Salinity legacy: Foliar microbiome's history affects mutualist-conferred salinity tolerance.

The rapid human-driven changes in the environment during the Anthropocene have placed extreme stress on many plants and animals. Beneficial interactions with microorganisms may be crucial for ameliorating these stressors and facilitating the ecosystem services host organisms provide. Foliar endophytes, microorganisms that reside within leaves, are found in essentially all plants and can provide important benefits (e.g., enhanced drought tolerance or resistance to herbivory). However, it remains unclear how important the legacy effects of the abiotic stressors that select on these microbiomes are for affecting the degree of stress amelioration provided to their hosts. To elucidate foliar endophytes' role in host-plant salt tolerance, especially if salinity experienced in the field selects for endophytes that are better suited to improve the salt tolerance of their hosts, we combined field collections of 90 endophyte communities from 30 sites across the coastal Everglades with a manipulative growth experiment assessing endophyte inoculation effects on host-plant performance. Specifically, we grew >350 red mangrove (Rhizophora mangle) seedlings in a factorial design that manipulated the salinity environment the seedlings experienced (freshwater vs. saltwater), the introduction of field-collected endophytes (live vs. sterilized inoculum), and the legacy of salinity stress experienced by these introduced endophytes, ranging from no salt stress (0 parts per thousand [ppt] salinity) to high salt stress (40 ppt) environments. We found that inoculation with field-collected endophytes significantly increased mangrove performance across almost all metrics examined (15%-20% increase on average), and these beneficial effects typically occurred when the endophytes were grown in saltwater. Importantly, our study revealed the novel result that endophyte-conferred salinity tolerance depended on microbiome salinity legacy in a key coastal foundation species. Salt-stressed mangroves inoculated with endophyte microbiomes from high-salinity environments performed, on average, as well as plants grown in low-stress freshwater, while endophytes from freshwater environments did not relieve host salinity stress. Given the increasing salinity stress imposed by sea level rise and the importance of foundation species like mangroves for ecosystem services, our results indicate that consideration of endophytic associations and their salinity legacy may be critical for the successful restoration and management of coastal habitats.

Plant water uptake from soil through a vapor pathway.

Water uptake from the soil via a vapor pathway was tested. Viburnum suspensum L. plants were divided into: (1) irrigated, (2) drought with vapor and (3) drought without vapor treatments. Each plant was placed into a larger bucket containing deuterium-labeled water as a vapor source (vapor treatment) or no water (drought and irrigation treatments). We also tested whether uptake via a vapor pathway could mitigate drought effects. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) were measured daily until the first visible signs of stress. Soil water content, stem water potential (Ψ) and the stable hydrogen isotope ratio (δ2 H) of soil and plant xylem water were then measured in all treatments. We show that water is taken up by plants through the vapor phase in dry soils. The δ2 H values of the soil water in the vapor treatment were highly enriched compared to the background isotope ratios of the non-vapor exposed irrigated and drought treatments. Stem water δ2 H values for the vapor treatment were significantly greater than those for irrigation and drought treatments not exposed to isotopically enriched vapor. In this experiment, movement of water to the plant via the vapor phase did not mitigate drought effects. A, E, plant Ψ and gs significantly decreased in the drought and vapor treatments relative to the controls, with no significant differences between vapor and drought treatments.

Leaf anatomical traits determine the 18 O enrichment of leaf water in coastal halophytes.

Anatomical adaptations to high-salinity environments in mangrove leaves may be recorded in leaf water isotopes. Recent studies observed lower 18 O enrichment (ΔL ) of leaf water with respect to source water in three mangrove species relative to adjacent freshwater trees, but the factors that govern this phenomenon remain unclear. To resolve this issue, we investigated leaf traits and ΔL in 15 species of true mangrove plants, 14 species of adjacent freshwater trees, and 4 species of semi-mangrove plants at five study sites along south-eastern coast of China. Our results confirm that ΔL was generally 3-4‰ lower for mangrove species than for adjacent freshwater or semi-mangrove species. We hypothesized that higher leaf water content (LWC) and lower leaf stomatal density (LS) both played important roles in reducing ΔL in mangroves relative to nearby freshwater plants. Both differences acted to elongate effective leaf mixing length (L) in mangroves by about 200% and lower stomatal conductance by about 30%. Péclet models based on both LWC and LS could accurately predict ΔL . Our findings highlight the potential species-specific anatomical determinants of ΔL (or L), which has important implications for the interpretation of environmental information from metabolites produced by leaf water isotopes in palaeoclimate research.

The impact of channel capture on estuarine hydro-morphodynamics and water quality in the Amazon delta.

Due to progressive erosion of the new Urucurituba Channel, the Amazon River has recently captured almost all discharge from the lower Araguari River (Amapá-AP, Brazil), which previously flowed directly to the Atlantic Ocean. These recent geomorphological changes have caused strong impacts on the landscape and hydrodynamic patterns near the Araguari River mouth, especially the alteration of the riverine drainage system and its water quality. Landsat images were used to assess the estuarine landscape morphodynamic, particularly the expansion of the Urucurituba Channel, 80km from the Araguari River mouth, chronicling its connection to the Amazon River. The results suggest that the Urucurituba developed by headward migration across the Amazon delta; this is perhaps the first observation of estuarine distributary network development by headward channel erosion. The rate of Urucurituba Channel width increase has been ≈5m/month since 2011, increasing drainage capacity of the channel. We also collected in situ hydrodynamic measurements and analyzed 17 water quality parameters. Having 2011 as baseline, the flowrate of Araguari River has been diverted by up to 98% through Urucurituba Channel, with substantial changes in net discharge recorded at 3 monitoring stations. Statistically significant differences in water quality (p<0.05) were observed between 2011 and 2015, associated with the change in the flow pattern. Estuarine salinity and solids concentrations have increased. Overall, we demonstrate changes in landscape, hydrodynamics and water quality of the lower Araguari River.

Arbuscular common mycorrhizal networks mediate intra- and interspecific interactions of two prairie grasses.

Arbuscular mycorrhizal fungi form extensive common mycorrhizal networks (CMNs) that may interconnect neighboring root systems of the same or different plant species, thereby potentially influencing the distribution of limiting mineral nutrients among plants. We examined how CMNs affected intra- and interspecific interactions within and between populations of Andropogon gerardii, a highly mycorrhiza dependent, dominant prairie grass and Elymus canadensis, a moderately dependent, subordinate prairie species. We grew A. gerardii and E. canadensis alone and intermixed in microcosms, with individual root systems isolated, but either interconnected by CMNs or with CMNs severed weekly. CMNs, which provided access to a large soil volume, improved survival of both A. gerardii and E. canadensis, but intensified intraspecific competition for A. gerardii. When mixed with E. canadensis, A. gerardii overyielded aboveground biomass in the presence of intact CMNs but not when CMNs were severed, suggesting that A. gerardii with intact CMNs most benefitted from weaker interspecific than intraspecific interactions across CMNs. CMNs improved manganese uptake by both species, with the largest plants receiving the most manganese. Enhanced growth in consequence of improved mineral nutrition led to large E. canadensis in intact CMNs experiencing water-stress, as indicated by 13C isotope abundance. Our findings suggest that in prairie plant communities, CMNs may influence mineral nutrient distribution, water relations, within-species size hierarchies, and between-species interactions.

Testing plant use of mobile vs immobile soil water sources using stable isotope experiments.

We tested for isotope exchange between bound (immobile) and mobile soil water, and whether there is isotope fractionation during plant water uptake. These are critical assumptions to the formulation of the 'two water worlds' hypothesis based on isotope profiles of soil water. In two different soil types, soil-bound water in two sets of 19-l pots, each with a 2-yr-old avocado plant (Persea americana), were identically labeled with tap water. After which, one set received isotopically enriched water whereas the other set received tap water as the mobile phase water. After a dry down period, we analyzed plant stem water as a proxy for soil-bound water as well as total soil water by cryogenic distillation. Seventy-five to 95% of the bound water isotopically exchanged with the mobile water phase. In addition, plants discriminated against 18 O and 2 H during water uptake, and this discrimination is a function of the soil water loss and soil type. The present experiment shows that the assumptions for the 'two water worlds' hypothesis are not supported. We propose a novel explanation for the discrepancy between isotope ratios of the soil water profile and other water compartments in the hydrological cycle.

Common mycorrhizal networks amplify competition by preferential mineral nutrient allocation to large host plants.

Arbuscular mycorrhizal (AM) fungi interconnect plants in common mycorrhizal networks (CMNs) which can amplify competition among neighbors. Amplified competition might result from the fungi supplying mineral nutrients preferentially to hosts that abundantly provide fixed carbon, as suggested by research with organ-cultured roots. We examined whether CMNs supplied (15) N preferentially to large, nonshaded, whole plants. We conducted an intraspecific target-neighbor pot experiment with Andropogon gerardii and several AM fungi in intact, severed or prevented CMNs. Neighbors were supplied (15) N, and half of the target plants were shaded. Intact CMNs increased target dry weight (DW), intensified competition and increased size inequality. Shading decreased target weight, but shaded plants in intact CMNs had mycorrhizal colonization similar to that of sunlit plants. AM fungi in intact CMNs acquired (15) N from the substrate of neighbors and preferentially allocated it to sunlit, large, target plants. Sunlit, intact CMN, target plants acquired as much as 27% of their nitrogen from the vicinity of their neighbors, but shaded targets did not. These results suggest that AM fungi in CMNs preferentially provide mineral nutrients to those conspecific host individuals best able to provide them with fixed carbon or representing the strongest sinks, thereby potentially amplifying asymmetric competition below ground.

Water uptake of Alaskan tundra evergreens during the winter-spring transition.

PREMISE OF THE STUDY: The cold season in the Arctic extends over 8 to 9 mo, yet little is known about vascular plant physiology during this period. Evergreen species photosynthesize under the snow, implying that they are exchanging water with the atmosphere. However, liquid water available for plant uptake may be limited at this time. The study objective was to determine whether evergreen plants are actively taking up water while under snow and/or immediately following snowmelt during spring thaw. METHODS: In two in situ experiments, one at the plot level and another at the individual species level, (2)H-labeled water was used as a tracer injected beneath the snow, after which plant stems and leaves were tested for the presence of the label. In separate experiments, excised shoots of evergreen species were exposed to (2)H-labeled water for ∼5 s or 60 min and tested for foliar uptake of the label. KEY RESULTS: In both the plot-level and the species-level experiments, some (2)H-labeled water was found in leaves and stems. Additionally, excised individual plant shoots exposed to labeled water for 60 min took up significantly more (2)H-label than shoots exposed ∼5 s. CONCLUSIONS: Evergreen tundra plants take up water under snow cover, some via roots, but also likely by foliar uptake. The ability to take up water in the subnivean environment allows evergreen tundra plants to take advantage of mild spring conditions under the snow and replenish carbon lost by winter respiration.

Effects of stomatal density and leaf water content on the ¹8O enrichment of leaf water.

Leaf water isotopic composition is imprinted in several biomarkers of interest and it is imperative that we understand the isotopic enrichment of leaf water. Here, we test the effect of stomatal density and leaf water content on the oxygen isotopic composition of leaf water in transgenic Arabidopsis plants expressing different stomatal densities, and several other species showing a range of stomatal density. We grew Arabidopsis plants hydroponically and collected other species in the field. Stomatal density and leaf water content were determined for each plant. We measured transpiration and extracted leaf water for isotopic determination. Using these measurements and the current leaf water isotope model, we calculated several of the parameters related to leaf water isotopic enrichment. High stomatal density promoted leaf water isotope enrichment. No conclusion, however, can be drawn regarding the effect of leaf water content on leaf water isotope enrichment. Factors such as transpiration might mask the effect of stomatal density on leaf water isotopic enrichment. We propose a method by which stomatal density can be incorporated in the current Peclet model of leaf water isotope enrichment. These findings have important applications in the use of plant-based metabolic proxies in paleoclimate studies.

Biochemical effects of salinity on oxygen isotope fractionation during cellulose synthesis.

The current isotope tree ring model assumes that 42% of the sucrose oxygen exchanges with stem water during cellulose synthesis and that the oxygen isotope biochemical fractionation is c. 27‰. However, previous studies have indicated that this model can overestimate the cellulose oxygen isotope ratio of plants under salinity or water stress. Saline stress increases soluble carbohydrates and osmolytes, which can alter exchange and biochemical fractionation during cellulose synthesis. To test the effect of salinity as well as the synthesis of osmolytes on exchange and biochemical fractionation, we grew wild-type and a transgenic mannitol synthesizer Arabidopsis thaliana hydroponically with fresh and saline water. We then measured the oxygen isotope ratios of leaf water, stem water and stem cellulose to determine the effects on exchange and biochemical fractionation. Biochemical fractionation did not change, but oxygen isotope exchange was twice as high for plants grown in saline water relative to freshwater-treated plants (0.64 and 0.3, respectively). Mannitol (osmolyte) synthesis did not affect exchange or biochemical fractionation regardless of salinity. Increases in salinity increased oxygen isotope exchange during cellulose synthesis, which may explain the overestimation of cellulose δ(18) O values under saline conditions.

Stomatal pore size and density in mangrove leaves and artificial leaves: effects on leaf water isotopic enrichment during transpiration.

We tested the hypothesis that the previously observed low isotopic enrichment of mangrove leaf water is caused by larger stomatal pores and lower densities compared with freshwater plants. First, we measured and compared pore size and density in mangroves, transitional and freshwater species in South Florida. We pooled this data with other reports encompassing 14 mangrove species and 134 freshwater species and tested for differences in pore size and density between mangroves and freshwater plants. Second, we built artificial leaves having different pore size and density and determined whether there were isotopic differences in their water after transpiration. Both the local survey and pooled data showed that mangrove leaves have significantly larger stomatal pores with lower densities compared with freshwater plants. Isotope enrichment of water from artificial leaves having larger less dense pores was lower than those having smaller and denser pores. Stomatal pore size and density has an effect on leaf water isotopic enrichment amongst other factors. Pore size and density probably affects key components of the Peclet ratio such as the distance advective flow of water must travel to the evaporative surface and the cross-sectional area of advective flow. These components, in turn, affect leaf water isotopic enrichment. Results from the artificial leaf experiment also mimic a recent finding that effective path length scales to the inverse of transpiration in real leaves. The implications of these findings further our understanding of leaf water isotope ratios and are important in applications of stable isotopes in the study of paleoclimate and atmospheric processes.

Are leaf physiological traits related to leaf water isotopic enrichment in restinga woody species?

During plant-transpiration, water molecules having the lighter stable isotopes of oxygen and hydrogen evaporate and diffuse at a faster rate through the stomata than molecules having the heavier isotopes, which cause isotopic enrichment of leaf water. Although previous models have assumed that leaf water is well-mixed and isotopically uniform, non-uniform stomatal closure, promoting different enrichments between cells, and different pools of water within leaves, due to morpho-physiological traits, might lead to inaccuracies in isotopic models predicting leaf water enrichment. We evaluate the role of leaf morpho-physiological traits on leaf water isotopic enrichment in woody species occurring in a coastal vegetation of Brazil known as restinga. Hydrogen and oxygen stable isotope values of soil, plant stem and leaf water and leaf traits were measured in six species from restinga vegetation during a drought and a wet period. Leaf water isotopic enrichment relative to stem water was more homogeneous among species during the drought in contrast to the wet period suggesting convergent responses to deal to temporal heterogeneity in water availability. Average leaf water isotopic enrichment relative to stem water during the drought period was highly correlated with relative apoplastic water content. We discuss this observation in the context of current models of leaf water isotopic enrichment as a function of the Péclet effect. We suggest that future studies should include relative apoplastic water content in isotopic models.

The role of effective leaf mixing length in the relationship between the δ18 O of stem cellulose and source water across a salinity gradient.

Previous mangrove tree ring studies attempted, unsuccessfully, to relate the δ(18) O of trunk cellulose (δ(18) O(CELL) ) to the δ(18) O of source water (δ(18) O(SW) ). Here, we tested whether biochemical fractionation associated with one of the oxygen in the cellulose glucose moiety or variation in leaf water oxygen isotope fractionation (Δ(LW) ) can interfere with the δ(18) O(SW) signal as it is recorded in the δ(18) O(CELL) of mangrove (saltwater) and hammock (freshwater) plants. We selected two transects experiencing a salinity gradient, located in the Florida Keys, USA. The δ(18) O(CELL) throughout both transects did not show the pattern expected based on that of the δ(18) O(SW) . We found that in one of the transects, biochemical fractionation interfered with the δ(18) O(SW) signal, while in the other transect Δ(LW) differed between mangrove and hammock plants. Observed differences in Δ(LW) between mangroves and hammocks were caused by a longer effective leaf mixing length (L) of the water pathway in mangrove leaves compared to those of hammock leaves. Changes in L could have caused the δ(18) O(CELL) to record not only variations in the δ(18) O(SW) but also in Δ(LW) making it impossible to isolate the δ(18) O(SW) signal.

Differential response to soil salinity in endangered key tree cactus: implications for survival in a changing climate.

Understanding reasons for biodiversity loss is essential for developing conservation and management strategies and is becoming increasingly urgent with climate change. Growing at elevations <1.4 m in the Florida Keys, USA, the endangered Key tree cactus (Pilosocereus robinii) experienced 84 percent loss of total stems from 1994 to 2007. The most severe losses of 99 and 88 percent stems occurred in the largest populations in the Lower Keys, where nine storms with high wind velocities and storm surges, occurred during this period. In contrast, three populations had substantial stem proliferation. To evaluate possible mortality factors related to changes in climate or forest structure, we examined habitat variables: soil salinity, elevation, canopy cover, and habitat structure near 16 dying or dead and 18 living plants growing in the Lower Keys. Soil salinity and elevation were the preliminary factors that discriminated live and dead plants. Soil salinity was 1.5 times greater, but elevation was 12 cm higher near dead plants than near live plants. However, distribution-wide stem loss was not significantly related to salinity or elevation. Controlled salinity trials indicated that salt tolerance to levels above 40 mM NaCl was related to maternal origin. Salt sensitive plants from the Lower Keys had less stem growth, lower root:shoot ratios, lower potassium: sodium ratios and lower recovery rate, but higher δ (13)C than a salt tolerant lineage of unknown origin. Unraveling the genetic structure of salt tolerant and salt sensitive lineages in the Florida Keys will require further genetic tests. Worldwide rare species restricted to fragmented, low-elevation island habitats, with little or no connection to higher ground will face challenges from climate change-related factors. These great conservation challenges will require traditional conservation actions and possibly managed relocation that must be informed by studies such as these.

Divergent biochemical fractionation, not convergent temperature, explains cellulose oxygen isotope enrichment across latitudes.

Recent findings based on the oxygen isotope ratios of tree trunk cellulose indicate that the temperature of biomass production in biomes ranging from boreal to subtropical forests converge to an average leaf temperature of 21.4°C. The above conclusion has been drawn under the assumption that biochemically related isotopic fractionations during cellulose synthesis are not affected by temperature. Here we test the above assumption by heterotrophically generating cellulose at different temperatures and measuring the proportion of carbohydrate oxygen that exchange with water during cellulose synthesis and the average biochemical fractionation associated with this exchange. We observed no variation in the proportion of oxygen that exchange with different temperatures, which averaged 0.42 as it has been observed in other studies. On the other hand, the biochemical oxygen isotope fractionation during cellulose synthesis is affected by temperature and can be described by a 2(nd) order polynomial equation. The biochemical fractionation changes little between temperatures of 20 and 30°C averaging 26‰ but increases at lower temperatures to values of 31‰. This temperature sensitive biochemical fractionation explains the pattern of cellulose oxygen isotope ratios of aquatic plants encompassing several latitudes. The observed temperature sensitive biochemical fractionation also indicates that divergent biochemical fractionation and not convergent leaf temperature explains the increase in oxygen isotope enrichment of cellulose across several biomes.

Aphids alter host-plant nitrogen isotope fractionation.

Plant sap-feeding insects and blood-feeding parasites are frequently depleted in (15)N relative to their diet. Unfortunately, most fluid-feeder/host nitrogen stable-isotope studies simply report stable-isotope signatures, but few attempt to elucidate the mechanism of isotopic trophic depletion. Here we address this deficit by investigating the nitrogen stable-isotope dynamics of a fluid-feeding herbivore-host plant system: the green peach aphid, Myzus persicae, feeding on multiple brassicaceous host plants. M. persicae was consistently more than 6‰ depleted in (15)N relative to their hosts, although aphid colonized plants were 1.5‰ to 2.0‰ enriched in (15)N relative to uncolonized control plants. Isotopic depletion of aphids relative to hosts was strongly related to host nitrogen content. We tested whether the concomitant aphid (15)N depletion and host (15)N enrichment was coupled by isotopic mass balance and determined that aphid (15)N depletion and host (15)N enrichment are uncoupled processes. We hypothesized that colonized plants would have higher nitrate reductase activity than uncolonized plants because previous studies had demonstrated that high nitrate reductase activity under substrate-limiting conditions can result in increased plant δ(15)N values. Consistent with our hypothesis, nitrate reductase activity in colonized plants was twice that of uncolonized plants. This study offers two important insights that are likely applicable to understanding nitrogen dynamics in fluid-feeder/host systems. First, isotopic separation of aphid and host depends on nitrogen availability. Second, aphid colonization alters host nitrogen metabolism and subsequently host nitrogen stable-isotope signature. Notably, this work establishes a metabolic framework for future hypothesis-driven studies focused on aphid manipulation of host nitrogen metabolism.

Leaf and root pectin methylesterase activity and 13C/12C stable isotopic ratio measurements of methanol emissions give insight into methanol production in Lycopersicon esculentum.

Plant production of methanol (MeOH) is a poorly understood aspect of metabolism, and understanding MeOH production in plants is crucial for modeling MeOH emissions. Here, we have examined the source of MeOH emissions from mature and immature leaves and whether pectin methylesterase (PME) activity is a good predictor of MeOH emission. We also investigated the significance of below-ground MeOH production for mature leaf emissions. We present measurements of MeOH emission, PME activity, and MeOH concentration in mature and immature tissues of tomato (Lycopersicon esculentum). We also present stable carbon isotopic signatures of MeOH emission and the pectin methoxyl pool. Our results suggest that below-ground MeOH production was not the dominant contributor to daytime MeOH emissions from mature and immature leaves. Stable carbon isotopic signatures of mature and immature leaf MeOH were similar, suggesting that they were derived from the same pathway. Foliar PME activity was related to MeOH flux, but unexplained variance suggested PME activity could not predict emissions. The data show that MeOH production and emission are complex and cannot be predicted using PME activity alone. We hypothesize that substrate limitation of MeOH synthesis and MeOH catabolism may be important regulators of MeOH emission.

Lake and watershed characteristics rather than climate influence nutrient limitation in shallow lakes.

Both nitrogen (N) and phosphorus (P) can limit primary production in shallow lakes, but it is still debated how the importance of N and P varies in time and space. We sampled 83 shallow lakes along a latitudinal gradient (5 degrees 55 degrees S) in South America and assessed the potential nutrient limitation using different methods including nutrient ratios in sediment, water, and seston, dissolved nutrient concentrations, and occurrence of N-fixing cyanobacteria. We found that local characteristics such as soil type and associated land use in the catchment, hydrology, and also the presence of abundant submerged macrophyte growth influenced N and P limitation. We found neither a consistent variation in nutrient limitation nor indications for a steady change in denitrification along the latitudinal gradient. Contrary to findings in other regions, we did not find a relationship between the occurrence of (N-fixing and non-N-fixing) cyanobacteria and the TN:TP ratio. We found N-fixing cyanobacteria (those with heterocysts) exclusively in lakes with dissolved inorganic nitrogen (DIN) concentrations of < 100 microg/L, but notably they were also often absent in lakes with low DIN concentrations. We argue that local factors such as land use and hydrology have a stronger influence on which nutrient is limiting than climate. Furthermore, our data show that in a wide range of climates N limitation does not necessarily lead to cyanobacterial dominance.

Oxygen stable isotope ratios of tree-ring cellulose: the next phase of understanding.

Analysis of the oxygen isotope ratio of tree-ring cellulose is a valuable tool that can be used as a paleoclimate proxy. Our ability to use this tool has gone through different phases. The first began in the 1970s with the demonstration of empirical relationships between the oxygen isotope ratio of tree-ring cellulose and climate. These empirical relationships, however, did not provide us with the confidence that they are robust through time, across taxa and across geographical locations. The second phase began with a rudimentary understanding of the physiological and biochemical mechanisms responsible for the oxygen isotope ratios of cellulose, which is necessary to increase the power of this tool. This phase culminated in a mechanistic tree-ring model integrating concepts of physiology and biochemistry in a whole-plant system. This model made several assumptions about leaf water isotopic enrichment and biochemistry which, in the nascent third phase, are now being challenged, with surprising results. These third-phase results suggest that, contrary to the model assumption, leaf temperature across a large latitudinal gradient is remarkably constant and does not follow ambient temperature. Recent findings also indicate that the biochemistry responsible for the incorporation of the cellulose oxygen isotopic signature is not as simple as has been assumed. Interestingly, the results of these challenges have strengthened the tree-ring model. There are several other assumptions that can be investigated which will improve the utility of the tree-ring model.

Ephemeral clonal integration in Calathea marantifolia (Marantaceae): Evidence of diminished integration over time.

A major advantage of clonal growth forms is the intergenerational transfer of resources through vascular connections (clonal integration). Connections linking ramets can be persistent or ephemeral. For species with ephemeral connections, whether the extent of clonal integration changes over time is unclear. To address this issue, we tracked water movement using an isotopic label and assessed the demographic performance of parent and offspring ramets over time in a severing experiment. Our study system was the understory herb Calathea marantifolia, which has parent ramets that produce vegetative bulbils (clonal offspring) that pass through distinct pre- and post-rooting stages. Little water was transported between parents and offspring, and the direction of movement was primarily from parent to pre-rooting offspring. Anatomical observations of inter-ramet connections showed that vascular bundles were twice as abundant in parent stems compared to inter-ramet connections. Severing inter-ramet connections reduced the growth of offspring ramets but not parents. Survival of pre-rooting offspring was reduced by 10% due to severing, but post-rooting offspring were not affected. Our results suggest that offspring ramets of C. marantifolia are weaned from their parent as they progress from pre- to post-rooting stages.