Xylem Structure of Successive Rings in the Stem of Abuta Grandifolia (Menispermaceae) a Statistical Approach

Home , Abuta, Menispermaceae, Tracheid, Vessel element

IAWA Journal, Vol. 31 (3), 2010: 309–316

XYLEM STRUCTURE OF SUCCESSIVE RINGS IN THE STEM OF ABUTA GRANDIFOLIA (MENISPERMACEAE) A STATISTICAL APPROACH

Neusa Tamaio* and Arno Fritz das Neves Brandes Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Diretoria de Pesquisa Científica, Rua Pacheco Leão 915, CEP 22460-030, Rio de Janeiro, RJ, Brazil *Corresponding author [[email protected]]

SUMMARY Abuta grandifolia (Mart.) Sandwith varies in habit and can be a shrub, tree or liana. Its stem shows successive cambia that produce secondary phloem and xylem. This study analyses qualitative and quantitative differences between the secondary xylem of lianas and shrubs of A. grandifolia, as well as between the successive rings within each habit category. The lianas had wider vessels, shorter vessel elements and thinner fibre-tracheid walls. In the first five successive rings, lianas showed an increase in vessel diameter and fibre-tracheid length, while fibre-tracheid wall thickness decreased. The shrubs showed an increase in the diameter and length of vessel elements and length of fiber-tracheids. In qualitative features the secondary xylem of lianas and shrubs did not differ, except in their type of parenchyma (axial parenchyma diffuse in the liana habit and diffuse to diffuse in-aggregates in the shrub habit). Since significant quantitative differences were found between the successive rings, the first vascular ring is recommended for comparing both habits. Whether this is generally valid in Menispermaceae requires further study. Key words: Abuta, liana, Menispermaceae, shrub, successive cambia.

INTRODUCTION

Abuta grandifolia (Mart.) Sandwith is a species that may occur as a tree, shrub or liana (Mennega 1982). As in most of the Menispermaceae, the cambial variants of the successive cambia type are preset in the stem. This arrangement consists of axial vascular segments flanked by wide vascular rays separated by residual conjunctive tissue (Jacques & de Franceschi 2007; Tamaio et al. 2009). This species is widely distributed in South America and has particular economic importance in Brazil for its use in phytotherapy and food dyes (e.g., Revilla 2001). A quantitative analysis of the secondary xylem of Abuta grandifolia, including the diameter and length of the vessel elements and length of fiber-tracheids, was carried out by Mennega (1982) on the tree, shrub and liana habit. She only carried out a descriptive statistical analysis without indicating which successive vascular ring was used in that evaluation. Nonetheless, she noted that the lianas had smaller vessel diameters and shorter fiber-tracheids.

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To compare the successive rings in the Menispermaceae family, the only quantitative analysis of the secondary xylem carried out to date has been that of Rajput and Rao (2003) with the shrub Cocculus hirsutus. In that study, the authors analyzed diameter and length of the vessel elements, frequency of vessels and length of fiber-tracheids in six successive rings. They showed decreasing vessel diameters, but increasing lengths of vessel elements and fiber-tracheids towards the most external ring. Based on the work of Rajput and Rao (2003), the following questions guided this study: 1) Are there qualitative and quantitative differences between the secondary xylem of lianas and shrubs of A. grandifolia? 2) Are there significant quantitative differences between the several successive rings? 3) Which successive ring should be used in anatomical comparisons of lianas and shrubs in Menispermaceae? To address these questions five successive rings in stems of both shrubs and lianas of of Abuta grandifolia were studied.

MATERIALS AND METHODS Plant materials Six samples of Abuta grandifolia (Mart.) Sandwith (Menispermaceae) were analyzed, three representing the liana habit, and three the shrub habit. The liana samples were obtained from the RB (RB 203548, Martinelli, G. 6924), INPA (INPA 45649, Prance 21712) and Uw (Uw 22771, Lindeman & Heyde 139). The shrub samples were from the INPA (INPA 68856, M. Silva 2136) and Uw (Uw 3341, Lindeman & Heyde 193; Uw 18757, Krukoff 10954 ). The samples from RB and INPA were identified by R. Barneby.

Microscopic features Small wood blocks (2 cm3) were cut and softened in boiling water. Transverse sections approximately 18 µm thick were prepared with a sliding microtome. The sections were cleared with sodium hypochlorite (60%), washed in water, stained with safranin and astrablue (Bukatsch 1972), dehydrated (Johansen 1940; Sass 1958) and mounted in synthetic resin. Macerations were prepared using the Franklin method (Jane 1956), stained with safranin and astrablue and mounted in a 50/50 mixture of glycerin and water (Strasburger 1924). The images were captured with the Image Pro Plus software, version 3.0 for Windows, linked to the microscope through a Media Cybernetics CoolSNAP-Pro video camera. This method was also used to perform the measuring and counting. The terminology used in the descriptions, as well as counting and measuring procedures, follow the recommendations of the IAWA list (IAWA Committee 1989). Tangential vessel diameter, vessel element length, and the length and wall thickness of fiber-tracheids were measured.

Statistical analyses Descriptive statistical analyses were performed to provide quantitative data for the characterization and comparison of the habits and successive xylem rings. Statisti- cal inference analyses was also conducted, using sample distribution analysis and Kolmogorov-Smirnov normality tests. To analyze the diameter of vessels, a sample dis-

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tribution analysis was performed, and 100 vessels of each individual were quantified, while in the other variants, the quantitative data are based on 25 counts by ring. Variance analysis was conducted (one-way ANOVA) to test for significant differences in the averages of the quantified characteristics in the comparison of the successive vascular rings. A t-test was conducted to compare a given habit with each one of the quantified variants. As such, the analyses were carried out with the measurements of all successive rings, as well as the first ring alone. All statistical analyses were carried out using the Statistic 7.0 program for Windows.

RESULTS

General description of the wood The wood of all samples shares the following features: cambial variant of the successive cambia type, indis- tinct growth ring boundaries (NB: the successive rings do not represent annual growth rings), diffuse porosity, exclusively solitary vessels, simple perforation plates; presence of vascular tracheids, fibers with simple to minutely bordered pits and fibers with distinctly bordered pits (fiber- tracheids); axial parenchyma diffuse in the liana habit, diffuse to diffuse- in-aggregates in the shrub habit, large multiseriate rays (ray height >1 mm), composed of procumbent, square and upright cells, with prismatic crystals in ray cells (Fig. 1 & 2).

Figures 1 & 2. Transverse section of Abuta grandifolia. – 1: Shrub. – 2: Liana. — Scale 1 2 bar = 100 µm.

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Xylem structure of successive rings The lianas of Abuta grandifolia show dimorphism in vessel diameter, while the shrubs do not. By the sample distribution analysis, it was observed that the diameter of vessels in the shrubs of Abuta grandifolia presented a normal distribution, while a bimodal distribution was present in the lianas. Based on these findings, two categories were established to classify the diameter of vessels in the lianas: <100 µm (small vessels) and >=100 µm (large vessels). The vessels in shrubs did not exceed 100 µm in diameter and are comparable to the small vessels of the lianas (<100 µm). Significant differences (p<0.01) were observed between the successive rings of the xylem in the lianas as well as in the shrubs. Using variance analysis, it was observed that the length of fiber-tracheids and the diameter of the vessels in the shrubs increased from the second ring, while vessel element length increased from the first ring. The wall-thickness of fiber-tracheids did not show significant differences (Table 1).

Table 1. Means and standard deviations of the quantitative anatomical features for each ring and for general descriptions of each habit. FL: fiber-tracheid length; FTW: fiber-tracheid wall thickness; VL: vessel element length; VD: vessel diameter (<100 µm and >=100 µm). — Mean; SD: standard deviation; p value for t-test between lianas and shrubs; *: p<0.01, ns: non-significant. Columns: General: all data from five successive rings; 1–5: data of each ring; p value forANOVA between five successive rings; *: p<0.01, ns: non-significant.

General 1 2 3 4 5 p FL Liana Mean 880 768 781 883 1040 982 0.000 * SD 295 261 285 278 302 258 Shrub Mean 1109 987 951 1068 1252 1286 0.000 * SD 326 298 321 297 275 298 p 0.000* 0.000* FTW Liana Mean 5 5 5 5 5 4 0.002 * SD 1.3 1.2 1.5 1.3 1.4 1.2 Shrub Mean 7 7 7 7 7 7 0.392 ns SD 1.6 1.9 1.6 1.3 1.5 1.7 p 0.000* 0.000* VL Liana Mean 352 347 331 352 374 359 0.343 ns SD 104 78 123 112 119 91 Shrub Mean 359 320 363 365 360 388 0.001 * SD 96 79 83 78 101 122 p 0.027 ns 0.036 ns VD Liana Mean 64 64 63 68 63 61 0.653 ns (<100 mm) SD 23 22 22 23 25 24 Shrub Mean 56 52 53 56 63 56 0.005 * SD 19 16 16 19 18 23 p 0.000* 0.000* VD Liana Mean 140 136 132 141 147 148 0.008 * (>=100 mm) SD 28 27 25 25 27 32

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In the lianas, the diameter of the large vessels (>=100 µm) tends to increase from the second xylem ring. The length of fiber-tracheids tends to increase from the second ring, and the wall thickness of fiber-tracheids shows a decrease after the second ring. The diameter of the smaller vessels (<100 µm) and length of vessel elements did not show significant differences among the successive rings (Table 1). Therefore, in the shrubs, as well as in the lianas of Abuta grandifolia, we observed an increasing diameter of the vessel elements and increasing length of the fiber-tracheids in the successive rings. There are quantitative differences in the secondary xylem of Abuta grandifolia between the liana and shrub habits. By the t-test, significant differences (p<0.01) were observed in the following variants: diameter of the smaller vessels, fiber-tracheid length and fiber-tracheid wall thickness. The lianas had small vessels (<100µ m) with a diameter larger than the shrubs and shorter fiber-tracheids with thinner walls (Table 1). When the analysis was limited to the first ring only, the same differences between lianas and shrubs were noted (Table 1).

DISCUSSION The secondary xylem of lianas and shrubs of Abuta grandifolia The general anatomy of the xylem of the lianas and shrubs of Abuta grandifolia showed no differences, except the type of axial parenchyma, which is diffuse in the liana habit and diffuse to diffuse in-aggregates in the shrub habit. A similar pattern was also reported by Mennega (1982) when comparing shrubs, trees and lianas of A. grandifolia. In the present study, lianas with dimorphism in vessel diameter (<100 µm and >100 µm) were observed. This phenomenon can be demonstrated by sample distribution analysis (e.g., Carlquist 1985). Our results showed that the diameter of the narrow vessels (<100 µm) is significantly smaller in the shrub habit. A similar pattern was also reported by Mennega (1982). This trend is in agreement with earlier studies showing that the lianas have wider vessel elements than trees or shrubs (e.g., ter Welle 1985; Iqbal 1994; Gasson & Dobbins 1991), usually in addition to more or less distinct popula- tions of narrow vessels (Baas et al. 1983, Baas & Schweingruber 1987). While reports focusing on the same species in different habits are rare, Vieira (1994) did study the wood anatomy in the liana and shrub habits of Bauhinia radiata Vell. and also found wider vessels in the climbing form. Our results on vessel element lengths pointed to the similarities in both habits, in agreement with Mennega (1982). Finally, we analyzed the length and wall thickness of fiber-tracheids and found shorter cells in the lianas, again supporting Mennega’s data. We also found thinner fiber-tracheid walls in lianas as compared to shrubs, although this parameter was not analyzed by Mennega.

Successive rings of lianas and shrubs of Abuta grandifolia Rajput and Rao (2003) observed a decrease in the diameter of vessels from the center towards the periphery for the lianas and shrubs. In this case, the authors only carried out a descriptive statistical analysis, but it would still have been possible to determine the different averages between the first (193 ± 10.53 µm) and the last (138 ± 9.24 µm) sampled ring. Therefore, to properly analyze cellular quantitative data, we

Downloaded from Brill.com09/23/2021 03:37:45PM via free access 314 IAWA Journal, Vol. 31 (3), 2010 need a method that measures the statistical differences between the xylem rings. To accomplish this, we carried out a variance analysis among the first five successive rings for all quantified characteristics inA . grandifolia. If the statistical results indicated the absence of significant differences between the rings, then any vascular ring could be used for the comparative xylem studies. On the other hand, the presence of significant differences would indicate the need for choice (standardization) of the vascular ring. Our data showed significant differences (p< 0.01) between the successive rings of the xylem in the lianas as well as in the shrubs. With the variance analysis, it was observed that the length of fiber-tracheids, in addition to the length and diameter of the vessels (<100 mm), increased in the shrubs. The other variables did not show significant differences (Table 1). The lianas showed an increase in the diameter of vessels (>=100 mm) and length of fiber-tracheids, but a decrease of wall thickness of fiber-tracheids. Another study that focused on quantitative analyses in successive rings was developed by Horak (1981) in Phytolaccaceae, but, again, only descriptive statistical analysis was applied. He describes a decrease in the length of the vessel elements in each of the seven successive rings of Stegnosperma, contrary to both our results and those of Rajput and Rao (2003). Our results demonstrate the necessity of establishing a standard by which to mea- sure the statistical differences between the vascular rings in wood anatomy studies for the Menispermaceae. The first ring is formed by the products of a single cambium originating from the procambium and pericycle (e.g., Tamaio et al. 2009), in addition to primary xylem and phloem. Successive rings are formed by a lateral meristem that produces conjunctive tissue, which, in turn, will form new cambia (Carlquist 1996; Tamaio et al. 2009). In the comparative analysis between lianas and shrubs, the first ring showed the same differences observed when we compared all rings, indicating that the first ring alone clearly demonstrates the differences between habits. For this reason and because the first rings are homologous in both habits, we recommend that the first ring be used. Standardization through the first ring may contribute to future studies of Menisper- maceae anatomy because materials deposited in herbaria can be used; however, a study with a higher number of taxa must be performed to test whether this standardization could be extended to other Menispermaceae. It should be pointed out that, when a given species has successive cambia, it is recommended that stems with at least two developed vascular rings be used, as indicated in the work of Tamaio et al. (2009) who confirmed that upon formation of the second ring, the differentiation of the first ring is already complete. Studies that examine size variation in vascular elements most frequently involve plants with a tree habit. Two types of analyses are normally investigated: axial and radial variation (Fan et al. 2009). Regarding the radial direction, the studies show that, in general, the size of the cell increases and the frequency decreases centrifugally, later stabilizing upon the formation of the adult wood (e.g., Giraud 1977; Bosman et al. 1994; Bosman 1996; Tsuchiya & Furukawa 2009). The results of the present study show that both lianas and shrubs of Abuta grandifolia have the same pattern of cell size increase as trees.

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CONCLUSION The anatomical differences of the stem of A. grandifolia are mainly quantitative, with the vessel diameter being notably higher in lianas. There are significant quantitative differences between the two habits as observed upon analysis of the five successive rings. This phenomenon is particularly highlighted by the increased diameter of the vessel elements towards pith-periphery orientation. Nonetheless, further studies involv- ing a higher number of taxa are needed to test whether the standardized “first-ring” method as proposed and described in this paper could be applied to the entire family of the Menispermaceae.

ACKNOWLEDGEMENTS

We thank the staff of the Laboratório de Botânica Estrutural, Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, for technical support, Dr. João Marcelo Alvarenga Braga for taxonomic identification and providing information about the species, Ms. Monique Figueiredo Neves for her help with the Abuta grandifolia measurements, Dra. Veronica Angyalossy and Dr. Pieter Baas for their valuable comments, and Programa Mata Atlântica (Petrobras) for funding.

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