Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda)

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.     

PHOTOADAPTATION AND THE “PACKAGE” EFFECT IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

In the marine unicellular chlorophyte, Dunaliella tertiolecta Butcher, the spectrally averaged m vivo absorption cross section, normalized to chlorophyll a (so-called a* values), vary two-fold in response to changes in growth irradiance. We used a kinetic approach to examine the specific factors which account for these changes in optical properties as cells photoadapt. Using Triton X-100 to solubilize membranes, we were able to differentiate between “package” effects and pigmentation effects. Our analyses suggest that 43–49% of the variability in a* is due to changes in pigmentation, whereas 51–57% is due to the “package” effect. Further analyses revealed that changes in cell sue did not significantly affect packaging, while thylakoid stacking and the transparency of thylakoid membranes were important factors. Our results suggest that thylakoid membrane protein/lipid ratios change during photoadaptation, and these changes influence the effective rate of light harvesting per unit chlorophyll a.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

Do Experience and Body Size Play a Role in Responses of Larval Ringed Salamanders,Ambystoma annulatum,to Predator Kairomones? Laboratory and Field Assays

Prey may experience ontogenetic changes in vulnerability to some predators, either because of changes in morphology or experience. If prey match their level of antipredator behavior to the level of predatory threat, prey responses to predators should reflect the appropriate level of threat for their stage of development. For larval salamanders, responses to predators may change with body size because larger larvae are less vulnerable to predation by gape‐limited predators or because fleeing responses by large salamanders may be more effective than for smaller salamanders. In a field experiment, small larval ringed salamanders, Ambystoma annulatum, responded to chemical stimuli (‘kairomones’) from predatory newts, Notophthalmus viridescens, with an antipredator response (decreased activity). Laboratory‐reared larvae decreased their activity following exposure to newt kairomones, indicating that larval ringed salamanders do not require experience with newts to recognize them as predators. In both experiments, larvae distinguished between chemical stimuli from newts and stimuli from tadpoles (non‐predators) and a blank control. In a third experiment, field‐caught (experienced) larvae showed a graded response to newt kairomones based on their body size: small larvae tended to decrease their activity while larger larvae showed no change or an increase in activity. This graded response was not observed for neutral stimuli, indicating that it is predator‐specific. Therefore, ringed salamander larvae exhibit threat‐sensitive ontogenetic changes in their response to chemical stimuli from predatory newts.     

The ecosystem approach to environmental assessment: moving from theory to practice

The ecosystem approach to environmental management is viewed by many as being fundamental to the development of appropriate management strategies. While this approach represents a major advance in the way researchers view environmental assessment, the approach in itself does not provide practical information as to what questions to ask and what tools to use in assessing and managing ecosystems. Similarly, the concept of ecosystem health, as it is usually defined, has little practical value for ecosystem managers. We suggest the next stage in environmental assessment will be the development of specific frameworks designed to assess individual ecosystems. Of primary importance is the need to consider the basic structure and function of the ecosystem itself. Such consideration, together with explicit identification of anthropogenic stresses particular to the system, serves to identify those components most at risk and those issues most deserving of attention. Researchers should explore critical linkages between environmental stressors and their observable, measurable and predictable effects on ecological parameters and use this understanding to develop a management strategy that incorporates appropriate ecological indicators. The importance of these considerations will be illustrated using examples from the Northern River Basins Study.     

Utility of field-based artificial streams for assessing effluent effects on riverine ecosystems

Experimentation using field-based artificial streams provides a promising, complimentary approach to biomonitoring assessments because artificial streams provide control over relevant environmental variables and true replication of treatments. We have used large and small artificial stream systems, based in the field, to examine the effect of treated bleached kraft pulp mill effluent (BKME) on the benthos of three large rivers in western Canada. Under natural regimes of temperature, water chemistry, and insolation, these artificial streams provide current velocities and substrata to food chains or food webs that are representative of those in the study river. With these tools we have shown that BKME stimulated mayfly growth in the Thompson River above that which could be accounted for by fertilization of their algal food supply. In contrast, moulting frequency was inhibited at high BKME concentrations. Results from artificial streams also indicate that increased algal biomass and abundances of benthic communities downstream of BKME outfalls were induced by nutrient enrichment from the effluent. BKME treatments did not change diatom species richness in the Fraser River, or diatom species diversity in either the Athabasca or Fraser Rivers. Artificial streams provide a means of understanding the mechanisms of stressor effects over a continuum ranging from single stressor effects on specific taxa to the effects of multiple stressors on communities and ecosystems. Because riverside deployment provides environmental realism within a replicated experimental design, this approach can (i) address questions that cannot be examined using laboratory tests or field observations, (ii) improve our mechanistic understanding of stressor effects on riverine ecosystems, and (iii) can contribute directly to the development, parameterization, and testing of models for predicting ecosystem-level responses.     

Biology and life cycle ofAtelocauda koae,an unusual demicyclic rust

Atelocauda koae, a rust of the native HawaiianAcacia koa, is considered as a demicyclic species, having spermogonial, aecial, and telial states, but is unusual in production of aeciospores simultaneously with teliospores rather than consecutively. Host inoculation with spores of each state separately confirmed that the life cycle was perpetuated by the telial state, but the aeciospores, while capable of germination and stomal penetration, did not produce detectable infection. This rust therefore behaves as a microcyclic species, and appears to be in evolutionary transition toward this reduced state. Teliospores produced vestigial, permanently attached basidiosopores which germinated to produce infective hyphae. The hyphae entered the host either through stomata or penetrated the epidermis directly, with the latter method being more common. Unusual nuclear associated with teliospore germination, in which meiosis occurs in more than one diploid nucleus was observed, in confirmation of an earlier study.     

GROWTH OF HETEROSIGMA CARTERAE (RAPHIDOPHYCEAE) ON NITRATE AND AMMONIUM AT THREE PHOTON FLUX DENSITIES: EVIDENCE FOR N STRESS IN NITRATE-GROWING CELLS

The marine alga Heterosigma carterae Hulburt (Raphidophyta) was grown in N-limiting batch cultures using either nitrate or ammonium as the N source, at photon flux densities (PFDs) of 50, 200, and 350 μmol·m^-2·s^-1 in a 12:12 h LD cycle. Carbon content could be estimated from biovolume (μg C = 0.278 × nL; R = 0.98) but not reliably from pigment content. During exponential growth, ammonium-grown cells (in comparison with nitrate-grown cells at the same PFD) attained higher growth rates by at least 20%, contained more N, and had a lower C:N ratio, higher concentrations of intracellular free amino acids, and higher ratios of glutamine: glutamate (Gln: Glu) and asparagine: aspartate (Asn:Asp). Growth was nearly light-saturated on ammonium at 200 μmol·m^-2·s^-1 (cell-specific growth rate of 1.2 d^-1) but probably not saturated in nitrate-grown cells at 350 μmol·m^-2·s^-1. PFD did not affect Gln: Glu or Asn: Asp for a given N source. These results indicate that the nitrate-growing cells were more N-stressed than those using ammonium (which in contrast were relatively C-stressed) and that this organism would show an enhanced competitive advantage against other species when supplied with a transient supply of ammonium rather than nitrate .