J. 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 and one variety of (Solanoidea, )

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 pruinosa Greenm., D. reburra A.S.Barclay and D. The genus Datura L. (Solanaceae) is endemic to arenicola. Datura stramonium var. tatula has been Mexico and adjacent areas of southwestern United classified as a different species but genetic States and the northern region of Central America evidence suggests it is a variety of D. stramonium [1]. It has become naturalized in Asia, Africa and [8, 9]. Australia [2, 3]. The grow mainly in dry, Species within the genus are used in traditional mild and subtropical regions [4]. The number of medicine and have economic importance due to recognized species varies from 12 to 14 [5, 6]. their content of amino acids, tannins, Recently, Bye and Sosa [7] described D. arenicola carbohydrates, phytic acid, clorogenic acids, Gentry ex Bye & Luna Baja California. Twelve lectins, albumins, as well as more than 70 alkaloids species have been recorded for Mexico, Datura of pharmaceutical importance [10, 11]. Research ceratocaula Jacq., D. innoxia Mill., D. lanosa has focused a few Datura species, mainly on A.S.Barclay ex Bye, D. metel L., D. wrightii Regel, genetic [12, 13] and biochemical aspects [14]. D. stramonium L. with two varieties stramonium L. Anatomical studies of Datura have focused on and tatula (L.) Torr., D. quercifolia Kunth, D. reproductive structures [15] and roots [16]. kymatocarpa A.S. Barclay, D. discolor Bernh., D. However, there is limited information on foliar *Corresponding author: Mahinda Martínez E-mail: [email protected]

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anatomy. Timmerman [17] described D. Processing and Analysis in Java). Stomata number stramonium, D. tatula, D. metel, D. innoxia and D. and glandular trichomes density on the abaxial leaf fastuosa, and pointed out that leaf indumentum can surface of D. ceratocaula was inferred using the help to discriminate species. Not with standing, for blade margin where they exhibited a glabrate D. stramonium and D. tatula, he only reported surface, as the density of simple trichomes in the differences in terms of trichomes size [15]. Thus, central portion of the leaf hindered direct we aimed to describe and compare the leaves of observation. five Datura taxa: D. ceratocaula, D. innoxia, D. Differences among Datura taxa for each variable stramonium var. stramonium, D. stramonium var. were tested by Kruskal-Wallis. Variables with tatula and D. quercifolia, which share a similar significant differences were further tested with a geographic distribution. Dunn test with a Holm correction for multiple comparisons [20, 21], the level of significance was MATERIALS & METHODS set at P< 0.05. Furthermore, an algorithm to build Description order for the five taxa follows the classification and regression trees (CART) was phylogeny of Datura proposed by Bye and Sosa [7] used on data of each leaf surface in order to who propose relationship as follow: Datura identify the variables that best separate the ceratocaula, D. innoxia, D. stramonium var. taxa[22]. To build the classification trees we used stramonium, D. stramonium var. tatula and D. the rpart package [23] in R [24]. quercifolia. Seeds of the five Datura taxa were collected from four sites in Santiago de Queretaro, RESULTS Mexico. Sampling sites were located between 20º Foliar structures of the five Datura taxa shared 31’ 38.91’’ - 20º 49’ 40.29’’ N and 100º 18’42.20’’ many anatomical characters. They presented - 100º 30’23.26’’ W. Site elevation varied from bifacial leaves with unstratified epidermis (Figure 1,814 to 2,156 m. Vegetation type, soil, and 1A), no wax, and amphistomatic leaves with conservation status were similar among sites, anisocytic stomata. Epidermal cells showed namely tropical deciduous forest with pellic evidence of slightly wavy walls, of variable size vertisol [18], and in ruderal areas. All species were among taxa (Table 1). Mesophyll was formed by a terrestrial except for the semiaquatic D. compact and unstratified palisade parenchyma, ceratocaula. except for D. ceratocaula that had two distinct lines Plants were grown in greenhouse conditions and of palisade cells (Figure 1B). Spongy parenchyma three to five leaf samples were sampled per taxon. had three to four cell layers, with abundant calcium Material was fixed overnight in Navashin's fixative, oxalate druses in all taxa (Figures 1A and 1B), dehydrated in ethanol, and embedded in paraffin. either as single prism-shaped crystals or grouped Transverse sections of 12 µm in width were crystals (Figure 1C). obtained with a rotary microtome (Leica, Both surfaces (adaxial and abaxial) of all taxa RM2125), stained with alcian blue and 50% showed two types of indumentum: simple safranin, and mounted with Permount (Fisher trichomes (Figure 1D) and glandular trichomes Chemicals). (Figure 1E). Some of the cells that form the Structures were described following microscopic trichomes exhibited prism-shaped crystals. Simple examination (Olympus, BX43) and scanning trichomes were uniseriate, filiform and electron microscopy (SEM, Carl Zeiss EVO 50). multicellular (from one to seven cells; Figure 1F). Tissue microphotographs were captured with Q- They were distributed throughout the lamina, but Capture Pro 7 software and optimized in Photoshop were most abundant along the nerves, with the (version CC2015) without altering structure highest density over the primary vein and, less information. Structure nomenclature followed Font abundantly, over the secondary veins, except for Quer [19]. Anatomical sections description was the abaxial surface of D. ceratocaula, in which the from the outer to the inner portions of the organs. main vein showed few simple trichomes. Glandular Density, size, distribution and ornamentation of 14 trichomes were multicellular with a secretory head, variables (see tables 1, 2 and 3) related to cell size, formed by six to seven cells, with two to three on stomata, and trichomes in both leaf surfaces were the base and four on the gland (Figures 2a and 2B). assessed under SEM using ImageJ software (Image In addition, D. ceratocaula exhibited trichomes 38

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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. Taxa/Structure D. ceratocaula D. innoxia D. quercifolia stramonium tatula

Length μm 46.3 ± 10.4 a 47.9 ±10. 7a 47.5 ± 9.0a 42.7 ± 8.8ab 37.7 ± 9.3b

a b bc ac b

Adaxial Width μm 29.3 ± 4.8 22.2 ± 3.9 22.5 ± 3.8 25.6 ± 3.6 19.4 ±5.0

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

Epidermal cells ab ac ab c b Abaxial Width μm 26.5 ± 7.7 22.0 ±5.3 24.7 ±5.9 18.5 ± 5.7 31.0 ±8.5

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

a a b bc ac Width μm 17.8 ± 8.5 15.9 ± 2.1 22.6 ± 2.1 19.9 ± 2.2 18.4 ± 2.7

Pore length μm 12.6 ± 1.8 ab 10.9 ± 1.7ac 18.9 ± 3.1d 12.8 ± 1.9b 11.1 ± 6.8c

Adaxial

Number/mm2 89.0 ± 8.0a 153.0± 26.0b 122.0 ± 21.0b 35.0 ± 7.0c 94.0 ± 21.0a

Stomata 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

a a a b ab

Abaxial Pore length μm 13.2 ± 3.5 13.5 ± 2.2 13.1 ± 2.6 12.5 ± 3.4 11.7 ± 2.8

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

*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. stramonium var. D. stramonium var. Structure D. ceratocaula D. innoxia D. quercifolia stramonium tatula 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

Number/mm2 11.0 ± 2.0 a 31.0 ± 9.0 b 0.10 ± 0.30c 9.0 ± 2.0 a 10.0 ± 1.0 a

Adaxial Scarce in lamina, Moderate in lamina, Scarce in lamina and Scarce in lamina and Moderate in lamina Distribution

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

Ssimplestrichome

Abaxial Abundant in lamina Moderate in lamina Scarce in lamina and Scarce in lamina and Moderate in lamina Distribution and nerve and nerve nerve nerve and nerve

Ornamentation Pustulate Pustulate Pustulate Pustulate Tuberculate

Estomas Both *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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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 D. quercifolia stramonium tatula

a b ab b Total length 76.1 ± 11.8 66.5 ± 8.9 69.9 ± 11.6 64.5 ± 6.3 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

a b b b b

Adaxial Gland width 41.2 ± 3.9 33.4 ± 4.7 36.1 ± 5.8 33.7 ± 6.0 35.5 ± 4.9

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

More abundant in More abundant in In nerve, scarce in In nerve, scarce in More abundant in Localization nerve nerve lamina lamina 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

Glandular trichomes 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

a a b b b Abaxial GlandAbaxial width 35.9 ± 2.4 40.8 ± 5.9 32.3 ± 5.3 32.0 + 6.0 31.0 ± 3.6

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 More abundant in In nerve, scarce in In nerve, scarce in Localization Not observed In lamina and nerve nerve lamina lamina

Ornamentation Smooth Smooth Smooth Smooth Rugulate

Both *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 trichomes. The 14 variables that were analyzed on were distributed similarly to the simple trichomes, both leaf surfaces revealed significant differences mainly along the length of the nerves. Their among the taxa here studied, except for the length observation was difficult in the abaxial surface of of glandular trichome stem in the adaxial surface. D.ceratocaula, as the lamina is practically covered Observed differences were in terms of the with simple trichomes, but could clearly be epidermal cell size, size and density of stomata, and observed on the main vein, which had few simple size, density and ornamentation of the trichomes.

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Length of the epidermal cells was similar among quercifolia (Figure 2E; Table 2). taxa, except for D. quercifolia, which exhibited the Datura ceratocaula and D. quercifolia had largest shortest and most narrow cells in the adaxial size glandular trichomes on the adaxial surface and surface while the widest cells observed were in D. the highest density per mm2 was observed for ceratocaula. Datura innoxia, while the lowest density was In terms of stomata, D. stramonium var. recorded for D. ceratocaula and D. stramonium stramonium had the largest. Stomata density was var. stramonium (Table 3). On the abaxial surface, highest for D. innoxia and D. stramonium var. the largest glandular trichomes were observed for stramonium, while lowest density was observed for D. ceratocaula and D. innoxia, and the highest D. stramonium var. tatula (Table 1). However, on density of glandular trichomes was observed for D. the abaxial surface epidermal cells were largest for quercifolia and D. stramonium var. tatula, while D. innoxia and D. quercifolia, whith also had the the lowest density was recorded for D. ceratocaula widest cells. The shortest and narrowest epidermal and D. innoxia. In both surfaces, glandular cells were found in D. stramonium var. tatula trichomes showed smooth ornamentation (Figure (Table 1). Regarding the average length and width 2A), except for D. quercifolia, that was rugulate of stomata, the highest measurements were (Figure 2B). Glandular trichomes of D. innoxia recorded for D. innoxia and the average density of were mostly covered by abundant secretion (Figure stomata was highest for D. quercifolia (Table 1). 2F). The largest simple trichomes were found in D. The Dunn tests and the classification tree (Figure quercifolia (Figure 1F) while the smallest were 3A) revealed that the anatomical traits of the observed for D. stramonium var. tatula. In the adaxial surface that best discriminate among adaxial surface, the highest trichome density was Datura taxa are stomata number, simple hairs recorded for D. innoxia and the lowest for D. number and simple hairs length formed by more stramonium var. stramonium (Table 2). In the than two cells. The validation test showed that the abaxial surface, the highest density of simple CART had an accuracy of 0.93 (p < 2.2 x 10-16). trichomes was recorded for D. ceratocaula, whose However, the CART (Figure 3B) for the abaxial entire laminar surface was practically covered by surface revealed that the anatomical traits that best them (Table 2). The lowest density was observed discriminate among Datura taxa were number and for both varieties of D. stramonium. It is worth length of simple trichomes and stomata number. mentioning that simple trichomes were most Here, the crossed validation test had an accuracy of abundant compared to the glandular trichomes in 0.98 (p < 2.2 x 10-16). both surfaces of D. ceratocaula and D. innoxia and Both analyses showed that some foliar anatomical on the adaxial surface of D. stramonium var. tatula characters allow a clear distinction among the and D. quercifolia. Glandular trichome density was Datura taxa studied. It was also obvious that highest in both surfaces of D. stramonium var. abaxial surface showed characters with high stramonium and on the abaxial surface of D. discriminatory power, in such a way that D. stramonium var. tatula and D. quercifolia (Tables 2 ceratocaula was characterized by the presence of and 3). The surface of the simple trichomes, in both abundant simple trichomes at a density higher than leaf surfaces, had a pustule-like appearance in D. 67 hairs/mm2. Datura innoxia varied in terms of the ceratocaula, D. stramonium var. tatula and D. number of simple trichomes (>17/mm2). Datura innoxia, which also was covered by an abundant stramonium var. stramonium varied in terms of secretion (Figure 2D). Datura stramonium var. simple trichomes length (<25.55 μm) and Datura stramonium had the simple trichome surface quercifolia showed a density of simple trichomes slightly postulate and densely-tuberculate in D. higher than two and lower than 17 trichomes/mm2.

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Figure 2. A. Glandular trichome of D. stramonium var. tatula with smooth ornamentation. B. Glandular trichome with rugulate ornamentation in the leaf epidermis of D. quercifolia. C. Glandular trichome with nine cells in a leaf of D. ceratocaula. D. Simple trichome with pustulate ornamentation and abundant secretion in an epidermal section of D.innoxia. E. Simple trichome with tuberculate ornamentation in the epidermis of D. quercifolia. F. Glandular trichome of D.innoxia with abundant secretion. ec: epidermal cell, gt: glandular trichome, sm: stomata, st: simple trichome.

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A

B

Figure 3. Classification tree (CART) of the anatomical characters of the compared Datura taxa. A. Adaxial surface. B. Abaxial surface.

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Finally, D. stramonium var. tatula was differed ceratocaula and D. innoxia revealed a higher from D. quercifolia in terms of the number of density. In terms of glandular trichomes, the largest stomata (> 112). were observed in D. innoxia, but D. quercifolia and D. stramonium var. tatula had the highest density. DISCUSSION The lowest density was recorded for D. ceratocaula Our study complements that of Wallis and Rohatgi and D. innoxia. [15] and Jackson and Wallis [16], who described in Based on the data here recorded, it is possible to great detail the anatomical floral and root differentiate D. ceratocaula by the presence of the characters of D. stramonium and D. tatula, abundant simple trichomes at a density higher than respectively, but did not include leaves. Equally, 67 hairs/mm2 on the abaxial surface and scarce our results help strengthen the observations made glandular trichomes with up to nine cells. Datura by Timmerman [17], who mentioned that innoxia differs from the rest of the taxa in terms of pubescence could help differentiate species, as we the number of simple trichomes (more than 17/mm2 confirmed that trichomes (especially simple and less than 67/mm2), and by having a more trichomes) are characters that help distinguish homogeneous distribution throughout the lamina at between taxa, as they show differences in size, both surfaces. Datura quercifolia can be identified distribution and ornamentation. by its larger epidermal cells in the adaxial surface, Species of Datura compared in this study showed larger simple trichomes at a density between 2/mm2 similar anatomical characters. However, the highest and 17/mm2. Moreover, it was the only taxon that number of traits (18) was shared by D. ceratocaula presented simple trichomes with a densely and D. innoxia, followed D. stramonium (17) and tuberculate ornamentation in both leaf surfaces, and D. ceratocaula that shared traits with D. rugulate glandular trichomes, while the rest of the stramonium (15). Taxa that had the highest number taxa showed postulate and smooth ornamentation, of distinct traits were D. innoxia and D. or lack of ornaments. Finally, both varieties of D. stramonium var. tatula (23), followed by D. stramonium revealed the lowest density of simple quercifolia when compared with D. ceratocaula, D. trichomes on the lamina and nerves. innoxia and D. stramonium var. stramonium. The main differences observed among the taxa here CONCLUSIONS included were: 1) epidermal cells size, 2) size and Anatomical characters were significantly different density of stomata, and 3) size, density, distribution among the Datura taxa, except for the glandular and ornamentation of the simple and glandular stem length in the adaxial surface. The grouping trichomes. observed after building the CART classification tree The studied taxa showed significant differences in showed that the anatomical characters of the abaxial size and density of stomata that allowed making surface of leaves had the highest discriminatory functional inferences. Datura innoxia showed a power. The anatomical character with the highest lower stomata density on the adaxial surface discrimination efficiency was the number of simple 2 (approximately 153/mm ) but with the smallest trichomes. There is still a need to increase our size. Datura. stramonium var. tatula had few knowledge on anatomical aspects of Datura species. 2 stomata (35/mm ), but they were larger. Abaxial However, this study provides information on foliar surface of D. quercifolia had the highest amount of anatomy of five Datura taxa as a basis for future 2 stomata (139 /mm ) and D. stramonium var. tatula anatomical and histological research on these and D. ceratocaula the lowest (approximately species. 2 38/mm ). Since D. ceratocaula grows in semiaquatic environments, we expected high AKNOWLEDGEMENTS numbers of stomata in one or both leaf surfaces in The authors thank the anonymous reviewers for order to eliminate water excess. However, this was their valuable suggestions to improve this not observed. manuscript. We are grateful for the financial Differences in size, density, and trichomes support bestowed by the Fondo para el localization are important traits for discerning Fortalecimiento Institucional of the Autonomous among the studied taxa. D. quercifolia exhibited a University of Queretaro (FOFI-UAQ2012). The higher number of simple trichomes. However, D. 46

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