual femeninos y masculinos respectivamente. Estas estructuras se caracterizan por su complejidad morfológica y utilidad en taxonomía y sistemática. En el presente trabajo se describen los detalles estructurales y ultraestructurales de la gametogénesis en Chara hydropitys. El material fértil del alga se recolectó en una quebrada tributaria del Río Meléndez en la ciudad de Cali, Colombia (3º21´23´´N - 76º32´5.2´´W). Los especímenes fueron fijados y procesados de acuerdo a los protocolos estándar para la inclusión en resina y obtención de secciones finas que se colorearon con toluidina O (0.3-0.7 μm) para su observación en microscopía fotónica y secciones ultrafinas (60-90 nm) para microscopía electrónica de transmisión (MET). Además, se procesaron muestras para microscopio electrónico de barrido (MEB). Los oosporangios están recubiertos por las células espirales que forman de 10-12 circunvoluciones y terminan en cinco células coronulares. La pared de los oosporangios inmaduros está formada por dos capas que corresponden a la pared de las células espirales y de la oosfera. Al madurar la pared del oosporangio tiene seis capas adicionales, tres de las cuales son aportadas por la oospora y las tres restantes por las células espirales. La oosfera aumenta progresivamente de tamaño a medida que las células espirales crecen y se dividen. En el citoplasma de la oosfera inmadura no se aprecian inclusiones citoplasmáticas conspicuas, pero con la maduración el número de gránulos de almidón aumenta llegando a ocupar la mayor parte del volumen celular. En las células espirales del oosporangio maduro se observan numerosos cloroplastos con prominentes depósitos de almidón entre las lamelas tilacoidales y una vacuola que ocupa casi toda la célula. En las observaciones con MEB se aprecia que la pared externa de la oospora, sobre la zona de la fosa presenta microornamentaciones de tipo verrucado. En los anteridios maduros las células del escudo están fuertemente pigmentadas de color naranja por la presencia de numerosos plastoglóbulos entre las lamelas tilacoidales. De las células del capítulo secundario se desarrollan los filamentos espermatógenos que por divisiones mitóticas unidireccionales y sincrónicas forman los espermatocitos. A partir de estas células haploides por espermiogénesis se desarrollarán los anterozoides biflagelados. Los eventos subcelulares relacionados con estos procesos de división y diferenciación celular incluyen inicialmente cambios en la condensación de la cromatina, pérdida del nucléolo y mayor actividad de los dictiosomas. Posteriormente, el citoplasma se retrae y los orgánulos se alinean a lo largo del núcleo condensado y del aparato flagelar. Los anterozoides maduros emergen a través de un poro lateral de la pared de los espermatocitos. Todos los eventos descritos indican que los procesos de gametogénesis y los detalles estructurales de los gametos son por lo general características ampliamente conservadas en este grupo de algas.

Abstract InCharophyceae, the oosporangia and antheridia are the respective female and male structures of sexual reproduction. These organs are characterized by their morphological complexity and usefulness in taxonomy and systematics. Here we described the structural and ultraestructural details of Chara hydropitys gametogenesis. The fertile material from the algae was collected in a tributary stream of the Río Meléndez in Cali, Colombia (3º21´23´´N - 76º32´5.2´´W) in March 2011. The specimens were fixed and processed following the standard protocols for inclusion in resin. Thin sections (0.3-0.5 μm) were stained with toluidine O, and were observed by photonic microscopy, and additional ultrathin sections (60-90 nm) were observed by transmission electron microscopy (TEM); other samples were processed and observed by scanning electron microscopy (SEM). We found that the oosporangia are covered with spiral cells, forming 10-12 convolutions and ends in five coronula cells. The immature oosporangia wall is formed by two layers that correspond to the wall of the spiral cells and to the oosphere. In mature stages, the oosporangia wall is composed by six additional layers, three of them are provided by the oosphere and the other three are provided by the spiral cells. Oosphere size increases progressively while the spiral cells grow and divide. The cytoplasm of the immature oosphere does not exhibit conspicuous cytoplasmic inclusions, nevertheless, with the maturation, the number of starch granules increases, occupying most of the cell volume. In the spiral cells of the mature oosporangia we observed large number of chloroplast with starch accumulations, between thylakoid lamellae and a vacuole that occupies almost the entire cell. By using SEM it was possible to appreciate, that the external wall of the oospore, more accurately, on the fossa area, shows verrucose micro-ornamentations with verrucae elevations. In mature antheridia, shield cells are strongly pigmented orange due to the presence of a large number of plastoglobules between thylakoid lamellae. The spermatogenous filaments are developed from cells of the secondary capitulum; those, by unidirectional and sincronic mitotic divisions develop the spermatocytes. The biflagellate antherozoids are developed from the haploid cells by spermiogenesis. The subcellular events related with these division and differentiation processes, include first, chromatin condensation, loss of nucleoli and more activity in dictyosomes. Subsequently, retracts the cytoplasm and the organelles are aligned along the condensed nucleus and flagellar apparatus. Mature antherozoids emerge through a side wall pore of the spermatocytes. All the described events showed that the gametogenesis processes and the gametes structural details in general, are widely conserved in this algae group.

[Sporogenesis, sporoderm and mature spore ornamentation in Lycopodiaceae]. / Esporogénesis, esporodermo y ornamentación de esporas maduras en 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 elec- tron 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 abun- dant 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, marked splitting of the family.

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.

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

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 0.2-0.5 microm 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 (3-5 microm) 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.

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.

[Sporogenesis and spores of Equisetum bogotense (Equisetaceae) from mountain areas of Colombia]. / Esporogénesis y esporas de Equisetum bogotense (Equisetaceae) de las áreas montañosas de 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 4500 m 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 (70-80 microm) 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.

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.

Ontogenia de los estróbilos, desarrollo de los esporangios y esporogénesis de Equisetum giganteum (Equisetaceae) en los Andes de Colombia

Ontogeny of strobili, sporangia development and sporogenesis in Equisetum giganteum (Equisetaceae) from the Colombian Andes. 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 2 200m 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. Rev. Biol. Trop. 59 (4): 1845-1858. Epub 2011 December 01.

Estudios sobre la ontogenia del estróbilo, los esporangios y la biología reproductiva de Equisetum son escasos, por lo tanto, para la especie E. giganteum, se estudiaron estos aspectos en especímenes recolectados a orillas del Río Frío, Santander, Colombia (2 200m). Los estróbilos en diferentes etapas de maduración fueron fijados, deshidratados, embebidos en parafina, seccionados en micrótomo rotatorio y teñidos con safranina O-fast green. Las observaciones se efectuaron mediante un microscopio óptico de alta resolución con contraste diferencial de interferencia (DIC) y microscopio de fluorescencia. Los estróbilos se inician a partir del meristemo apical, tanto en el eje principal como en los laterales, sin diferencias en el proceso de ontogenia y esporogénesis entre estróbilos de diferentes ejes. Sucesivas mitosis y diferenciación celular conducen al crecimiento del estróbilo, y a la formación de los esporangióforos peltados, formados por el manubrio, o porción basal con aspecto de pedicelo, el escutelo, o porción apical aplanada y las iniciales del esporangio, los cuales se diferenciarán para formar la pared del esporangio, los esporocitos y el tapete. No se forma arquesporio y los esporocitos experimentan meiosis para formar tétradas de esporas. El tapete mantiene la integridad histológica hasta la formación de las tétradas y en esa etapa forma un plasmodio que invade la cavidad esporangial la cual rodea parcialmente las tétradas y luego las esporas, y aparecen las cámaras plasmodiales, un término propuesto aquí para las formaciones designadas en inglés "tapetal gaps". La pared del esporangio queda reducida a dos capas celulares: una externa con engrosamientos lignificados en todas las paredes celulares y una interna picnótica. Al finalizar la esporogénesis, el tapete degenera, y las esporas, con exosporio, perisporio delgado, casi membranáceo y eláteres quedan libres en la cavidad esporangial. El esporodermo, los núcleos y nucléolos presentan fluorescencia roja, inducida por coloración con safranina O, mientras que los eláteres y las células de la pared del esporangio presentan autofluorescencia amarillo-naranja.

[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.

Ontogenia del esporangio y esporogénesis del licopodio Huperzia brevifolia (Lycopodiaceae) de las altas montañas de Colombia

Sporangia ontogeny and sporogenesis of the lycopodium Huperzia brevifolia (Lycopodiaceae) from the high mountains of Colombia. Huperzia brevifolia is one of the dominant species of the genus Huperzia living in paramos and superparamos from the Colombian Andes. A detailed study of the sporangium’s ontogeny and sporogenesis was carried out using specimens collected at 4200m above sea level, in Parque Natural Nacional El Cocuy, Colombia. Small pieces of caulinar axis bearing sporangia were fixed, dehydrated, paraffin embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made, stained with aqueous Toluidine Blue (TBO). The sporangia develops basipetally, a condition that allows observation of all the developmental stages taking place throughout the caulinar axis of adult plants. Each sporangium originates from a group of epidermal cells, axilar to the microphylls. These cells undergo active mitosis, and produce new external and internal cellular groups. The sporangium wall and the tapetum originate from the external group of cells, while the internal cellular group leads to the sporogenous tissue. Meiosis occur in the sporocytes and produce simultaneous types tetrads, each one giving rise four trilete spores, with foveolate ornamentation. During the sporangium ripening, the outermost layer of the wall develops anticlinally, and inner periclinal thickenings and the innermost one perform as a secretory tapetum, which persists until the spores are completely mature. All other cellular layers colapse. Rev. Biol. Trop. 57 (4): 1141-1152. Epub 2009 December 01.

Se describe la ontogenia y la esporogénesis en H. brevifolia, en material recolectado en el Parque Nacional Natural El Cocuy (Boyacá-Colombia) a 4200m de altitud. Los esporangios se desarrollan de forma basípeta sobre el eje caulinar: los iniciales y juveniles se localizan en el ápice y los adultos a maduros, en la base. El desarrollo se inicia a partir de un grupo de células epidérmicas localizadas en las axilas que forman los microfilos con el eje caulinar. Estas células se dividen activamente por mitosis formando una masa celular externa y otra interna. La primera da origen a la pared del esporangio, de varios estratos celulares; de éstos, el estrato externo desarrolla engrosamientos en las paredes anticlinales y en la periclinal interna. El estrato celular interno se diferencia para formar el tapete secretor. Los demás estratos celulares de la pared se degradan durante la maduración del esporangio. La masa celular interna da origen al tejido esporógeno que forma los esporocitos, que experimentan la meiosis I hasta la etapa de díada. La meiosis II concluye con la formación de tétradas, constituidas por esporas en disposición tetraédrica. Las esporas son foveoladas con abertura trilete y son liberadas del esporangio a través de la dehiscencia.