XYLEM CHARACTERISTICS AND ECOPHYSIOLOGY OF TROPICAL TREES FROM FOUR CLADES OF SPERMATOPHYTES

Jonathan Chua En Zhe, Ng Yu Song, Chung Ray Ern, Chew Zichun1, Jeffrey Lee2

1Raffles Institution, One Raffles Institution Lane, 575954 2Raffles Science Institute, One Raffles Institution Lane, Singapore 575954

ABSTRACT The structure and function of xylem vessels in different clades of vascular woody reflect trade- offs during evolution between conductive efficiency and conductive safety. We studied four tropical tree species representing main clades of spermatophytes to trace xylem phylogenesis and see how this impacts transport efficiency versus risk of cavitation. Vessel anatomy was studied with light microscopy; vessel resistance was determined by the single-vessel resistance method; and transpiration pull was assessed using the Scholander pressure chamber. We described three phylogenetically consistent xylem anatomy patterns, viz. gnetophyte, magnoliid-eudicot, and monocot. The efficiency indices of lumen conductivity diameter and vulnerability index were useful in describing a trend in xylem anatomy among the four species - with tigillarium and Gnetum gnenom representing two ends of an efficiency-safety trade-off spectrum, respectively. Unlike the magnoliid iners, the eudicot Pometia pinnata, and the monocot O. tigillarium, the gnetophyte G. gnemon was the only species with significantly different high and low vessel resistance readings; possibly due to the unique gnetophyte pattern which lacks vessel groups, resulting in water not being able to bypass end walls via lateral movement through pits into neighbouring vesssels to reduce resistance.

INTRODUCTION No two plants have the exact same wood anatomy, allowing us to identify wood to the genus, or even species, of the [1]. Being a product of evolution, the structure of wood matches its function under particular ecological conditions, and is thus a record of phylogenetic history[1]. The xylem of plants is adapted to the plants’ habitat through variations in anatomical features such as vessel diameter, vessel density or inter-vessel wall thickness. Wood must accomplish three functions: mechanical strength, conductive efficiency and conductive safety. Studies indicate that the structure and function of xylem, after providing for enough mechanical strength, reflect a trade-off between efficiency (greater flow rates) and safety (reduced vulnerability to cavitation). This is because to produce efficient sap flow in xylem under negative tension, vessels should be wide and offer little resistance. Wide vessels, however, are vulnerable to cavitation, where the resulting gas embolism breaks the continuity of the water column and prevents the transport of water[2]. In order to achieve conductive safety, xylem can evolve in different ways[1]. Vessels may become narrower with thicker walls to increase surface tension and capillary action, reducing the risk of cavitation. Vessels may also group together so that water can be rerouted to other vessels in the event of cavitation. These changes, however, would increase xylem resistance at the expense of efficiency. An illustration of such a trade-off occurring within the same plant can be seen in temperate ring-porous wood species like oak. In spring when water is abundant, the plant risks cavitation by developing early wood containing vessels with large lumen and thin walls for maximal flow and minimal resistance. In summer when there is more water stress and increased possibility of cavitation, late wood with thicker vessel walls and smaller lumen is produced that has less efficient water flow, higher resistance, but greater safety in terms of less vulnerability to cavitation[3]. Such relationships have been formalized using principles of fluid dynamics. Xylem hydraulic resistivity is determined by the lumen resistivity (RL) and end wall resistivity (RW). Lumen resistivity (RL ) can be calculated by the Hagen-Poiseuille equation:

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4 RL = 128 η/(πD ) where η is the viscosity at 20°C and D is the diameter of the xylem conduit. End walls in plant xylems, which can be studded with pits, cause end wall resistivity. A longitudinal file of conduits creates an axial flow path through overlapping end walls. The total end wall resistivity (RW) is a function of flow resistance through the connecting pits and the distance between successive end walls. The total [4] resistivity of the conduit (Rc) is roughly the sum of the two values RL and RW . In some plants, the total resistivity is close to the value of the lumen resistivity, suggesting negligible end wall resistivity. Other plants have high end wall resistivity, with short conduits that have end walls close together and less area for pits in the overlapping regions. In general, high end wall resistivity suggests limited conduit conductivity, in exchange for increased safety against cavitation. Low end-wall resistivity promotes increased conductivity, but with higher risk of cavitation. As mentioned, vessel grouping is another indicator of water stress in the environment. Plants under significant water stress tend to group xylem vessels together, as water can be conducted via adjacent vessels through the pits in the vessels if cavitation occurs, or an end wall adds resistance to the flow of the water. In summary, plants under significant water stress will tend to evolve vessels with smaller diameter, thicker vessel walls, higher end wall resistivity and greater vessel grouping. Conversely, plants under less water stress will tend to develop wider vessels, thinner vessel walls, lower end wall resistivity and fewer vessel groupings.

To assess the relative contribution of various xylem properties to conductive efficiency and safety for interspecific comparisons, we used four indices described in Lens et al. (2011)[8]. The two efficiency indices are the lumen conductivity diameter (DLC) - a measure of the vessel lumen conductivity determined from the Hagen-Poiseuille equation; and the vulnerability index (VI) of the vessel to cavitation - which combines the effects of vessel diameter and grouping on cavitation rates. Higher DLC indicates larger lumen, which correlates with higher efficiency in transporting water. But this would result in high VI correlated with lower safety and greater danger of cavitation. Plants with such attributes are expected to live in environments with low water stress[8]. The two safety indices are the vessel grouping index (GI) - a measure of the frequency of vessel groups; and the thickness-to-span ratio of the vessels (TSR) - which combines the effects of inter-vessel wall thickness and vessel lumen conductivity diameter on safety from cavitation. Plants under higher water stress will tend to group vessels together (higher GI) and develop thicker walls and narrower vessels (higher TSR). Definitions and calculations of these indices are detailed in Table 1.

The Scholander pressure chamber is a reliable method for determining the water status and stress of [12] plants . The stem water potential (ΨS) indicates the capacity of the plant to conduct water from the soil to the atmosphere, and has been successfully applied as a water deficit indicator in tree orchards[12]. ΔΨ, the difference between leaf water potential (ΨL) and stem water potential (ΨS) measured simultaneously on the same plant, was shown to be an indicator of instantaneous shoot transpiration[12].

Research on temperate plants has revealed much about how environmental factors have shaped the structure of xylem vessels during evolution, but similar studies on tropical plants have been relatively lacking. We aim to take a first look at the xylem characteristics and ecophysiology of representatives of tropical plants in four major clades of native spermatophytes: Gnetum gnemon (gnetophyte), Cinnamomum iners (magnoliid), Pometia pinnata (eudicot) and (monocot). G. gnemon and C. iners are found in evergreen forests, the latter a common pioneer tree in secondary forests. O. tigillarium, grows near swamps. There are two subspecies of P. pinnata: f. alnifolia from swamp forests, and f. glabra from well-drained soil[13]. Fig. 1 summarizes the evolutionary relationship among the species. We hope to obtain an overview of the divergence of xylem structure and function during phylogenesis in tropical trees, and contribute to better knowledge of our native plants. We hypothesize that the crown group consisting of the three angiosperm clades would share synapomorphies that distinguish them from their sister group, the gnetophytes.

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Spermatophytes (seed plants) “Gymnosperms” Angiosperms (flowering plants) “Dicotyledons” Clade Gnetophytes Monocots Order Gnetales Magnoliales Family Gnetaceae (Berlinjau family) (Laurel family) ( family) (Palm family) Species Gnetum gnemon Cinnamomum iners Pometia pinnata Oncosperma tigillarium (Berlinjau) (Wild Cinnamon) (Kasai Daun Besar) (Nibung Palm) Phylogeny

Fig. 1. Evolutionary relationship among Gnetum gnemon, Cinnamomum iners, Pometia pinnata and Oncosperma tigillarium. Clades, order and family according to Judd et al. 2008[10]. Common names in brackets according to Boo et al. 2006[11].

By correlating xylem structure and function with environmental factors, we may gain a better picture of how plants may be affected in the face of climate change; particularly local changes in factors which affect transpiration, such as rainfall, temperature and humidity. In this way, we may be in a better position to predict clades of plants with particular xylem attributes that might be severely affected, and devise timely measures to protect such vulnerable groups.

MATERIALS AND METHODS Plant material Branches were obtained from four species of plants located in open, unshaded areas of the school campus: 15 and 10 2 m-tall potted saplings of G. gnemon and P. pinnata, respectively, bought from a local nursery and placed behind the science laboratories; a coppiced individual of C. iners located near the school gate; and an O. tigillarium cluster planted in 1997 beside the school library. All the specimens were planted in well-drained soil under similar conditions in the same vicinity. This minimized any developmental changes that might have accrued so we can assume that the differences we observed would be due more to descent and the influence of their respective natural habitat over evolutionary time.

Xylem anatomy Thin transverse sections of the secondary stem of each species were cut by hand using a Gillette razor blade (with the exception of O. tigillarium which only possesses a primary stem due to its growth form). Following Schoch et al. (2004)[5], the sections were softened in formalin-acetic acid-alcohol (FAA), soaked in 20% bleach, stained with 1% safranin, rinsed in water, dehydrated through a sequence of 50%, 96% (twice) and 100% ethanol, cleared in Histo-clear solution (National Diagnostic, Atlanta, GA, USA) and mounted in Euparal (ANSCO Laboratories, Manchester, UK). The anatomical parameters listed in Table 1 from sections of three different branches for each species were measured, derived and analyzed under a 100x light microscope using Applied Vision imaging system (Ken-A-Vision, Kansas City, MO, USA) and ImageJ software[6].

Single-Vessel resistance A modified procedure of Zwieniecki et al. (2001)[7] was used to measure single-vessel resistance. Segments of 1.5–4.5 cm length of the secondary branch of the plant (or primary branch for O. tigillarium) were cut and soaked to saturate the vessel conduits with water. The length, and the average of three diameter readings taken from the two ends and centre of the segments were measured. A borosilicate glass capillary tube (O.D. 1.5 mm; I.D. 0.86 mm) was pulled into a fine tip with a P30 micropipette puller (Sutter Instrument, Novato, CA, USA) and inserted into the open lumen of a single

3 vessel using a 67.5x stereomicroscope (Olympus SZ61, Tokyo, Japan) and a micromanipulator consisting of a motion controller (New Focus, San Jose, CA, USA) manipulating a precision stage and picomotor-driver (Newport, Irvine, CA, USA). The micropipette tip was fixed in place to the vessel by a liquid cyanoacrylate glue (3M Scotch, St Paul, MN, USA) and setting was hastened with a glue accelerator (Bob Smith Industries, Atascadero, CA, USA) Compressed nitrogen gas was forced through the vessel from the same direction as water flowed to the leaves and the pressure when steady bubbling was seen at the opposite end of the stem submerged in water was recorded.

Water stress and transpiration pull A Scholander pressure chamber (PMS Instrument Model 670, Albany, OR, USA) was used to compare the water status of our specimens. Leaf samples were collected at or shortly after midday (1230-1430 h). To determine the leaf water potential (ΨL), a leaf was covered with a plastic bag and cut off directly at the base of the petiole. To find stem water potential (ΨS), a leaf was covered with an opaque, reflective bag for 15 min before cutting to allow the water potential in the leaf and stem to reach equilibrium. Air temperature, humidity, light intensity and soil temperature at the moment of leaf collection were recorded with a digital hygrothermometer, lux meter (Lutron LX-101, Taiwan) and soil thermometer, respectively.

RESULTS AND DISCUSSION Xylem anatomy Figure 2 shows the transverse sections of the xylem for the four species. The anatomical parameters and indices obtained from light microscopy are defined in Table 1 and summarized in Table 2. Three distinct patterns of xylem anatomy could be discerned: (1) the gnetophyte pattern of G. gnemon with small, densely-packed, single, ungrouped vessels arranged radially; (2) the magnoliid-eudicot pattern of C. iners and P. pometia with single or grouped, intermediate-sized vessels, radially arranged and more separated from each other; (3) the monocot pattern of O. tigillarium with widely separated scattered bundles of large, single or grouped, vessels. The scattered vascular bundles of O. tigillarium arise from the unique form of diffuse secondary growth of monocots[3] resulting in the vessel density for O. tigillarium being an order of magnitude lower than that of the rest.

Fig. 2. Photomicrographs of transverse sections of xylem in four species of plants.

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Table 1. List of measured/derived xylem anatomical parameters and calculated indices, with definitions and units employed, modified from Lens et al. 2011[8]. Symbol Name Definition & calculation Units Measured parameters 2 AT vessel area transverse area of vessel µm Davg vessel diameter (D) measured as that of a circle with the same area as the vessel µm diameter lumen;

average diameter of vessel =

TW inter-vessel inter-vessel wall thickness = double thickness of vessel wall making contact µm wall with an adjacent vessel thickness Den vessel = vessel frequency = number of vessels per wood area mm-2 density Calculated efficiency and safety indices th DLC lumen to reflect the fact that lumen conductivity increases with diameter to the 4 µm conductivity power, we calculate the vessel diameter corresponding to the average lumen diameter conductivity determined from the Hagen-Poiseuille equation = (Σ(D4/n))0.25 VI vulnerability vessel diameter corresponding to average lumen conductivity divided by - -1 Index vessel density = DLC (Den)

GI vessel = ; a solitary vessel counts as one vessel group - grouping index TSR thickness- = inter-vessel wall thickness divided by vessel lumen conductivity diameter - -1 to-span ratio = TW DLC Table 2. Xylem anatomy parameters (mean + standard deviation) and calculated efficiency and safety indices (defined in Table 1) for four species of plants. More than 100 xylem vessels in total were measured for each species. Species Parameters Indices Efficiency Safety Vessel area Vessel Intervessel Vessel Lumen Vulnerability Vessel Thickness- 2 AT / µm diameter wall density conductivity index grouping to-span ratio -1 Davg / µm thickness Den / mm diameter VI index TSR TW / µm DLC / µm GI Gnetum 1.25 + 0.53 x103 42.2 + 10.6 no vessel 137.5 + 9.0 53.4 0.39 no vessel no vessel gnemon groups groups groups Cinnamomum 1.52 + 0.71 x103 46.5 + 12.1 9.8 + 6.0 57.3 + 5.7 66.4 1.16 1.35 0.21 iners Pometia 3.41 + 2.0 x103 67.8 + 22.2 14.4 + 18.1 34.4 + 8.4 72.8 2.12 1.65 0.45 pinnata Oncosperma 3.66 + 1.4 x103 71.4 + 13.6 25.3 + 4.5 2.8 + 0.7 110.5 39.46 1.49 0.29 tigilarium

The efficiency indices imply a clear and consistent trend of increasing conductivity and vulnerability to cavitation along the sequence of species: G. gnemon < C. iners < P. pinnata < O. tigillarium. This is a consequence of the increasing trends in vessel area (AT) and vessel diameter (Davg), accompanied by the opposite decreasing trend in vessel density (Den) for the respective species. The combination of large lumen (DLC) with low vessel density (Den) for O. tigillarium yielded a VI an order of magnitude higher than that of the rest, underscoring it’s particularly high efficiency and vulnerability to cavitation. These results indicate that O. tigillarium has adapted to conduct water extremely efficiently at the risk of cavitation. In contrast, G. gnemon, with its converse characteristics of small vessel diameter, small vessel area and high vessel density, appears to possess characteristics adapted to maintain conductive safety at the expense of conductive efficiency. In fact, G. gnemon has no vessel groups at all, having only single vessels, packed at a density highest amongst the species. The absence of vessel groups for G. gnemon meant that we could not calculate safety indices for this species. The safety indices for the remaining three species did not show a consistent trend and the GI appeared to be not much different from each other. On the whole the safety indices were not as useful as the efficiency indices in showing trends. 5

Single-Vessel Resistance The results obtained from the single-vessel resistance measurements were standardized using ANCOVA[14] to remove the effect that varying lengths and diameters of the stem segments might have on single-vessel resistance. The frequency distributions of the standardized readings are shown in fig. 3. Though the four graphs all appear to be bimodal, ANCOVA revealed that only G. gnemon had two statistically different modes (Levene’s test: F=7.4725; p = 0.026), while C. iners (F=0.06925; p=0.803), P. pinnata (F=0.0002572; p=0.988) and O. tigillarium (F=0.9943; p=0.357) did not. Therefore, we present the single-vessel resistance values for the four species in Table 3 as five categories: G. gnemon (RL), G, gnemon (RC), and C. iners, P. pinnata, O. tigillarium (RC). This is aligned with the idea mentioned earlier that total conduit resistivity (RC) = lumen resistivity (RL) + end wall resistivity (RW).

Fig. 3. Frequency distribution of single-vessel resistance measurements from four species of plants.

There was only marginal statistical difference among the resistance values of the four species based on Levene’s test (p=0.06). Among the pairwise differences, only G. gnemon RL was significantly different from RC as already shown. All other pairwise differences were statistically insignificant. Thus although G. gnemon (RC) had the highest single-vessel resistance, followed by C. iners, O. tigillarium, and P. pinnata, these interspecific comparison results were inconclusive.

Table 3. Single-vessel resistance values [mean + standard error (sample size)] for four species of plants with pairwise comparison p values from ANCOVA analysis (Levene’s test for equality of error variance: F=2.5435, p=0.06). Species Single-Vessel ANCOVA pairwise comparison p Resistance / MPa values

Gnetum gnemon (RL) 5.6 + 8.0 (6) 0.01; 0.06; 1; 0.22

Gnetum gnemon (RC = RL+ RW) 43.1 + 9.0 (4) 0.01; 1; 0.31; 1

Cinnamomum iners (RC) 35.4 + 5.9 (7) 0.06; 1; 0.32; 1

Pometia pinnata (RC) 17.4 + 5.3 (10) 1; 0.31; 0.32; 0.35

Oncosperma tigillarium (RC) 34.9 + 6.7 (8) 0.22; 1; 1; 0.35

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Water stress and transpiration pull Data obtained using the Scholander pressure chamber were standardized using ANCOVA analysis[14] to remove the effects of the covariables humidity, air temperature, soil temperature and light intensity, [12] and are summarized in Table 4. Using ΨS as a water deficit indicator , P. pinnata was under the highest (most negative) water stress followed by O. tigillarium, G. gnemon, and C. iners. Using ΔΨ as an indicator of instantaneous shoot transpiration[12], G. gnemon had the most negative transpiration pull, followed by O. tigillarium, C. iners, and P. pinnata.. However, due to the small sample size of readings, the Levene’s test showed no significant differences between the ΔΨ of the four species, so the data on water stress and transpiration pull were inconclusive as well.

Table 4. Standardized leaf (ΨL) and stem (ΨS) water potential (mean + standard error) obtained from Scholander pressure chamber measurements for four species of plants. Sample size = 3 readings from 3 individuals for G. gnemon and P. pinnata; 3 readings from 1 individual for C. iners and O. tigillarium. The ANCOVA pairwise comparison p values (Levene’s test for equality of error variance: F=1.8803, p=0.211) and the calculated transpiration pull (ΔΨ = ΨL - ΨS) are shown. Species Leaf water Stem water Transpiration ANCOVA pairwise potential ΨL / potential ΨS / pull ΔΨ / MPa comparison of ΔΨ p MPa MPa value Gnetum gnemon -172.6 + 50.8 -30.4 + 41.0 -142.2 + 10.6 0.008; 0.005; 0.011

Cinnamomum -132.9 + 37.9 -114.7 + 30.6 -18.2 + 7.9 0.008; 1; 0.980 iners Pometia pinnata -128.6 + 36.6 -117.5 + 29.6 -11.1 + 7.7 0.005; 1; 0.564

Oncosperma -146.0 + 35.8 -110.8 + 28.9 -35.1 + 7.5 0.011, 0.981; 0.564 tigillarium CONCLUSION Figure 4 summarizes the overall results of our study. The efficiency indices showed a clear trend of trade-off for the four species. At one end of the spectrum was G. gnemon - with its gnetophyte pattern of small, single, densely-packed vessels adapted to maintain conductive safety at the expense of efficiency. At the other extreme was O. tigillarium - with its monocot pattern of large, grouped, widely scattered vessels adapted to conduct water very efficiently but at the risk of vessel cavitation. C. iners and P. pinnata – with their magnoliid-eudicot pattern of intermediate-sized, grouped, intermediate density vessels – would, by this reasoning have moderate efficiency and safety, balancing the respective demands of both. Data from resistance and water potential measurements could have shown the ecophysiological evidence for these conclusions: Plants under high water stress would be predicted to have high resistance and high transpiration pull while plants under low water stress would have conversely low values. Our ecophysiological data, though not statistically significant, offered both corroborative support (high resistance and transpiration pull in G. gnemon) as well as conflicting evidence (resistance and transpiration pull of O. tigillarium not the lowest but higher than that of P. pinnata). More work on these two aspects would be needed to clarify matters, in particular, a complete leaf and stem water potential profiling of the plants over the course of the day. On a side note, it is interesting to point out our finding of P. pinnata having the lowest resistance and least negative transpiration pull of all, may imply that the specimens we examined were the swamp (P. pinnata f.alnifolia) rather than the road-side tree (P. pinnata f. glabra) form.

The anatomical findings were consistent with the habitats of the angiosperm clades: the swamp- dwelling O. tigillarium can probably afford to have large vessels of high efficiency but greater risk of cavitation because water would not be a limiting factor in its swamp habitat. The lower efficiency indices of C. iners compared to P. pinnata may indicate the need for increased provision for safety in the drier secondary forest habitat of C. iners compared to the swamp or well-drained soil habitats of P.

7 pinnata. The rainforest G. gnemon, however, would not be expected to have such low efficiency indices for its habitat, and we suspect that the results reflect a phylogenetic constraint. Phylogenetically, our results are consistent with the accepted evolutionary relationships among the four clades. Among angiosperms, the magnoliid and eudicot shared plesiomorphic characteristics whilst the monocots evolved distinctive apomorphic characters of large vessels arranged in scattered vascular bundles. The gnetophytes, unique among gymnosperms, have independently and convergently evolved vessels similar to that of angiosperms[10]. Our results show that these gnetophyte vessels were, as expected for a convergent trait, different from the angiosperm pattern in being small, densely packed, and notably, occurring singly and never grouped.

Gnetum gnemon Cinnamomum iners Pometia pinnata Oncosperma tigillarium

Fig. 4. Summary of efficiency and safety indices derived from xylem anatomical characteristics and ecophysioloical parameters for four species of plants: Gnetum gnemon, Cinnamomum iners, Pometia pinnata and Oncosperma tigillarium.

Did this difference in type and grouping of vessels between gnetophytes and angiosperms have any ecophysiological consequences? We found that only G. gnemon possessed two statistically distinct resistance readings. We suggest three possible explanations for this phenomenon. Firstly, the difference may be due to the absence of vessel groups in gnetophytes. Water cannot be transferred to neighboring vessels through pits but must be forced through end walls which present greater resistance to flow[4]. On the other hand, water in the angiosperms may be transferred to neighboring vessels through pits, by- passing the end walls, and substantially reducing flow resistance. A prediction from this hypothesis would be for the angiosperms, the higher the vessel grouping index (GI), the lower the resistance (RC), as there would be more neighboring vessels for water to flow through. This is indeed what we saw in our results: C. iners (GI 1.35; RC 35.4), O. tigillarium (GI 1.49; RC 34.9) P. pinnata (GI 1.65; RC 17.4). The second explanation is that the angiosperm results did consist of distinct groups of readings:

8 specifically a lumen resistance, a lumen + pit resistance, and a lumen + end wall resistance. This can account for the visible hint of bimodal distribution for the angiosperm resistance readings in fig. 3. However, due to the small sample size, we could not detect a statistically significant bimodal distribution, let alone a multimodal one as hypothesized. The last possible explanation is that the resistivity of the end walls for each species is different due to different types of end wall perforations [9]. This hypothesis would be consistent with the noted trend of simplification of end walls during xylem vessel evolution which increases flow efficiency[1]. The end walls of the angiosperms may have evolved to give less resistance, causing the end wall and lumen readings to differ less and thus be indistinguishable. Again, more data on resistance would allow us to make better informed conclusions. The best test of these three hypotheses would be to examine the characteristics of the pits and end walls of the plants using electron microscopy and correlate them with vessel resistance. Without such resources, however, our alternative would be to conduct cell maceration studies to study the longitudinal and end walls of the vessels in the four species by light microscopy.

We have obtained a very preliminary glimpse of the diversity of xylem characteristics among the major clades of our native tropical plants. By continued collection of data and follow-up investigation of the tantalizing questions thrown up, we can test our initial conclusions and discover more about the adaptation of xylem to the water demands in tropical habitats.

ACKNOWLEDGEMENTS We are very grateful to the following people for all the help given to us throughout the project: Prof N. Michele Holbrook and Mr James Wheeler of Harvard University for starting us off on this project; Dr Shawn Lum of the National Institute of Education, Dr Adrian Loo of the Raffles Science Institute and Mr Ang Wee Foong of the National Parks Board for advice on local botany and ecology; Mr Johnny Wee for teaching us the plant histology methods; Mr William Tan of Chemoscience Pte Ltd for useful histology tips; Mr Jeremy Woon of the National Biodiversity Centre for granting us permission to collect plant material around RI; Ms Tan Beng Chiak for helpful contacts; our school scientists and lab technologists, particularly Dr Abigayle Ng, Mr Tan Cheng Leng, Mdm Neo Heok Tee, Ms Musta'inah Binte Suratman, Ms Hay Chay Seam and Mr Goh Chun Lian for help with the equipment, chemicals and procedures; and all others who have helped us in one way or other. Field work was conducted under NParks permit NP/RP12-099.

REFERENCES [1] Carlquist, S. (2001). Comparative wood anatomy. (2nd ed.). Berlin: Springer Verlag. [2] Tyree, M. T., & Sperry, J. S. (1989). Vulnerability of xylem to cavitation and embolism. Ann. Rev. Plant Phy. Mol. Biol. 40: 19-38. [3] Esau, K. (1977). Anatomy of Seed Plants (2nd ed.). Santa Barbara, CA: John Wiley & Sons, Inc. [4] Sperry, J. S., Hacke, U. G., & Wheeler, J. K. (2005). Comparative analysis of end wall resistivity in xylem conduits. Plant, Cell and Environment. 28:456–465. [5] Schoch,W.,Heller,I.,Schweingruber,F.H.,Kienast,F. (2004). Wood anatomy of Central European Species. Online version: www.woodanatomy.ch [6] Schindelin, J., I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak and A. Cardona (2012) Fiji: an open-source platform for biological-image analysis, Nature Methods 9(7): 676-682 PDF Supplement [7] Zwieniecki, M.A., P.J. Melcher & N.M. Holbrook. (2001). Hydraulic properties of individual xylem vessels of Fraxinus americana. J. Expt. Biol. 52(355): 257-264. [8] Lens, F., Sperry, J. S., Christman, M. A., Choat, B., Rabaey, D., & Jansen, S. (2011). Testing hypotheses that link wood anatomy to cavitation resistance and hydraulic conductivity in the genus acer. New Phytologist. 190:709-723 [9] Christman, M. A. & Sperry, J. S. (2010). Single-vessel flow measurements indicate scalariform perforation plates confer higher flow resistance than previously estimated. Plant, Cell & Environment . 33: 431-443. [10] Judd, W.S., Campbell, C.S., Kellogg, E.A., Stevens, P.F., Donoghue, M.J. (2008). Plant Systematics: A Phylogenetic Approach. 3rd Ed. Sunderland, MA. USA: Sinauer Associates. [11] Boo, C.M., Omar-Hor, K., Ou-Yang, C.L. (2006). 1001 Garden Plants in Singapore. 2nd Ed. Singapore: National Parks Board. [12] Choné, X., van Leeuwen, C., Dubourdieu, D., Gaudillère, J.P. (2001). Stem water potential is a sensitive indicator of grapevine water status. Annals of Botany. 87: 477-483. [13] O’Dempsey, T. (undated). Flora Singapura. Last retrieved on 29 Jul 2013 from www.florasingapura.com. [14] MedCalc Statistical Software Version 12.7.0(2013). MedCalc Software, Ostend, Belgium.

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