J. Plant Bio. Res. 2016, 5(2):37-47
Journal of Plant Biology Research 2016 5(2): 37-47
eISSN:2233-0275 pISSN:2233-1980 http://www.inast.org/jpbr.html
REGULAR ARTICLE
Leaf anatomy of four species and one variety of Datura (Solanoidea, Solanaceae) Olga Lidia Gómez-Nucamendi, Luis Hernández-Sandoval, Mónica Figueroa-Cabañas and Mahinda Martínez* Facultad de Ciencias Naturales. Universidad Autónoma de Querétaro. Avenida de las Ciencias S/N Juriquilla, C.P. 76230. Querétaro, Mexico.
ABSTRACT Foliar anatomy of five taxa of the genus Datura is described to identify the anatomic characters that can help distinguish them. Three to five individual leaf samples per taxon were collected and processed using conventional techniques. Foliar characters were measured and analyzed by Kruskal-Wallis and posthoc Dunn tests. To define rules of classification, regression and classification trees (CART) were created. Studied taxa share anatomical characters, such as bifacial leaves with unstratified epidermis, amphistomatic leaves with anisocytic stomata, and the presence of simple and glandular trichomes. Epidermal cells have slightly wavy walls. Mesophyll is composed by an unstratified compact palisade parenchyma, except for D. ceratocaula, that had two strata. Spongy parenchyma was compact, with three to four cell layers and with abundant calcium oxalate druses. Anatomical characters were significantly different between species, except for the length of the glandular trichome stem of the adaxial surface. Anatomical characters of the abaxial surface had the highest discriminating power. Characters with the best discriminating efficiency were the number of simple trichomes. Other relevant differences were the distribution and ornamentation of glandular and simple trichomes. The sum of anatomical differences allows the recognition the Datura taxa studied. Keywords: Anatomy leaf, Datura, Querétaro, Mexico.
INTRODUCTION The genus Datura L. (Solanaceae) is endemic to Mexico and adjacent areas of southwestern United States and the northern region of Central America [1]. It has become naturalized in Asia, Africa and Australia [2, 3]. The plants grow mainly in dry, mild and subtropical regions [4]. The number of recognized species varies from 12 to 14 [5, 6]. Recently, Bye and Sosa [7] described D. arenicola Gentry ex Bye & Luna Baja California. Twelve species have been recorded for Mexico, Datura ceratocaula Jacq., D. innoxia Mill., D. lanosa A.S.Barclay ex Bye, D. metel L., D. wrightii Regel, D. stramonium L. with two varieties stramonium L. and tatula (L.) Torr., D. quercifolia Kunth, D. kymatocarpa A.S. Barclay, D. discolor Bernh., D. author: Mahinda Martínez E-mail:
[email protected] *Corresponding
pruinosa Greenm., D. reburra A.S.Barclay and D. arenicola. Datura stramonium var. tatula has been classified as a different species but genetic evidence suggests it is a variety of D. stramonium [8, 9]. Species within the genus are used in traditional medicine and have economic importance due to their content of amino acids, tannins, carbohydrates, phytic acid, clorogenic acids, lectins, albumins, as well as more than 70 alkaloids of pharmaceutical importance [10, 11]. Research has focused a few Datura species, mainly on genetic [12, 13] and biochemical aspects [14]. Anatomical studies of Datura have focused on reproductive structures [15] and roots [16]. However, there is limited information on foliar
J. Plant Bio. Res. 2016, 5(2):37-47
anatomy. Timmerman [17] described D. stramonium, D. tatula, D. metel, D. innoxia and D. fastuosa, and pointed out that leaf indumentum can help to discriminate species. Not with standing, for D. stramonium and D. tatula, he only reported differences in terms of trichomes size [15]. Thus, we aimed to describe and compare the leaves of five Datura taxa: D. ceratocaula, D. innoxia, D. stramonium var. stramonium, D. stramonium var. tatula and D. quercifolia, which share a similar geographic distribution.
MATERIALS & METHODS Description order for the five taxa follows the phylogeny of Datura proposed by Bye and Sosa [7] who propose relationship as follow: Datura ceratocaula, D. innoxia, D. stramonium var. stramonium, D. stramonium var. tatula and D. quercifolia. Seeds of the five Datura taxa were collected from four sites in Santiago de Queretaro, Mexico. Sampling sites were located between 20º 31’ 38.91’’ - 20º 49’ 40.29’’ N and 100º 18’42.20’’ - 100º 30’23.26’’ W. Site elevation varied from 1,814 to 2,156 m. Vegetation type, soil, and conservation status were similar among sites, namely tropical deciduous forest with pellic vertisol [18], and in ruderal areas. All species were terrestrial except for the semiaquatic D. ceratocaula. Plants were grown in greenhouse conditions and three to five leaf samples were sampled per taxon. Material was fixed overnight in Navashin's fixative, dehydrated in ethanol, and embedded in paraffin. Transverse sections of 12 µm in width were obtained with a rotary microtome (Leica, RM2125), stained with alcian blue and 50% safranin, and mounted with Permount (Fisher Chemicals). Structures were described following microscopic examination (Olympus, BX43) and scanning electron microscopy (SEM, Carl Zeiss EVO 50). Tissue microphotographs were captured with QCapture Pro 7 software and optimized in Photoshop (version CC2015) without altering structure information. Structure nomenclature followed Font Quer [19]. Anatomical sections description was from the outer to the inner portions of the organs. Density, size, distribution and ornamentation of 14 variables (see tables 1, 2 and 3) related to cell size, stomata, and trichomes in both leaf surfaces were assessed under SEM using ImageJ software (Image
Processing and Analysis in Java). Stomata number and glandular trichomes density on the abaxial leaf surface of D. ceratocaula was inferred using the blade margin where they exhibited a glabrate surface, as the density of simple trichomes in the central portion of the leaf hindered direct observation. Differences among Datura taxa for each variable were tested by Kruskal-Wallis. Variables with significant differences were further tested with a Dunn test with a Holm correction for multiple comparisons [20, 21], the level of significance was set at P< 0.05. Furthermore, an algorithm to build classification and regression trees (CART) was used on data of each leaf surface in order to identify the variables that best separate the taxa[22]. To build the classification trees we used the rpart package [23] in R [24].
RESULTS Foliar structures of the five Datura taxa shared many anatomical characters. They presented bifacial leaves with unstratified epidermis (Figure 1A), no wax, and amphistomatic leaves with anisocytic stomata. Epidermal cells showed evidence of slightly wavy walls, of variable size among taxa (Table 1). Mesophyll was formed by a compact and unstratified palisade parenchyma, except for D. ceratocaula that had two distinct lines of palisade cells (Figure 1B). Spongy parenchyma had three to four cell layers, with abundant calcium oxalate druses in all taxa (Figures 1A and 1B), either as single prism-shaped crystals or grouped crystals (Figure 1C). Both surfaces (adaxial and abaxial) of all taxa showed two types of indumentum: simple trichomes (Figure 1D) and glandular trichomes (Figure 1E). Some of the cells that form the trichomes exhibited prism-shaped crystals. Simple trichomes were uniseriate, filiform and multicellular (from one to seven cells; Figure 1F). They were distributed throughout the lamina, but were most abundant along the nerves, with the highest density over the primary vein and, less abundantly, over the secondary veins, except for the abaxial surface of D. ceratocaula, in which the main vein showed few simple trichomes. Glandular trichomes were multicellular with a secretory head, formed by six to seven cells, with two to three on the base and four on the gland (Figures 2a and 2B). In addition, D. ceratocaula exhibited trichomes
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Table 1. Mean ± standard deviation epidermal cells and stomatic characters (density and size) at the adaxial and abaxial surface of the leaves of five Datura species. D. stramonium var.
D. stramonium var.
stramonium
tatula
47.9 ±10. 7a
47.5 ± 9.0a
42.7 ± 8.8ab
37.7 ± 9.3b
29.3 ± 4.8 a
22.2 ± 3.9 b
22.5 ± 3.8 bc
25.6 ± 3.6 ac
19.4 ±5.0 b
Length μm
45.3 ± 8.1 ab
54.8 ± 11.1 c
52.7 ± 9.0 ac
37.0 ± 11.2 b
53.9 ± 8.0 c
Width μm
26.5 ± 7.7 ab
22.0 ±5.3 ac
24.7 ±5.9ab
18.5 ± 5.7c
31.0 ±8.5 b
Length μm
25.9 ± 9.2 ab
23.5 ± 2.2 a
29.0 ± 3.2c
26.6 ± 2.8 bc
23.8 ± 2.5a
Width μm
17.8 ± 8.5a
15.9 ± 2.1a
22.6 ± 2.1b
19.9 ± 2.2bc
18.4 ± 2.7ac
Pore length μm
12.6 ± 1.8 ab
10.9 ± 1.7ac
18.9 ± 3.1d
12.8 ± 1.9b
11.1 ± 6.8c
Number/mm2
89.0 ± 8.0a
153.0± 26.0b
122.0 ± 21.0b
35.0 ± 7.0c
94.0 ± 21.0a
Length μm
24.9 ± 3.6 a
32.3 ± 3.9 b
31.3 ± 4.1 b
24.6 ± 2.8 a
24.7 ± 2.8 a
Width μm
16.4 ± 2.8 a
24.4 ± 2.2 b
21.1 ± 2.4 c
17.1 ± 2.8 a
20.8 ± 2.7 c
Pore length μm
13.2 ± 3.5a
13.5 ± 2.2a
13.1 ± 2.6a
12.5 ± 3.4b
11.7 ± 2.8ab
Number/mm2
40.0 ± 6.0 a
58.0 ± 03.0 b
109.0 ± 10.0 c
57.1 ± 25.2 ab
139.0 ± 14.0 c
Adaxial Abaxial Abaxial
Stomata
Adaxial
Epidermal cells
Taxa/Structure
D. ceratocaula
D. innoxia
Length μm
46.3 ± 10.4 a
Width μm
D. quercifolia
*Anatomical characters that share the same letter (a, b or c) were not significantly different after Kruskal-Wallis and Dunn tests with Holm correction for multiple comparisons
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Table 2. Mean ± standard deviation of simple trichomes of the adaxial and abaxial surface of the leaves of five Datura species. D. ceratocaula
D. innoxia
D. stramonium var. stramonium
D. stramonium var. tatula
D. quercifolia
Trichome < 3 cells
233.0 ± 63.0 a
0.0 + 0.0b
0.0 + 0.0b
368.0 ± 159.0 ac
545.0 ± 241.0 c
Trichome > 2 cells
138.0 ± 30.0 ab
142.0 ± 45.0ab
0.0 + 0.0c
108.0 ± 33.0 a
146.0 ± 49.0b
11.0 ± 2.0 a
31.0 ± 9.0 b
0.10 ± 0.30c
9.0 ± 2.0 a
10.0 ± 1.0 a
Scarce in lamina,
Moderate in lamina,
Scarce in lamina and
Scarce in lamina and
Moderate in lamina
moderate in nerve
abundant in nerve
nerve
nerve
and nerve
Trichome < 3 cells
237.17 ± 51.4 a
NA
291.3 ± 100.8 ac
418.5 ± 183.2c
225.0 ± 129.6 a
Trichome > 2 cells
127.5 ± 64.7a
141.8 ± 28.6 a
NA
137.9 ± 72.4 a
71.3 ± 12.5 c
Number/mm2
131.6 ± 20.8 a
28.6 ± 2.6 a
0.2 ± 0.3 b
1.0 ± 3.5 b
4.9 ± 2.6c
Abundant in lamina
Moderate in lamina
Scarce in lamina and
Scarce in lamina and
Moderate in lamina
and nerve
and nerve
nerve
nerve
and nerve
Pustulate
Pustulate
Pustulate
Pustulate
Tuberculate
Adaxial
Structure
Number/mm2
Abaxial
Ssimplestrichome
Distribution
Estomas Both
Distribution
Ornamentation
*Anatomical characters that share the same letter (a, b or c) were not significantly different after Kruskal-Wallis and Dunn tests with Holm correction for multiple comparisons. 0.0 = the structures were absent of sampled.
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Both
D. quercifolia
Total length
76.1 ± 11.8 a
66.5 ± 8.9 b
69.9 ± 11.6 ab
64.5 ± 6.3 b
72.5 ± 13.3 ab
Stem length
31.7 ± 9.2 a
27.8 ± 6.7 a
30.7 ± 7.2 a
28.2 ± 4.0a
29.8 ± 8.6a
Gland length
44.4 ± 4.4 a
38.6 ± 4.4 bc
39.2 ± 5.8 bc
36.3 ± 5.8 b
42.7 ± 9.8 ac
Gland width
41.2 ± 3.9 a
33.4 ± 4.7 b
36.1 ± 5.8 b
33.7 ± 6.0 b
35.5 ± 4.9b
Number/mm2
3.0 ± 2.0 a
9.0 ± 2.0 b
2.0 ± 2.0 a
6.0 ± 2.0 c
6.0 ± 2.0 c
Localization
More abundant in nerve
More abundant in nerve
In nerve, scarce in lamina
In nerve, scarce in lamina
More abundant in nerve
Total length
89.7 ± 8.0 a
89.3 ± 7.7 a
68.7 ± 9.8 b
56.9 + 3.7 c
55.3 ± 5.7 c
Stem length
43.6 ± 4.2 a
43.4 ± 3.3 a
33.7 ± 5.5b
25.3 + 6.1 c
22.3 ± 5.8 c
Gland length
46.1 ± 6.1 a
45.9 ± 6.4a
35.0 ± 7.1 b
33.5 + 6.7 b
33.0 ± 2.4 b
Gland width
35.9 ± 2.4 a
40.8 ± 5.9 a
32.3 ± 5.3 b
32.0 + 6.0 b
31.0 ± 3.6 b
Number/mm2
3.1 ± 2.0 ab
2.0 ± 1.1 ac
0.7 ± 1.0 b
5.9 + 3.0 c
9.4 ± 2.4 c
Localization
Not observed
More abundant in nerve
In nerve, scarce in lamina
In nerve, scarce in lamina
In lamina and nerve
Smooth
Smooth
Smooth
Smooth
Rugulate
Abaxial
Abaxial
Glandular trichomes
Adaxial
Table 3. Mean ± standard deviation of glandular trichomes of the adaxial and abaxial surface of the leaves of five Datura species. D. stramonium var. D. stramonium var. Structure D. ceratocaula D. innoxia stramonium tatula
Ornamentation
*Anatomical characters that share the same letter (a, b or c) were not significantly different after Kruskal-Wallis and Dunn tests with Holm correction for multiple comparisons.
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Figure 1. Transverse leaf section of: A. D. quercifolia, B. D. ceratocaula, C. D. innoxia, with grouped crystals of calcium oxalate, D. Simple trichome in the epidermis of D. stramonium var. tatula. E. Glandular trichome in the epidermis of D. innoxia, F. Trichomes of D. quercifolia. ade: adaxial epidermis, abe: abaxial epidermis, co: calcium oxalate druse, cw: cell wall, ep: epidermis, gt: glandular trichome, nu: nucleus, pc: prism-shaped crystals, pp: palisade parenchyma, sp: spongy parenchyma, st: simple trichome, vb: vascular bundle.
with nine cells (Figure 2C). Glandular trichomes were distributed similarly to the simple trichomes, mainly along the length of the nerves. Their observation was difficult in the abaxial surface of D.ceratocaula, as the lamina is practically covered with simple trichomes, but could clearly be observed on the main vein, which had few simple
trichomes. The 14 variables that were analyzed on both leaf surfaces revealed significant differences among the taxa here studied, except for the length of glandular trichome stem in the adaxial surface. Observed differences were in terms of the epidermal cell size, size and density of stomata, and size, density and ornamentation of the trichomes.
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Length of the epidermal cells was similar among taxa, except for D. quercifolia, which exhibited the shortest and most narrow cells in the adaxial surface while the widest cells observed were in D. ceratocaula. In terms of stomata, D. stramonium var. stramonium had the largest. Stomata density was highest for D. innoxia and D. stramonium var. stramonium, while lowest density was observed for D. stramonium var. tatula (Table 1). However, on the abaxial surface epidermal cells were largest for D. innoxia and D. quercifolia, whith also had the widest cells. The shortest and narrowest epidermal cells were found in D. stramonium var. tatula (Table 1). Regarding the average length and width of stomata, the highest measurements were recorded for D. innoxia and the average density of stomata was highest for D. quercifolia (Table 1). The largest simple trichomes were found in D. quercifolia (Figure 1F) while the smallest were observed for D. stramonium var. tatula. In the adaxial surface, the highest trichome density was recorded for D. innoxia and the lowest for D. stramonium var. stramonium (Table 2). In the abaxial surface, the highest density of simple trichomes was recorded for D. ceratocaula, whose entire laminar surface was practically covered by them (Table 2). The lowest density was observed for both varieties of D. stramonium. It is worth mentioning that simple trichomes were most abundant compared to the glandular trichomes in both surfaces of D. ceratocaula and D. innoxia and on the adaxial surface of D. stramonium var. tatula and D. quercifolia. Glandular trichome density was highest in both surfaces of D. stramonium var. stramonium and on the abaxial surface of D. stramonium var. tatula and D. quercifolia (Tables 2 and 3). The surface of the simple trichomes, in both leaf surfaces, had a pustule-like appearance in D. ceratocaula, D. stramonium var. tatula and D. innoxia, which also was covered by an abundant secretion (Figure 2D). Datura stramonium var. stramonium had the simple trichome surface slightly postulate and densely-tuberculate in D.
quercifolia (Figure 2E; Table 2). Datura ceratocaula and D. quercifolia had largest size glandular trichomes on the adaxial surface and the highest density per mm2 was observed for Datura innoxia, while the lowest density was recorded for D. ceratocaula and D. stramonium var. stramonium (Table 3). On the abaxial surface, the largest glandular trichomes were observed for D. ceratocaula and D. innoxia, and the highest density of glandular trichomes was observed for D. quercifolia and D. stramonium var. tatula, while the lowest density was recorded for D. ceratocaula and D. innoxia. In both surfaces, glandular trichomes showed smooth ornamentation (Figure 2A), except for D. quercifolia, that was rugulate (Figure 2B). Glandular trichomes of D. innoxia were mostly covered by abundant secretion (Figure 2F). The Dunn tests and the classification tree (Figure 3A) revealed that the anatomical traits of the adaxial surface that best discriminate among Datura taxa are stomata number, simple hairs number and simple hairs length formed by more than two cells. The validation test showed that the CART had an accuracy of 0.93 (p < 2.2 x 10-16). However, the CART (Figure 3B) for the abaxial surface revealed that the anatomical traits that best discriminate among Datura taxa were number and length of simple trichomes and stomata number. Here, the crossed validation test had an accuracy of 0.98 (p < 2.2 x 10-16). Both analyses showed that some foliar anatomical characters allow a clear distinction among the Datura taxa studied. It was also obvious that abaxial surface showed characters with high discriminatory power, in such a way that D. ceratocaula was characterized by the presence of abundant simple trichomes at a density higher than 67 hairs/mm2. Datura innoxia varied in terms of the number of simple trichomes (>17/mm2). Datura stramonium var. stramonium varied in terms of simple trichomes length ( 112).
DISCUSSION Our study complements that of Wallis and Rohatgi [15] and Jackson and Wallis [16], who described in great detail the anatomical floral and root characters of D. stramonium and D. tatula, respectively, but did not include leaves. Equally, our results help strengthen the observations made by Timmerman [17], who mentioned that pubescence could help differentiate species, as we confirmed that trichomes (especially simple trichomes) are characters that help distinguish between taxa, as they show differences in size, distribution and ornamentation. Species of Datura compared in this study showed similar anatomical characters. However, the highest number of traits (18) was shared by D. ceratocaula and D. innoxia, followed D. stramonium (17) and D. ceratocaula that shared traits with D. stramonium (15). Taxa that had the highest number of distinct traits were D. innoxia and D. stramonium var. tatula (23), followed by D. quercifolia when compared with D. ceratocaula, D. innoxia and D. stramonium var. stramonium. The main differences observed among the taxa here included were: 1) epidermal cells size, 2) size and density of stomata, and 3) size, density, distribution and ornamentation of the simple and glandular trichomes. The studied taxa showed significant differences in size and density of stomata that allowed making functional inferences. Datura innoxia showed a lower stomata density on the adaxial surface (approximately 153/mm2) but with the smallest size. Datura. stramonium var. tatula had few stomata (35/mm2), but they were larger. Abaxial surface of D. quercifolia had the highest amount of stomata (139 /mm2) and D. stramonium var. tatula and D. ceratocaula the lowest (approximately 38/mm2). Since D. ceratocaula grows in semiaquatic environments, we expected high numbers of stomata in one or both leaf surfaces in order to eliminate water excess. However, this was not observed. Differences in size, density, and trichomes localization are important traits for discerning among the studied taxa. D. quercifolia exhibited a higher number of simple trichomes. However, D.
ceratocaula and D. innoxia revealed a higher density. In terms of glandular trichomes, the largest were observed in D. innoxia, but D. quercifolia and D. stramonium var. tatula had the highest density. The lowest density was recorded for D. ceratocaula and D. innoxia. Based on the data here recorded, it is possible to differentiate D. ceratocaula by the presence of the abundant simple trichomes at a density higher than 67 hairs/mm2 on the abaxial surface and scarce glandular trichomes with up to nine cells. Datura innoxia differs from the rest of the taxa in terms of the number of simple trichomes (more than 17/mm2 and less than 67/mm2), and by having a more homogeneous distribution throughout the lamina at both surfaces. Datura quercifolia can be identified by its larger epidermal cells in the adaxial surface, larger simple trichomes at a density between 2/mm2 and 17/mm2. Moreover, it was the only taxon that presented simple trichomes with a densely tuberculate ornamentation in both leaf surfaces, and rugulate glandular trichomes, while the rest of the taxa showed postulate and smooth ornamentation, or lack of ornaments. Finally, both varieties of D. stramonium revealed the lowest density of simple trichomes on the lamina and nerves. CONCLUSIONS Anatomical characters were significantly different among the Datura taxa, except for the glandular stem length in the adaxial surface. The grouping observed after building the CART classification tree showed that the anatomical characters of the abaxial surface of leaves had the highest discriminatory power. The anatomical character with the highest discrimination efficiency was the number of simple trichomes. There is still a need to increase our knowledge on anatomical aspects of Datura species. However, this study provides information on foliar anatomy of five Datura taxa as a basis for future anatomical and histological research on these species. AKNOWLEDGEMENTS The authors thank the anonymous reviewers for their valuable suggestions to improve this manuscript. We are grateful for the financial support bestowed by the Fondo para el Fortalecimiento Institucional of the Autonomous University of Queretaro (FOFI-UAQ2012). The
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first author was funded by a PhD studentship from Consejo Nacional de Ciencia y Tecnología, CONACYT. We thank Gustavo Rodríguez Hernández for his invaluable help with plant tending, Ana Lucia Tovar Alvarez for obtaining the SEM images, and Patricia Herrera Paniagua for comments that greatly improved the manuscript.
[12] [13]
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