Differential effects of water depth and sediment type on clonal growth of the submersed macrophyte <Emphasis Type="Italic">Vallisneria natans</Emphasis>

The response of clonal growth and ramet morphology to water depth (from 60 to 260 cm) and sediment type (sand versus organic clay) was investigated for the stoloniferous submersed macrophyte Vallisneria natans in an outdoor pond experiment. Results showed that water depth significantly affected clonal growth of V. natans in terms of clone weight, number of ramets, number of generations, clonal radius and stolon length. V. natans showed an optimal clonal growth at water depths of 110–160 cm, but at greater depths clonal growth was severely retarded. A high allometric effect was exhibited in ramet morphology. Along the sequentially produced ramet generations, ramet weight and plant height decreased while stolon length and the root:leaf weight ratio increased. When using ramet generations as covariate, sediment type rather than water depth more strongly affected the ramet characteristics. For plants grown in clay, ramet weight, ramet height and stolon length were greater, and plants exhibited lower root:leaf weight ratio. These data suggest that water depth and sediment type have differential effects on clonal growth of V. natans: Water depth appears primarily to affect numerical increase in ramets and spatial spread, whereas sediment type mainly affects biomass accumulation and biomass allocation. Handling editor: S. M. Thomaz     

Proteomic approach to substrates related to MAPK pathway in 293T cells

Abundant evidence indicates that potential scaffold proteins and adaptor or linker molecules organize and specify various MAP kinase cascades. In the present study, proteomic methodologies were applied to screen these potential molecules in combination with cell morphology and cell cycle analysis. MEK1E, MKK3b, MKK5D and MKK7D were selected as representative MKKs of four main MAPK pathways. Our results showed that similar morphological transformation and G(2)/M cell cycle arrest were promoted by the over-expressed four kinases. Furthermore, global change in response to the over-expressed four kinases was characterized by differential proteomics. Eleven distinctly changed proteins were detected, in which four proteins (serine/threonine kinase 4, glutathione S-transferase p1-1, glycoprotein IX and soluble inorganic pyrophosphatase) were reported to be relative to MAPK pathways, while the other seven proteins may be new elements of substrates of the kinases. In our experiment, the expression of platelet glycoprotein IX precursor, glutathione S-transferase p1-1, peroxiredoxin 6, Ras-related protein Rab-34 and arginase II, mitochondrial precursor was up-regulated, while the expression of serine/threonine kinase 4 (MST1) was down-regulated by the four kinases. These results suggest that these six proteins may be common targets of all the MAPK pathways in 293T cell line. Interestingly, the expression of splicing factor 3B subunit 4 and soluble inorganic pyrophosphatase (Ppase) was specifically up-regulated by MEK1E and MKK5D, and by MEK1E, MKK3b and MKK5D, respectively. The expression of methylglyoxalase was down-regulated by MEK1E and MKK7D. Furthermore, the expression of ADP-ribosylation factor-like protein 1 was up-regulated by MKK5D but down-regulated by MKK3b and MKK7D. These findings revealed the characteristic molecular responses to four MKKs. In conclusion, our study not only confirms that MST1, glutathione S-transferase p1-1, glycoprotein IX and soluble PPase belong to MAPK pathways, but also provides seven novel molecules for the further study of the pathways.     

Phenotypic plasticity and contemporary evolution in introduced populations: evidence from translocated populations of white sands pupfish (<Emphasis Type="Italic">Cyrpinodon tularosa</Emphasis>)

Contemporary evolution has been shown in a few studies to be an important component of colonization ability, but seldom have researchers considered whether phenotypic plasticity facilitates directional evolution from the invasion event. In the current study, we evaluated body shape divergence of the New Mexico State-threatened White Sands pupfish (Cyprinodon tularosa) that were introduced to brackish, lacustrine habitats at two different time in the recent past (approximately 30 years and 1 year previously) from the same source population (saline river environment). Pupfish body shape is correlated with environmental salinity: fish from saline habitats are characterized by slender body shapes, whereas fish from fresher, yet brackish springs are deep-bodied. In this study, lacustrine populations consisted of an approximately 30-year old population and several 1-year old populations, all introduced from the same source. The body shape divergence of the 30-year old population was significant and greater than any of the divergences of the 1-year old populations (which were for the most part not significant). Nonetheless, all body shape changes exhibited body deepening in less saline environments. We conclude that phenotypic plasticity potentially facilitates directional evolution of body deepening for introduced pupfish populations.     

Tree dynamics and co‐existence in the montane–sub‐alpine ecotone: the role of different light‐induced strategies

Questions: Is light availability the main factor driving forest dynamics in Pyrenean sub‐alpine forests? Do pines and firs differ in growth, mortality and morphological response to low light availability? Can differences in shade tolerance affect predictions of future biome changes in Pyrenean sub‐alpine forests in the absence of thermal limitation? Location: Montane–sub‐alpine ecotones of the Eastern Pyrenees (NE Spain). Methods: We evaluated morphological plasticity, survival and growth response of saplings of Scots pine, mountain pine and silver fir to light availability in a mixed forest ecotone. For each species, we selected 100 living and 50 dead saplings and measured size, crown morphology and light availability. A wood disk at root collar was then removed for every sapling, and models relating growth and mortality to light were obtained. Results: Fir had the lowest mortality rate (<0.1) for any given light condition. Pines had comparable responses to light availability, although in deep shade Scots pine risked higher mortality (0.35) than mountain pine (0.19). Pines and fir developed opposing strategies to light deprivation: fir employed a conservative strategy based on sacrificing height growth, whereas pines enhanced height growth to escape from shade, but at the expense of higher mortality risk. Scots pine showed higher plasticity than mountain pine for all architectural and morphological traits analysed, having higher adaptive capacity to a changing environment. Conclusions: Our results support the prediction of future biome changes in Pyrenean sub‐alpine forests as silver fir and Scots pine may find appropriate conditions for colonizing mountain pine‐dominated stands due to land‐use change‐related forest densification and climate warming‐related temperature increases, respectively.     

<Emphasis Type="Italic">Ophiocordyceps pulvinata</Emphasis> sp. nov., a pathogen of ants with a reduced stroma

Ophiocordyceps pulvinata, a pathogen of ants, is formally described as a new species. Genus level designation of this species is difficult due to several apparently conflicting morphological and ecological characters. Affinity with Ophiocordyceps is suggested by the dark color stroma and ascospore morphology. However, the species was included in a book of entomopathogenic fungi of Japan as Torrubiella sp. due to the production of perithecia on an astipitate stroma. Phylogenetic analyses of molecular data support a close relationship with O. unilateralis, a finding consistent with morphological characteristics of the color, asci and ascospores and ecological traits of host affiliation. Thus, O. pulvinata represents another example of the loss of stipe for the hypocrealean arthropod pathogenic fungi and highlights the utility of asci and ascospore morphology as taxonomically informative characters of closely related taxa.     

Photosynthetic and morphological characters of Caragana microphylla in different slope aspects and positions

In order to find out how small scale topographical factors affect growth and physiological characters of Caragana microphylla, which is a widely distributed shrub species and has an important role in restoring degraded grassland in natural ecosystem, a natural population of C. microphylla was chosen in a typical steppe community in June, 2009. The population was 34 km to the southeast of Xilinhot City, China, and a total of 54 shrubs were selected from different slope aspects and positions. We investigated the photosynthetic and morphological characters of these shrubs and analyzed the relationship between plant traits of C. microphylla and soil nitrogen and phosphorus availability. Moreover, the relationship between plant traits of C. microphylla and herbaceous aboveground biomass was studied. (1) The maximum net photosynthetic rate (Pn_max) was significantly lower on shady slopes than that on sunny slopes and higher on upper slopes than that on lower slopes. Stomatal conductance (Gs), net photosynthetic rate/intercellular CO₂ concentration (Pn/Ci) and intercellular CO₂ concentration (Ci) under saturated irradiance showed similar trends with slope aspect and position. Likewise, the maximum photochemical efficiency of PSII (Fv/Fm), PSII potential activity (Fv/Fo) and the first-year shoot morphological characters of C. microphylla were also correlated with slope aspect and position. (2) Soil nitrogen availability showed no significant effect on photosynthetic or morphological traits of C. microphylla, however, there were several significant relationships between soil phosphorus availability and plant traits. Dry weight, shoot length, compound leaf size, and leaflet length of first-year shoots of C. microphylla were significantly negatively correlated with soil C:P ratio. Though not significant, photosynthetic parameters under saturated light and other morphological characters of first-year shoots were negatively correlated with soil C:P ratio, i.e., these traits increased with increasing soil phosphorus availability. These suggested that the difference of soil phosphorus availability played an important role in making C. microphylla having different photosynthetic and morphological characters on different slope aspects and positions. The individuals grown in relatively P-rich site had longer shoots and larger leaves and grew better. Low phosphorus content was thought to limit photosynthetic activity through several different mechanisms, including both stomatal and non-stomatal limitations, the latter being more likely in the present study. (3) Photosynthetic and morphological characters of C. microphylla were all negatively correlated with herbaceous aboveground biomass, though only Pn/Ci and length of first-year shoot were significantly correlated with it. This indicated that the difference in plant community was a factor making C. microphylla have different growth and physiological characters on different aspects and positions of slope. A number of studies showed that grazing of the herbaceous layer promoted the establishment and proliferation of woody species, and then led to grassland deterioration; but in arid and semi-arid ecosystems, some widely distributed shrub species like C. microphylla created resource islands and provide favorable microhabitat for grass species. In the present study, we found negative correlations between traits of C. microphylla and herbaceous aboveground biomass. We suggested that the removal of livestock grazing result in the decrease of the distribution C. microphylla and increase of grass coverage, and lead to the restoration of the typical steppe.     

Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

Morphological examination during in vitro cartilage formation by human mesenchymal stem cells

The formation of the skeleton through endochondral ossification is one of the most complex processes in development. One approach to resolving this complexity is to examine simplified systems. In vitro cartilage formation by mesenchymal stem cells (MSCs) is observed when the cells are cultured as a micromass. Several studies have confirmed the molecular events, showing the usefulness of these cells as a differentiation model. We have elucidated the process of cartilage formation in MSCs from the morphological point of view by light and transmission electron microscopy and immunohistochemical examination. The morphology of the MSCs changed from spherical to spindle-shaped, and the cells aggregated and formed junctional complexes during Day 1. At Day 7, three layers were observed. The superficial zone consisted of several layers of elongated cells with junctional complexes. The middle zone was composed of apoptotic bodies, and the deep zone was occupied by chondrocyte-like cells excreting extracellular matrices. At Day 14, the middle zone had disappeared, and the chondrocyte-like cells in the deep zone were detected within cartilage lacuna. They were covered by cartilage matrices containing collagen types I, II, and X and chondroitin sulfate. By Day 21, the outer layer consisting of spindle-shaped cells had disappeared in places. As the pellet grew, the outer layer seemed to be unable to stretch to maintain a constant covering around the pellet. Our findings have thus revealed that MSCs change their morphology depending upon their microenvironment during differentiation. In vitro cartilage formation by MSCs makes it possible to clarify the detailed morphological events that occur during chondrogenesis. S. Ichinose and I. Sekiya contributed equally to this study