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South African Journal of Botany 81 (2012) 61 - 70
www.elsevier.com/locate/sajb
Water use strategies of seedlings of three Malagasy Adansonia species
under drought
T. Randriamanana a *, F. Wang a, T. Lehto b, P.J. Aphalo a
a Department of Biosciences, P.O. Box 65 (Viikinkaari 1), 00014 University of Helsinki, Finland b School of Forest Sciences, University of Eastern Finland, P.O. Box 111, 80101, Finland
Received 21 August 2011; received in revised form 17 May 2012; accepted 25 May 2012
Abstract
Adansonia species, known by the common name "baobab," have a very low regeneration rate in Madagascar. In order to determine if Malagasy Adansonia seedlings' vulnerability to drought may account for this low rate of regeneration, we compared growth, photosynthetic behavior and water use strategy of three species of Malagasy Adansonia (A. grandidieri, A. madagascariensis, A. rubrostipa). Our results indicated that drought depressed the growth, net assimilation rate, stomatal conductance and transpiration rate of Adansonia seedlings but increased their water use efficiencies. Adansonia species are able to withstand drought by reducing water loss through stomatal closure and their ability to store water within roots. Interspecific differences were attributed to diversity in water-use strategies, relative water content and biomass allocation. A. rubrostipa and A. grandidieri appeared to be more adapted to arid environments than A. madagascariensis. Ecological implications of these results are discussed. © 2012 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords: Baobabs; Growth; Madagascar; Photosynthetic responses; Water stress; Water use
1. Introduction
Tropical dry forests have received less conservation and scientific attention in comparison with tropical wet forests (Fajardo et al., 2005; WWF, 2010). Over 97% of Madagascar's western dry deciduous forests have been lost and are consequently considered as "Critical or Endangered" ecoregion according to the Global 200 representation of WWF (Olson and Dinerstein, 1998; WWF, 2010).
Adansonia (BOMBACACEAE sensu stricto, MALVACEAE sensu lato), commonly known as "Baobab", is a xeric genus that symbolizes Madagascar's floral biodiversity, as six out of eight world-wide species are endemic to the island. It was chosen as a model species in our studies because of its predominance in
* Corresponding author at: Natural Product Research Laboratory, Department of Biology, University of Eastern Finland, PO Box 111, 80101 Joensuu, Finland. Tel.: +358 504423402.
E-mail address: tendry.randriamanana@uef.fi (T. Randriamanana).
Malagasy western forests. Species of the genus Adansonia are classified as "keystone mutualists" due to their likely role in stabilizing dry forest ecosystems (Baum, 1996). Apart from their ecological role (Baum, 1995, 1996; Metcalfe and Trevelyan,
2007), they are multipurpose trees and have numerous economic functions in some Malagasy rural communities (Baum, 1996; Marie et al., 2009; Seddon et al., 2000; Wickens and Lowe,
2008). In Madagascar, Adansonia fruit pulps which are rich in vitamin C and with high antioxidant activity (Assogbadjo et al., 2008; Chadare et al., 2009) are consumed by local communities. Moreover, Malagasy Adansonia leaves possess higher nutritional value in terms of leaf vitamin and crude protein contents when compared to African baobab A. digitata L. (Maranz et al., 2007), and can be consequently used to improve local population diet. In Madagascar, dried leaves of Adansonia are used for food seasoning (Marie et al., 2009). Besides, Adansonia leaves are used in Malagasy traditional pharmacopeia, its bark and fibers are confectioned to make rope, mats, baskets and roofing materials, their seeds produce edible oil, and medicines (Baum, 1996; Marie
0254-6299/$ -see front matter © 2012 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2012.05.005
et al., 2009; Seddon et al., 2000; Wickens and Lowe, 2008). Their trunks are sometimes hollowed out as tanks for water storage in dry areas of Madagascar (Baum, 1996; Marie et al., 2009). In addition, due to their impressive appearance, species of the genus Adansonia are associated with some Malagasy superstitions and folklore which consider certain trees as sacred (Baum, 1995; Marie et al., 2009).
Field observations have reported a noticeably low survival of seedlings in all species of Malagasy Adansonia especially in arid areas of Madagascar (Baum, 1995, 1996; Miège and Morat, 1974; Razanameharizaka, 2009; Wickens and Lowe, 2008). 75% of newly established seedlings (particularly A. rubrostipa Jum. & H. Perr.) die before their first rainy season in their natural environment, apparently due to combined water deficit and high light intensity (Razanameharizaka, 2009). Studies undertaken by Venter and Witkowski (2010) on African A. digitata reported that the predicted drop in rainfall attributed to climate change may increase Adansonia trees rate of mortality in the future.
The goal of this study is to investigate the physiological responses of Malagasy Adansonia seedlings to water stress and whether such responses may explain their low regeneration rate and their pattern of distribution across the island. We tested the hypothesis that species-specific differences influence the effects of drought on seedling growth and photosynthetic activity in three species of Adansonia endemic to Madagascar, and predicted that seedlings of Adansonia madagascariensis Baill. from the northern dry deciduous and subhumid forests of Madagascar would be less drought tolerant during water stress when compared to A. rubrostipa and A. grandidieri Baill. which are from the arid spiny thickets and succulent woodlands of the south. To test this hypothesis, we assessed under controlled conditions the photo-synthetic behavior and water use strategy of these three species of Adansonia.
2. Materials and methods
Seeds from three species of Adansonia (A. rubrostipa, A. grandidieri, and A. madagascariensis) were collected from the western part of Madagascar at Ifaty, Marofandilia and Mahajanga (Table 1; Fig. 1) by "URP Forêts et Biodiversité" (Madagascar).
2.1. Plant material establishment and experimental design
The experiment was carried out at the Department of Biosciences, University of Helsinki (Finland). Seeds of A. rubrostipa and A. madagascariensis were scalded in boiling water for 60 s while seeds of A. grandidieri were soaked in tap
water for 12 h. Seeds were then sown at 1 cm depth in pots (at a density of 30 seeds per pot of 50 cm x 30 cm dimension) containing sterile sand moistened with distilled water. The pots were kept at 30± 1 °C under a 12 h photoperiod, illuminated with 175 pmol m- 2 s-1 photosynthetic photon flux from fluorescent lamps at ambient CO2. After 2 to 3 days, radicules emerged from almost 95% of the seeds and the germinating seeds were transplanted to 410 mL pots containing vermiculite and white Sphagnum peat 1:1 (v/v). All pots were filled with the same quantity of dry mixture (40 g per pot) and were watered every other day, ensuring that excess water drained through the bottom of the pots. The content of fertilizer already in the limed peat (White 420 W, Kekkila, Vantaa, Finland) was 1 g L 1 of N-P2O5-K2O (14:9:24) plus micronutrients (Kekkila starter fertilizer 1) plus 6.2 g L-1 of dolomite lime. At the beginning of the experiment, each seedling was watered with 40 mL liquid fertilizer (Taimi Superex®, Kekkila, Vantaa, Finland) containing 19% N, 4.4% P, 20.2% K, 1.2% Mg, 0.03% B, 0.001% Co, 0.008% Cu, 0.17% Fe, 0.08% Mn, 0.005% Mo, and 0.012% Zn (w/w) at a concentration of 1 g L-1.
Versatile MLR-350 294L test chambers (Sanyo Electric Biomedical Ltd., Japan) were used to grow the seedlings under controlled environmental conditions. Photosynthetic photon irradiance inside the growth chambers was measured using a LI-190 quantum sensor plus a LI-250A meter (LI-COR®, Biosciences, Lincoln, NE, USA). Photosynthetically Active Radiation (PAR) photon irradiance was set at 175 pmol m- 2 s-1 with a photoperiod of 12 h. Temperatures were held at 29± 1 °C during the day and 24 ± 1 °C during the night, while air humidity ranged between 26% during the day and 31% at night. Temperature and relative humidity were logged at 10-15 cm above plant height in all chambers (iButton, DS1922L and DS1923, Maxim Integrated Products, Sunnyvale, CA, USA).
Pots were assigned to three different complete randomized blocks based on seedling size so that all chambers would have equal numbers of same-height seedlings. After transplantation in pots, seedlings were allowed to grow for 28 days before watering treatment so that they acquire well developed leaves and roots. Three replicates of 10 seedlings per species and per watering regime were grown in five separate growth chambers; each chamber contained two species.
2.2. Watering treatment
Starting 28 days after germination, the seedlings were subjected to two water regimes: watering to 100% ("control") and to 50% field capacity ("water stressed"). Field capacity was determined in
Seed origin, altitude, annual rainfall, mean annual maximum and minimum temperatures for the three species of Adansonia collected in the western part of Madagascar in 2004.
Seed origin Altitude (m) Annual rainfall2 (mm) Mean annual maximum Mean annual minimum
temperature a (°C) temperature a (°C)
A. madagascariensis Mahajanga (15°40'S, 47°03'E) 89 2236 31.93 18.2
A. grandidieri Ifaty (20°08'S, 44°33'E) 36 1156 31.72 20.85
A. rubrostipa Marofandilia (23°08'S, 43°36') 16 302 30.65 20.19
a Source: National meteorology service, 2004.
Fig. 1. Map of Madagascar's primary vegetation with occurrence zones (black symbols) of Adansonia rubrostipa (represented as stars), A. madagascariensis (represented as circles) and A. grandidieri (represented as triangles). Sources: adapted from Moat and Du Puy (1997) "With the permission of the Trustees of the Royal Botanic Gardens, Kew" and MBG (2009).
pots containing overwatered mixture and allowed to drain overnight. To reach field capacity, 200 mL water per pot was needed. For water-stressed and control treatments, pots were enclosed within plastic bags sealed at the stem base to allow measurement of water use. The control seedlings were weighed every second day, checked and watered to reach the 100% field capacity by replacing the amount of water transpired. Stressed plants were re-watered to reach only 50% of field capacity. This water content was reached in most pots.
2.3. Stable carbon isotope analysis
Leaf samples from well-watered seedlings of all three species (n = 12) were oven-dried at 60 °C and then used for stable carbon isotope analysis. Carbon isotope composition (513C) was measured
in the bulked dried and ground leaf blades at the Waikato Stable Isotope Unit (University of Waikato, New Zealand) using a gas chromatograph and magnetic sector mass spectrometer GC-MS (Europa Scientific Ltd., England). The main objective of this measurement was to confirm that all three species use the C3 photosynthetic pathway.
2.4. Water use measurements
Pots and their contents (soil plus plant) were weighed, then were re-watered every second day for 6 days. For each seedling, the difference between the weight after watering the pot in the previous weighing and the weight before watering in the current weighing was used to calculate water use in the intervening 2 days. Water use was measured by recording the amount of water needed
to restore initial soil water content (50% or 100% field capacity depending on the watering treatment). Water use measurements were done three times during the watering treatment: after 2, 4, and 6 days of water stress.
2.5. Gas exchange measurements
For the gas exchange measurements, 35- and 42-day old seedlings were used (n=48). Light response curves were first measured in order to determine optimal conditions of measurements. Light response curves were obtained by measuring net assimilation rate on the youngest fully expanded leaves at varying increasing levels of PAR (from 0 to 1000 pmolm 2s 1). Stomatal conductance (gs), net photosynthesis rate (A), and transpiration rate (EE) were measured using a GFS-3000 gas exchange system (Heinz Walz GmbH, Germany), with a 4 cm2 aperture plate in the leaf cuvette, and red plus blue LEDs light source (3040-L, Heinz Walz GmbH). Gas exchange measurements were performed on the youngest fully expanded leaves of the seedlings from 1000 h to 1300 h after 1 week and after 2 weeks of watering treatment. Measurements were undertaken at 370 prnol mol-1 CO2, flow rate of 750 prnol s-1, a saturation photon flux density of 1000 |imol m-2 s-1, 30 °C, 54% relative humidity (RH) and 20.5 Pa/kPa leaf to air vapor pressure difference (VPD). Water Use Efficiency (WUE) was calculated as WUE = A/E.
2.6. Chlorophyll content measurements (Chl)
Chlorophyll content of fresh leaves was determined using a SPAD-502 chlorophyll meter (Konica Minolta sensing Inc., Japan) at the beginning and at the end of the watering treatment. A calibration curve for the SPAD readings was established from spectrophotometry chlorophyll measurements in N, N-dimethylformamide extracts from discs 6.4 mm in diameter obtained from one leaf per seedling of the three species (n = 144). The equation (Chl) = 10A (SPADA0.267) was used and the calibration curve was fitted using the procedure of Markwell et al. (1995), yielding a calibration coefficient which is very similar to that obtained by Markwell et al. for other species (Glycine max and Zea mays).
2.7. Leaf water status measurements (RWC)
Fourteen days after the start of watering treatment, leaf relative water content for each species (n = 60) was measured according to the formula:
RWC = 100 (Fresh weight - Dry weight) / (Full-turgor weight - Dry weight)
Discs of 6.4 mm diameter were cut from leaves and weighed (fresh weight), then incubated in darkness and at room temperature in Petri dishes containing distilled water for 12 h and weighed again to obtain the full-turgor weight. Finally, disks were oven-dried at 85 °C for 24 h and weighed to obtain dry weight.
2.8. Growth measurements
Stem diameter was measured with digital calipers at 7 and 14 days of drought conditions for all species and both watering regimes (n = 144). Seventeen days after the beginning of watering treatments, stem, leaves, and roots of all the seedlings were harvested and oven dried at 105 °C for 48 h to a constant weight.
Thereafter, leaf weight ratio (LWR), stem weight ratio (SWR), and root weight ratio (RWR) were calculated according to the equations:
LWR = dry weight of leaves/total dry weight SWR = dry weight of stems/total dry weight RWR = dry weight of roots/total dry weight
Specific leaf area was calculated from the same samples as used for RWC measurements according to SLA=area/ dry weight.
2.9. Statistical analysis
Two-way analysis of variance (ANOVA), for testing significance of main effects and interactions, were undertaken with SPSS 16.0 software. For significant effects, differences between species were compared using Tukey's HSD test for every growth and photosynthetic parameter. Differences were considered significant at the P< 0.05 level. Error bars represent standard error of mean (±SE).
3. Results
3.1. Carbon isotope composition
The ô 13C of the three species differed significantly (P < 0.001). The ô13C of well-watered seedlings belonging to the three Adansonia species were typical of C3 photosynthetic metabolism. The ô 13C of A. rubrostipa (- 27.01 %o) was lower than those of A. madagascariensis (— 24.35%), and A. grandidieri (— 25.65%).
3.2. Water use (WU)
Water use values varied from 4.94 to 32.96 mL per day for A. rubrostipa, from 10.91 to 41.67 mL per day for A. madagascariensis and from 6.74 to 33.15 mL per day for A. grandidieri (from water-stressed to well-watered seedlings). Interaction between species and watering treatment was significant (P < 0.05). WU was significantly higher with well-watered seedlings (P < 0.001). The average WU of A. grandidieri and A. rubrostipa were significantly lower than that of A. madagascariensis (Fig. 2).
3.3. Gas exchange measurements
Light curves showed light compensation points of about 20 pmolrn—2 s — 1 that were similar for the three species as shown in Fig. 3A-C. For all gas exchange measurements, interaction effects between main factors were not statistically significant (P=0.05) except for the drought durationx watering treatment
Fig. 2. Water use per seedling (WU) of three Adansonia species. Each measurement is the mean of three measurements after 2, 4, and days of drought. Mean±SE (n = 144 observations on 24 seedlings per species and treatment).
significantly higher in well-watered seedlings compared to water-stressed ones when subjected to drought during 7 or 14 days (Fig. 4A-B-C). The same effect was found with transpiration rate (P< 0.001; Fig. 4D-E-F). Additionally, transpiration rate of all species slightly increased from the first to the second week of drought but such changes were not statistically significant. In a species comparison, the lowest transpiration rate was found in A. rubrostipa regardless of watering treatment. Stomatal conductance decreased markedly (P< 0.001) with drought for all species (Fig. 4G-H-I). Decrease in all those three parameters led to an increase in Water Use Efficiency which was then significantly higher (P<0.001) in all water-stressed conditions of all three species (Fig. 4J-K-L).
interaction of the assimilation rate parameter (Table 2) which was significant (P< 0.01). Moreover, no statistical effects of either drought duration (7 vs. 14 days) or interspecific variations were observed. In addition to that, net assimilation rate (P < 0.001) was
Fig. 3. Typical response of net assimilation rate to PAR irradiance in Malagasy Adansonia grandidieri (A), A. madagascariensis (B) and A. rubrostipa (C) seedlings under well-watered conditions.
3.4. Chlorophyll content (Chl)
Species x watering treatment x drought duration interaction as well as species x watering treatment interaction were not statistically significant. Species x drought duration interaction was significant at P=0.03. A. grandidieri displayed the highest amount of chlorophyll among the three species (Fig. 5A-B-C, P< 0.001). Moreover, chlorophyll content increased after 14 days for both watering regimes (P< 0.001).
3.5. Growth and leaf water status measurements
For all growth and leaf water status parameters, drought duration x species x watering treatment interaction, species x watering treatment interaction, and species x drought duration interaction were not statistically significant. Watering treatment x drought duration interaction was statistically significant (P < 0.001). The stem diameter increased significantly with chronological age among the well-watered seedlings (P < 0.001), while it remained invariable in water-stressed seedlings whatever their ages (Fig. 6A-B-C). A. grandidieri and A. madagascariensis displayed significantly higher stem diameters than A. rubrostipa (P < 0.001). As interaction between species and watering treatment factors was not significant for RWC and all biomass related parameters, data were pooled and the variation attributed to species (Table 3) and drought (Table 4) were separately assessed. The highest RWC was found with A. rubrostipa (62.62%). A. grandidieri and A. rubrostipa invested significantly more biomass in roots and in stems than did A. madagascariensis (P < 0.001). This latter species allocated more biomass to leaves compared to the two other species (P < 0.001). Relative water content was higher for well-watered seedlings (P < 0.01). Compared to water-stressed seedlings, well-watered ones allocated significantly higher amounts of biomass to stems than they did to the other parts of the plants (P < 0.01). Drought did not affect SLA but the three species differed significantly from each other. A. madagascariensis had the highest SLA, A. grandidieri had intermediate SLA and A. rubrostipa the smallest SLA (Table 3). SLA was 50% larger in A. madagascariensis than in A. rubrostipa, while SLA in A. grandidieri differed from that in A. rubrostipa by only 9%.
Comparison of P-values from factorial ANOVA of Net assimilation rate (A), Transpiration rate (E), Stomatal conductance (gs), Water Use Efficiency (WUE) in terms of drought duration, species, watering treatments, and drought duration x species, drought duration x watering treatment, species x watering treatment, drought duration x species x watering treatment interactions in Adansonia.
A (p.mol m 2 s !) E (mmol m 2 s 1) gs (mol m 2 s 1) WUE (mol mol 1)
Drought duration 0.57 ns 0.89 ns 0.09 ns 0.02*
Species 0.16 ns 0.07 ns 0.09 ns 0.32 ns
Watering treatment 0.000*** 0.000*** 0.000*** 0.000***
Drought duration x Species 0.17 ns 0.74 ns 0.65 ns 0.91 ns
Drought duration x Watering treatment 0.008** 0.72 ns 0.84 ns 0.94 ns
Species x Watering treatment 0.58 ns 0.26 ns 0.19 ns 0.50 ns
Drought duration x Species x Watering treatment 0.91 ns 0.86 ns 0.94 ns 0.59 ns
* 0.01 <P>0.05; **, 0.001 <P>0.01 ; ***, P < 0.001 ; ns, non-significant.
Fig. 4. Net assimilation rate (A), Transpiration rate (E), Stomatal conductance (gs), Water Use Efficiency (WUE) in four well-watered and four drought-stressed seedlings/treatment/species of three Adansonia species: A. grandidieri (A—D—G—J), A. madagascariensis (B-E-H-K), A. rubrostipa (C—F—I—L) after 7 days and 14 days of drought treatment. Mean±SE (n=48). P-values shown in Table 2.
Fig. 5. Leaf chlorophyll content (Chl) in well-watered and water-stressed seedlings of three Adansonia species: A. grandidieri (A), A. madagascariensis (B), A. rubrostipa (C) after 7 days and 14 days of drought. Mean±SE (n = 144). P-values for watering regime (W) and for species (S) are shown in the upper right of the figure.
Fig. 6. Seedling stem-base diameter in well-watered and drought-stressed seedlings of three Adansonia species: A. grandidieri (A), A. madagascariensis (B), A. rubrostipa (C) after 7 days and 14 days of drought. Mean± SE (n = 144). P-values for watering regime (W) and for species (S) are shown in the upper right of the figure.
4. Discussion
Previous studies reported that Malagasy adult Adansonia are structurally and morphologically adapted to drought (Chapotin et al., 2005,2006a, b); however, despite several studies that focused on responses of Adansonia digitata to drought (Cuni Sanchez et al., 2011; De Smedt et al., 2012; Sanchez et al., 2010), little is known about Malagasy Adansonia seedling vulnerability and adaptation to water stress. To our knowledge, this is the first paper dealing experimentally with such issue.
The use of isotope discrimination analysis ascertained that the three Adansonia species are C3 plants. Light response curves in
seedlings of all three species showed a light compensation point (20 pmolm-2 s-typical of C3 plants at 30 °C (Sharp et al., 1984).
Like in most C3 species for which there are tradeoffs between maximizing growth and acclimation to survive (see for example Bacelar et al., 2007; França et al., 2000; Hessini et al., 2009; Wu et al., 2008; Yin et al., 2005) drought depressed Adansonia seedling growth. As reflected by biomass measurements, seedlings invested less carbon in building aboveground organs particularly stems that are not actively involved in photosynthesis to optimize their water balance during water stress. In our study, stem diameters of well-watered seedlings
Comparison of relative water content (RWC), biomass, leaf weight ratio (LWR), stem weight ratio (SWR), root weight ratio (RWR), specific leaf area (SLA) and leaf area per seedling (L) between the three Adansonia species (using marginal means, because species x watering treatment interaction was not significant). Leaf area was not measured directly.
Species RWC (%) Biomass (g) LWR SWR RWR SLA (cm2 g-') L (cm2)
A. grandidieri 42.91 2.34 0.26 0.35 0.39 78 47
A. madagascariensis 37.05 2.10 0.49 0.30 0.21 113 116
A. rubrostipa 58.39 1.59 0.30 0.34 0.35 85 41
*0.01 <P>0.05; **0.001 <P>0.01; ***P< 0.001; ns, non significant; -: not tested.
FS: species effect; P>FS indicates comparisons between three species.
were significantly bigger than that of water-stressed seedlings after 14 days of drought. In African A. digitata, stems were reported to contain up to 20% of Adansonia seedlings total water content (De Smedt et al., 2012). Thus we might suggest that such differences are the result of a mere variation in stem water status and cell turgor. More precisely, stem water content of droughted seedlings might be lower than well-watered ones, resulting in a smaller stem diameter. Incidentally the same behavior is also found in adult Adansonia trees whose stem diameter decreased during the leaf flushing period in dry seasons when stored water in stems is used to maintain leaf turgor (Chapotin et al., 2006a, 2006b).
Chlorophyll content was unaffected by drought in our experiment. This result is consistent with results from a related species, Ceibapentandra (according to Baum et al., 2004) within a rainforest mesocosm; C. pentandra showed changes neither in pigment (e.g., chlorophyll a, chlorophyll b, violaxanthin) concentrations nor in chlorophyll a/b ratio during watering treatment (Rascher et al., 2004). Drought had markedly reducing effects on all photosynthetic parameters and increasing effect on WUE in the three Adansonia species. Same trends have also been recorded in A. digitata (De Smedt et al., 2012), Pseudobombax septenatum (Hogan et al., 1995), Ceiba pentandra (Rascher et al., 2004), Ceiba samauna (Slot and Poorter, 2007) that are all included in MALVACEAE family. As in our studies, De Smedt et al. (2012) did not find photosynthetic differences in seedlings from different geographic provenances.
Our results suggest that the main cause behind the observed depression in photosynthetic rate was the reduced stomatal conductance resulting from partial closure of stomata. One way for Adansonia seedlings to avoid water loss is then to close their stomata to limit water loss and to maintain a RWC high enough
Table 4
Comparison of relative water content (RWC), biomass, leaf weight ratio (LWR), stem weight ratio (SWR), root weight ratio (RWR) between well-watered and water-stressed seedlings ofthe three Adansonia species (using marginal means because species x watering treatment interaction was not significant).
Watering treatment RWC (%) Biomass (g) LWR SWR RWR
Well-watered 49.50 2.43 0.34 0.34 0.30
Water-stressed 42.74 1.59 0.34 0.32 0.32
P > FT * *** ns * ns
* 0.01 <P>0.05; **, 0.001 <P>0.01 ; ***, P < 0.001 ; ns, non-significant. FT: Watering treatment effect; P> FT indicates comparisons between watering regimes of all three species.
for their survival. Adjustment of stomatal conductance allows for optimization of carbon assimilation in relation to water supply (WUE). Slight increase in assimilation rate, stomatal conductance, and transpiration rate in 14 days water-stressed seedlings when compared with 7 days water-stressed seedlings argues for seedlings' ability to acclimatize through time.
We observed that well-watered A. rubrostipa seedlings had a slightly less negative 513C than the other two species considered; A. grandidieri displayed an intermediate behavior, while A. madagascariensis had the lowest 513C, which would suggest that A. rubrostipa had lower WUE than A. grandidieri and A. madagascariensis. However, we did not find any differences in WUE between the species. Because 513C increases with growth irradiances (Farquhar et al., 1989) we suggest that the reason why 513C failed to predict accurately WUE is the relatively low-light conditions of the growth chambers contrasting with the saturating irradiance that we used for gas exchange measurements.
A. rubrostipa used the smallest amount of water, A. madagascariensis the greatest, with A. grandidieri in between. These are whole-plant WU values which depend both on stomatal conductance and the amount of foliage per plant. Due to technical issues, we did not scale WU with leaf area (L), but from leaf dry weights and SLA, it can be inferred that A. madagascariensis had approximately twice as much leaf area than the other two species (Table 3). The leaf dry weights were 0.83-1.19 g for A. madagascariensis, 0.36-0.58 g for A. rubrostipa and 0.51-0.71 g for A. grandidieri. As SLA are supposed to be the same across foliage in seedlings of the same species, multiplying leaf dry weights by SLA values enabled us to approximate the value of L. Moreover, specific leaf area (SLA) calculated from leaf discs that we used from water content data showed that SLA significantly differed between species. The larger leaf area (L) of A. madagascariensis than of A. rubrostipa and A. grandidieri together with small and non-significant differences among species in light-saturated stomatal conductance, both in well-watered and drought conditions, allow us to infer that the differences in WU were mainly a consequence of differences in the amount of foliage. In relative terms, the differences between species were largest under drought when WU by A. rubrostipa was approximately one half that by A. madagascariensis.
The effect of drought on growth was similar for all species. Plant dry mass at the end of the experiment was largest for A. madagascariensis, indicating a faster growth rate, which can be explained by a larger allocation of growth to foliage than in the other two species. This was accompanied by a reduced allocation
to roots, results that are in accordance with A. digitata's responses to drought (Cuni Sanchez et al., 2011; De Smedt et al., 2012). Apart from developing deep fine roots to access underground water (like most drought-tolerant species), Adansonia seedlings develop thick taproots enabling them to store water (De Smedt et al., 2012; Wickens and Lowe, 2008). A. grandidieri and A. rubrostipa which both originate from drier environments allocated more resources to such root development in comparison with A. madagascariensis. A. madagascariensis also had the lowest RWC in leaves, suggesting that the other two species had some degree of leaf water retention. Our results are in accordance with studies on Adansonia digitata which reported that Adansonia morphology and biomass allocation depend on seedlings provenances, and that seedlings from drier provenances have higher water content and invest more biomass in their root system but less in foliage (Cuni Sanchez et al., 2011; De Smedt et al., 2012).
A. rubrostipa stands out with a significantly higher RWC and A. madagascariensis with the highest WU. A. rubrostipa and A. grandidieri probably used water slightly more efficiently than A. madagascariensis. A. grandidieri had water use similar to A. rubrostipa but RWC and transpiration pattern similar to A. madagascariensis. The differences between species seem to be due mainly to differences in biomass allocation; higher allocation to roots should be even more effective for drought tolerance in plants growing in the open ground than in pots. All these results suggest that A. madagascariensis is the least drought tolerant of the three species and A. rubrostipa the most tolerant one, with A. grandidieri having intermediate tolerance. Even though our results are limited by the fact that drought duration during our experiment was very short compared to the three- to five-month long drought under natural conditions these relative degrees of drought tolerance correlate with the water availability in the natural ranges ofthe species: A. madagascariensis is mostly found in subhumid regions (dry deciduous forests between Mahajanga and Antsiranana, Sambirano) and the two other species are found in drier areas, with A. grandidieri subjected to higher rainfall than A. rubrostipa (Baum, 1995, 1996). The fact that A. rubrostipa is the most tolerant species among the three might explain why it has greater ecological plasticity and a larger area of distribution than the other two species. Therefore, drought might be a factor influencing the distribution patterns of Adansonia in Madagascar Island.
From our results, we suggest that even though water stress limits growth and photosynthesis in Adansonia seedlings, their efficient water use strategy and biomass allocations enable them to withstand drought. Moreover, in their natural environments A. rubrostipa has the lowest rate of regeneration while A. madagascariensis and A. grandidieri have medium rates of regeneration (Razanameharizaka, 2009), which is in contrast with our results where A. rubrostipa is the most adapted to drought. Therefore other environmental factors might explain the natural regeneration of Adansonia. For example, the environments where Adansonia occur are subjected to frequent slash and burn practices. Even though surveys undertaken by Marie et al. (2009) found evidence that people are protecting adult and juvenile Adansonia trees, the probability of increased seedling mortality due to fire
cannot be ruled out. Dispersal elements may also be another factor that may account for this low rate of regeneration. For instance, it might be linked to limited seed dispersion (probably due to the lack of suitable agents of dispersion) or high seed predation rate. Another possibility also is that regeneration of A. rubrostipa depends on light brought by occasional canopy gaps as suggested by Metcalfe and Trevelyan (2007).
In conclusion, Malagasy Adansonia seedlings of different species use the same mechanism, mainly partial stomatal closure, to withstand drought events; however, seedlings of the more drought-tolerant species are structurally different with larger root systems and proportionally less foliage. The short-term response to drought leads to reduced stem diameter, biomass and growth when seedlings face a need to maximize water use efficiency. With their higher root biomass, compound leaves and lower WU, however, A. rubrostipa and A. grandidieri seedlings are better adapted to the arid region in the southwestern part of the Island where they naturally co-exist; A. madagascariensis which does not present such characteristics is present in less arid areas of the northwestern region.
Acknowledgments
We are grateful to the "University of Helsinki's exchange program" which provided T. Randriamanana with a grant for her stay in Helsinki. The authors also thank "URP Forêts et Biodiversité" Madagascar, especially Dr. Pascal DANTHU for having generously provided the seeds and helped with administrative processes needed for seeds exportation. We also thank Dr. Aro Vonjy RAMAROSANDRATANA, (Laboratory of Plant Physiology, University of Antananarivo, Madagascar) for his valuable comments and Sharon Bowen, E.L.S. for her edits on the earlier versions of the manuscript.
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Edited by OM Grace
