Limits of thermal and hydrological tolerance in a foundation tree species (Populus fremontii) in the desert southwestern United States.

Populus fremontii is among the most dominant, and ecologically important riparian tree species in the western United States and can thrive in hyper-arid riparian corridors. Yet, P. fremontii forests have rapidly declined over the last decade, particularly in places where temperatures sometimes exceed 50°C. We evaluated high temperature tolerance of leaf metabolism, leaf thermoregulation, and leaf hydraulic function in eight P. fremontii populations spanning a 5.3°C mean annual temperature gradient in a well-watered common garden, and at source locations throughout the lower Colorado River Basin. Two major results emerged. First, despite having an exceptionally high Tcrit (the temperature at which Photosystem II is disrupted) relative to other tree taxa, recent heat waves exceeded Tcrit , requiring evaporative leaf cooling to maintain leaf-to-air thermal safety margins. Second, in midsummer, genotypes from the warmest locations maintained lower midday leaf temperatures, a higher midday stomatal conductance, and maintained turgor pressure at lower water potentials than genotypes from more temperate locations. Taken together, results suggest that under well-watered conditions, P. fremontii can regulate leaf temperature below Tcrit along the warm edge of its distribution. Nevertheless, reduced Colorado River flows threaten to lower water tables below levels needed for evaporative cooling during episodic heat waves.

Evidence of climate-driven selection on tree traits and trait plasticity across the climatic range of a riparian foundation species.

Selection on quantitative traits by heterogeneous climatic conditions can lead to substantial trait variation across a species range. In the context of rapidly changing environments, however, it is equally important to understand selection on trait plasticity. To evaluate the role of selection in driving divergences in traits and their associated plasticities within a widespread species, we compared molecular and quantitative trait variation in Populus fremontii (Fremont cottonwood), a foundation riparian distributed throughout Arizona. Using SNP data and genotypes from 16 populations reciprocally planted in three common gardens, we first performed QST -FST analyses to detect selection on traits and trait plasticity. We then explored the environmental drivers of selection using trait-climate and plasticity-climate regressions. Three major findings emerged: (1) There was significant genetic variation in traits expressed in each of the common gardens and in the phenotypic plasticity of traits across gardens, both of which were heritable. (2) Based on QST -FST comparisons, there was evidence of selection in all traits measured; however, this result varied from no effect in one garden to highly significant in another, indicating that detection of past selection is environmentally dependent. We also found strong evidence of divergent selection on plasticity across environments for two traits. (3) Traits and/or their plasticity were often correlated with population source climate (R2 up to .77 and .66, respectively). These results suggest that steep climate gradients across the Southwest have played a major role in shaping the evolution of divergent phenotypic responses in populations and genotypes now experiencing climate change.

Simple methods to remove microbes from leaf surfaces.

Endophytes have been defined as microorganisms living inside plant tissues without causing negative effects on their hosts. Endophytic microbes have been extensively studied for their plant growth-promoting traits. However, analyses of endophytes require complete removal of epiphytic microorganisms. We found that the established tests to evaluate surface sterility, polymerase chain reaction, and leaf imprints, are unreliable. Therefore, we used scanning electron microscopy (SEM) as an additional assessment of epiphyte removal. We used a diverse suite of sterilization protocols to remove epiphytic microorganisms from the leaves of a gymnosperm and an angiosperm tree to test the influence of leaf morphology on the efficacy of these methods. Additionally, leaf tissue damage was also evaluated by SEM, as damaging the leaves might have an impact on endophytes and could lead to inaccurate assessment of endophytic communities. Our study indicates, that complete removal of the leaf cuticle by the sterilization technique assures loss of epiphytic microbes, and that leaves of different tree species may require different sterilization protocols. Furthermore, our study demonstrates the importance of choosing the appropriate sterilization protocol to prevent erroneous interpretation of host-endophyte interactions. Moreover, it shows the utility of SEM for evaluating the effectiveness of surface sterilization methods and their impact on leaf tissue integrity.

Genotypic variation in phenological plasticity: Reciprocal common gardens reveal adaptive responses to warmer springs but not to fall frost.

Species faced with rapidly shifting environments must be able to move, adapt, or acclimate in order to survive. One mechanism to meet this challenge is phenotypic plasticity: altering phenotype in response to environmental change. Here, we investigated the magnitude, direction, and consequences of changes in two key phenology traits (fall bud set and spring bud flush) in a widespread riparian tree species, Populus fremontii. Using replicated genotypes from 16 populations from throughout the species' thermal range, and reciprocal common gardens at hot, warm, and cool sites, we identified four major findings: (a) There are significant genetic (G), environmental (E), and GxE components of variation for both traits across three common gardens; (b) The magnitude of phenotypic plasticity is correlated with provenance climate, where trees from hotter, southern populations exhibited up to four times greater plasticity compared to the northern, frost-adapted populations; (c) Phenological mismatches are correlated with higher mortality as the transfer distances between provenance and garden increase; and (d) The relationship between plasticity and survival depends not only on the magnitude and direction of environmental transfer, but also on the type of environmental stress (i.e., heat or freezing), and how particular traits have evolved in response to that stress. Trees transferred to warmer climates generally showed small to moderate shifts in an adaptive direction, a hopeful result for climate change. Trees experiencing cooler climates exhibited large, non-adaptive changes, suggesting smaller transfer distances for assisted migration. This study is especially important as it deconstructs trait responses to environmental cues that are rapidly changing (e.g., temperature and spring onset) and those that are fixed (photoperiod), and that vary across the species' range. Understanding the magnitude and adaptive nature of phenotypic plasticity of multiple traits responding to multiple environmental cues is key to guiding restoration management decisions as climate continues to change.

Plant origin authentication of Sonoran Desert propolis: an antiproliferative propolis from a semi-arid region.

The main chemical composition of Sonoran propolis (SP), as well as its antiproliferative activity on cancer cells through apoptosis induction, has been reported. The chemical constitution of SP remained qualitatively similar throughout the year, whereas the antiproliferative effect on cancer cells exhibited significant differences amongst seasonal samples. The main goal of this study was to provide phytochemical and pharmacological evidence for the botanical source of SP and its antiproliferative constituents. A chemical comparative analysis of SP and plant resins of species found in the surrounding areas of the beehives was carried out by HPLC-UV-DAD, as well as by 1H NMR experiments. The antiproliferative activity on cancerous (M12.C3.F6, HeLa, A549, PC-3) and normal cell lines (L-929; ARPE-19) was assessed through MTT assays. Here, the main polyphenolic profile of SP resulted to be qualitatively similar to Populus fremontii resins (PFR). However, the antiproliferative activity of PFR on cancer cells did not consistently match that exhibited by SP throughout the year. Additionally, SP induced morphological modifications on treated cells characterised by elongation, similar to those induced by colchicine, and different to those observed with PFR treatment. These results suggest that P. fremontii is the main botanical source of SP along the year. Nevertheless, the antiproliferative constituents of SP that induce that characteristic morphological elongation on treated cells are not obtained from PFR. Moreover, the presence of kaempferol-3-methyl-ether in SP could point Ambrosia ambrosioides as a secondary plant source. In conclusion, SP is a bioactive poplar-type propolis from semi-arid zones, in which chemical compounds derived from other semi-arid plant sources than poplar contribute to its antiproliferative activity.

Interactive effects of water, light and heat stress on photosynthesis in Fremont cottonwood.

Fremont cottonwood seedlings are vulnerable to water stress from rapid water-table decline during river recession in spring. Water stress is usually cited as the reason for reduced establishment, but interactions of water stress with microclimate extremes are more likely the causes of mortality. We assessed photosynthetic responses of Fremont cottonwood seedlings to water, light and heat stresses, which commonly co-occur in habitats where seedlings establish. Under moderate temperature and light conditions, water stress did not affect photosynthetic function. However, stomatal closure during water stress predisposed Fremont cottonwood leaves to light and heat stress, resulting in greatly reduced photosynthesis beginning at 31 °C versus at 41 °C for well-watered plants. Ontogenetic shifts in leaf orientation from horizontal to vertical, which occur as seedlings mature, reduce heat and light stress, especially during water stress. When compared with naturally occurring microclimate extremes, seedling stress responses suggest that reduced assimilation and photoprotection are common for Fremont cottonwood seedlings on exposed point bars where they establish. These reductions in photosynthesis likely have negative impacts on growth and may predispose young (<90-day-old) seedlings to early mortality during rapid water-table declines. Interactions with heat and light stress are more important in these effects than water stress alone.

Variation in carbon isotope composition among years in the riparian tree Populus fremontii.

Water availability is an important factor limiting the productivity of desert plants but little is known about the impact of water-limiting conditions on the physiology of plants in mesic environments. Riparian ecosystems of the western US receive significantly more water than the surrounding desert environments but experience dramatic interannual fluctuations in water availability because both stream flow and precipitation are highly variable over time. This variability results in different growing conditions each year which may influence the physiology of riparian species such as Fremont cottonwood (Populus fremontii), the dominant, native canopy tree species in lowland southwestern US river systems. We wished to determine if the physiology of this species varies among years, what climatic parameters are related to any observed physiological variation and if individuals within a P. fremontii population differ in their physiological response to variation through time. We collected tree ring cores from a central New Mexico cottonwood population and analyzed carbon isotope composition (δ13C) in each year from 1981 to 1995. We used δ13C analysis in this study because it allowed us to obtain multi-year estimates of physiological activity. During these years, mean stream flow at our study site ranged over two orders of magnitude from 0.82 to 80.94 m3 s-1, precipitation ranged fourfold from 49 to 215 ccmm and mean temperature ranged from 20.5 to 22.6°C during the growing season. δ13C varied from a low of -26.7‰ in 1984 to a high of -24.7‰ in 1981. Low δ13C values were associated with years in which stream flow and/or precipitation were high and temperature was low. The opposite was true of years with high δ13C values. We observed a strong linear relationship between δ13C and stream flow during years when stream flow was <25 m3 s-1 but no significant relationship between these variables when stream flow was >25 m3 s-1. Additionally, there was a linear relationship between δ13C and precipitation during years when stream flow was <25 m3 s-1 but not in years when stream flow was >25 m3 s-1. These data suggest that above a threshold of total stream flow, increased flow does not influence physiology. Below this threshold, precipitation can be an important water source. The ten individuals within our study population varied significantly in mean δ13C values but responded to interannual variation in a similar manner (i.e., all individuals had low δ13C values when water was abundant). These results suggest that precipitation as well as stream flow are important factors influencing the physiology of this riparian tree.

THE EFFECTS OF HOST-PLANT GENOTYPE, HYBRIDIZATION, AND ENVIRONMENT ON GALL-APHID ATTACK AND SURVIVAL IN COTTONWOOD: THE IMPORTANCE OF GENETIC STUDIES AND THE UTILITY OF RFLPS.

Using restriction fragment length polymorphisms (RFLPs) we show how host-plant genotype and hybridization in cotton wood, Populus sp., affects the attack and survival of the gall-forming aphid, Pemphigus betae. Fremont cottonwoods, hybrid ∗∗∗F1's and backcross ∗∗∗1's were found to be highly resistant, while backcross 2's, 3's, 4's and pure narrowleafs ranged from highly resistant to highly susceptible with only a few trees being highly resistant. Results from our genetic analysis also suggest that resistance is likely polygenic, not the result of single gene resistance. In addition, genetic analysis showed that studies based upon leaf morphology alone give an inaccurate assessment of host-plant genotype ∗∗∗and the extent of hybridization, altering previous views of the relationship between plant hybridization and pest attack. Previous studies assumed that narrowleafs were more resistant than backcross genotypes based upon comparisons of overall levels of resistance between the hybrid zone and the "pure" narrowleaf zone. Results from RFLP analyses, however, show that there are no significant differences in the levels of resistance between backcross genotypes (BC2's-4's) and pure narrowleafs. Furthermore, results show that the "pure" narrowleaf zone is in fact a mixture of pure and backcross genotypes, extending the zone of introgression previously reported. Experiments in combination with RFLP analyses suggest that resistance traits are differentially expressed along an environmental gradient partially explaining the previously reported differences in resistance between these two regions. In light of our results it is clear that genetic studies will be necessary to discern the true relationship between hybridization and pest resistance. Until such studies are widely conducted generalizations regarding the effects of hybridization on the structure and dynamics of pest populations will be premature at best.

MITOCHONDRIAL INHERITANCE PATTERNS ACROSS A COTTONWOOD HYBRID ZONE: CYTONUCLEAR DISEQUILIBRIA AND HYBRID ZONE DYNAMICS.

In this study we examine the cytoplasmic inheritance patterns of an interspecific hybridizing population of Fremont and narrowleaf cottonwoods, using mitochondrial DNA. Three mitochondrial probes showing polymorphisms were used to distinguish between trees of known nuclear inheritance. Every tree screened had only one cytoplasmic genotype, either Fremont or narrowleaf. Thus, these results demonstrate that mitochondria are uniparentally inherited in these trees. Previous studies of the nuclear inheritance of this interspecific hybridizing population of cottonwood trees indicated an asymmetry in the frequency of parental genes. Using mitochondrial markers we tested one hypothesis potentially responsible for this asymmetric distribution (i.e., trees of mixed genotypes will be sterile or will not survive if their cytoplasm is derived from one or the other parent). Our results, however, show that both Fremont and narrowleaf mitochondrial markers are found in trees with mixed nuclear genotypes. Thus, nuclear-cytoplasmic incompatibilities do not appear to account for the asymmetric distribution of nuclear genotypes within the hybrid swarm. An alternative explanation for the observed asymmetric distribution of nuclear genotypes is advanced. Although nuclear-cytoplasmic incompatibilities do not appear to explain the asymmetric distribution of nuclear alleles within the hybrid zone, nonrandom associations between nuclear and cytoplasmic genotypes do exist. For example, all F1 hybrids had Fremont mitochondrial genotypes. Furthermore, backcrosses between F1 hybrid and narrowleaf trees have a higher than expected proportion of heterozygous loci and a higher than expected proportion of Fremont mitochondria. We propose that seeds, seedlings, or trees with high proportions of heterozygous loci are at a disadvantage unless they also have the Fremont mitochondrial genotype. While it is generally difficult to infer dynamic processes from static patterns, studies such as ours enable one to gain new insights to the dynamics of plant hybrid zones. A hybridization pattern of decreasingly complex backcrosses as one proceeds from higher to lower elevation within the hybrid swarm, a residue of Fremont cytoplasmic DNA within the pure narrowleaf population, and the unidirectional nature of these crosses suggest that the narrowleaf population may be spreading down the canyon and the Fremont population receding. The eventual fate of the hybrid zone, in relation to these processes, is discussed.