The Influence of Tree Size and Microenvironmental Changes on the Wood Anatomy of Roupala Rhombifolia

88 IAWAIAWA Journal Journal 34-1 34-1 (2013) (2013) 88–106

THE INFLUENCE OF TREE SIZE AND MICROENVIRONMENTAL CHANGES ON THE WOOD ANATOMY OF ROUPALA RHOMBIFOLIA

Viviane Jono*, Giuliano Maselli Locosselli and Gregório Ceccantini Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, CEP 05508-090, Cidade Universitária, São Paulo, SP, Brazil *Corresponding author; e-mail: [email protected]

Abstract We analysed how variation in microenvironmental conditions and stem size affects the wood anatomy of Roupala rhombifolia in three contrasting habitats in the same study area: open field, hilltop forest and riparian forest. The wood anatomy features analysed were: vessel area and density, vessel element length, fibre length, and ray width and height. Vegetation cover and soil attributes were also quantified and integrated into the analyses. Separate analyses were performed on i) raw anatomical data and ii) residuals from linear fits between wood anatomical features and plant height and stem diameter. Raw data showed a clear difference between specimens from riparian forest and open fields, which represented the most mesic and xeric anatomical features respectively. After residual extraction to correct size-related variation, only fibre length and vessel area differed between habitats. Vessel areas in riparian forest differed from those in hilltop forest, but were similar to those in open fields. This result can be explained when vegetation cover and soil are considered together. While open field and hilltop forest have similar soils and lower moisture availability when compared to riparian forest, water demand in open fields is lower, presumably resulting in higher water availability. Keywords: Proteaceae, ecological anatomy, stem size, wood maturity, residual extraction.

Introduction

The anatomical structure of wood records environmental influences and ontogenetic processes experienced throughout its development. Decades of ecological wood anatomy studies have demonstrated that the relative influences of environment and plant size on wood anatomy are often difficult to disentangle (Baaset al. 1984). While it has been suggested that plants of similar size should be studied to remove the influence of stem diameter and plant height on anatomical data (Lev-Yadun & Aloni 1995), it is frequently impracticable to restrict plant size along environmental gradients since the latter may modulate both anatomical variation and plant size. For example, both wood anatomy and plant height vary with elevation in Rhododendron spp. (Noshiro et al. 1995).

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Over the years, many classical studies of ecological wood anatomy have demonstrated that species show different phenotypes as a result of different selective pressures from the environment (e.g. Baas et al. 1983; Barajas-Morales 1985; Carlquist 1975, 1988; Van den Oever et al. 1981). These responses reflect the organization of conductive tissues, which results from a trade-off between safety and efficiency in water transport (Carlquist 1977). This balance, taking into account phenotypical plasticity within the conductive tissue and phylogenetic aspects, allows plants to survive in different environments, including those in which conditions are suboptimal (Walter 1985). These trends have been found at a wide range of altitudes and latitudes, and under a variety of different macro- and microclimatic conditions (e.g. Baas 1973; Roig 1986; Baas & Schweingruber 1987; Lindorf 1994; Alves & Angyalossy-Alfonso 2000; Bosio et al. 2010; Noshiro et al. 2010; Sonsin 2012). However, environmental influences on wood anatomy may not be completely iso- lated from other factors, such as plant size. This sampling artifact can result in biased data which interfere with tests of ecological hypotheses (Lev-Yadun & Aloni 1995). It is known that plant size influences wood anatomy (e.g. Preston et al. 2006; Fichtler & Worbes 2012). Many studies that have used age variation of wood anatomical characters have shown that they vary with stem diameter, to a larger degree in softwoods and a lesser degree in hardwoods. Stem diameter is not the only plant size variable that may influence wood anatomy. Plant height has also been shown to introduce a bias in wood anatomical attributes, as shown by a study of dwarf trees (Baas et al. 1984). Even when only mature wood is analysed, correlations between plant size and wood anatomy may be found (Noshiro & Suzuki 2001). By examining wood anatomy, plant size, latitude and altitude of provenance, Noshiro et al. (2010) showed a correlation between vessel area ratio (%) and stem diameter and height. In that study the dependence was not considered important, since vessel area ratio was not correlated with altitude, which was the focus of the study. The aim of our study was to analyse the effect of microenvironmental and stem size variation on the wood anatomy of Roupala rhombifolia Mart. ex Meisn. (Proteaceae). The study site included three different habitats, which allowed us to sample in distinct microenvironmental conditions while avoiding macroclimatic variation in factors such as precipitation and incident solar radiation. To better understand the results, we used a simple method of data analysis to remove the influence of stem diameter and height. Besides, plant density/biomass was quantified via multispectral satellite images and soils were sampled in order to characterise each habitat.

Material and Methods

Sampling took place in the Serra do Cipó (Minas Gerais state, Brazil), a mountain chain rich in endemism and characterised by a vegetation type known as campo rupestre (rocky field). The region shows a broad range of edaphic conditions (Meguro et al. 1996) and also harbours forested vegetation types. These are semideciduous montane forests with trees up to 15 metres tall, which grow both along rivers and distant from water bodies. Mean temperature is 19.3 ºC and total annual precipitation is 1312 mm

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(data from the Diamantina climate station, National Institute of Meteorology - INMET). The rainy season is between October and April, and the dry season between May and September (Fig. 1). We studied two different forest habitats and the open fields surrounding them, all three closely located within the Serra do Cipó National Park (Santana do Riacho, Brazil – 19º 15' 36" S, 43º 32' 37" W). These habitats have different microenvironmental conditions but identical macroclimates (Fig. 2). Sampling sites differed in proximity to water and slightly in altitude. Although water availability varies between these habitats, none of them suffers flooding. In order to better characterise the habitats, satellite images were used to produce a normalised difference vegetation index (NDVI) image. A multispectral image from the CBERS2 satellite, acquired in July 2005, was analysed with IDRISI ANDES software. NDVI values were used to estimate biomass and vegetation cover (Thiam & Eastman 2006) from the remote sensing data. Additionally, soils were characterised via three samples collected at a depth of 15 cm in each habitat, in July 2006. Soil samples were analysed by the Agronomic Institute of Campinas (IAC) via standard methods for determining water content at saturation, field capacity, wilting point, soil density, and organic matter content. A multivariate cluster analysis was used to group similar soil samples by Ward’s hierarchical method applied to standardised data in the R program (R Development Core Team 2011). The study species was Roupala rhombifolia Mart. ex Meisn. (Proteaceae), which usually grows in seasonal higher-elevation environments (Prance & Plana 1998). Our observations suggest that this evergreen species is a pioneer that probably plays a role in the formation and expansion of forest islands in the Serra do Cipó. Although R. rhombifolia seems to behave as a pioneer species, the wood density of Roupala spp. is reported to be relatively high ranging from 0.8 to 1.2 g/cm3 (Record & Hess 1943). First, wood maturity was analysed in a branch of a forest specimen by measuring the length of 30 fibres and vessel elements at each millimetre from cambium to pith after maceration (Franklin 1945). This preliminary analysis was helpful to indicate the limits of trunk size for sampled specimens, especially the open field specimens that were relatively small. Wood samples of the five biggest open field individuals were obtained at their base. For the other habitats, samples were taken from the trunk at breast height, using a handsaw, a chisel and a hammer. Wood samples of eight specimens from each forest formation and five specimens from the open field were collected and deposited at the University of São Paulo xylarium (SPFw). The height and diameter of each sampled specimen were recorded. For these wood samples the following anatomical variables were measured: fibre and vessel element length, vessel area, vessel density, and ray height and width. Sam- ples near the cambium were macerated (Franklin 1945) to measure fibre and vessel element length. Samples of approximately 1.5 × 1.5 × 2 cm were sectioned to measure other variables. In addition, wood samples were softened with 10% ethylenediamine (Carlquist 1982 adapted) and soaked in PEG 1500 (Rupp 1964) to produce transverse and tangential sections using a Leica SM 2000R sliding microtome. Thirty measurements were taken for each variable analysed, except for ray height and width, for which

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300

50 100

40 80

30 60

20 40 Temperature (°C)Temperature Precipitation (mm)

10 20

0 0 J A S O N D J F M A M J Month Figure 1. Climatic diagram from the climate station of Diamantina, MG, Brazil (National Institute of Meteorology - INMET).

0.10 0.40 0.70

Figure 2. A: Study site location in southeastern Brazil (black dot), with an upper dashed line indicating the Equator and a lower dashed line indicating the Tropic of Capricorn. – B: A detail of the study site in a higher resolution panchromatic image (resolution = 2.5 m). – C: Normalised difference vegetation index (NDVI) image (resolution = 20 m) of the study site. Darker shades of gray indicate lower levels of vegetation cover/biomass, and lighter shades indicate higher levels. Circles show the location of sampling sites in the open field (Fi), hilltop forest (HF) and riparian forest (RF) habitats. The black line shows a road near the study site. — Scale bar = 500 m.

Downloaded from Brill.com10/04/2021 11:42:55AM via free access 92 IAWA Journal 34-1 (2013) only 20 measurements were taken due to a lower number of multiseriate rays per slide. Lengths were measured with Zeiss KS100 3.0 software, and other variables were measured using ImageJ version 1.38x (Rasband 2011). Raw data with no previous statistical treatment were analysed using Kruskall- Wallis tests to verify if significant differences in anatomical data between habitats could be found. Differences in wood anatomy between habitats were identified by a non-parametric post hoc test which was performed according to Siegel and Castelan (1988), using the “pgirmess” package (Giraudoux 2011) in R (R Development Core Team 2011). To understand how stem size influenced overall xylem anatomy, linear regressions were fitted among anatomical data and stem height and diameter. When a linear fit was statistically significant, 2R values indicated the amount of anatomical data variation explained by one of the plant dimensions. The difference between the predicted linear regression values (represented by the line) and the actual anatomical values are known as residuals (Farawey 2005), and indicate the amount of data variation not explained by the stem size. Althought anatomical features might be influenced by both, stem height and diameter, only one predictor variable was used in the regression analyses due to the fact that the two stem size variables were highly correlated (high degree of colinearity). Only the predictor which had the highest R2 value was considered in the residual extraction analyses. Kruskal-Wallis and the non-parametric post hoc analyses were re-performed with anatomical data residuals. The purpose of these analyses was to test the effect of environmental variation on xylem anatomy independent of plant size.

Results Environmental attributes The study site was composed of three habitats: riparian forest, hilltop forest, and a matrix of open fields (see maps in Fig. 2). Since the NDVI is an estimation of vegetation biomass and cover, it showed that the open field has a lower vegetation biomass and

Cluster Dendrogram

Height 4

2 HF3 RF2 0 F3 HF2 F1 RF1 RF3 F2 HF1 Figure 3. Cluster analysis of similarity of habitat based on soil data. RF: riparian forest, HF: hilltop forest and F: open field. The numbers indicate sample labels. Soils of hilltop forest and open field are similar to each other and both are distinct to the riparian forest soil.

Downloaded from Brill.com10/04/2021 11:42:55AM via free access Jono et al. – Tree size, environment, anatomy of Roupala 93 cover (dark gray) than the two forest formations (light gray). Differences between the two forest types are not evident in this analysis due to their similar vegetation structure. Differences between habitats were also found in the soil analyses. The cluster analysis indicated that open field and hilltop forest soils were similar to each other, and that both were different from riparian forest soils (Fig. 3). The similarity of open field and hilltop forest soils is evident in the physical soil properties (water content at saturation, field capacity, permanent wilting point, estimated bulk density), being distinct only for organic matter (Table 1). As expected, riparian forest soils presented the highest values for all attributes, with the exception of estimated bulk density. Since all these attributes have a strong influence on water retention capacity of soils, the riparian forest soils had the highest moisture availability for plants.

Table 1. Mean and standard deviation of soil attributes from each habitat: open field, hilltop forest and riparian forest.

Habitat Mean Standard deviation

Open field 0.56 0.04 Water content at saturation (m3/m3) Hilltop forest 0.54 0.03 Riparian forest 0.72 0.06

Open field 0.20 0.04 Field capacity 30 kPa (m3/m3) Hilltop forest 0.17 0.02 Riparian forest 0.37 0.02

Open field 0.17 0.05 Permanent wilting point 1500 kPa (m3/m3) hilltop forest 0.14 0.01 Riparian forest 0.33 0.06

Open field 1.28 0.17 Estimated bulk density (g/dm3) Hilltop forest 1.36 0.12 Riparian forest 0.84 0.12

Open field 29.0 2.65 Organic matter (g/dm3) Hilltop forest 67.3 25.8 Riparian forest 70.3 11.9

Qualitative characterisation of wood anatomy Roupala rhombifolia has scalariform axial parenchyma in a festooned pattern, with large multiseriate rays, similar to most Proteaceae. The axial parenchyma bands are usually associated with vessels (Fig. 4A, B). The ray composition is heterocellular, with body procumbent and some upright to square marginal and sheath cells (Fig. 4C, F). Ray height and width increase with distance from the pith, and the procumbent cells vary in size changing the body rays from homocellular to heterocellular (Fig. 4E, F).

Wood maturity The maturity of Roupala rhombifolia wood was established at a radius greater than one centimetre (Fig. 5). Beyond that, fibres and vessel element lengths tend to stabilise

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Figure 4. Wood anatomy of Roupala rhombifolia. A: Transverse section. – B: Detail of vessels (V), with vessel tails (vt), associated with parenchyma cells (P) and tracheids (T). – C & D: Ray parenchyma with upright marginal and sheath cells (arrows). – E: Body ray parenchyma with same-dimension cells in tangential section. – F: Body ray parenchyma with different-dimension cells. – G: Conductive elements of secondary xylem: tracheids and vessel elements. Perfora- tion plates of narrower vessel elements are indicated by arrows. — Scale bars: A = 150 µm; B = 50 µm; C, D, E = 100 µm; F = 200 µm; G = 75 µm.

Downloaded from Brill.com10/04/2021 11:42:55AM via free access Jono et al. – Tree size, environment, anatomy of Roupala 95 length (µm) Vessel element Vessel Fibre length (µm) 0 200 400 0 200 600 1200 1800 5 10 15 Distance from pith (mm) Figure 5. Length-on-age curves for fibres and vessel elements of Roupala rhombifolia (gray line = fibre length, black line = vessel element length).

in the stem, and wood anatomy does not appear to be influenced by tree age and size. Specimens from open fields, which were among the smallest individuals, were sampled with a radius equal to or greater than 1 cm with the exception of specimen “Jono 24” which had a radius equal to 0.95 cm (Table 2).

Ecological anatomy Descriptive statistics of all anatomical features measured, stem height and diameter from each Roupala rhombifolia individual is presented in Table 2. These values of stem dimensions were used as predictor variables of anatomical data in the linear regressions analyses. The results of linear regressions and residuals between wood anatomical variables and stem sizes are shown in Figure 6, where, on the left side, linear regressions using raw data are presented while on the right side there are scatterplots of the extracted residuals. Vessel area and ray width are positively correlated with stem diameter. Fibre length and ray height are positively correlated with stem height. Vessel element length is negatively correlated with stem height. Vessel density was the only studied anatomical variable correlated with neither stem diameter nor height. Stem diameter explained 46% and 28% of vessel area and ray width data variability, respectively, while stem height explained 46%, 31% and 26% of ray height, fibre length and vessel element length data variability, respectively. These results are summarised in Table 3 along with data from the literature on different species from several provenances. For significance tests, R2 values ranged from 0.08 to 0.60 in the different species. Concerning the residuals of the selected wood anatomical variables in Figure 6, the linear fit parallel to the x-axis indicates a complete absence of stem size influence on them. With Kruskal-Wallis tests (Table 4) of the raw data it was possible to statistically distinguish some associations between habitat and ray height, ray width, fibre length, vessel area, and vessel density. Vessel element length was the only non-informative character, which did not distinguish any habitat. As seen in Figure 7, vessel density, vessel area, ray height, ray width, and fibre length were lower in open field specimens than in those from other habitats. Vessel area, ray height, ray width, and fibre length were higher for riparian forest than the other habitats. Thus, mean values for plants from the hilltop forest were generally intermediate between the other two habitats, except

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A area Residuals – Vessel B Stem diameter (cm) Stem diameter (cm) Vessel area (µm Vessel

C D Plant height (m) Plant height (m) Fibre length (µm) Vessel Fibre length (µm) Vessel element lenght (µm) Residuals – Fibre length Residuals – Vessel element lengh Residuals – Fibre length Vessel

E F Plant height (m) Plant height (m)

Figure 6 (for legends, see the next page).

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G H Stem diameter (cm) Stem diameter (cm) Ray Ray length (µm) width (µm) Residuals – Ray length Residuals – Ray width

I J Plant height (m) Plant height (m)

Figure 6. Scatterplots of raw data and size-corrected wood anatomical variables versus plant size. Linear regressions of vessel (A), vessel element (C), fibre (E) and ray (G and I) dimensions versus plant height and stem diameter demonstrate positive or negative correlations with plant size, while the extracted residuals (B, D, F, H and J) show no correlation with plant size. Symbols indicate locality of provenance: open field (squares), hilltop forest (triangles) and riparian forest (circles).

for vessel density, which were the highest in hilltop forest. However, when size information was removed from these data (by analysing the residuals), the pattern changed (Table 4). Kruskal-Wallis tests only indicated statistical differences between habitats in vessel area and fibre length, both variables that showed the strongest significance in the raw data analysis. Ray height, ray width, and vessel element length showed no significant difference between habitats (Table 4). For residuals of vessel area data, hilltop and riparian forests differed from each other but not from the open field. For residuals of fibre length, riparian forest differed from the other two habitats. Vessel density was not analysed by its residuals because it showed no correlation with stem size (stem height and diameter).

Downloaded from Brill.com10/04/2021 11:42:55AM via free access 98 IAWA Journal 34-1 (2013) Std 739 954 732 550 633 476 724 440 959 894 438 592 509 416 542 1120 1227 1410 1026 1056 1559

Ray height 1959 2562 3254 3160 2887 2722 1984 1935 2880 2521 1493 2224 1702 2999 2773 1992 1332 1704 1240 1086 1423 Mean ––––––––––––––

Std 111 114 154 194 125 103 155 121 107 125 148 168 138 315 83.3 96.3 89.6 61.9 77.7 52.9 81.4

Ray width 381 435 630 369 393 360 456 385 334 319 334 351 341 522 491 384 274 334 282 182 275 –––––––––––––– Mean m). Additionally, data on stem height Additionally, m). µ

Std 421 330 420 328 463 307 375 237 306 332 216 369 233 212 182 381 147 178 148 196 109

Fibre length 2023 1854 1815 2123 2134 1993 1829 2139 1773 1438 1363 1764 1823 1694 1559 1679 1421 1353 1509 1512 1228 Mean –––––––––––––– m) and ray height ( µ

5.7 7.5 5.1 6.8 8.1 9.3 7.2 8.4 8.1 9.0 8.5 5.0 6.4 5.3 5.6 5.6 Std 11.7 14.5 12.5 12.8 12.8

Vessel density Vessel 16.1 29.8 18.8 20.6 15.4 14.8 18.5 18.3 35.0 20.0 19.9 27.6 16.7 28.0 24.3 25.7 15.3 16.8 18.8 16.3 15.0 m), ray width ( Mean –––––––––––––– µ

Std 67.9 50.2 68.8 67.0 46.7 42.0 40.7 38.6 46.1 50.8 45.1 52.4 52.0 48.9 48.6 37.8 50.6 60.6 69.5 56.9 49.5

), fibre length ( 2 220 232 234 269 250 227 194 228 240 200 132 258 229 220 234 230 222 331 324 277 285 Mean –––––––––––––– Vessel element length Vessel

Roupala rhombifolia

Std 377 4497 3794 6252 2879 2531 2922 3088 3593 1534 1637 2066 2258 2074 1240 1339 2021 2064 1075 1676 1239

Vessel area Vessel 9966 Mean –––––––––––––– 8841 6351 5804 6836 8865 9043 4254 4793 4960 5860 5699 4929 1020 3727 3912 4595 3213 4009 3515 10980

m), vessel density (vessels /mm µ 7.3 8.6 9.2 6.4 9.2 7.6 7.3 5.4 6.4 1.9 2.5 2.2 2.9 2.5 12.7 10.2 17.5 10.5 12.7 12.7 19.1 Stem diameter

5 4 6 6 4 3 8 4 3 5.5 6.5 4.5 4.5 4.5 3.5 4.5 1.2 1.3 1.1 1.5 1.1 eight H

Site ), vessel element length ( 2 illtop forest illtop forest illtop forest illtop forest illtop forest illtop forest illtop forest illtop forest m Riparian forest Riparian forest Riparian forest Riparian forest Riparian forest Riparian forest Riparian forest Riparian forest Open field Open field Open field Open field Open field

h h h h h h h h able 2. Descriptive statistics of each wood specimen Specimen

Jono 16 Jono 17 Jono 18 Jono 19 Jono 20 Jono 21 Jono 22 Jono 23 Jono 2 Jono 29 Jono 3 Jono 30 Jono 31 Jono 32 Jono 33 Jono 5 Jono 24 Jono 25 Jono 26 Jono 27 Jono 28

T area ( Vessel (m) and diameter (cm) are given.

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Table 3. Summary of R2 values calculated in linear regressions between stem dimension and wood anatomy variables of Roupala rhombifolia. Results from a set of relevant studies from the literature are also summarised. The presence of (-) indicates a negative influence of stem size on the wood anatomical parameter, ns for non-significant and “–“ for not tested. VD = vessel diameter, VA = vessel area, VF = vessel frequency, VL = vessel element length, FL = fibre length, RW = ray width, and RH = ray height.

Species Wood anatomical features Stem size Provenance ––––––––––––––––––––––––––––––––––––––––––––––––––––––––– parameter References VD VA VF VL FL RW RH

Roupala rhombifolia Brazil Current paper Diameter – 0.18 ns (-) 0.26 0.25 0.28 0.15 Height – 0.44 ns (-) 0.26 0.31 0.18 0.46 111 tropical species Africa, South America Fichtler & Worbes (2012) Diameter 0.37 0.38 (-) 0.08 – – – – Height 0.32 0.32 (-) 0.08 _ _ _ _ Cornus controversa Japan Noshiro & Baas (2000) Diameter 0.19 – (-) 0.09 0.07 ns – – Height 0.23 _ ns 0.15 ns – – Cornus kousa Japan Noshito & Baas (2000) Diameter 0.16 – ns ns ns – – Height 0.29 – ns ns ns – – Cornus macrophylla Japan Noshito & Baas (2000) Diameter – – (-) 0.19 ns ns – – Height 0.26 – ns ns ns – – Alnus nepalensis Nepal Noshiro et al. (1994) Diameter ns ns ns ns ns – ns Height 0.26 0.6 (-) 0.12 ns ns – ns Rhododendron spp. Nepal Noshiro et al. (1995) Diameter 0.56 0.60 (-) 0.20 0.31 0.34 0.16 ns Height 0.56 0.55 (-) 0.19 0.40 0.48 0.19 ns 42 rainforest species Bolivia Poorter et al. (2010) Diameter – – – – – – – Height 0.24 – (-) 0.15 – – – –

Discussion

Roupala rhombifolia wood anatomy Roupala rhombifolia wood possesses features typical of Proteaceae, namely vessels that are solitary or in small tangential bands/clusters, festooned axial parenchyma bands associated with vessels and additional bands independent of them, larger rays, and ground tissue fibres with tracheids (Record & Hess 1943; Johnson & Briggs 1975). Mennega (1966), who compared Roupala with Euplassa, described R. rhombifolia as having many vessels (mean = 50 vessels/mm2), more than twice the number found in our study. However, she used only one specimen in her analysis. It is important to

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Table 4. Mean, standard deviation (Std) and comparative analyses (Kruskall-Wallis and multiple-comparison tests) of six wood anatomical features in Roupala rhombifolia from three habitats. The analyses were performed with raw data and residuals of linear regression between anatomical variables and plant size. Different letters in the multiple comparison test indicate significant differences between habitats. — χ2 = calculated chi-square value, DF = degrees of freedom, p = probability of p value, * significant at α = 0.05.

Raw date Residuals ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– habitat Mean Std Kruskal-Wallis Multiple Kruskal-Wallis Multiple comparison comparison

Vessel Open field 3849 525 χ2 14.7 B χ2 13.8 A-B area hilltop forest 4405 1534 DF 2 B DF 2 B (µm2) Riparian forest 8336 1824 p 0.0006* A p 0.001* A

Ray Open field 1357 231 χ2 10.3 B χ2 1.0 A height hilltop forest 2323 561 DF 2 A DF 2 A (µm) Riparian forest 2558 542 p 0.006* A p 0.6 A

Ray Open field 270 55 χ2 11.1 B χ2 3.0 A width hilltop forest 385 78 DF 2 A-B DF 2 A (µm) Riparian forest 426 89 p 0.004* A p 0.22 A

Fibre Open field 1405 119 χ2 15.6 B χ2 10.0 B length hilltop forest 1637 167 DF 2 B DF 2 B (µm) Riparian forest 1989 140 p 0.0004* A p 0.006* A

Vessel Open field 288 44 χ2 5.8 A χ2 2.8 A element hilltop forest 218 38 DF 2 A DF 2 A length Riparian forest 232 22 p 0.05 A p 0.24 A (µm)

Vessel Open field 16 1.5 χ2 8.1 A density hilltop forest 25 5.8 DF 2 B (mm2) Riparian forest 19 4.7 p 0.02* A-B

highlight that vasicentric tracheids were found in the wood of Roupala rhombifolia. To our knowledge, it is the first time that this feature is reported for this genus. Vasicentric tracheids are cavitation resistant elements that may serve as subsidiary conducting system when vessels are embolised (Carlquist 1985, 1989; Rosell et al. 2007). The upright ray cells seem to originate from fusiform initials in the vascular cambium, as suggested by the cell contacts with axial parenchyma, which resembles sheath cells (Wheeler et al. 1989). Besides, except for the occasional sheath cells, the heterogene- ous rays have smaller cells in the outer part (Fig. 4F), in contrast to some other genera of Proteaceae such as Cardwellia and Orites (Metcalfe & Chalk 1950).

The effects of wood maturity and plant size Plant growth has an important influence on wood anatomical characteristics. Age trends are a result of the cambial initial maturation, and juvenile wood exhibits a period of fast increase in axial cell length, followed by a stabilisation phase (Baas et al. 1986). The increase in axial cell length is less marked in hardwood species, while in softwood

Downloaded from Brill.com10/04/2021 11:42:55AM via free access Jono et al. – Tree size, environment, anatomy of Roupala 101 species like Pinus longaeva (Pinaceae) tracheid length tends to show a rapid initial increase followed by a slower increase that does not stabilise even after 2000 years of age (Baas et al. 1986; Lachenbruch et al. 2011). Hardwood species like Dodonaea viscosa (Sapindaceae) and Cornus controversa (Cornaceae) show a more moderate

Figure 7. Wood sections of specimens from the open field (A, B), hilltop forest (C, D) and riparian forest (E, F). — Scale bars: 500 µm (transverse sections) and 800 µm (tangential sections).

Downloaded from Brill.com10/04/2021 11:42:55AM via free access 102 IAWA Journal 34-1 (2013) increase in vessel element and fibre length, with an overall stabilisation trend closer to the pith (Noshiro & Baas 2000; Liu & Noshiro 2003). Wood of Cornus controversa was considered mature after the 10th growth ring by Noshiro and Baas (2000), while the wood of Dodonea viscosa was considered mature just 10 mm away from the pith, for both fibre and vessel elements (Liu & Noshiro 2003). Although the wood of Roupala rhombifolia was considered mature in specimens with a diameter equal to or greater than 20 mm by using vessel element and fibre length as indicators, it was not clear if samples from specimens of that size could be used as mature wood, since correlations between wood anatomical features and plant size were still found within the sampled specimens. There are also other wood anatomical features that are related to wood maturity, such as ray size. Rays tend to increase in size with increasing distance from pith and young leaves (Lev-Yadun & Aloni 1995). The extent influence of stem size parameters on wood anatomical features ofR. rhombifolia goes up to 46% of anatomical data variability. Even higher values of stem size influence were reported in the literature. Noshiro et al. (1995) showed that 56% of vessel diameter data variability of Rhododendron spp. was explained by stem diameter and height while 60% of vessel area was explained by stem diameter. Other researches like Noshiro et al. (1994), Noshiro & Baas (2000), Poorter et al. (2010) and Fichtler & Worbes (2012) also reported in their results correlations between wood anatomical features and stem size. It is important to realise that these results were found in different species or groups of species at distinct provenances (all cited in Table 3). This fact indicates that it is probably a common trend and stem size information should always be recorded. From all anatomical parameters analysed by these researches, stem size seems to have the highest influence on vessel area and vessel diameter, the most commonly measured anatomical parameters in ecological wood anatomy analyses. Probably, it reflects the fact that functional advantages of these vessel attributes are well established (Wheeler et al. 2007), and they show a trade-off between safety and efficiency in water transport (Carlquist 1977). The residual analysis used in the present research was intended to remove all size information from the wood anatomy variables of Roupala rhombifolia. Relevant variables for determining wood maturity, such as vessel element length, fibre length and ray size, were not correlated with stem size after residual extraction. These residuals data can thus be considered to reflect patterns from similarly-sized specimens in the three studied habitats.

Ecological anatomy Raw data analysis showed the expected results, considering previous literature. The higher moisture availability in the riparian forest was associated with mesic wood anatomical characteristics, in contrast to those found in plants from the two other habitats. As seen in Roupala rhombifolia, wider vessels, lower vessel frequency, and higher fibre length were found in 11 species from a gallery forest when compared to the same species from a nearby cerrado area (Brazilian savanna) studied by Sonsin et al. (2012). Preston et al. (2006) also found that vessel area was positively correlated with

Downloaded from Brill.com10/04/2021 11:42:55AM via free access Jono et al. – Tree size, environment, anatomy of Roupala 103 soil water content in 51 angiosperm species from the California coast. It appears that soil moisture availability plays an important role in wood anatomical features, especially vessel area. When the results of the Kruskal-Wallis test of vessel area residuals are compared with the soil analysis, it appears that soil features alone do not explain the results, although they do explain the results of the raw data analysis. Open field and hilltop forest soils are similar to each other and both different from riparian forest, corroborating other studies such as Barij et al. (2007), showing that moisture in montane soils are higher at lower elevations than at higher elevations, as it was the case for the riparian forest and hilltop forest we studied, which differed in altitude but were located on the same side of the hill. The NDVI data also failed to explain the vessel area residual results, once the NDVI from hilltop forest and riparian forest are similar to each other and higher than the open field. This means that both forests have a higher plant density than the open field.Even though open field is submitted to higher evaporative load, one of the most relevant environmental stresses (Thomas 1997), R. rhombifolia seems not to perceive this environmental difference. However, analysing soil and NDVI data together suggests a new hypothesis. If soil moisture availability in open fields and hilltop forest are similar but the open fields have a lower plant density, it is possible that soil moisture available for plants in the open fields is higher due to lower plant demand or competition. Conse- quently, this relative water abundance might be recorded in wood as higher values of vessel area in similarity to those found in the wood of riparian forest individuals. The proposed hypothesis may also be supported by the fact that individuals from hilltop forest showed the highest raw vessel density values, and that this wood variable was not correlated with stem size. Usually, higher vessel density as well as narrow vessels are found in more xeric environments (Wheeler et al. 2007). Another aspect to take into account is that root competition could reduce the availability of soil resources, such as nutrients, space and water (Casper & Jackson 1997; Schenk 2006). This competition is so important in plant communities that in 70% of the studies evaluated in a meta-analysis (Wilson 1988) root competition had a stronger impact than shoot competition. Likewise, there is strong evidence that the intensity of belowground competition increases with depletion of resources (Schenk 2006). This might be one reason for the differences in the residuals of vessel area found in Roupala rhombifolia specimens from open fields and hilltop forest, once cluster and NDVI analyses showed that both areas have similar soil properties with low moisture availability and different plant densities.

Conclusion

Despite the proximity of the habitats we studied, it was possible to identify differences associated with each locality (open fields, hilltop forest and riparian forest) via quantita- tive analyses of soil data, plant cover/biomass and raw wood anatomy data of Roupala rhombifolia. When variation due to plant size was removed from the anatomical data, only vessel area and fibre length were found to differ between habitats, in concert with

Downloaded from Brill.com10/04/2021 11:42:55AM via free access 104 IAWA Journal 34-1 (2013) soil data and plant cover/biomass. These dissimilarities may reflect differences in water availability between habitats, as determined by soil attributes and possibly intensified by root competition. Finally, from an ecological wood anatomy point of view, we encourage to disentangle the effect of stem size on wood anatomical data in future studies. By doing so, not only new insights arise but also this approach may help resolving common problems in field sampling designs like the lack of homogeneity in sample size and wood maturity in natural ecosystems.

Acknowledgements

The authors thank Marisa Dantas Bitencourt for providing satellite image facilities and instructions; Gisele Costa and Paulo Cesar Fernandes for laboratory support; Pieter Baas, Guillermo Angeles, Marcelo Pace, Vitor Barão, José Hernandez, Luiza Costa and Nara Oliveira for valuable input on the manuscript; Flávio Roscor for allowing access to the study site; the Brazilian Institute of Environ- ment and Natural Resources (IBAMA) for providing facilities; and the National Council for Scientific and Technological Development (CNPq – 131418/2006-8) and the São Paulo Research Foundation (FAPESP – 03/10277-8) for financial support.

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Accepted: 10 January 2013

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