Botany A - CSIRO Publishing

18 downloads 0 Views 3MB Size Report
Most species within the genus Eucalyptus exhibit heteroblastic leaf development. In particular, Eucalyptus globulus Labill. develops strikingly different seedling,.
P u b l i s h i n g

Australian Journal of Botany Volume 49, 2001 © CSIRO 2001

An international journal for the publication of original research in plant science All enquiries and manuscripts should be directed to: Australian Journal of Botany CSIRO Publishing PO Box 1139 (150 Oxford St) Collingwood, Vic. 3066, Australia Telephone: +61 3 9662 7613 Fax: +61 3 9662 7611 Email: [email protected] Published by CSIRO Publishing for CSIRO and the Australian Academy of Science

w w w. p u b l i s h . c s i ro . a u / j o u r n a l s / a j b

Aust. J. Bot., 2001, 49, 259–269

Leaf morphological and anatomical characteristics of heteroblastic Eucalyptus globulus ssp. globulus (Myrtaceae) Shelley A. JamesAB and David T. BellA A

B

Department of Botany, The University of Western Australia, Nedlands, WA 6907, Australia. Corresponding author. Present address: Department of Botany, University of Hawaii, 3190 Maile Way, Honolulu, HI 96822, USA; email: [email protected]

Abstract. Leaf characteristics of Eucalyptus globulus Labill. ssp. globulus vary in response to plant genotype, ontogenetic position and environmental conditions. Glasshouse-grown seedlings from provenances at St Marys, Tasmania, and Wilsons Promontory, Victoria, produced seedling leaves for 10 nodes before producing leaves of juvenile form. Tasmanian provenance seedlings began to produce juvenile leaves after 18 weeks, 4 weeks earlier than Wilsons Promontory seedlings. Tasmanian seedlings continued to produce juvenile foliage, whereas Wilsons Promontory seedlings began producing transitional leaves at 33 weeks. Successive transitional leaves ranged from the juvenile to the adult leaf form owing to variability in the rate of change of particular morphological and anatomical leaf characteristics. Retention of broad, thin, sessile, horizontally oriented, dorsiventral, hypostomatal juvenile leaves of Tasmanian seedlings assists in increasing growth rates under mesic conditions. Early production of thick, narrow, petiolate, vertically oriented, isobilateral, amphistomatal adult leaves by Wilsons Promontory seedlings appears to be related to the stressful conditions within its local habitat. An increase in amphistomy and the distribution of palisade mesophyll on both leaf surfaces with ontogenetic development was strongly related to leaf orientation and light interception, increasing the supply of CO2 for photosynthesis. BT9 04 SE.uAca.lJaypmteus aglnodbuDl.uTsle.Balfstructure

Introduction SheleyA. JamesandDavdi .TBel

Heteroblastic development is a progressive, environmentally independent change in the size and structure of successive organs (Nobel and Walker 1985), resulting in distinctly different juvenile and adult phases of shoot development. Most species within the genus Eucalyptus exhibit heteroblastic leaf development. In particular, Eucalyptus globulus Labill. develops strikingly different seedling, juvenile, transitional and adult leaf forms during successive life stages. Broad, thin, blue-grey, glaucescent, dorsiventral and hypostomatal juvenile leaves shift to narrow, thick, green, isobilateral and amphistomatal leaves with the ontogenetic development of the tree. Although the heteroblastic leaf forms of eucalypts are often described, no studies have documented in detail the ontogenetic changes in leaf morphology and anatomy. Morphological and anatomical differences between the major E. globulus leaf forms have been described in other studies (Johnson 1926; Ito and Suzaki 1990), but none has detailed the gradual and successive changes in leaf characteristics. This present study documents spatial and chronological ontogenetic leaf changes, and describes the morphology and anatomy of © CSIRO 2001

successive ontogenetic leaf forms of two E. globulus ssp. globulus provenances that have strikingly different rates of vegetative phase change. The first provenance from St Marys, Tasmania (Tasmanian provenance), produces leaves of juvenile form for 2–3 years in field trials (James 1998). The second provenance from Wilsons Promontory, Victoria (Wilsons Prom.), produces leaves of transitional and adult form within 9 months. Finally, the potential advantage of structural leaf changes under different environmental conditions is discussed. Methods Ontogenetic change Eucalyptus globulus ssp. globulus seedlings were used to determine the change from the seedling to transitional leaf morphology. Seed from Tasmanian (CSIRO forest research seedlot number 16474 CL002) and Wilsons Prom. (16399 DFC 219) provenances were germinated and grown in the Department of Botany, University of Wyoming, for 11 months. Temperatures were maintained between 15–18°C (night) and 27–32°C (day) with a relative humidity of 40–90%. Light availability at noon ranged from 170 to 1500 µmol m–2 s–1. Seedlings were watered to field capacity twice daily, fertilised every 3 weeks with an allpurpose fertiliser, and rotated around the growth room weekly to minimise variation in light and temperature. 10.1071/BT99044

0067-1924/02/02259

260

Leaf dimensions were determined for 11 seedlings of both provenances. Lamina length and maximum width were measured for fully expanded main-stem leaves produced during 11 months, and the ratio of leaf length to width was calculated. As adult leaves were not produced by the seedlings during the 11 months, a single 2.5-year-old Wilsons Prom. sapling grown under the conditions described above was used to determine the change from juvenile to adult leaf morphology. All branches were removed and the main stem lopped above the 38th node to induce resprouting and juvenile leaf production. Leaf length, maximum width and petiole length were measured for a fully expanded leaf at each node along 11 resprout branches. The mean and standard error of the mean was calculated for the characteristics at each leaf node by using Minitab (release 10.51, Minitab Inc., State College, PA). The relationship between leaf dimension and node number was compared between the two provenances by regression analysis and F-tests. Leaf characteristics Eucalyptus globulus ssp. globulus leaves were collected from a 5-yearold provenance trial at the Vasse Research Station, 13 km south of Busselton, Western Australia. Three pairs of Tasmanian and Wilsons Prom. trees were sampled from the centre of the block trial. Leaves were collected from the north side of the trees. Tasmanian trees had juvenile, transitional and adult foliage within the canopy. Wilsons Prom. canopies consisted only of adult foliage. Tasmanian trees were almost twice the height (12.5 ± 0.8 cf. 6.6 ± 0.9 m) and diameter (13.2 ± 0.9 cf. 7.7 ± 1.1 cm) of Wilsons Prom. trees. Tasmanian leaves were visually and subjectively classified into juvenile, transitional and adult forms, on the basis of the insertion point along the branch, petiole length and leaf shape. Fully expanded 1- and 2-year-old adult leaves of the Wilsons Prom. provenance were collected. Twelve juvenile, 25 transitional and 44 adult Tasmanian leaves, and 74 Wilsons Prom. adult leaves were measured. Fresh leaf mass was determined within 24 h of collection and dry mass was determined after drying at 70°C. Leaf area was determined by using a leaf area meter (LI3000, LI-COR Inc., Lincoln, NE). Total blade length, length from the leaf base to the point of maximum width (length to maximum width), maximum width, width at the leaf midlength (midwidth), and petiole length were measured. The angle between the leaf margin and midvein at the base of the leaf blade (basal angle) was measured with a protractor. Anatomical characteristics were determined for four juvenile, four transitional and eight Tasmanian adult leaves, and 14 Wilsons Prom. adult leaves. Pieces of lamina (1 cm2) were sampled between the margin and midvein at the leaf midlength. Tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, dehydrated in an alcohol series, and embedded in glycol methacrylate (Feder and O’Brien 1968). Transverse sections of 3-µm thickness were stained with toluidine blue (pH 4.4). Leaf, cuticle, epidermis, palisade mesophyll and spongy mesophyll thickness were measured at the midpoint of each transverse section. The length and width of five palisade mesophyll cells within the first cell layer were measured and averaged. Epidermal characteristics were measured for four juvenile, nine transitional and nine adult leaves from the Tasmanian provenance, and 18 adult Wilsons Prom. leaves. Surface impressions of the region between the margin and midvein at the leaf midlength were obtained by the methods outlined in James and Bell (1995). Adaxial and abaxial stomatal density (d) was determined as the average number of stomata in ten 0.06-mm2 fields of view. Stomatal complex length (l) and width (w), and stomatal pore length were measured for 10 replicate stomata and averaged. Stomatal complex area as a percentage of leaf surface area (As) was calculated for the two leaf surfaces as: As = d × ¼π(l × w) × 100. Total stomatal complex area per unit leaf area was the sum of adaxial and abaxial complex areas. Surface oil gland density was determined as the average number of oil glands in ten 1.06-mm2 fields of view.

S. A. James and D. T. Bell

For each leaf, the ratios of length to maximum width, midwidth to maximum width, and length to maximum width to total length were calculated. Leaf specific mass was calculated as the leaf dry mass per unit area. The ratio of the adaxial to abaxial value was calculated for characteristics measured on both leaf surfaces. Cuticle, epidermis, palisade mesophyll and spongy mesophyll thickness, and palisade cell length as a proportion of total leaf thickness were determined. The ratio of adaxial to the sum of adaxial and abaxial stomatal densities (total stomatal density) was calculated for each leaf. The mean and standard error of the mean was determined for all ontogenetic leaf characteristics by using Minitab. Confidence intervals between values were determined by using Fisher’s least significant difference. One-way analysis of variance and multivariate analyses were used to determine statistical differences between the leaf forms and surfaces for each of the measured characteristics. Regression analysis was used to determine statistical relationships between leaf characteristics. Morphological, anatomical and epidermal characteristics were grouped for each analysed leaf. Partial detrended canonical correspondence analysis (DCA) was completed for the three data sets by using CANOCO (version 3.11, Agricultural Mathematics Group, Wageningen, The Netherlands). Individual values for each characteristic were range standardised. Subjective, visually determined designations of juvenile, transitional and adult were imposed over the objective ordination values for each leaf. Oksanen and Minchin (1997) have reported an order-dependent programming error in the non-linear rescaling procedure that forms part of DCA within the program CANOCO. Ordination results reported here were regarded as sufficiently accurate, as only the first two axes were considered in detail and axis eigenvalues were substantially different for all ordinations.

Results Ontogenetic change First-formed E. globulus ssp. globulus seedling leaves were linear in shape, 25 mm long and 8 mm wide (Fig. 1). Juvenile E. globulus ssp. globulus leaves were blue-green, oblongacuminate in shape, bicoloured, sessile, opposite and horizontal in orientation. Gradual development from the juvenile to adult leaf form was most clearly indicated by an increase in intranodal extension, longer and narrower leaves, and an increase in petiole length (Fig. 2). Adult leaves were concolourous, falcate-lanceolate in shape, petiolate, alternate and vertically oriented. Seedling leaf length and width of both provenances increased from the first to the 10th main-stem node (Fig. 1A, B). With further node development, dimensions of the Tasmanian juvenile leaves remained constant, whereas Wilsons Prom. leaf width declined (Fig. 1B). For both provenances, the ratio of leaf length to width decreased significantly (r = 0.81, P < 0.01) from the first leaf pair to node 12 (Fig. 1C). From the 18th node, the ratio remained constant at about 1.8 for the Tasmanian juvenile leaves, but increased significantly for Wilsons Prom. seedlings as transitional leaves were produced (P < 0.01). Leaf length and width of Wilsons Prom. resprout branches initially increased linearly with node number, from the juvenile to transitional leaf form (Fig. 3A, B). From node 47, leaf length remained at about 150 mm but maximum leaf

Eucalyptus globulus leaf structure

261

Leaf characteristics

Fig. 1. Leaf dimensions with increasing main-stem node of Tasmanian (䉭) and Wilsons Prom. (䊏) Eucalyptus globulus ssp. globulus seedlings. (A) Leaf length; (B) maximum width; and (C) ratio of leaf length to width. Seedling (S), juvenile (J), Tasmanian juvenile (JTas) and Wilsons Prom. transitional (TWP) leaf forms have been indicated. Values are means and bars are the standard error of the mean for 1–11 replicates.

width declined. Consequently, the ratio of leaf length to width increased exponentially with node production until adult leaves were developed at node 53 (Fig. 3C). Petiole length of transitional leaves increased from node 38 until node 48 when a petiole length of about 16 mm was maintained.

The gradual change in leaf structure with E. globulus ssp. globulus development was depicted clearly by DCA ordinations of leaf characteristics. More than 90% of variation was described by the first and second ordination axes of each analysis. The ontogenetic leaf forms were distributed in a similar pattern whether morphological, anatomical or epidermal data sets were considered (Fig. 4). Juxtaposition of the visual determination of leaf form onto the ordinations showed a clear distinction between the E. globulus leaf forms. Juvenile and adult leaves were placed at opposite ends of the ordination hyperspace, and transitional leaves were positioned between the juvenile and adult leaves. Axis values of the juvenile, transitional and adult Tasmanian leaves within each ordination were significantly different (P < 0.01). This objective separation lent support to the visual determination employed in the field. Axis values of the morphological and epidermal ordinations were significantly different (P < 0.01) for the Tasmanian and Wilsons Prom. adult leaves. Petiole length and leaf dimensions strongly separated the leaf forms in the morphological ordination (Fig. 4A). Total leaf thickness, spongy mesophyll thickness and epidermal cell dimensions had the greatest influence on the distribution of the leaf forms within the anatomical ordination hyperspace (Fig. 4B). Stomatal dimensions, abaxial stomatal density and surface oil gland density were the epidermal characters that most strongly differentiated the juvenile, transitional and adult leaf forms (Fig. 4C). Adult leaves of the Tasmanian provenance were the longest (210 mm) of the leaf forms (Table 1). Leaf width decreased significantly with ontogenetic development. Wilsons Prom. adult leaves were the narrowest (30 mm) of the leaf forms. The leaf length to width ratio was significantly greater for adult leaves than for juvenile or transitional leaves. While most juvenile leaves lacked a petiole, adult leaves of both E. globulus provenances had a petiole of about 30 mm. Basal angle increased significantly from the juvenile to adult leaves. All Tasmanian ontogenetic leaf forms had a similar fresh mass (Table 1). Adult Wilsons Prom. leaves had a significantly lower fresh and dry mass than adult leaves of the Tasmanian provenance. Tasmanian juvenile leaves had the largest (95 cm2) and Wilsons Prom. adult leaves the smallest (34 cm2) area. Leaf specific mass increased significantly from the juvenile to the adult leaves of the Tasmanian provenance. Adult Wilsons Prom. leaves had the greatest leaf specific mass. Water content per gram of dry mass was above 50% (Table 1). Leaf water content per unit leaf area increased significantly with ontogenetic development, corresponding to the increase in leaf specific mass. Juvenile E. globulus leaves were dorsiventral in structure, with adaxial palisade mesophyll and abaxial

262

S. A. James and D. T. Bell

Fig. 2. Representative ontogenetic leaf forms of Eucalyptus globulus ssp. globulus. Juvenile leaves (far left) to adult leaves (far right) with a gradation of transitional leaves. Scale bar = 5 cm.

spongy mesophyll (Fig. 5A). Transitional leaves were largely isobilateral in structure (Fig. 5B). Adult leaves of both the Tasmanian and Wilsons Prom. provenances were isobilaterally symmetrical, with multiple palisade mesophyll layers and no obvious distinction between palisade and spongy mesophyll (Fig. 5C). Both juvenile and adult E. globulus leaves were heterobaric, having collenchyma and sclerenchyma extending from the bundlesheath cells of major veins to the epidermis (Fig. 5). Oil glands were found within the mesophyll of all ontogenetic leaf forms. Leaf thickness increased significantly with ontogenetic development from the juvenile to adult leaf form (Fig. 5, Table 1). Juvenile leaves had one or two adaxial palisade mesophyll layers (Fig. 5). Juvenile leaves had a similar thickness of palisade (182.5 ± 48.3 µm) and spongy (156.4 ± 55.7 µm) mesophyll. Transitional leaves had multiple palisade cell layers (8.5 ± 0.6 layers), with limited spongy mesophyll development. Adult leaves of Tasmanian (9.3 ± 0.9 layers) and Wilsons Prom. (9.9 ± 0.3 layers) trees had the greatest number of palisade mesophyll cell layers. Palisade mesophyll thickness was significantly and positively related to the number of palisade cell layers (r = 0.94, F = 532.4, P < 0.01) and total leaf thickness (r = 0.91, F = 128.2, P < 0.01).

Adaxial palisade mesophyll cells were significantly longer than abaxial palisade cells for all ontogenetic leaf forms (Table 1). Juvenile adaxial palisade cells were longer than the palisade cells of transitional and adult leaves. Tasmanian juvenile leaves had significantly broader palisade cells than adult leaves. Wilsons Prom. adult leaves had the thickest cuticle and epidermal cells. Tasmanian juvenile and transitional leaves had the thinnest cuticle but were more glaucous (Fig. 2, Table 1). Eucalyptus globulus stomata were largely located in interveinal areas because of the heterobaric leaf anatomy. The density of stomata was greater on the abaxial surface (70–445 stomata mm–2) than on the adaxial surface (0–52 stomata mm–2) (Table 1). A gradient of increasing amphistomy was found with ontogeny from the juvenile to adult leaf form. Juvenile leaves were hypostomatal, often with less than 10% of the stomata on the adaxial surface. In contrast, the Wilsons Prom. adult leaf form was most amphistomatal, with a similar density of adaxial and abaxial stomata (Table 1). Total stomatal density was greatest for juvenile and transitional leaves. Stomatal pore length, complex length and complex width increased from the juvenile to adult ontogenetic leaf form (Table 1). Adult leaves of the Wilsons Prom. trees had the largest stomata. Juvenile leaves alone had a significantly

Eucalyptus globulus leaf structure

Fig. 3. Dimensions of resprout Wilsons Prom. Eucalyptus globulus ssp. globulus leaves with node from the base of the main stem. (A) Leaf length; (B) maximum width; (C) ratio of leaf length to width; and (D) petiole length. Juvenile (J), transitional (T) and adult (A) leaves have been indicated. Values are means and bars are the standard error of the mean for 1–11 replicates.

263

Fig. 4. Axes 1 and 2 of DCA ordinations of (A) morphological; (B) anatomical; and (C) epidermal characteristics of the juvenile (J), transitional (T) and adult (A) leaves of the Tasmanian Eucalyptus globulus ssp. globulus provenance, and adult (W) leaves of the Wilsons Prom. provenance.

264

S. A. James and D. T. Bell

Table 1.

Leaf morphological, anatomical and epidermal characteristics of juvenile, transitional and adult Tasmanian provenance leaves, and adult Wilsons Prom. leaves Values are mean and standard error of the mean. Values for a characteristics with the same letter are not statistically significant at P < 0.05

Leaf characteristic

Morphology Leaf total length (mm) Maximum width (mm) Length:width Area (cm2) Petiole length (mm) Basal angle (°) Fresh mass (g) Dry mass (g) Leaf specific mass (g cm–2) Water content mass (%) area (%) Anatomy (µm) Leaf thickness Palisade mesophyll thickness Palisade cell length adaxial abaxial Palisade cell width adaxial abaxial Cuticle thickness adaxial abaxial Epidermal thickness adaxial abaxial Epidermis Stomatal density (mm–2) adaxial abaxial total Adaxial:total stomatal density Pore length (µm) adaxial abaxial Complex length (mm) adaxial abaxial Complex width (µm) adaxial abaxial Total complex area (%) Gland density (mm–2) adaxial abaxial

Tasmanian provenance

Wilsons Prom.

Juvenile

Transitional

Adult

Adult

148.6 ± 5.4a 86.9 ± 3.7a 1.72 ± 0.06a 94.7 ± 8.4a 1.21 ± 0.041a 12.3 ± 3.9a 4.02 ± 0.40a 1.84 ± 0.17ac 1.94 ± 0.03a

144.0 ± 4.6a 65.9 ± 2.3b 2.24 ± 0.10a 66.3 ± 3.5b 15.64 ± 2.13b 63.8 ± 6.4b 3.19 ± 0.15a 1.43 ± 0.06b 2.18 ± 0.04a

212.8 ± 6.3b 40.8 ± 2.0c 5.57 ± 0.22b 60.5 ± 4.0b 28.81 ± 1.10c 131.1 ± 2.2c 3.58 ± 0.29a 1.62 ± 0.13bc 2.64 ± 0.06b

153.4 ± 2.7a 30.2 ± 0.8d 5.28 ± 0.15b 33.7 ± 1.0c 30.42 ± 1.08c 163.0 ± 1.6c 2.17 ± 0.07b 1.01 ± 0.03d 2.99 ± 0.04c

53.9 ± 0.6ab 2.27 ± 0.05a

55.1 ± 0.4a 2.67 ± 0.04b

54.5 ± 0.4a 3.16 ± 0.06c

53.6 ± 0.2b 3.46 ± 0.04c

385.0 ± 16.2a 182.5 ± 48.3a

423.5 ± 12.1a 369.5 ± 10.4b

490.9 ± 18.2b 420.4 ± 17.9c

519.9 ± 10.7b 445.5 ± 10.3c

82.1 ± 4.2a –

67.9 ± 1.5b 45.9 ± 3.4a

64.3 ± 1.6b 53.0 ± 1.7b

63.8 ± 1.3b 50.1 ± 1.0ab

11.3 ± 0.4a 12.6 ± 0a

10.7 ± 0.4bc 10.6 ± 0.7b

3.5 ± 0.3a 4.75 ± 0.8a

6.5 ± 0.9b 6.75 ± 1.1a

8.5 ± 1.4b 11.6 ± 1.8b

12.6 ± 1.2c 12.6 ± 0.7b

21.3 ± 1.2a 18.5 ± 1.4ab

19.5 ± 0.96a 17.3 ± 0.9a

20.9 ± 0.9a 20.5 ± 0.5b

23.5 ± 0.5b 22.5 ± 0.9c

19.5 ± 5.8a 171.1 ± 15.2a 191 ± 17a 0.10 ± 0.03a

54.3 ± 7.0b 166.2 ± 14.5a 220 ± 14a 0.25 ± 0.03b

51.6 ± 4.6b 88.7 ± 9.8b 140 ± 13b 0.37 ± 0.02c

61.3 ± 2.9b 70.4 ± 3.5b 132 ± 5b 0.47 ± 0.01d

23.3 ± 0.6a 17.5 ± 1.4a

23.0 ± 1.2a 22.0 ± 1.2a

32.9 ± 1.4b 31.7 ± 1.6b

39.7 ± 0.9c 38.6 ± 1.1c

28.6 ± 1.0a 21.9 ± 1.5a

30.0 ± 1.4a 26.8 ± 1.7a

42.9 ± 1.8b 40.1 ± 2.0b

50.8 ± 0.9c 48.8 ± 1.1c

24.8 ± 0.6a 18.2 ± 1.4a 6.3 ± 0.4a

25.3 ± 1.2a 22.4 ± 1.4a 10.9 ± 0.9b

38.2 ± 1.6b 35.9 ± 1.7b 16.0 ± 1.0c

45.2 ± 0.8c 43.8 ± 1.0c 22.6 ± 0.7d

2.70 ± 0.40ab 0.92 ± 0.20a

3.55 ± 0.48b 2.40 ± 0.32b

2.21 ± 0.31a 1.09 ± 0.21a

0.84 ± 0.16c 0.35 ± 0.06a

larger adaxial than abaxial stomatal pore length. Juvenile leaves had the lowest, and Wilsons Prom. adult leaves had the highest proportion of leaf surface area occupied by stomatal complexes (Table 1). Stomatal pore length increased with

9.9 ± 0.4bcd 9.3 ± 0.2c

9.7 ± 0.2cd 9.4 ± 0.2c

increasing amphistomy. A significant and positive linear relationship was found between the ratio of adaxial to total stomatal density, and the adaxial (r = 0.90, F = 284.9, P < 0.01) and abaxial (r = 0.89, F = 391.9, P < 0.01) pore

Eucalyptus globulus leaf structure

265

Fig. 5. Photomicrographs of transverse sections of representative Tasmanian provenance Eucalyptus globulus ssp. globulus leaf forms. (A) Juvenile; (B) transitional; and (C) adult leaves. Sections have been stained with periodic acid-Schiff’s reagent and/or toluidine blue. Scale bars = 100 µm.

length for the E. globulus leaves. The most hypostomatal leaves were also the thinnest leaves. A significant, positive linear relationship was found between the degree of amphistomy and leaf specific mass (r = 0.79, F = 62.8, P < 0.01). The density of surface oil glands was significantly lower than the density of stomata (Table 1). Oil gland density was greatest for transitional leaves, and Wilsons Prom. adult leaves had the lowest density of oil glands. Discussion Eucalyptus globulus ssp. globulus leaf morphology and anatomy showed variation due to plant genotype, ontogenetic position on the plant, and microenvironment during leaf

development. Measurements within this study provide further evidence that the transition from the juvenile to the adult phase of vegetative growth is a progressive rather than an abrupt, single-event change (Thomas and Vince-Prue 1984; Hackett 1985). Seedling E. globulus leaves showed a continuous and gradual change in leaf characteristics from the cotyledons to the juvenile leaf form. Similarly, transitional leaves formed a continuum between the juvenile and adult leaf forms. The rate of ontogenetic change varied with E. globulus ssp. globulus genotype. Wilsons Prom. seedlings maintained juvenile ratios of leaf length to width for only a limited number of nodes and rapidly underwent vegetative phase change. In contrast, Tasmanian seedlings produced juvenile

266

S. A. James and D. T. Bell

Table 2.

Suggested rates of change of leaf morphological, anatomical and epidermal characteristics for leaves of the Tasmanian Eucalyptus globulus ssp. globulus provenance Rate of change is based on the statistical difference between juvenile, transitional and adult leaves of the Tasmanian provenance

⇐ later change ‘Juvenile’ characteristics

Rate of characteristic change ‘Transitional’ characteristics

earlier change ⇒ ‘Adult’ characteristics

Leaf length Leaf length:width Leaf thickness

Maximum width Midwidth Mid:maximum width Length to max. width:total length Petiole length Base angle Leaf specific mass % Water per unit leaf area

Length to maximum width Leaf area Palisade thickness Palisade number Spongy thickness

Cuticle thickness—abaxial Palisade cell length—adaxial:abaxial Stomatal density—abaxial, total Stomatal length—adaxial, abaxial Stomatal width—adaxial, abaxial Stomatal pore length—adaxial

Stomatal density—adaxial:total

Stomatal pore length—abaxial

leaves for a considerable period of development. The initiation of vegetative phase change by Wilsons Prom. seedlings ranged from Nodes 16 to 30, indicating genotypic differences within the seed source. Similar variability in vegetative phase change has been reported for E. tenuiramis seedlings (Wiltshire and Reid 1992). In addition to the timing of vegetative phase change, adult leaves of the Tasmanian and Wilsons Prom. provenances were different in a number of morphological and epidermal characteristics. This result further suggests taxonomic differences between the Wilsons Prom. provenance and core E. globulus ssp. globulus, represented by the Tasmanian provenance, as found by Jordan et al. (1994) and Nesbitt et al. (1995). Changes in phase-specific traits are not always completely correlated, with transitional leaves having characteristics of more than one phase (Gould 1993; Wiltshire et al. 1994; Lawson and Poethig 1995; BongardPierce et al. 1996). The transition from the juvenile to adult leaf form of E. globulus ssp. globulus is a gradual, progressive change, with leaf characteristics changing at different rates. Leaf area, palisade mesophyll characteristics, and adaxial stomatal density underwent change at a more rapid rate than the ratio of leaf length to width, leaf thickness and stomatal characteristics (Table 2). Hence, a transitional leaf may have an array of both juvenile and adult leaf characteristics in varying proportions, depending on the stage of vegetative phase change. Eucalyptus globulus leaf characteristics do not change uniformly, nor in a parallel fashion with vegetative phase change, which provides further evidence that there are independent control mechanisms for various leaf characteristics (Borchert 1976). Measurements of juvenile, transitional and adult leaf characteristics of the two E. globulus ssp. globulus

Cuticle thickness—adaxial Palisade cell length Palisade cell width—abaxial Stomatal density—adaxial Stomatal length—adaxial:abaxial Stomatal width—adaxial:abaxial Stomatal pore length—adaxial:abaxial Gland density—adaxial:abaxial

provenances were consistent with the findings of previous descriptive studies of eucalypt ontogenetic leaf forms (Johnson 1926; Cameron 1970; Kirkpatrick 1975; Pereira and Kozlowski 1976; Ridge et al. 1984; Ito and Suzaki 1990; James and Bell 1995). Leaf size and dimension directly affects the thickness of the leaf boundary layer, energy exchange and the diffusion of water vapour at the leaf surface (Gates 1980; Smith et al. 1997). Shorter adult leaves of E. globulus are found on trees in regions with shallow soils or low precipitation (Kirkpatrick 1975). Shorter and narrower adult E. globulus leaves may have increased convective heat dissipation and, hence, remain cooler and be more advantageous under conditions of moisture limitation than the broader juvenile leaf form. Leaf thickness increased with ontogenetic development from the juvenile to adult E. globulus form. This trend has been documented for numerous broadleaf plant species (Doorenbos 1965; Hoflacher and Bauer 1982; Hackett 1985; Gould 1993; Day et al. 1997). Eucalyptus globulus leaf specific mass increased with ontogenetic development because of an increase in leaf thickness, and possibly because of an accumulation of secondary compounds, such as tannins, phenols, lignins and starch (Dijkstra 1990). Corresponding with the ontogenetic increase in leaf thickness, distinct differences were found in the mesophyll structure. Spongy mesophyll of the adult leaves was reduced to a layer of shorter, less compact cells resembling the adjacent palisade tissues, as found by Johnson (1926). Increased leaf thickness was due to an increase in the number of palisade cell layers and palisade thickness, as found by others (Cameron 1970; Doley 1978; Ashton and Turner 1979; Hoflacher and Bauer 1982; James and Bell 1995; Nobel and Walker 1985). Although thicker mesophylls tend to reduce CO2 assimilation per gram of

Eucalyptus globulus leaf structure

tissue, through internal self-shading and CO2 diffusion limitation (Parkhurst 1986), the increased internal cell wall surface area with E. globulus ontogenetic leaf development would reduce liquid phase transport of CO2. Combined with the increase in chloroplast density and enzyme content per unit leaf area with increased leaf thickness, assimilation per unit leaf area would increase with ontogenetic development from the juvenile to adult leaf form (James et al. 1999). Palisade distribution and cell length between the adaxial and abaxial leaf surfaces of the ontogenetic leaf forms may be a response to the proportion of light incident to each leaf surface. A greater proportion of light is incident to the adaxial surface of horizontal, dorsiventral juvenile leaves, whereas light is more equally distributed between the two surfaces of the vertical, isobilateral adult leaf form (James and Bell 2000). Columnar palisade mesophyll facilitates the penetration of direct light into leaf mesophyll (Vogelmann et al. 1996). The adjustment of palisade thickness, geometry and packing according to ambient light conditions may act to control the distribution of internal light, and maximise light absorption and carbon fixation within the leaf. Longer palisade cells of juvenile E. globulus leaves may assist in the penetration of light into the leaf without the development of further palisade cell layers. Highly light-scattering and reflecting spongy mesophyll found in the juvenile leaves would be particularly advantageous under low-light conditions (Vogelmann et al. 1996). Cuticle thickness of the E. globulus leaves increased with ontogenetic development from the juvenile to adult leaf form. Juvenile leaves had greater epicuticular wax development than adult leaves. The corresponding increase in cuticular thickness with leaf thickness has previously been reported for E. globulus (Johnson 1926) and E. camaldulensis (James and Bell 1995). Thicker epidermal cells and the uneven epidermal cell-wall thickening of adult leaves could enhance the focusing of collimated light within these leaves (Vogelmann et al. 1996; James 1998). Stomata of the E. globulus ssp. globulus ontogenetic leaf forms varied in distribution, density and size. Juvenile Eucalyptus leaves are typically hypostomatal, whereas adult leaves are amphistomatal (Johnson 1926; Cameron 1970; Ridge et al. 1984; Ito and Suzaki 1990). Stomatal densities for the two E. globulus provenances were within the ranges found for other Eucalyptus species (Cameron 1970; Ridge et al. 1984; James and Bell 1995). An increase followed by a decrease in stomatal density is often found with the successive development of leaves on a plant (Ticha 1982). The total density of stomata per unit area of Tasmanian E. globulus leaves increased from the juvenile to transitional leaf form, then declined with the development of the adult leaf form. The change in stomatal density from the juvenile to adult leaf form could be interpreted as a common ontogenetic developmental sequence.

267

Stomatal size usually changes inversely with stomatal density (Ticha 1982; Willmer and Fricker 1996). Stomata of the juvenile leaves of E. globulus ssp. globulus were substantially smaller and had a greater density than those of the adult leaf form. Adaxial stomata of the juvenile leaves were also larger than the more numerous abaxial stomata, whereas adult leaf stomata were of a similar size on the two leaf surfaces, as found for leaves of other eucalypt species (Johnson 1926; Cameron 1970; Ridge et al. 1984; Ito and Suzaki 1990). Although stomatal size increased inversely with density, the adult E. globulus leaves had a significantly greater stomatal complex area per unit leaf area than the juvenile leaf form. This suggests a greater external supply of CO2 within the thicker adult leaves. Amphistomy is found primarily in species from highlight, arid habitats (Mott et al. 1982; Gibson 1998). It could, therefore, be hypothesised that the hypostomatal juvenile eucalypt leaves are better adapted to mesic conditions, whereas the adult leaves are more xeromorphic. It has been suggested that morphology primarily determines stomatal distribution, as amphistomy increases with mesophyll thickness (Parkhurst 1978; Beerling and Kelly 1996). The change from the dorsiventral juvenile leaf structure to the isobilateral adult E. globulus ssp. globulus leaf structure was correlated strongly with the distribution of stomata on the adaxial leaf surface. Thick-leaved species with high maximum photosynthetic rates (Willmer and Fricker 1996) and species with vertically oriented leaves and/or isobilateral organisation of palisade mesophyll (Mott et al. 1982; Smith et al. 1998) are typically amphistomatous. The diffusion pathway through the mesophyll of thick leaves may be too long and inefficient if stomata occurred only on one surface (Parkhurst 1978; Mott et al. 1982). Although it has often been reported that the juvenile E. globulus leaves lack stomata on the adaxial surface (Johnson 1926; Ito and Suzaki 1990), the juvenile leaves studied here did have a limited number of stomata within the adaxial leaf epidermis. This may have been a consequence of a greater thickness of the juvenile leaves than that reported in other studies. Leaves of most Eucalyptus species have small glands distributed immediately beneath the epidermis or deeper within the mesophyll which are filled with oils composed of odorous terpenes (Boland et al. 1991; Bolhár-Nordenkampf and Draxler 1993). The number and size of oil glands increases with ontogenetic leaf development of E. regnans (Ashton and Turner 1979). In contrast, the E. globulus leaves studied here had fewer surface oil glands than the leaves of E. regnans, and showed no ontogenetic trend. The adult Wilsons Prom. leaves had a lower number of oil glands than the Tasmanian adult leaves, indicating a genotypic difference in the quantitative production of oil between the Tasmanian and Wilsons Prom. provenances. The morphological differences between juvenile and adult leaves, and between the Tasmanian and Wilsons Prom.

268

E. globulus ssp. globulus provenances, may have an adaptive significance. The lower leaf specific mass of the juvenile leaves confers rapid establishment, as a high leaf area index can be obtained with a low investment in biomass (Ticha 1982; Linder 1985). The numerous small stomata found on juvenile leaves would allow for rapid gas exchange and growth. Both internal and external competition for water may increase with increasing plant size, resulting in more sclerophyllous and drought-resistant leaves, as found for Juniperus occidentalis (Miller et al. 1995). Increased leaf thickness and structural development result in a higher specific leaf mass of the adult leaf form. Palisade cell development and amphistomy would increase the carbon gain of the adult leaf form without greatly increasing water loss. A thick, reflective cuticle and vertical leaf orientation further assist in the reduction of heat loading and water loss of the adult leaves. Rapid vegetative phase change by the Wilsons Prom. seedlings may confer an adaptive advantage within the native habitat, which is exposed to strong winds and salt spray (Potts and Jordan 1994). Acknowledgments The authors thank Professor William K. Smith and the Department of Botany, University of Wyoming; the Western Australian Department of Conservation and Land Management for access to the E. globulus field trial and seed; Dr William Loneragan for his advice; and Professor Ian James at Murdoch University for statistical assistance. This research was supported by an Australian Postgraduate Award and a UWA Graduates Association Postgraduate Research Travel Award. References Ashton DH, Turner JS (1979) Studies on the light compensation point of Eucalyptus regnans F. Muell. Australian Journal of Botany 27, 589–607. Beerling DJ, Kelly CK (1996) Evolutionary comparative analyses of the relationship between leaf structure and function. The New Phytologist 134, 35–51. Boland DJ, Brophy JJ, House APN (1991) ‘Eucalyptus leaf oils: use, chemistry, distillation and marketing.’ (Inkata Press: Melbourne) Bolhár-Nordenkampf HR, Draxler G (1993) Functional leaf anatomy. In ‘Photosynthesis and production in a changing environment: a field and laboratory manual’. (Eds J Coombs, DO Hall, SP Long, JMO Scurlock) pp. 91–112. (Chapman and Hall: London) Bongard-Pierce DK, Evans MMS, Poethig RS (1996) Heteroblastic features of leaf anatomy in maize and their genetic regulation. International Journal of Plant Sciences 157, 331–340. Borchert R (1976) The concept of juvenility in woody plants. Acta Horticulturae 56, 21–36. Cameron RJ (1970) Light intensity and the growth of Eucalyptus seedlings. I. Ontogenetic variation in E. fastigata. Australian Journal of Botany 18, 29–43. Day JS, Gould KS, Jameson PE (1997) Vegetative architecture of Elaeocarpus hookerianus. Transition from juvenile to adult. Annals of Botany 79, 617–624. Dijkstra P (1990) Cause and effect of differences in specific leaf area. In ‘Causes and consequences of variation in growth rate and

S. A. James and D. T. Bell

productivity of higher plants’. (Eds H Lambers, ML Cambridge, H Könings, TL Pons) pp. 125–140. (SBS Academic: The Hague) Doley D (1978) Effects of shade on gas exchange and growth in seedlings of Eucalyptus grandis Hill ex Maiden. Australian Journal of Plant Physiology 5, 723–738. Doorenbos J (1965) Juvenile and adult phases in woody plants. In ‘Encyclopedia of plant physiology’. (Ed. W Ruhland) pp. 1222– 1235. (Springer-Verlag: Berlin) Feder N, O’Brien TP (1968) Plant microtechnique: some principles and new methods. American Journal of Botany 55, 123–142. Gates DM (1980) ‘Biophysical ecology.’ (Springer Verlag: New York) Gibson AC (1998) Photosynthetic organs of desert plants. BioScience 48, 911–920. Gould KS (1993) Leaf heteroblasty in Pseudopanax crassifolius: functional significance of leaf morphology and anatomy. Annals of Botany 71, 61–70. Hackett WP (1985) Juvenility, maturation and rejuvenation in woody plants. Horticultural Reviews 7, 109–155. Hoflacher H, Bauer H (1982) Light acclimation in leaves of the juvenile and adult life phases of ivy (Hedera helix). Physiologia Plantarum 56, 177–182. Ito S, Suzaki T (1990) Morphology and water relations of leaves of Eucalyptus globulus sprouts. Bulletin of the Kuyshu University Forests 63, 37–53. James SA (1998) Comparative leaf structure and function, and the growth of two provenances within Eucalyptus globulus ssp. globulus. PhD Thesis, The University of Western Australia, Perth. James SA, Bell DT (1995) Morphology and anatomy of leaves of Eucalyptus camaldulensis clones: variation between geographically separated locations. Australian Journal of Botany 43, 415–433. James SA, Bell DT (2000) Leaf orientation, light interception, and conductance of Eucalyptus globulus ssp. globulus leaves. Tree Physiology 20, 815–823. James SA, Smith WK, Vogelmann TC (1999) Mesophyll structure and chlorophyll distribution in ontogenetically different leaves of Eucalyptus globulus ssp. globulus (Myrtaceae). American Journal of Botany 86, 198–207. Johnson ED (1926) A comparison of the juvenile and adult leaves of Eucalyptus globulus. The New Phytologist 25, 202–212. Jordan GJ, Borralho NMG, Tilvard P, Potts BM (1994) Identification of races in Eucalyptus globulus ssp. globulus based on growth traits in Tasmania and geographic distribution. Silvae Genetica 43, 292–298. Kirkpatrick J (1975) Geographical variation in Eucalyptus globulus. Forestry and Timber Bureau Bulletin 47, 1–64. Lawson EJR, Poethig RS (1995) Shoot development in plants: time for a change. Trends in Genetics 11, 263–268. Linder S (1985) Potential and actual production in Australian forest stands. In ‘Research for forest management’. (Eds JJ Landsberg, W Parson) pp. 11–35. (CSIRO: Melbourne) Miller PM, Eddleman LE, Miller JM (1995) Juniperus occidentalis juvenile foliage: advantages and disadvantages for a stress-tolerant, invasive conifer. Canadian Journal of Forest Research 25, 470–479. Mott KA, Gibson AC, O’Leary JW (1982) The adaptive significance of amphistomatic leaves. Plant, Cell and Environment 5, 455–460. Nesbitt KA, Potts BM, Vaillancourt RE, West AK, Reid JB (1995) Partitioning and distribution of RAPD variation in a forest tree species, Eucalyptus globulus (Myrtaceae). Heredity 74, 628–637. Nobel PS, Walker DB (1985) Structure of leaf photosynthetic tissue. In ‘Photosynthetic mechanisms and the environment’. (Eds J Barber, NR Baker) pp. 503–536. (Elsevier: Amsterdam) Oksanen J, Minchin PR (1997) Instability of ordination results under changes in input data order: explanations and remedies. Journal of Vegetation Science 8, 447–454.

Eucalyptus globulus leaf structure

269

Parkhurst DF (1978) The adaptive significance of stomatal occurrence on one or both surfaces of leaves. Journal of Ecology 66, 367–383. Parkhurst DF (1986) Internal leaf structure: a three-dimensional perspective. In ‘On the economy of plant form and function’. (Ed. TJ Givnish) pp. 215–249. (Cambridge University Press: Cambridge) Pereira JS, Kozlowski TT (1976) Leaf anatomy and water relations of Eucalyptus camaldulensis and E. globulus seedlings. Canadian Journal of Botany 54, 2868–2880. Potts BM, Jordan GJ (1994) The spatial pattern and scale of variation in Eucalyptus globulus ssp. globulus: variation in seedling abnormalities and early growth. Australian Journal of Botany 42, 471–492. Ridge RW, Loneragan WA, Bell DT, Colquhoun IJ, Kuo J (1984) Comparative studies in selected species of Eucalyptus used in rehabilitation of the northern jarrah forest, Western Australia. II. Wood and leaf anatomy. Australian Journal of Botany 32, 375–386. Smith WK, Vogelmann TC, DeLucia EH, Bell DT, Shepherd KA (1997) Leaf form and photosynthesis. Do leaf structure and orientation interact to regulate internal light and carbon dioxide? BioScience 47, 785–793. Smith WK, Bell DT, Shepherd KA (1998) Associations between leaf structure, orientation, and sunlight exposure in five Western Australian communities. American Journal of Botany 85, 56–63.

Thomas B, Vince-Prue D (1984) Juvenility, photoperiodism and vernalisation. In ‘Advanced plant physiology’. (Ed. MB Wilkins) pp. 408–412. (Longman Scientific: Essex) Ticha I (1982) Photosynthetic characteristics during ontogenesis of leaves. 7. Stomata density and sizes. Photosynthetica 16, 375–471. Vogelmann TC, Nishio JN, Smith WK (1996) Leaves and light capture: light propagation and gradients of carbon fixation within leaves. Trends in Plant Science 1, 65–70. Willmer C, Fricker M (1996) ‘Stomata.’ (Chapman and Hall: London) Wiltshire RJE, Murfet IC, Reid JB (1994) The genetic control of heterochrony: evidence from developmental mutants of Pisum sativum L. Journal of Evolutionary Biology 7, 447–465. Wiltshire RJE, Reid JB (1992) The pattern of juvenility within Eucalyptus tenuiramis Miq. saplings. In ‘Mass production technology for genetically improved fast growing forest tree species. AFOCEL-IUFRO Symposium, Bordeaux’. pp. 37–49. (Association Forêt Cellulose: Nangis, France)

Manuscript received 21 June 1999, accepted 25 September 2000

http://www.publish.csiro.au/journals/ajb