Christopher Baraloto, François Morneau, Damien Bonal, Lilian Blanc, and Bruno Ferry. 2007. Seasonal water stress tolerance and habitat associations within four neotropical tree genera. Ecology 88:478–489.

Appendix B. Juvenile traits in response to drought and inundation.

Experimental Design

Seeds of the eight species were collected at the Paracou experimental plots in March 2003 from within a 10-m radius of each of a minimum of five adult trees per species and mixed with respect to maternal source. For the two morpho-species of Symphonia, herbarium vouchers were collected for the adults from which seeds were gathered and deposited at the INRA herbarium at Kourou, French Guiana. Seeds were planted directly into 6.5-L pots (15 cm square, 40 cm height) filled with a 2:1 mixture of a brown ferralitic clay soil from terra firme forest at Paracou, and a white sand of podzolic origin. The experiment was conducted in a shadehouse at the INRA research facilities in Kourou. A neutral density shadecloth and a plastic cover were used to reduce light levels to about 7.9 ± 1.3 % full sun and to prevent rainfall from entering the shadehouse. This light level was chosen to approximate that of a small single treefall gap (Baraloto et al. 2005) to best evaluate the effects of soil resources where deep shade does not limit growth (e.g., Bloor and Grubb 2003).

Soil in the pots was maintained at field capacity (0.25 m³ m^-3) with water added every two–three days, for 18 months from planting, when juveniles for most species reached the minimum size for sapling inventories (150 cm tall). At this time, eight juveniles of each species were harvested as described below. Subsequently, juveniles were subjected to one of three watering treatments designed to mimic the potential water stresses that distinguish seasonally-flooded and terra firme forest. Juveniles in the water limitation treatment received one-third of the water necessary to keep pots at saturation, to achieve soil water potentials similar to those reached in the middle of the dry season in terra firme forest at Paracou (Bonal et al. 2000; Baraloto et al. 2005). Juveniles in the inundated treatment were placed into PVC containers of 20 cm diameter that completely enclosed the pots in which they were planted and in which water levels were maintained above the soil surface with water added every two days, to simulate the most extreme conditions occurring in seasonally-flooded forest during the four-month rainy season. Juveniles in the control treatment continued to be watered as before. Between six and ten juveniles of each species were assigned to each treatment at random, and groups of one juvenile per species × treatment were arranged in the shadehouse in a completely randomized design.

Methods for trait measurement

About every four weeks after the treatments were imposed, leaf gas exchange measurements were conducted under non-limiting environmental conditions (PAR = 600 ± 10 µmol m^-2 s^-1; vapor pressure deficit = 1.2 ± 0.4 kPa; air temperature = 30.0 ± 2.1 °C) using a portable photosynthesis system (IRGA, CIRAS1, PP-Systems, Hoddesdon, UK) operating in open mode and fitted with a Parkinson leaf cuvette. Gas exchange measurements were conducted on at least two leaves of each juvenile at each campaign. Equations of Caemmerer and Farquhar (1981) were used to calculate net carbon assimilation rate (A, µmol m^-2 s^-1), stomatal conductance for water vapor (gs, mol m^-2 s^-1) and intrinsic water use efficiency (WUE = A/gs). Pots in the drought treatment were weighed each time gas exchange measurements were made, and volumetric moisture content was calculated from soil weight at harvest, corrected for plant size using allometric formulas relating plant diameter and height to biomass for each species in previous harvests. Plants were assigned to the treatment of drought stress on an individual basis if they were exposed to conditions of less than 0.10 m³ m^-3 for at least four weeks.

Plants were harvested after 16 weeks, when juveniles were of about 22 months age. For each harvested plant, stem diameter at soil surface, height, and leaf number were recorded, and biomass was partitioned into leaf, stem and petiole, root, and remaining cotyledons. A separate sample of leaf tissue was obtained from the most recent fully-expanded growth unit, for the calculation of specific leaf area (SLA). Leaf surface area was measured immediately with a LI-COR 3000 leaf area meter (LI-COR Inc, Lincoln, Nebraska, USA). All plant parts were then dried to constant mass at 50 C and weighed to a precision of 0.1 mg.

Based on these measurements, we calculated SLA as the ratio of leaf area to leaf biomass in the subsample, and root-shoot ratios (R-S) as the ratio of total root biomass to total shoot biomass (Hunt 1978). We then determined RGR during the six-month period over which treatments were applied, using the equation RGR = (ln m₂ - ln m₁)/(t₂ - t₁), in which m is the total dry mass (g), and the denominator represents the number of days between the initial planting date and harvest dates for each individual. We used a randomized pairing (with replacement) of individuals at the final harvest with one of the eight juveniles sampled at the eighteen month inventory; this approach is conservative as it maximizes the variance in the initial juvenile size.

TABLE B1. Summary of experimental watering regimes on juvenile performance (relative growth rate during treatment period, RGR) and morphological (root-shoot ratio, RS; specific leaf area, SLA) and ecophysiological (photosynthetic capacity, A; stomatal conductance, gs; and water use efficiency, WUE) parameters under controlled conditions of inundation (I), field capacity (FC), and drought stress (D), for four congeneric species pairs.

   Notes: Seedlings were grown in a shadehouse at 8% of full sun, and watering treatments were imposed on six juveniles per treatment from 18 – 22 months age. The top rows present the summary of a three-way MANOVA testing for interactions between genus, habitat preference type and treatment, including univariate F tests for each dependent variable. The bottom rows show summaries of one-way MANOVA for each species, including univariate F tests and a summary of post-hoc Tukey’s tests between the treatment means.

ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001
† No survivors in inundated treatment

Principal Components Analysis

To examine associations among traits of seasonally-flooded forest and terra-firme forest species, we conducted a principal components analysis (PCA) using data from the shadehouse experiment of 22-month old juveniles grown under controlled conditions. To describe the general morphology of the species, we included the mean value for the control treatment at field capacity (CTL). To describe a species response to water stress treatments, we calculated the response ratios of those variables for which significant effects were found for any species (see Table 1), as the mean proportion change in that variable between the control treatment and either the inundation treatment (IN:CTL) or the drought treatment (D:CTL). The analysis was conducted in Statistica v. 6.0 based on a correlation matrix among variables, using a varimax normalized rotation of the axes.

TABLE B2.  Results of a principal components analysis for juvenile morphological and physiological traits and their response to experimental water stress treatments including inundation (IN) and drought stress (D).

   Notes: Shown are the loadings of each variable along each principal component axis with eigenvalue greater than 1. Variables highly correlated with each axis (a loading greater than 0.65) are shown in bold. The two axes explained 36.5 and 31.1 % of variation in the system.

LITERATURE CITED

Baraloto, C., D. E. Goldberg, and D. Bonal. 2005. Performance trade-offs among tropical tree seedlings in contrasting microhabitats. Ecology 86:2461–2472.

Bloor, J. M. G., and P. J. Grubb. 2003. Growth and mortality in high and low light: trends among 15 shade-tolerant tropical rain forest tree species. Journal of Ecology 91:77–83.

Bonal, D., C. Atger, T. S. Barigah, A. Ferhi, J. Guehl, and B. Ferry. 2000. Water acquisition patterns of two wet tropical canopy trees of French Guiana as inferred from H218O extraction profiles. Annals of Forest Science 57:717–724.

Caemmerer Von, S., and G. D. Farquhar. 1981. Some relationships between the biochemistry of photosynthesis and the gas exchange rates of leaves. Planta 153:376–387.

Hunt, R. 1978. Plant Growth Analysis. Edward Arnold, London, UK.