Macroscopic-microscopic characteristics and AFLP ingerprint for identi ication of Erythroxylum novogranatense, E. cambodianum and E. cuneatum endemic to Thailand

Erythroxylum novogranatense (Morris) Hieron, E. cambodianum Pierre and E. cuneatum (Miq.) Kurz in family Erythroxylaceae was traditionally used as an antipyretic, general stimulant and gastrointestinal diseases. Due to their morphological similarity, the correct identi ication was necessary for the quality control in herbal medicine. E. novogranatense (Morris) Hieron, E. cambodianum Pierre and E. cuneatum (Miq.) Macroscopic andmicroscopic characteristics evaluated Kurz endemic to Thailand according to WHO standard guideline and ampli ied fragment length polymorphism (AFLP) ingerprint. Morphological characters of E. novogranatense, E. cambodianum and E. cuneatum were similar in their lower, fruit and seed but different in stem and leaf. Microscopic characteristics from these three species, including constant leaf numbers, showed individual values. The stomata were classi ied as paracytic type. The midrib transverse section showed distinct characters of the epidermis, palisade cell, stomata, spongy cell, parenchyma, xylem vessel, phloem tissue and collenchyma. AFLP ingerprint showed highly polymorphisms 97.42% with the number of bands (349 bands) ranging between 50-750 bands. Primer E+ACG/M+CTT had the highest number of AFLP band (91 bands). The dendrogram generated from UPGMA could separate these three species. In summary, the combination of morphological characteristics, microscopic investigation and AFLP ingerprinting can be used to identify plant species and determine the genetic relationship among three Erythroxylum species.


INTRODUCTION
The Erythroxylum (Erythroxylon) is the most well-known genus in the family Erythroxylaceae.
Approximately 230 species are widely distributed in South America, Africa and Asia, including some parts of India, China and Thailand (Aynilian et al., 1974;Bieri et al., 2006). The presence of tropane alkaloids characterised the herbal plants in Erythroxylum species, tannins, lavonoids, diterpenes and phenylpropanoids (Bohm et al., 1988;Ansell et al., 1993;Zuanazzi et al., 2001). These plants have been used in folk medicine in South America as a general stimulant and for various gastrointestinal ailments (Bonefeld et al., 1986). In Thailand, E. novogranatense (Morris) Hieron known as Coca, E. cambodianum Pierre known as Hun-Hai and E. cuneatum (Miq.) Kurz known as Krai-Thong were used as folk medicine. Three species are closely related with their morphological characteristics such as stem, lower, fruit and seed ( Figure 1). As a medicinal used, these species were also well known in Thai traditional medicine for anti-fever as well as an anti-in lammatory agent. However, scienti ic standards and pharmacognostic parameters have not been reported to ascertain the identity of these herbs.
The quality control of herbal plants is essential for assurance on quality, safety and ef icacy of herbal medicines as recommended by the World Health Organization (WHO). Nowadays, various methods have been used for medicinal plants identi ication. The pharmacognostic speci ication is a useful technique for identi ication of herbal plant identity. The DNA-based techniques have been widely used for genetic diversity of herbal drug. This technique is being developed to more advantages over typical phenotype markers and reliable for informative polymorphisms (Joshi et al., 2004). Ampliied fragment length polymorphism (AFLP) is one of molecular technique has been used in DNA markers as its strengths on reproducibility, high genomic abundance, highly polymorphic bands and replicable tags. This method is widely used for genetic diversity studies and phylogenetic relationship for identi ication of species level (K et al., 2005).
The similarities morphology of these 3 Erythroxylum led to confusion in their identi ication. Several characteristics, as well as molecular techniques, have been employed in this study for taxonomic identi ication and quality control of these plants.

Plant materials
Both fresh mature and fresh young leaves of 3 species of genus Erythroxylum were collected from various geographical areas in Thailand. Plant specimens were authenticated by botanist, compared to the herbarium specimens and voucher specimens were kept at College of Public Health Sciences (CPHS), Chulalongkorn University, Thailand. Table 1 is a list of three Erythroxylum species used in this study.

Macroscopic-microscopic determination
Macroscopic-microscopic examinations of three Erythroxylum species were examined. Macroscopic characters were considered on the shape, size, colour and other visual inspections. Morphological characteristics were observed and described as botanical characters. Microscopic characters were physiologically seen under of microscope according to WHO standard guideline (WHO, 1998). For leaf preparation, the representative pieces of the sample were selected and cut into suitable length (1 square centimetre) for anatomical character study under a microscope. For leaf measurement, matured leaves were immersed in 3% sodium hypochlorite solution for chlorophyll removal and gentry warming with chloral hydrate solution until it transparent (Mukherjee, 2008) and then mounted in 50% glycerin on a glass slide and observed under the microscope. Leaf constant numbers were examined with some modi ication according to the method previously described by Evans (Evans and Trease, 2009). Thirty ields for an individual specimen which were representative plant areas of photographs were determined in constant values. The city of each picture was calculated in square millimetre using Axio Vision program and then recorded. Leaf constant numbers in each parameter were analysed and presented as mean ± standard deviation (SD).

DNA isolation and AFLP ingerprint
Each sample (50-100 mg) of fresh young leaves were ground in liquid nitrogen to obtain a ine powder for isolation of DNA. Genomic DNA was individually extracted using a modi ied CTAB technique (Doyle and Doyle, 1990) and employed a DNA puri ication step to remove a large amount of polysaccharide. The genomic DNA was measured using a spectrophotometer (NanoDrop Technologies, USA) and stored for DNA template to qualify the DNA.
AFLP ingerprint was performed as described by Vos et al., (Vos et al., 1995) with some modi ications. EcoRI digested genomic DNA (100 ng/µl) (10 U/µl) and Tru9I (10 U/µl) (Boehringer Mannheim, Germany) in buffer A (Promega, USA) at 37 ºC for one h. The ligation process was carried out at 37 ºC for three h to generate DNA template-adapter for ampli ication. Pre-ampli ication using EcoRI+A and MseI+C primers (Euro ins MWG Operon, Germany) and followed by selective ampli ication using three selective nucleotides of EcoRI and MseI primers were performed using PCR Thermal Cycler (Thermo Electron Corporation, USA). The particular ampli ication products of primers combination were sizefractionated on 6% denaturing polyacrylamide gel electrophoresis, stained with silver nitrate and AFLP ingerprints were analysed (Bassam et al., 1991).
For data analysis, the presence or absence of AFLP polymorphic bands was visually scored to generate a binary data set. The Jaccard's coef icient similarity matrix was computed (Jaccard, 1908) and the Unweighted Pair Group Method with Arithmetic mean (UPGMA) dendrogram was constructed using FreeTree software (Pavlicek et al., 1999). Bootstrap replication of 1,000 re-sampling data subset was

Macroscopic Evaluation
Herbal medicine has been practised worldwide and now recognised by WHO as essential for primary healthcare. Herbal based drugs play a crucial role in the healthcare management system. The correct identi ication of medicinal plants is the irst step in quality control. Pharmacognostic speci ication and authentication are needed for reliable identi ication to ensure the safety and ef icacy of their medicinal properties. Macroscopic and microscopic is one of the methods for the examination of plant identi ication.
The macroscopic examinations are based on their morphological features are always used to distinguish various species or evaluate their quality. Morphological characters of E. novogranatense, E. cambodianum and E. cuneatum were similar in lower, fruit and seed, but they were different in stem and leaf shape. The stem of E. novogranatense and E. cambodianum were shrub, but E. cuneatum was shrub or small tree. While the shape of E. novogranatense is obovate, E. cambodianum is elliptic or obovate, and E. cuneatum is obovate or elliptic to elliptic-lanceolate. The important character of these three species is more conspicuous of two longitudinal curved lines (false midrib) on each side of the midrib on the lower face of the leaf. The morphological characteristics of three Erythroxylum species are summarised in Table 2.

Microscopic Evaluation
Microscopic observation is based on the optical system by the microscope. This method refers to the analysis of size, shape and relative structure of different cells and internal features in medicinal plants. It can provide supporting evidence for identi ication and standardisation of herbal drugs.
Anatomical and histological studies of the leaf can be used for discrimination of differential internal structures in each species. Midrib transverse section of E. novogranatense (Figure 2), E. cambodianum ( Figure 3) and E. cuneatum (Figure 4) showed distinct characters of the epidermis, palisade cell, stomata, spongy cell, parenchyma, xylem vessel, phloem tissue and collenchyma. Three Erythroxylum species demonstrated distinguished arrangement of vessel members. The arrangement of xylem, phloem and ibre of E. cuneatum and E. cambo- Seed in fertile locule, lattened to planoconvex, 1-3 mm wide and 5-9 mm long Seed in fertile locule, lattened, 1.5-3 mm wide and 6-10 mm long Seed in fertile locule, lattened, 1-2.5 mm wide and 5-10 mm long dianum were shown circular arrangement but not shown this character in E. novogranatense. These internal characteristics can be used for classi ication of these three Erythroxylum species. According to the results, microscopic evaluation of these three species revealed the different morphological characteristics but contained almost similar cell components.
The form and surrounding cells arrangements of the stomata is one of microscopic character that can be distinguished each plant species. Four types of stomata; anomocytic, anisocytic, diacytic, and paracytic are often available for matured leaves that classify the differentially of the species (WHO, 1998). The type of stomata in each Erythroxylum species was classi ied as paracytic type or rubiaceous type (two subsidiary cells are long parallel to the axis of the stomata) and presented only on the lower (abaxial) epidermis of these three Erythroxylum species. The structure and shape of the epidermis and stomatal type are the irst investigations in the microscopic analysis of leaf identi ication on surface area (Jones, 1986;Baranova, 1992). Based on characteristics of epidermal cells on lower epidermis, E. cambodianum showed polygonal cells shape, whereas E. novogranatense and E. cuneatum were irregular shape ( Figure 5). Constant values of leaves are used to evaluation of character for different species or some closely related species by microscopic examination. Leaf measurement is one method that is a constant number used to examine the identi ication of each plant and useful parameters to distinguish in species level by qualitative microscopic evaluation (WHO, 1998). Leaf constant numbers were examined under the microscope. Leaf constant numbers of these three species    showed individual values. The constant numbers in this study were presented in Table 3.
As the results in a recent study, determination of leaf constants was considered as one useful parameter for species identi ication. The reviews of E. novogranatense, E. cambodianum and E. cuneatum have not been previously reported in constant numbers. This recent study is the irst report of these three species. However, these constant values have been widely used for Thai medicinal plants identi ication and previous research used for identi ication of Datura species (Issaravanich et al., 2013), Cassia species (Sihanat et al., 2015) as well as Mangifera indica (Palanuvej et al., 2016).
In the current study, the different of macroscopicmicroscopic evaluation can be served as an important tool for species identi ication.

Genetic Relationship
For AFLP ingerprint in a recent study, a total of 48 AFLP primers combination were screened. For the genetic relationship, the genetic diversity  estimates (GDEs) were used for UPGMA clustering by calculated from Jaccard's similarity matrix (Table 5). The similarity index varied from 0.27 to 0.99. The highest similarity index (0.988) was found between E. novogranatense in location 2 (ENO-2) and E. novogranatense in location 3 (ENO-3). In contrast, the lowest similarity index (0.270) was found between E. cuneatum in location 1 (ECU-1) and E. novogranatense in location 3 (ENO-3).
According to the dendrogram, plant samples in genus Erythroxylum; E. cambodianum, E. cuneatum and E. novogranatense collected from 3 different locations were classi ied as three distinct groups. Cluster A was composed of E. cambodianum from three locations (ECA-1, ECA-2 and ECA-3). Cluster B was composed of E. cuneatum from two loca-tions (ECU-1 and ECU-2). Cluster C was composed of E. novogranatense from three locations (ECA-1, ECA-2 and ECA-3) and Strychnos nux-vomica (SNV) was used as an out-group plant which separated from three Erythroxylum species (Figure 7). The result of genetic similarity index showed that these three species could be clustering into three groups from their differentiated morphological characteristics and leaf constant numbers.
Therefore, AFLP marker is a powerful approach to detect DNA polymorphism for identi ication of species level, applicable to all organisms without previous sequence information and generally results in highly informative ingerprints. However, another DNA-based method such as sequence characterised ampli ied region (SCAR) marker, or another technique should be further developed for simpler to identi ication in these three species.
DNA ingerprinting method such as AFLP marker can be used for improvements over botanical characteristics and chemotaxonomic analysis in terms of time, cost, and analyse genetic diversity within among relatively plant species level (Welsh and McClelland, 1990). AFLP analysis was a powerful tool and very effective to analyse genetic variation in the medicinal plant as well as Erythroxylum samples in the previously reported to characterise and can identify the varieties of coca between the cultivated taxa of Erythroxylum (Johnson et al., 2003). AFLP method has more advantages in the ability to work without sequence information, multi-locus detection, small amount of DNA needed, highly informative ingerprints and high reproducibility (Karihaloo, 2015). This technique is a valuable essential marker for evaluation of identi ication and genetic relationships. AFLP ingerprint has been successful when used for plants diversity (Mba and Tohme, 2005). In other words, discrimination between closely related species and authentication of some other medicinal plants such as Zingiber species (Ghosh et al., 2011), Capparis species Aichi-Yous i et al. (2016) and Boesenbergia species (Techaprasan et al., 2008).

CONCLUSIONS
In this study can be concluded that morphological characteristics, leaf constant numbers and AFLP ingerprinting have successfully used for identi ication and distinguish of these three Erythroxylum species. The correlation was found between the morpho-logical and molecular analysis. The results of the recent study can use in genetic variation analysis among closely related species. The combination of morphological characteristics, microscopic investigation, and DNA ingerprinting can be useful for identi ication of plant species to provide supporting evidence for the quality control of medicinal plants.