Graeme P. Berlyn

Toward a new concept of the evolution of symbiotic nitrogen fixation in the Leguminosae

It is generally believed that only the nodulating species of the Leguminosae fix atmospheric nitrogen; however, anatomical, ecological and taxonomic considerations indicate that non-nodulating legume species may also fix nitrogen. To test whether nitrogen-fixing symbioses in the Leguminosae might extend to the non-nodulating species, a survey of the Leguminosae was conducted: living plants of non-nodulating species were assayed using acetylene reduction. Ethylene evolution, indicating apparent nitrogenase activity, was detected in non-nodulating species representing the major taxonomic groups of Caesalpinioideae as well as in non-nodulating species of the Papilionoideae and Mimosoideae. Non-nodules nitrogen fixation appears to have provided evolutionary precursors for the nodular symbiosis in the Leguminosae.

Spectral reflectance and photosynthetic properties of Betula papyrifera (Betulaceae) leaves along an elevational gradient on Mt. Mansfield, Vermont, USA.

We studied relationships between spectral reflectance and photosynthesis of mountain paper birch, Betula papyrifera var. cordifolia (Regel) Fern., leaves from three different elevations on Mt. Mansfield (summit elevation 1339 m above sea level) in the Green Mountains of Vermont, USA. The different reflectance indices we used all suggested progressively increasing stress with increasing elevation. The photochemical reflectance index (PRI) indicated lower photosynthetic radiation use efficiency at higher elevations, the red edge position (λRE) indicated lower chlorophyll concentrations at higher elevations, and the structure-independent pigment index (SIPI) indicated a higher carotenoid : chlorophyll a ratio at higher elevations. The rate of change in these indices with changes in elevation was much higher than we have observed in our studies of red spruce and balsam fir reflectance along a similar elevational gradient; we take this to be an indicator of the greater susceptibility of paper birch to elevation-related stressors compared to the very stress-tolerant conifers. At all light levels, photosynthesis decreased with increasing elevation; this pattern was most noticeable in the light-saturated rate of photosynthesis (A(sat)), which was nearly twice as high in low-elevation leaves (17.0 ± 1.0 μmol·m(-2)·s(-1)) than in high-elevation leaves. The quantum yield of photosynthesis (Φ) exhibited a similar trend. Furthermore, the highest elevation leaves showed a much sharper transition from the light-limited to the light-saturated part of the light response curve than did the lowest elevation leaves. The photochemical reflectance index was highly correlated with A(sat) (r(2) = 0.99) and Φ (r(2) = 0.96). In addition to contributing to our knowledge of the ecophysiology of paper birch along a steep environmental gradient, these results are further evidence of the usefulness of reflectance measures for the rapid and noninvasive detection of plant stress, especially when used in conjunction with direct measurements of photosynthesis.

Cytophotometric differentiation of high elevation spruces: physiological and ecological implications.

Red and black spruce and their hybrids can be determined by morphological indices; however, the criteria are somewhat subjective and increasingly difficult to use at higher elevations. Although the chromosome number is identical (2n = 24), red spruce has twice as much nuclear DNA (48 pg) than black spruce (24 pg) and thus the species and their hybrids can also be separated by cytophotometry. This is relevant to spruce decline studies because black spruce is much more resistant to high elevation environmental stresses, both natural and anthropogenic. It also has implications for the effect of climatic changes on the composition of high elevation spruce-fir forests because red spruce can outcompete black spruce under more mesic conditions. Four elevation transects sampling spruce on the east and west sides of Mount Washington (New Hampshire) and Camels Hump (Vermont) and a single transect on the southwest side of Whiteface Mountain (New York) were made to investigate the degree of hybridization and introgression between these two species. A positive correlation was found between increased elevation and increased black spruce genes on Mount Washington and Camels Hump. Pure black spruce was found on Mount Washington from 1356 m to 1582 m. No pure black or red spruce was found on Camels Hump although the proportion of red spruce alleles was significantly greater on Camels Hump. All trees sampled at all elevations on Whiteface Mountain were pure red spruce. Thus the proportion of black spruce alleles in high elevation spruce populations decreases from east to west. This closely parallels the increase in spruce decline which increases from east to west.

Feulgen Cytophotometry of Pine Nuclei; Effects of Fixation, Role of Formaline

The effects of 5 fixatives: FAA, Carnoys, Craf III, formalin and glutaraldehyde were analyzed for use in quantitative Feulgen cytophotometry of pine embryo tissues. Craf III and glutaraldehyde had serious deficiencies because they depressed the absorption peak, severely interfered with DNA extraction and in the case of glutaraldehyde there was considerable cytoplasmic dye-binding. Neutral 10% formalin gave good tissue fixation but did not permit the degree of enzymatic or acid extraction of DNA as did Carnoys solution. Haupts adhesive, with the usual 4% formalin as a hardener, at temperatures of 45-56 C completely prevented the enzymatic extraction of nuclear DNA by DNase and also greatly increased the resistance of the DNA to mineral acid hydrolysis. Denaturation of DNA by formalin appeared to be responsible for these results. Absorption was linearly related to both section thickness and DNA concentration per nucleus.

Seedling growth of three co-occurring Entandrophragma species (Meliaceae) under simulated light environments: implications for forest management in central Africa

It has been hypothesized that niche partitioning of the light resource by tree seedlings helps maintain species diversity in tropical forests. This paper describes a study where three co-occurring Entandrophragma spp. were grown under simulated light environments to test for a shift in species rank that would support the niche partitioning hypothesis. The species described represent some of the highest value and most heavily exploited timber species in central Africa and data on seedling light requirements has direct application to silviculture. Results showed that Entandrophragma cylindricum and E. utile performed as well as E. angolense under conditions simulating the light environment of small forest gaps. E. angolense out-performed congeners under intermediate light conditions and all three species performed poorly under full sunlight. The results provide some support for the niche partitioning hypothesis but additional studies that include understory light conditions are necessary. The growth performance results of E. utile under low light are particularly important given its extremely poor performance under a variety of fertility-moisture combinations in recent studies. These results suggest that the best way to favor recruitment of Entandrophragma spp. seedlings in natural forest would be through a series of small to intermediate sized gaps.

Isolation and culture of protoplasts from cotyledons of Pinus coulteri D. Don.

A protocol was developed for the isolation and culture of protoplasts from the cotyledons of seedlings of Pinus coulteri D. Don. Incubation of cotyledon pieces in a mixture consisting of cellulase Onozuka R10 2%, Pectolyase Y-23 0.1%, mannitol 10%, CaCl2 500 mg/l and other macro and micro-nutrients yielded viable protoplasts. After 24 hours of culture in a complex nutrient medium, the protoplasts regenerated new cell walls and the first divisions were observed within 7–10 days. Small cell colonies were formed within 15–20 days, but these started to accumulate phenolics and no further growth of the colonies was observed.

Feulgen cytophotometry of pine nuclei. II : Effect of pectinase used in cell separation

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.

Seedling performance of four sympatric Entandrophragma species (Meliaceae) under simulated fertility and moisture regimes of a Central African rain forest

Relative densities of mahogany species vary across tropical Africa and correspond with changes in soil fertility and moisture status. Seedling growth of four co-occurring African mahoganies (Entandrophragma spp.) was studied in relation to soil nutrient and moisture status in a shade-house experiment. On naturally occurring forest soils, E. cylindricum and E. utile exhibited increased relative growth rate (RGR) and decreased root mass ratio (RMR) with an increase in soil fertility while E. angolense and E. candollei did not. Changes in leaf morphology with fertility did not correspond to changes in species performance. On moist, fertile soils, E. angolense outperformed congeners but E. candollei performed equally well on moist infertile soils. Entandrophragma cylindricum performed as well as E. angolense and E. candollei on two of three soil moisture stress treatments but E. utile consistently performed poorly. Comparative seedling performance fitted well with limited available data on the distribution of Entandrophragma spp. in relation to soil fertility and moisture gradients and suggests that within-forest microsite heterogeneity may help explain the distribution of Entandrophragma species within the Dzanga-Sangha Dense Forest Reserve, Central African Republic.

Variation in Nuclear DNA Content in Pinus taeda L. Tissue Cultures of Diploid Origin

Summary Despite prolonged efforts to obtain plant regeneration from pine callus cultures, little attention has been given to the genetic condition of callus cultures. We measured nuclear DNA content in loblolly pine (Pinus taeda L.) callus during six months of continuous culture. Absolute DNA content was measured quantitatively using microspectrophotometry in conjunction with an internal standard. Twenty to thirty percent of the cells in the cultures contained nuclear DNA contents in excess of the 4C amount with the remainder having the 2C-4C amount, equivalent to cells in a diploid cell cycle. Cells with increased DNA contents arose early and were present throughout the culture period. Examination of occasional shoot meristems that formed on «callus-induction» medium revealed that shoots formed which contained cells with abnormal nuclear DNA content as compared with controls. Because polyploidy is deleterious to pines, these results demonstrate the importance of screening tissue cultures for genetic stability in an in vitro propagation program.

Regulation of foliar plasticity in conifers: developmental and environmental factors.

Foliar plasticity is widespread among woody plants, and historically most observations have been made in angiosperms. This review concentrates on examples from gymnosperms, particularly the Pinaceae, since there have been a number of recent studies on coniferous forest species. Foliar plasticity can be defined as variation in the morphology and physiological function of foliage produced over time and space within a single individual. For example, leaves produced in the sun have a higher leaf mass area (LMA) than leaves produced in the shade, and differ in their photosynthetic capacities. Sun and shade leaves are examples of heterophylly, or variation in leaf morphology in response to environmental variation in the immediate space surrounding the apical meristem producing the leaf primordia. The LMA of coniferous foliage also tends to increase with age, independently of the external environment, and this variation, called heteroblasty, appears to be a result of maturation of the apical meristem, which occurs over time. The regulation of variation due to heterophylly and heteroblasty appears to be very different. Heterophylly in response to light appears to vary linearly with available light. More massive sun foliage produced by a given apical meristem may be a response to available Photosynthetically Active Radiation (PAR) and its effects on net photosynthesis, or may be a photomorphogenetic response to the ratio of red to far red light. In contrast, heteroblastic variation can occur independently of available light, and may be the result of the developmental age of the apical meristem. It appears to vary curvilinearly with age, with the most rapid change occurring during the earliest life stages of the plant. Heteroblastic variation appears to be less plastic than heterophyllic, since grafted scions from mature or juvenile donors retain LMA characteristics of donor whether grafted onto juvenile or mature rootstocks.