Fitness of an allopolyploid rupicolous fern compared with its diploid progenitors: from sporogenesis to sporophyte formation.

PREMISE: When two populations of related cytotypes grow in sympatry, the rarer cytotype tends to be excluded due to a frequency-dependent mating disadvantage. Evolutionary models predict that polyploids, which are typically the rarer cytotype upon first formation, should have higher relative fitness and/or higher selfing rates to establish and then coexist with diploid parents. METHODS: Performance during early recruitment was compared among three co-occurring rupicolous fern species: the allotetraploid Cheilanthes tinaei and its diploid ancestors, C. hispanica and C. maderensis. In culture experiments, fresh spores and samples of soil spore banks were tested for variation among cytotypes in germination, survival, fecundity, and mating system of gametophytes. RESULTS: Compared with its diploid parents, C. tinaei fresh spores had higher abortion percentages, lower dispersal ability as a result of its larger spores, and similar vigor at germination. For gametophytes from soil spore banks, C. tinaei had high survival similar to C. maderensis, but its sex expression resembled that of C. hispanica, with a high proportion of males. Patterns of sporophyte formation by females and bisexuals indicate that the polyploid does not have an increased gametophytic selfing rate. Gametophytes were larger in C. tinaei, but its reproductive success (sporophyte formation) was intermediate relative to diploids. CONCLUSIONS: Our results show no evidence of higher selfing or fitness advantage of the allopolyploid over both diploid parents at any stage of early recruitment. These two unexpected findings suggest that further factors, such as niche differentiation, play a more important role in cytotype coexistence.

Cyclic AMP induction of Dictyostelium prespore gene expression requires autophagy.

Dictyostelium discoideum amoebas display colonial multicellularity where starving amoebas aggregate to form migrating slugs and fruiting bodies consisting of spores and three supporting cell types. To resolve the cell signalling mechanism that control sporulation, we use insertional mutagenesis of amoebas transformed with fusion constructs of spore genes and red fluorescent protein. We identified the defective gene in a mutant lacking spore gene expression as the autophagy gene Atg7. Directed knock-out of atg7 and of autophagy genes like atg5 and atg9 yielded a similar phenotype, with lack of viable spores and excessive differentiation of stalk cells. The atg7-, atg5- and atg9- cells were specifically defective in cAMP induction of prespore genes, but showed enhanced cAMP stimulation of prestalk genes at the same developmental stage. The lack of prespore gene induction in the autophagy mutants was not due to deleterious effects of loss of autophagy on known components of the cAMP pathway, such as cAMP receptors and their cAMP-induced phosphorylation and internalization, PKA and the transcription factors SpaA and GbfA, or to lack of NH3 production by proteolysis, which was previously suggested to stimulate the spore pathway. Our continued mutagenesis approach is the most likely to yield the intriguing link between autophagy and prespore gene induction.

Deletion of Htt cause alterations in cAMP signaling and spatial patterning in Dictyostelium discoideum.

In the present study, we have analyzed in detail the functions of Htt during growth and development of the protist, Dictyostelium discoideum by creating mutants (both overexpressor and knockout). The mRNA was present at all stages of growth and development. Overexpression of htt did not show any major anomaly, while deletion resulted in delayed aggregation territory formation and showed asynchronous development especially after slug stage. The slugs formed by htt - cells showed aberration in anterior-posterior boundary, showing increased prestalk region. DdHtt regulates STAT transcription factors in the tip organizer region that help maintain patterning and culmination. In chimeras with the wild-type, htt - cells preferentially localized to the tip of the slug and basal disc regions of the fruiting body showing prestalk/stalk bias, while the overexpressing cells majorly populated the prespore/spore region showing spore bias. These differences could be attributed to protein kinase A (PKA)-regulated cyclic adenosine monophosphate (cAMP) signaling.

A new species of Myxosporea, Henneguya quelen, from silver catfish Rhamdia quelen (Siluriforme: Pimelodidae) in the Amazonian region.

Henneguya quelen n. sp. (Myxosporea) is described from the kidney of the silver catfish, Rhamdia quelen (Quoy & Gaimard, 1824), from Marajó Island. In all, 40% of the 57 silver catfish collected from the Paracauari River in the municipality of Salvaterra presented cysts in the kidney. The cysts were whitish, spherical, and 0.45 mm in diameter. The histological analyses revealed tubular renal compression and cellular degeneration in the area adjacent to the xenomas. The spores are ellipsoid, with well-marked valves, caudal projection, and two elongated polar capsules of equal size. The mature spores measure approximately 15.6 µm in length, with the caudal appendage split posteriorly; 24.3 µm in length, for a total spore length of 40.0 µm; and a width of 4.1 µm. The polar capsules are pyriform, 5.5 µm long and 1.7 µm wide. A phylogenetic analysis based on Bayesian inference confirmed that the specimens represented a new species, which was denominated Henneguya quelen n. sp.

Structure and Development of the Auxospore in Ardissonea crystallina (C. Agardh) Grunow Demonstrates Another Way for a Centric to Look Like a Pennate.

Reproductive development in Ardissonea crystallina revealed a unique mode of enlargement involving a combination of novel and known structures. In light microscopy, auxospores of this elongated polar centric diatom were superficially similar to the auxospores of pennates. With SEM we found three different components in the auxospore wall. In the youngest, nearly spherical cell-stage, the wall consisted only of a delicate veil containing minute siliceous spherules. Incunabular elements developed underneath this layer. Second, a previously unknown form of specifically modified incunabular scales shaped the subsequent ellipsoidal-capsule auxospore stage. Third, there was a clear contribution of scales to the development of scaly transverse perizonial bands (or scaly bands, for brevity). Such bands, although noted by previous researchers, have not been fully appreciated for the evolutionary information they may convey: possibly common among polar centrics but not pennates. Finally, we propose maintaining the term transverse perizonium to refer to these bands in polar diatoms, but to introduce the differentiation of scaly bands described here from pinnate bands (currently known as typical of pennates). Further research into band types among polar centrics may provide new insights into the relationship between the groups within polar centrics that are currently unresolved by molecular methods.

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

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

Henneguya laseeae n. sp. from flathead catfish (Pylodictis olivaris) in the upper Mississippi River.

A novel species of Henneguya was isolated from flathead catfish (Pylodictis olivaris) captured in the upper Mississippi River near Lansing (Allamakee County), IA, and La Crosse (La Crosse County), WI. Designated Henneguya laseeae n. sp., this novel species is described using critical morphological features, histology, and 18S ribosomal RNA gene sequence. Ovoid cysts, ranging from 1200 to 1800 µm in width, tended to be at filament tips or in the distal third, often directly on the filament midline, but occasionally paramedian. Lanceolate-shaped myxospores were consistent with those of the genus Henneguya. The spore body was 16.2 ± 0.5 µm (mean ± standard deviation; range = 15.1-17.0 µm) in length, 6.0 ± 0.4 µm (5.1-6.6 µm) in width, and 4.7 ± 0.2 µm (4.4-4.9 µm) thick. The two polar capsules at the anterior of the spore body were 5.9 ± 0.3 µm (5.3-6.3 µm) in length and 1.8 ± 0.1 µm (1.6-2.1 µm) in width and contained six to seven turns in the polar filament. The caudal processes tapered to fine points and were 54.3 ± 2.9 µm (49.1-61.7 µm) in length. Total spore length was 70.4 ± 3.3 µm (64.5-79.4 µm). The spores and plasmodium of this species are of similar size and morphology to other species of Henneguya from ictalurid fishes. Additionally, the 18S rRNA gene sequences placed this isolate within a clade populated by Henneguya spp. from North American ictalurids. This is the first reported species of Henneguya from flathead catfish.

Henneguya paraensis n. sp. (Myxozoa; Myxosporea), a new gill parasite of the Amazonian fish Cichla temensis (Teleostei: Cichlidae): morphological and molecular aspects.

The present study describes light microscopy, transmission electron microscopy, and molecular analyses of a myxosporid found parasitizing the gill region of the teleost fish Cichla temensis, collected from the Tocantins River, near Cametá, Pará State, Brazil. The prevalence of infection was 60 %. The spore-containing cysts that were located in the gill lamellae were oval and whitish. The spores had a mean length of 42.3 ± 0.65 µm; fusiform body, 12.8 ± 0.42-µm long and 8.6 ± 0.32-µm wide; each of the two valves exhibited a tapering tail of 29.5 ± 0.73 µm length. The spores had two polar capsules, 7.4 ± 0.16-µm long by 2.6 ± 0.08-µm wide, containing a polar filament with 5-7 twists. The spores differ from the species previously described, and phylogenetic analysis based on spore morphology and molecular aspects indicated that the fish parasite Henneguya sp. has a strong trend to form clades mainly based on the environment and host order/family. Thus, we conclude that the species belongs to the family Myxobolidae, genus Henneguya, which comprises a new species: Henneguya paraensis n. sp.

Between sexual and apomictic: unexpectedly variable sporogenesis and production of viable polyhaploids in the pentaploid fern of the Dryopteris affinis agg. (Dryopteridaceae).

BACKGROUND AND AIMS: In ferns, apomixis is an important mode of asexual reproduction. Although the mechanisms of fern reproduction have been studied thoroughly, most previous work has focused on cases in which ferns reproduce either exclusively sexually or exclusively asexually. Reproduction of ferns with potentially mixed systems and inheritance of apomixis remains largely unknown. This study addresses reproduction of the pentaploid Dryopteris × critica, a hybrid of triploid apomictic D. borreri and tetraploid sexual D. filix-mas. METHODS: Spore size, abortion percentage and number of spores per sporangium were examined in pentaploid plants of D. × critica grown in an experimental garden. The sporangial content of leaf segments was cultivated on an agar medium, and DNA ploidy levels were estimated by DAPI flow cytometry in 259 gametophytes or sporophytes arising from the F2 generation of the pentaploid hybrid. KEY RESULTS: The hybrid is partly fertile (89-94% of aborted spores) and shows unstable sporogenesis with sexual and apomictic reproduction combined. The number of spores per sporangium varied from approx. 31 to 64. Within a single sporangium it was possible to detect formation of either only aborted spores or various mixtures of aborted and well-developed reduced spores and unreduced diplospores. The spores germinated in viable gametophytes with two ploidy levels: pentaploid (5x, from unreduced spores) and half of that (approx. 2·5x, from reduced spores). Moreover, 2-15% of gametophytes (both 2·5x and 5x) formed a viable sporophyte of the same ploidy level due to apogamy. CONCLUSIONS: This study documents the mixed reproductive mode of a hybrid between apomictic and sexual ferns. Both sexual reduced and apomictic unreduced spores can be produced by a single individual, and even within a single sporangium. Both types of spores give rise to viable F2 generation gametophytes and sporophytes.

Morfología y anatomía de ejes caulinares, licofilos y esporangios de Phlegmariurus phylicifolius: un aporte a la sistemática de las Lycopodiaceae neotropicales / Morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius: a contribution to the systematics of Neotropical Lycopodiaceae

Phlegmariurus is the only genus of Lycopodiaceae with the species grouped in 22 informal groups. Species level relationships within Phlegmariurus are poorly understood and their circumscriptions require a thorough molecular and morphological review. A detailed study of morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius was carried out in order to contribute to the elucidation of species circumscription in the informal group Phlegmariurus phlegmaria. Small pieces of caulinar axes bearing trophophylls, sporophylls and sporangia were fixed, dehydrated, Histowax (paraffin) embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made and stained with the same technique. P. phylicifolius includes slender, pendulous plants up to 40cm long. Shoots heterophyllous, in the basal divisions ca. 10-20(-25)mm in diameter including the trophophylls, then abruptly constricted to (l-) 1.5-2(-2.5)mm in diameter including the imbricate, reduced sporophylls. Trophophylls are borne in alternating whorls of three, or decussate, subdecussate, or alternate, widely spaced in alternate leaved caulinar axes portions, perpendicular to the caulinar axes to falcately ascending, lanceolate to linear-lanceolate, with flat to slightly revolute margins. Each lycophyll is supplied by a single central vascular bundle, connected to a protoxylem pole in the stele. At the site of leaf-trace departure, no leaf (lycophyll) gap is present. Caulinar axes excluding leaves 0.7-1.2mm thick at the base, upward tapering to ca. 0.5mm. Caulinar axes present unistratified epidermis and endodermis, the cortex is characterized by the presence of a trabecular structure of lisigenous origin formed in the parenchimatous tissue next to the endodermis. The vascular tissue occupies the central part of the caulinar axes, forming a plectostele of subradiate organization, with five poles of protoxylem. The epidermal cells present sinuous anticlinal walls; invaginations in the inner side of external periclinal wall of the epidermal cells could be probably adaptive morphological feature of a water deficient environment. Leaves of constricted terminal divisions are decussate, or subdecussate, continuously or discontinuously sporangiate, appressed, abaxially rounded to carinate, widely lanceolate to widely ovate or subcordate, acute to mucronate or cuspidate, shorter than the sporangia. Each sporangium originates from a group of epidermal cells, axilar to the sporophylls. The cell walls of epidermal cell of the sporangia are Huperzioideae type. The morphological studies of trophophylls contribute to confirm the differences between P. phylicifolius and P. subulatus. Rev. Biol. Trop. 62 (3): 1217-1227. Epub 2014 September 01.

Phlegmariurus es el único género de Lycopodiaceae con las especies reunidas en 22 grupos informales. Las relaciones a nivel de especie dentro de Phlegmariurus están pobremente estudiadas y la circunscripción de las mismas requiere profundos exámenes moleculares y morfológicos. Se ha llevado a cabo un estudio detallado de la morfología y la anatomía de ejes caulinares, licofilos y esporangios de P. phylicifolius, con el fin de contribuir al esclarecimiento en la delimitación de las especies en el grupo Phlegmariurus phlegmaria. Segmentos de ejes caulinares con trofofilos, esporofilos y esporangios fueron fijados, deshidratados, incluidos en Histowax (parafina), cortados con un micrótomo rotatorio y coloreados usando la técnica tradicional Safranina O-Verde Rápido; además se hicieron cortes a mano alzada y se colorearon con la misma técnica. P. phylicifolius incluye plantas colgantes y péndulas de hasta 40cm de longitud. Los ejes son heterofilos, de aproximadamente 10-20(-25)mm de diámetro en las divisiones basales incluyendo los trofofilos, luego abruptamente reducidos a (l-) 1.5-2(-2.5)mm de diámetro incluyendo los esporofilos reducidos e imbricados. Los trofofilos están dispuestos en anillos alternantes de a tres, o decusados, subdecusados o alternos, dispuestos en forma espaciada en los ejes caulinares, perpendiculares al tallo hasta falcadamente ascendentes, lanceolados a lineal-lanceolados, con márgenes lisos o levemente revolutos. Cada licofilo está provisto de un haz vascular simple y central, conectado a un polo de protoxilema de la estela y sin laguna foliar. Los tallos poseen un ancho de 0.7-1.2mm en la base, excluyendo los licofilos, estrechándose hasta cerca de 0.5mm hacia el ápice. Los ejes caulinares presentan una epidermis uniestratificada y endodermis, la corteza se caracteriza por la presencia de una estructura trabecular de origen lisígeno formada en el tejido parenquimático próximo a la endodermis. El tejido vascular ocupa la parte central del eje caulinar, formando una plectostela de organización subradiada, con cinco polos de protoxilema. Las células epidérmicas presentan paredes anticlinales sinuosas; las invaginaciones en la cara interna de la pared periclinal externa podrían ser probablemente un característica morfológica adaptativa a un ambiente con períodos de sequía. Las hojas de las porciones apicales son decusadas o subdecusadas, con esporangio de disposición continua o discontinua, adpresas, abaxialmente redondeadas a carinadas, ampliamente lanceoladas a ovadas o subcordadas, ápice agudo a mucronado o cuspidado, más corto que el esporangio. Cada esporangio se origina de un grupo de células epidérmicas, en la axila de los esporofilos con el eje caulinar. Las paredes celulares de las células epidérmicas del esporangio son de tipo Huperzioideae. El estudio de la morfología de los trofofilos contribuye a confirmar las diferencias entre P. phylicifolius y P. subulatus.

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.

Two novel myxosporean species (Myxosporea: Bivalvulida), Myxobolus marumotoi n. sp. and Cardimyxobolus japonensis n. sp., from the dark sleeper, Odontobutis obscura, in Japan.

Two new myxosporean species, Myxobolus marumotoi n. sp. and Cardimyxobolus japonensis n. sp. (Myxozoa: Myxosporea: Bivalvulida), are described from the dark sleeper, Odontobutis obscura, in Japan, based on their morphological and molecular characterizations. Plasmodia of M. marumotoi n. sp. (Myxobolidae) grew in the myofiber of trunk muscles, forming pseudocysts. The rounded spore was relatively large, measuring 13.3-15.0 µm (average 13.8) in length, 14.2-15.0 µm (14.6) in width, and 10.0-11.7 µm (10.8) in thickness, with two subspherical polar capsules of 7.9-9.6 µm (8.4) in length by 5.4-6.3 µm (5.9) in width (n = 15). The polar capsules were directed toward the apex of the spore, packing five to six spirals of the polar filament. Plasmodia of C. japonensis n. sp. (Ortholineidae) were surrounded by thin fibrous tissue, forming cysts in the lamina propria of the alimentary tract. The spore was ovoid, wider than long, in valvular view and spindle-shaped in sutural view. It measured 8.8-10.4 µm (9.4) in length, 11.3-12.5 µm (11.9) in width, and 5.2-6.7 µm (5.8) in thickness, with two ovoid polar capsules of 4.2-5.0 µm (4.7) in length by 2.9-3.8 µm (3.3) in width (n = 15). The shell valves of spores often showed a flattened anterior border and semicircular posterior border, and the two polar capsules were directed toward opposite lateral sides. In addition, the sporoplasm contained an iodinophilous vacuole. Almost complete small-subunit (SSU) rDNA sequences, except for primer flanking regions, were obtained for both species; 1,996 bp long for the former and 1,588 bp long for the latter. On phylogenetic trees based on the SSU rDNA sequences of representative species of Bivalvulida, M. marumotoi n. sp. and C. japonensis n. sp. formed a distinct branch in the Henneguya/Myxobolus clade or near but outside this clade, respectively. This study is the first report of the genetic characterization for the genus Cardimyxobolus.

Genome size evolution in Ontario ferns (Polypodiidae): evolutionary correlations with cell size, spore size, and habitat type and an absence of genome downsizing.

Genome size is known to correlate with a number of traits in angiosperms, but less is known about the phenotypic correlates of genome size in ferns. We explored genome size variation in relation to a suite of morphological and ecological traits in ferns. Thirty-six fern taxa were collected from wild populations in Ontario, Canada. 2C DNA content was measured using flow cytometry. We tested for genome downsizing following polyploidy using a phylogenetic comparative analysis to explore the correlation between 1Cx DNA content and ploidy. There was no compelling evidence for the occurrence of widespread genome downsizing during the evolution of Ontario ferns. The relationship between genome size and 11 morphological and ecological traits was explored using a phylogenetic principal component regression analysis. Genome size was found to be significantly associated with cell size, spore size, spore type, and habitat type. These results are timely as past and recent studies have found conflicting support for the association between ploidy/genome size and spore size in fern polyploid complexes; this study represents the first comparative analysis of the trend across a broad taxonomic group of ferns.

Structure and function of CrACA1, the major PM-type Ca2+-ATPase, expressed at the peak of the gravity-directed trans-cell calcium current in spores of the fern Ceratopteris richardii.

Spores of the fern Ceratopteris richardii have proven to be a valuable single-cell system for studying gravity responses. The earliest cellular change directed by gravity in these cells is a trans-cell calcium current, which peaks near 10 h after the spores are induced to germinate. This current is needed for gravity-directed axis alignment, and its peak is coincident with the time period when gravity polarises the direction of subsequent nuclear migration and rhizoid growth. Transcriptomic analysis of genes expressed at the 10-h time point revealed several that encode proteins likely to be key components that either drive the current or regulate it. Notable among these is a plasma membrane (PM)-type Ca(2+) ATPase, CrACA1, whose activity pumping Ca(2+) out of cells is regulated by gravity. This report provides an initial characterisation of the structure and expression of this protein, and demonstrates its heterologous function complementing the K616 mutant of yeast, which is deficient in PM-type Ca(2+) pump activity. Gravity-induced changes in the trans-cell Ca(2+) current occur within seconds, a result consistent with the hypothesis that the force of gravity can rapidly alter the post-translational state of the channels and pumps that drive this current across spore cells. This report identifies a transporter likely to be a key driver of the current, CrACA1, and characterises the role of this protein in early germination and gravity-driven polarity fixation through analysis of expression levels, functional complementation and pharmacological treatments. These data, along with newly available transcriptomic data obtained at the 10-h time point, indicate that CrACA1 is present, functional and likely a major contributing component of the trans-cell Ca(2+) efflux. CrACA1 is not necessary for polar axis alignment, but pharmacological perturbations of it disrupt rhizoid development. These data support and help refine the post-translational modification model for gravity responses.

Myxobolus leafa sp. nov. (Myxozoa: Bivalbulida) from the gill filament of Labeo bata (Hamilton) from Manipur, India.

OBJECTIVE: The aim of this study was to investigate the gill filaments of Labeo bata in India parasitologically and to describe the new Myxozoa parasite species, Myxobolus leafa sp. nov., which was determined. METHODS: Fish were collected alive from fish farms from Manipur (India). Sporogonic plasmodia were removed with sterile forceps and observed under phase contrast microscope. For permanent preparations, air dried smears were stained with Giemsa after fixation in acetone free absolute methanol. RESULTS: Mature spores of the new species appear as leaf-like structures in valvular or frontal view, tapering at both ends. The anterior end of the spore is broader than the posterior end. Spores measure 15.3-11.8 µm (16.91 ± 1.16) in length and 13.6-15.3 µm (14.15 ± 0.67) in breadth. CONCLUSION: The new gill parasite species, Myxobolus leafa sp. nov., was described from fish in India. The prevalence of the parasite was 3.3% and two stages (cyst and spore) of the parasite existed in its life cycle.

PsVPS1, a dynamin-related protein, is involved in cyst germination and soybean infection of Phytophthora sojae.

Plant pathogens secrete effector proteins to suppress plant immunity. However, the mechanism by which oomycete pathogens deliver effector proteins during plant infection remains unknown. In this report, we characterized a Phytophthora sojae vps1 gene. This gene encodes a homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene vps1 that mediates budding of clathrin-coated vesicles from the late Golgi, which are diverted from the general secretory pathway to the vacuole. PsVPS1-silenced mutants were generated using polyethylene glycol-mediated protoplast stable transformation and were viable but had reduced extracellular protein activity. The PsVPS1-silenced mutants showed impaired hyphal growth, and the shapes of the vacuoles were highly fragmented. Silencing of PsVPS1 affected cyst germination as well as the polarized growth of germinated cysts. Silenced mutants showed impaired invasion of susceptible soybean plants regardless of wounding. These results suggest that PsVPS1 is involved in vacuole morphology and cyst development. Moreover, it is essential for the virulence of P. sojae and extracellular protein secretion.

Kudoa ogawai n. sp. (Myxozoa: Multivalvulida) from the trunk muscle of Pacific barrelfish Hyperoglyphe japonica (Teleostei: Centrolophidae) in Japan.

Kudoa ogawai n. sp. (Myxozoa; Multivalvulida) is described from the trunk muscle of Pacific barrelfish Hyperoglyphe japonica caught in Japan. Oval to ellipsoidal cysts, white to yellowish in colour, 1-2 mm in size, were filled with numerous spores having four polar capsules. Spores were stellate with rounded peripheral edges in apical view, and garlic shaped with four prominent apical projections in side view. Average (range) spore size was 13.3 (12.0-14.2) µm in width and 9.0 (8.3-9.7) µm in length. The spore dimensions and the apical projections of K. ogawai n. sp. were clearly distinguishable from the other muscle-infecting species. Molecular analyses of 18S rDNA and 28S rDNA sequences showed that K. ogawai n. sp. is closely related to Kudo iwatai and Kudoa hemiscylli (95% and 92% genetic similarity, respectively), but the present species is morphologically distinct from the two species. Phylogenetic analysis of 18S and 28S rDNA placed K. ogawai n. sp. sister to all Kudoa spp. available in GenBank.

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

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

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

[Cytogenetic peculiarities of cell genesis in apical meristems under gametophytic apomixis (using autonomous apomicts of the Asteraceae as an example)].

Cytogenetic peculiarities of cell genesis in apical meristems of apomicts has been analyzed using a series of the Asteraceae species as an example. The extent to which aneu- and mixoploids are spread among plants in the investigated populations of the Asteraceae species is so high (up to 30-60% of the studied plants and their offspring), that it seems reasonable to suppose that their rise is a natural phenomenon. It has been shown that in the aposporous facultative apomict Pilosella officinarum microgametophyte is a relatively stable element of the seed reproduction system from the point of view of caryotypical variation.

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

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

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