Conducting Pathways in North Temperate Deciduous Broadleaved Trees

IAWA Journal, Vol. 29 (3), 2008: 247–263

CONDUCTING PATHWAYS IN NORTH TEMPERATE DECIDUOUS BROADLEAVED TREES

Toshihiro Umebayashi1, Yasuhiro Utsumi2,*, Shinya Koga2, Susumu Inoue2, Seizo Fujikawa3, Keita Arakawa3, Junji Matsumura4 and Kazuyuki Oda4

SUMMARY The interspecific variation of dye ascent in the stems of 44 broadleaved deciduous species growing in Japan was studied using freeze-dried samples after dye injection. The dye ascending pattern differed both within and between ring-porous and diffuse-porous species. In large earlywood vessels of all ring-porous species, the dye ascended only in the outermost annual ring, and the inner annual rings had lost their water transport function. The dye ascending pattern within the inner annual rings in the ring-porous species was categorized into three types: i) the dye ascended both in the many latewood vessels throughout the latewood and small earlywood vessels; ii) the dye ascended in the many vessels throughout the latewood; and iii) the dye ascended mainly in the late latewood vessels. In diffuse-porous species, the dye ascending pattern within the annual rings also was categorized into three types: i) the dye ascended throughout the annual rings; ii) the dye ascended mainly in the earlywood vessels; and iii) the dye ascended mainly in the latewood vessels. Xylem water distribution was also examined by cryo-SEM in three ring-porous and three diffuse-porous species that had different dye ascending patterns. The water distribution pattern within annual rings was correlated with the dye ascending pattern except for one diffuse- porous species (Salix gracilistyla). In this case, water was distributed in the whole region of the annual rings although dye was mainly distributed in the earlywood. These results showed that the functional area of water transport within annual rings differed among ring-porous species and diffuse-porous species. Key words: Dye, cavitation, diffuse-porous, ring-porous, deciduous trees.

1) Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 812-8581, Japan. 2) Shiiba Research Forest, Graduate School of Agriculture, Kyushu University, Shiiba 883-0402, Japan. 3) Laboratory of Wood Biology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. 4) Laboratory of Wood Science, Faculty of Agriculture, Kyushu University, Hakozaki 812-8581, Japan. *) Corresponding author [E-mail: [email protected]]. Associate Editor: John Sperry

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INTRODUCTION

Transpiring woody plants move the water from the soil to the leaves through the conduits (vessels and tracheids) of their stems, and the anatomical characteristics of the stem water-conducting pathway can have a profound impact on the efficiency of hydraulic conductivity (Tyree & Zimmermann 2002). In stems of broadleaved trees growing in northern temperate zones, ring-porous species have considerably larger diameter vessels in the earlywood, and the earlywood vessels in the outermost annual ring have a major role in water transport (Chaney & Kozlowski 1977; Ellmore & Ewers 1986; Granier et al. 1994). On the other hand, diffuse-porous species have similar diameter vessels scattered throughout the whole region of an annual ring, and the vessels in the outer several annual rings have a role in water transport (Baker & James 1933; Kozlowski & Winget 1963; Anfodillo et al. 1993). These results indicate that the functional region for water transport in the stem xylem is obviously different between ring-porous trees and diffuse-porous trees. However, in broadleaved trees, there are many ring-porous and diffuse-porous species and their xylem structures vary widely (Wheeler et al. 1989; Carl- quist 2001). The interspecific variation of the water transport pathways in ring-porous and diffuse-porous species is not fully understood. To evaluate the xylem sap flow of the stems of transpiring woody plants, various methods have been used. Thermal methods such as the heat pulse method (Swanson 1994) and the thermal dissipation method (Granier 1987; Lu et al. 2004) have revealed the radial profile of sap flow in the trunk (Čermáket al. 1992; James et al. 2002). Tracer injection methods such as dye injection (Baker & James 1933; Koslowski et al. 1965; Sakamoto & Sano 2000; Kuroda 2005), radioactive isotope injection (Postlethwait & Rogers 1958; Thomas 1967), and deuterium injection (Meinzer et al. 2003, 2006) have been used to describe the sap pathway. To visualize water distribution in the xylem, nuclear magnetic resonance imaging (Holbrook et al. 2001; Clearwater & Clark 2003; Utsuzawa et al. 2005) and cryo-scanning electron microscopy (cryo-SEM, Ohtani & Fujikawa 1990; Utsumi et al. 1996, 2003) have also been used. In the various methods mentioned above, the dye injection method is relatively easy to conduct and has been widely used to visualize the water-conducting pathway at the tissue level in trees (Rudinsky & Vité 1959; Harris 1961; Essiamah & Eschrich 1986; Barnett et al. 1993; De Faÿ et al. 2000), woody shrubs (Hargrave et al. 1994) and herbaceous plants (Tang & Boyer 2002). However, the difficulty in clarifying the functional region of water transport at the cellular level has also been pointed out (Čermák et al. 2002). To prevent the diffusion or flow of dye-carrying water after sampling of the stem, Sano et al. (2005) proposed the dye injection method, which enabled the visualization of the water-conducting xylem tissue of living trees at the cellular level using freeze drying and dye insoluble solution when samples were embedded, and optimal conditions for visualizing water-conducting pathways by this method was determined (Umebayashi et al. 2007). In this study, we examined the patterns of dye ascent of 44 deciduous broadleaved species with ring-porous and diffuse-porous wood structure, using the improved dye injection method. We especially focused on the interspecific variation of the water

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transport region between ring-porous and diffuse-porous species and within ring- porous and diffuse-porous species at the cellular level. In addition, we examined the correspondence between tissue regions that remained sap-filled throughout the winter and tissue regions taking up dye.

MATERIALS AND METHODS Plant materials Forty-four deciduous broadleaved species (eight ring-porous and 36 diffuse-porous species) growing in the Shiiba Research Forest of Kyushu University (32° 22' N, 131° 09' E; elevation 880–1150 m) in Japan were used in this study (Table 1). According to the temperature and precipitation data from 2003 to 2005 in the Shiiba Research Forest (elevation 600 m), the yearly mean air temperature was 12.7 °C. The daily maximum temperature for 2003, 2004, and 2005 was 33.0, 32.5, and 33.4 °C, respectively, and the minimum temperature for the three years was -9.1, -8.5, and -8.3 °C, respectively. The average yearly mean precipitation for the three years was 2918 mm.

Introduction of the dye solution and collection of samples Dye injection experiments were performed from 11:00 to 14:00 on sunny days from mid-July to August in 2003, 2004, and 2005. A watertight collar was set at a stem height of 1.0 m and filled with distilled water. The southern half of the stem was cut with a fine hand saw under water and the water was then replaced with an aqueous 0.2% acid fuchsin solution that had been filtered through a 0.22μ m filter (GV, Millipore, Billerica, USA) before the experiment. To keep water on the cut surface and avoid the adhesion of debris, the stem was wrapped with a fine cotton fabric during the replacement. After perfusion for 30 min, sample discs of 2 cm thickness were taken at 0, 10, 30, and 50 cm above the dye injection height, and at 50 cm intervals up to 1.0 m to 10 cm below the maximum height of the stained xylem. All discs were frozen by liquid nitrogen (LN2) immediately, transferred to the laboratory, and freeze-dried at -50 °C (Umebayashi et al. 2007). Two experiments were carried out for each species.

Observation of the dye distribution in the xylem The surface of the sample discs was trimmed with a razor blade and the dye distribution in the cross section of stem xylem was observed macroscopically. Following this, the stained portion of the disc was split into small blocks, the surfaces of the cross section were planed with a sliding microtome, and the dye distribution at the tissue level was observed with a stereomicroscope (SMZ800, Nikon, Tokyo, Japan). Subsequently, the small blocks containing at least one annual ring were trimmed and embedded with an epoxy resin (Sano et al. 2005). Finally, thin sections of 4–7 μm were cut from the embedded blocks with a semi-ultramicrotome (RM2045, Leica, Wetzlar, Germany) and the dye distribution in each xylem cell was observed by light microscopy (Optiphoto-2, Nikon, Tokyo, Japan). Images were obtained using a digital microscope camera system (DP70, Olympus, Tokyo, Japan) attached to the stereomicroscope and to the light microscope.

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Table 1. Tree height, diameter at breast height, the maximum dye ascent height and the stained annual rings. Number of stained annual ring was counted from the outermost annual ring to the pith. Asterisks show the species that have coloured heartwood.

Max. Stained annual rings dye (total rings in sapwood) Tree ascent –––––––––––––––––––––––––––––––––– Species height DBH height 10 cm above Maximum (m) (cm) (m) dye injection dye height height Ring-porous species Acanthopanax sciadophylloides* 5.2 4.3 3.3 1–13 (17) 1–2 (4) 4.8 5.0 3.1 1–11 (15) 1–2 (6) Aralia elata* 2.8 3.5 2.1 1–3 (4) 1 (1) 2.7 4.9 1.8 1–4 (4) 1 (1) Elaeagnus umbellata 5.0 3.0 1.9 1–6 (7) 1–2 (4) 6.4 5.3 0.9 1–21 (26) 1 (25) Kalopanax pictus* 5.8 5.8 4.1 1–15 (15) 1 (1) 4.9 4.7 3.6 1–24 (46) 1 (4) Morus australis* 7.5 5.4 2.3 1–5 (12) 1–2 (11) 7.4 5.5 1.5 1–4 (12) 1–2 (10) Quercus crispula* 9.2 7.0 6.3 1–25 (26) 1 (6) 8.6 5.1 6.1 1–22 (22) 1–2 (6) Rhus javanica var. roxburghii* 5.1 6.4 2.1 1–4 (4) 1–2 (2) 7.8 6.4 6.2 1–3 (3) 1 (1) Rhus trichocarpa* 5.5 4.2 2.8 1–11 (11) 1 (12) 3.9 4.2 1.9 1–18 (20) 1 (14) Diffuse-porous species Acer carpinifolium 5.5 6.0 0.9 1–25 (25) 1–14 (19) 6.1 5.6 1.6 1–15 (21) 1–5 (16) Acer mono var. marmoratum 5.9 7.2 1.6 1–28 (29) 1–10 (27) 7.0 7.2 2.3 1–31 (31) 1–4 (21) Acer palmatum 5.3 4.5 1.1 1–29 (30) 1–3 (10) 3.5 3.3 1.8 1–22 (23) 1–4 (21) Acer sieboldianum 9.4 9.7 1.6 1–52 (54) 1–9 (43) 6.8 9.0 1.4 1–49 (52) 1–5 (46) Betula grossa 9.5 8.6 2.1 1–23 (23) 4–9 (19) 8.0 6.8 2.9 1–18 (18) 1–2, 6–7 (11) Carpinus laxiflora 6.6 4.9 2.8 1–8 (10) 2–3 (4) 5.4 5.1 1.1 1–9 (10) 1–4 (10) Carpinus tschonoskii 6.9 5.0 1.6 1–10 (10) 1–3 (6) 4.6 5.0 1.6 1–10 (10) 1–2 (4) Cercidiphyllum japonicum 6.2 3.5 1.1 1–6 (9) 1 (8) 5.6 6.3 0.8 1–19 (22) 3–5, 8–9, 14, 16 (21) Clethra barbinervis 4.5 3.9 1.1 1–15 (17) 1–13 (15) 7.1 5.1 1.2 1–17 (19) 1–2, 9–10 (16) Cornus macrophylla 4.4 3.7 2.7 1–8 (11) 3–4 (4) 5.2 7.4 2.3 1–9 (11) 1 (5) Deutzia crenata 4.2 2.5 1.4 1–7 (7) 1–2 (3) 4.3 2.9 1.6 1–6 (9) 1–3 (4) Euonymus alatus* 5.1 6.4 1.8 1–19 (45) 1–4 (38) 5.0 4.0 1.6 1–19 (44) 1–11 (39) Euptelea polyandra 7.3 4.0 1.3 1–11 (11) 1–3 (9) 7.4 4.1 0.9 1–15 (15) 2–4 (14) – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

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(Table 1 continued) Max. Stained annual rings dye (total rings in sapwood) Tree ascent ––––––––––––––––––––––––––––––––– Species height DBH height 10 cm above Maximum (m) (cm) (m) dye injection dye height height Fagus japonica 5.6 5.6 1.9 1–11 (16) 1 (11) 6.4 5.2 2.1 1–12 (17) 2–3, 5–6 (12) Hydrangea paniculata* 4.6 7.0 0.5 1–8 (11) 1–4 (11) 5.5 6.5 1.8 1–8 (12) 1–5 (12) Hydrangea sikokiana 2.8 2.0 0.3 1–6 (9) 1–2 (7) 3.5 2.6 0.4 1–14 (16) 1–13 (16) Ilex serrata 4.1 3.0 0.8 1–34 (40) 1–8 (35) 3.8 2.2 1.1 1–23 (28) 1–4 (19) Lindera erythrocarpa 7.0 5.6 1.8 1–17 (19) 1–5 (15) 6.3 9.7 1.9 1–20 (26) 1–14 (21) Lindera praecox 5.5 3.8 1.1 1–14 (14) 1–4 (12) 4.0 2.6 1.1 1–15 (18) 1 (15) Lindera triloba 6.9 6.0 2.1 1–23 (27) 2–5 (20) 5.2 2.9 1.5 1–14 (15) 1 (12) Lyonia ovalifolia var. elliptica 7.3 8.5 1.7 1–32 (33) 2 (27) 7.7 8.0 1.2 1–29 (31) 1–4 (27) Magnolia obovata* 5.4 5.0 1.4 1–13 (14) 1–2 (13) 5.8 4.8 1.2 1–12 (14) 1–3 (12) Meliosma myriantha 6.5 6.9 0.9 1–14 (16) 1–3 (14) 6.0 7.5 1.1 1–22 (23) 1–5 (23) Pourthiaea villosa var. laevis 7.0 7.2 1.6 1–18 (31) 1–12 (23) 5.2 4.5 1.6 1–12 (35) 1–2 (29) Prunus jamasakura* 7.2 8.0 1.4 1–13 (26) 1 (21) 8.2 8.3 1.4 1–17 (25) 1–2, 5–6 (23) Pterocarya rhoifolia 9.0 6.5 2.1 1–7 (8) 1–2 (7) 8.1 8.3 1.6 1–7 (7) 1–2, 4 (6) Pterostyrax corymbosa 6.2 8.5 2.1 1–20 (21) 1–2 (17) 5.1 5.0 1.3 1–19 (19) 1–5 (15) Rhododendron reticulatum 3.7 5.0 0.8 1–37 (37) 9–11 (28) 3.8 7.0 1.5 1–37 (37) 1–3 (33) Salix gracilistyla* 5.2 6.2 2.4 1–13 (20) 1–2 (7) 4.0 3.3 1.8 1–10 (16) 1–2 (6) Sapium japonicum 6.5 5.4 1.6 1–18 (23) 1–12 (16) 6.2 4.2 1.2 1–9 (16) 3–5 (12) Sorbus gracilis 4.9 1.8 1.8 1–26 (29) 2–4 (19) 5.8 2.0 0.6 1–18 (23) 1–15 (23) Stewartia monadelpha 6.1 6.4 2.8 1–17 (20) 1–3 (17) 5.8 5.8 3.6 1–20 (28) 1–2 (14) Styrax japonica 5.5 5.5 2.7 1–22 (22) 1–3 (10) 5.8 6.2 1.4 1–18 (22) 1–3 (17) Styrax shiraiana 3.8 2.9 1.6 1–7 (9) 1–2 (5) 5.8 3.7 1.5 1–8 (9) 1–2 (5) Viburnum plicatum 5.3 4.0 1.8 1–23 (23) 1–12 (12) 5.3 3.9 0.8 1–27 (27) 1–6 (19) Weigela japonica* 6.5 6.5 3.9 1–15 (15) 1 (6) 7.1 7.1 3.8 1–11 (15) 1–2 (6)

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Cryo-SEM In three ring-porous species (Quercus crispula Blume, Rhus javanica var. roxburghii (DC.) Rehd. & Wils., and Acanthopanax sciadophylloides Franch. & Sav.) and three diffuse-porous species (Salix gracilistyla Miq., Fagus japonica Maxim., and Acer palmatum Thunb.), two sample stems were collected in February 2006 for cryo-SEM observation. A watertight collar with a plastic funnel was fitted to the stem at a height of 100 cm and filled with LN2 to freeze the stem before sunrise (Utsumi et al. 2003). Three-centimeter-thick discs were cut out with a hand saw from the frozen portion of the stem and placed in LN2 (Utsumi & Sano 2007). The sample stem blocks were trimmed and planed on a sliding microtome (Sano et al. 1995) in a cold room at -23°C. The frozen samples were slightly etched, coated with platinum and carbon in the cryo- SEM system (JSM840-a equipped with CRU-7000, JEOL, Tokyo, Japan; Fujikawa et al. 1988), and observed at 5 kV to assess the xylem water distribution of the outer three or more annual rings (Utsumi et al. 1996; Utsumi & Sano 2007).

Definition of vessel arrangement in an annual ring In ring-porous species, we described the earlywood as the initial zone of an annual ring that contained tangentially one or several rows of larger diameter vessels in cross section (Fig. 1a, E), and described the latewood as the other part of an annual ring. In the earlywood of ring-porous species, two species (Acanthopanax sciadophylloides and Kalopanax pictus (Thunb.) Nakai) have some clusters of small vessels (Itoh 1998). In this case, we differentiated the small earlywood vessels (Fig. 1b, arrows) from the large earlywood vessels (Fig. 1b, V). In the latewood of ring-porous species, we determined the early latewood to be the early half region of latewood (Fig. 1a, EL), and the late latewood to be the late half region (Fig. 1a, LL). In diffuse-porous species, we described the earlywood as the early half region of an annual ring (Fig. 1c, E) and the latewood as the other zone (Fig. 1c, L).

RESULTS

Dye distribution in ring-porous species At 10 cm above the dye injection height, the outer 3–25 annual rings from the cam- bium in all ring-porous species were stained (Table 1). The samples of seven species had colored heartwood (Table 1, asterisks), and the heartwood and innermost region of the sapwood of these samples had no dye distribution (Fig. 2a). Quercus crispula had the largest number of stained annual rings, and Rhus javanica var. roxburghii and Aralia elata Miq. had the lowest number of stained rings. The stained annual ring number in all ring-porous species decreased with an increase in stem height (Fig. 2a). At the maximum dye ascent height, the outermost annual ring was stained in all species (Fig. 2b), and some vessels in the late latewood (see Fig. 1) of the second annual ring were also stained in five species (Fig. 2b, arrow; Table 1, Acanthopanax sciadophylloides, Elaeagnus umbellata Thunb., Morus australis Poir, Q. crispula and R. javanica var. roxburghii). At 10 cm above the dye injection height, many large earlywood vessels in the outermost annual ring were stained, but the vessels in the inner annual rings were not stained

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Figure 1. Definition of earlywood and latewood in ring-porous and diffuse-porous species in this study. – a: Cross section of Kalopanax pictus. E, earlywood; EL, early latewood; LL, late latewood. – b: Earlywood of K. pictus. V, large earlywood vessels; arrows, small earlywood vessels. – c: Cross section of Lindera erythrocarpa. E, earlywood; L, latewood. — Scale bars: a, c = 200 μm; b = 100 μm.

in all species (Fig. 2c). In A. sciadophylloides and Kalopanax pictus, many small earlywood vessels in all stained annual rings had dye in their lumina (Fig. 2d & e, arrows). Many vessels throughout the entire latewood in the outermost annual ring were stained in all species. In the inner rings, many or some vessels in the whole latewood were stained in Q. crispula, E. umbellata, A. sciadophylloides, and K. pictus (Fig. 2d),

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Figure 2. Micrographs of the stained xylem, cross section. – a: Discs from 10 cm above injection to the maximum dye height in Rhus javanica var. roxburghii. The numbers give the distance (m) from the dye injection height. – b: Outer two annual rings at the 150 cm height level in Quercus crispula. Arrow, stained late latewood. – c: Outer two annual rings at the 10 cm height level in R. javanica var. roxburghii. – d: Outer two annual rings at the 10 cm height level in Kalopanax pictus. – e: Earlywood of the second annual ring at the 10 cm height level in K. pictus. Arrows, dye in the small earlywood vessel lumina. – f: Outer two annual rings at the 10 cm height level in Q. crispula. – g: Earlywood of the second annual ring at the 10 cm height level in Q. crispula. Arrows, dye in the cells surrounding the vessel lumina. – h: Discs from 0.1 m to the maximum dye height in Prunus jamasakura. The numbers give the distance (m) from the dye injection. – i: Outer four annual rings at the 150 cm height level in Weigela japonica. Arrowheads, annual ring boundary. – j–m: Outer three or four annual rings at the 10 cm height level in W. japonica (j), Betula grossa (k), Fagus japonica (l), and Salix gracilistyla (m). Arrowheads, annual ring

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and in the other species, only some late latewood vessels were stained (Fig. 2c). The dye distribution pattern between each annual ring was similar in all species with the exception of that for the outermost annual ring. Thus, we categorized the ring-porous species into three types based on the dye distribution pattern within the annual ring (Table 2). The first type is where small earlywood vessels and many latewood vessels of the whole ring were stained (Fig. 2d & e; Table 2, r1). The second type is where many vessels throughout the latewood were stained (Fig. 2f; Table 2, r2). The third type is where mainly late latewood vessels were stained (Fig. 2c; Table 2, r3). In all stained xylem at all heights, the tissues adjacent to stained vessels (e.g., vasicentric tracheids and wood fibers) contained dye in their lumina in four species (Fig. 2g, arrows; Table 2, Q. crispula, Aralia elata, M. australis and R. javanica var. roxburghii). Ray parenchyma cells were not stained in any species.

Dye distribution in diffuse-porous species At 10 cm above the dye injection height, the outer 6–52 annual rings from the cam- bium in each diffuse-porous species were stained (Table 1). Samples of six species had colored heartwood (Table 1, asterisks), and in these samples, the heartwood and innermost region of the sapwood had no dye (Fig. 2h). In this study, Acer sieboldianum Miq. had the largest number of stained annual rings, and three species (Cercidiphyllum japonicum Sieb. & Zucc., Hydrangea sikokiana Maxim., and Deutzia crenata Sieb. & Zucc.) had the lowest number of stained annual rings. The stained annual ring number in all diffuse-porous species decreased with an increase in stem height (Fig. 2h). At the maximum dye ascent height, the outer several annual rings were stained uniformly in most samples. Only one sample from six species was discontinuously stained across annual rings (Table 1, Betula grossa Sieb. & Zucc., Cercidiphylllum japonicum, Clethra barbinervis Sieb. & Zucc., Fagus japonica, Prunus jamasakura Sieb. ex Koidz. and Pterocarya rhoifolia Sieb. & Zucc.). The dye distribution pattern within the annual rings of inner rings was the same at all heights for all species (Fig. 2i & j). In most species, a similar pattern of the stained region was also observed between the outermost annual ring and inner annual rings (Fig. 2j–l). In the outermost annual ring of two species (Salix gracilistyla and Clethra barbinervis), vessels of the whole region were stained, and in the inner annual rings the latewood was mainly stained (Fig. 2m). In the inner annual rings, the dye ascent pattern was classified into three types. In species of the first pattern, most or some vessels of the whole ring were stained (Fig. 2j & k; Table 2, d1). In species of the second pattern, mainly earlywood vessels were stained (Fig. 2l; Table 2, d2). In species of the third pattern, mainly latewood vessels were stained (Fig. 2m; Table 2, d3).

← boundary. – n: Earlywood of the second annual ring at the 10 cm height level in Pterostyrax corymbosa. Arrow, dye in a pit between vessel and surrounding tissue. – o: Earlywood of the second annual ring at the 10 cm height level in Magnolia obovata. Arrow, the dye in a ray parenchyma cell. — Scale bars: a, h = 1 cm; b, c, i–m = 1 mm; d, f = 500 μm; e, g = 50 μm; n, o = 25 μm.

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Table 2. Staining pattern within annual rings. – r: ring-porous species; d: diffuse-porous species; rl: whole latewood and small-earlywood vessels were stained; r2: whole latewood vessels were stained; r3: mainly late latewood vessels were stained; d1: vessels of whole ring were stained; d2: mainly earlywood vessels were stained; d3: mainly latewood vessels were stained; none: the tissues adjacent to the vessels were not stained; portion: several tissues adjacent to vessels were stained; whole: many tissues adjacent to vessels were stained.

Species Species Staining pattern of maximum dye height Stained tissue adjacent to vessels Staining pattern of maximum dye height Stained tissue adjacent to vessels

Ring-porous species Hydrangea paniculata – none Hydrangea sikokiana – none r1: Ilex serrata – portion Acanthopanax sciadophylloides r3 none Lindera erythrocarpa – none Kalopanax pictus r3 none Lindera praecox – portion r2: Lindera triloba – portion Elaeagnus umbellata r3 none Magnolia obovata – none Quercus crispula r3 whole Meliosma myriantha – none Pourthiaea villosa var. laevis – none r3: Prunus jamasakura – portion Aralia elata – portion Pterocarya rhoifolia – portion Morus australis – portion Pterostyrax corymbosa – portion Rhus javanica var. roxburghii – portion Sapium japonicum – none Rhus trichocarpa – none Sorbus gracilis – none Stewartia monadelpha – portion Diffuse-porous species Styrax japonica – none d1: Styrax shiraiana – none Acer carpinifolium – none Viburnum plicatum – none Acer mono var. marmoratum – none Weigela japonica – portion Acer palmatum – none d2: Acer sieboldianum – none Euptelea polyandra – portion Betula grossa – none Fagus japonica – portion Carpinus laxiflora – portion Lyonia ovalifolia var. elliptica – none Carpinus tschonoskii – portion Rhododendron reticulatum – portion Cercidiphyllum japonicum – none Cornus macrophylla – whole d3: Deutzia crenata – portion Clethra barbinervis – portion Euonymus alatus – portion Salix gracilistyla – none

In 13 species of the d1 type, three species in d2 type and one species in d3 type, dead xylem cells adjacent to stained vessels had dye in their lumina (Fig. 2n, arrow, Table 2). Furthermore, in Lindera triloba Blume, Magnolia obovata Thunb., Sapium japonicum Pax & K.Hoffm., Cercidiphyllum japonicum, Ilex serrata Thunb., and Weigela japonica Thunb., the lumina of parenchyma cells adjacent to the vessels was also stained (Fig. 2o, arrow).

Water distribution Of the six species examined by cryo-SEM (Rhus javanica var. roxburghii, Quercus crispula, Acanthopanax sciadophylloides, Acer palmatum, Fagus japonica, and Salix

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Figure 3. Cryo-scanning electron micrographs of xylem cross section. – a: The second and third annual rings of Acanthopanax sciadophylloides. Arrow, small earlywood vessel. – b: The second annual ring of Quercus crispula. Arrow, water-filled latewood vessel. – c & d: The second and third annual rings of Rhus javanica var. roxburghii. – e: The second annual ring of Acer palmatum. – f: The fourth and fifth annual rings of Fagus japonica. – g: The fourth annual ring of Salix gracilistyla. – e–g: Arrowhead, annual ring boundary. — Scale bars: a–c, e–g = 100 μm; d = 50 μm.

gracilistyla), all except for S. gracilistyla showed similar patterns of water distribution in the outer three annual rings. In all three ring-porous species, most large earlywood vessels did not have water in their lumina (Fig. 3a–c). In A. sciadophylloides, most small earlywood vessels (Fig. 3a, arrow) and all latewood vessels had water in their

Downloaded from Brill.com10/05/2021 06:51:38AM via free access 258 IAWA Journal, Vol. 29 (3), 2008 Umebayashi et al. — Conducting pathways in temperate trees 259 lumina. The wood fibers were rarely water-filled. InQ. crispula, some latewood vessels had water in their lumina (Fig. 3b, arrow), and many cells adjacent to the vessels also had water in their lumina. In R. javanica var. roxburghii, no early latewood vessels had water (Fig. 3c), and some late latewood vessel clusters had water in their lumina (Fig. 3d). The wood fibers were rarely water-filled, except for the tissues adjacent to late latewood vessels. In A. palmatum and F. japonica, most vessels throughout an annual ring had water in their lumina (Fig. 3e & f). In S. gracilistyla, most outermost annual ring vessels were filled with water. In the second and inner annual ring, only latewood vessels had water in their lumina (Fig. 3g). In the three diffuse-porous species, many wood fibers had water in their lumina (Fig. 3e–g).

DISCUSSION

The stem water-conducting pathway in 44 deciduous broadleaved species varied among ring-porous and diffuse-porous species as well as between these two groups (Table 2). In ring-porous species, some reports indicate that the water in tree stems is transported mainly through the outermost annual ring (Greenidge 1955; Kramer & Kozlowski 1979; Kubler 1991; Kozlowski & Pallardy 1997). However, other reports indicate that inner annual rings as well as the outermost annual ring have water transport functions (Kozlowski & Winget 1963; Granier et al. 1994). In all ring-porous species we studied, we found that large earlywood vessels of the outermost annual ring functioned in water transport, but there was no water transport function in the second and inner annual rings (Fig. 2b–g & 3a–c). In general, cavitation, the rupture of water continuity in the water transport system, is induced by water stress and freezing stress (Tyree & Sperry 1989). It is unlikely that water stress-induced cavitation occurred in most large earlywood vessels, because many large earlywood vessels in the outermost annual ring of all ring-porous species in this study maintained their water transport function during the growing season when most leaves transpired and the water potential decreased. On the other hand, some reports indicate that the hydraulic conductivity of ring-porous species declines rapidly after the first frost (Cochard & Tyree 1990; Sperryet al. 1994) and that earlywood vessels in the outermost annual ring are cavitated (Utsumi et al. 1999). It is suggested that the freeze-thaw cycle causes embolism in conduits when the diameter is greater than 43 μm at the -0.5 MPa condition (Davis et al. 1999; Pittermann & Sperry 2003). The water in dead xylem cells freezes at several degrees below zero (Zimmermann 1964), although cold acclimated living tissue freezes commonly between -8 and -10 °C (Sakai & Larcher 1987). The large earlywood vessels of the examined species were over 43 μm in diameter (Fig. 3a–c). And the minimum yearly temperature at our study site was less than -8 °C during the experiment. Therefore, freeze-induced cavitation would occur in the large earlywood vessels of the outermost annual ring, which would lose their water transport function during winter. We showed that small earlywood and latewood vessels in some ring-porous species maintained their water transport system over several years (Fig. 2c–e). There is a positive correlation between vulnerability to cavitation caused by freezing and conduit diameter (Ewers 1985; Cavender-Bares 2005), and smaller conduits tend to experience

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less freeze-thaw embolism than larger ones (Cochard & Tyree 1990; Feild & Brodribb 2001). Functioning periods for water transport will differ between large earlywood vessels and small earlywood and latewood vessels. These small vessels would contrib- ute less to water transport under normal conditions. However, ring-porous deciduous species leaf out later than many diffuse-porous species (Wang et al. 1992) and expand their leaves before the maturation of earlywood vessels in the outermost annual ring (Suzuki et al. 1996). In the earliest stage of leaf expansion, small vessels of the second and inner annual ring would have a major role in water transport. In the diffuse-porous species studied, the water-conducting pattern within an annual ring was categorized into three types. Many previous reports have only shown the conducting annual ring number macroscopically (e.g., Kozlowski & Winget 1963; Čermák et al. 2002), and only a few studies analyzed variation of the water-conducting pathway within individual annual rings (Baker & James 1933; Chaney & Kozlowski 1977). Our study is the first report showing the variation of the water transport pathway of annual rings for various diffuse-porous species. Species in which all vessels in each annual ring conduct water (d1 type) probably avoid embolism throughout the growing season or reverse it by a mechanism such as root pressure. It is known that the water transport system in Betula spp. and Acer spp. generates high positive pressures in the stems and roots (Merwin & Lyon 1909; Kramer & Kozlowski 1960; Milburn & OʼMalley 1984). All species of Betula and Acer studied showed the d1 type. Positive high pressure may enable the vessels in the d1 type to be functional in the whole vessel region. In species that only transport water in the earlywood (d2 type), the whole vessel region was filled with water (Fig. 3f). In this case, the water ascending region and water distribution in xylem did not correspond. Relatively small latewood vessels would have a water transport function, but the amount of moving water would be small when large vessels are functioning because the vessel diameter strongly affects water transport efficiency according to the Hagen-Poiseuille law (Tyree & Zimmermann 2002). In the d3 type, all vessels in the outermost annual ring and the latewood vessels in the inner annual rings had water in their lumina and a transport function (Fig. 2m & 3g). These results indicate that the earlywood vessels of the outermost annual ring maintained the water transport function during the first growing season, but lost water during the first dormant season. Freeze-induced cavitation would occur in the larger earlywood vessels of the outermost annual ring in winter. A clear relationship between the dye distribution pattern of the tissue surrounding the vessel lumen and the tissue cell type was not observed. In ring-porous trees, Quercus species have vasicentric tracheids (Wheeler et al. 1989) and the surrounding tissue of Q. crispula was stained with dye in this study. On the other hand, the species that have no vasicentric tracheids and where the vessels are surrounded by axial parenchyma such as Morus australis (Itoh 1995), were also stained. Even in the same ge- nus, the surrounding tissue of Rhus javanica var. roxburghii was stained, but that of R. trichocarpa Miq. was not stained. In diffuse-porous species, the surrounding tissue of Lindera praecox (Sieb. & Zucc.) Blume and Lindera triloba was stained but that of Lindera erythrocarpa Makino was not stained. These congeneric species had vasicentric parenchyma and showed no apparent structural difference under the light

Downloaded from Brill.com10/05/2021 06:51:38AM via free access 260 IAWA Journal, Vol. 29 (3), 2008 Umebayashi et al. — Conducting pathways in temperate trees 261 microscope. The staining property of the cells surrounding the vessel lumina may not be determined by the cell type but regulated by the porosity and chemical property of the pit membranes between the vessel and the adjacent cells. In ring-porous species, most of the water was transported in the outermost annual ring, although many annual rings of sapwood had a water transport function. This is because the large earlywood vessels can maintain their water transport function only during the first growth season. On the other hand, the radial sap flow pattern in diffuse- porous species was unclear in this study. In a sample of Cercidiphyllum japonicum, Fagus japonica, Cornus macrophylla Wall., Lindera praecox, L. triloba and Weigela japonica Thunb., the fastest water ascent region was in the outermost annual ring, while in a sample of Rhododendron reticulatum D. Don ex G. Don, water ascended mainly in the inner annual ring (Table 1). In diffuse-porous and coniferous species, the sap flow varies radially (Čermák & Nadezhdina 1998; Jiménez et al. 2000). Some reports indicate that the middle portion of the sapwood transports water rapidly (Čermák et al. 1992), and in other studies, it was suggested that the outer part of the sapwood was the fastest region (Čermák et al. 2002; Nadezhdina et al. 2002). In diffuse-porous trees, the radial sap flow pattern might differ between species and even within a species. However, our results only show the water conducting pathways because dye was introduced from a notch in the stem. To clarify the actual radial distribution pattern of sap flow among ring-porous and diffuse-porous species, further qualitative and quantitative studies such as dye injection from the root are needed.

ACKNOWLEDGEMENTS

The authors thank Hisami Nagasawa, Katsuyoshi Kubota, Sachiko Inoue, Shigeru Osaki, Yasuki Shiiba and Toshiaki Itoh for their technical support. This work was supported in part by a Grant-in-Aid for Scientific Research (No. 19780121) from the Ministry of Education, Science and Culture, Japan.

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