Morpho-anatomical characterization and DNA barcoding of Artemesia vulgaris L.

Caracterização morfoanatômica e código de barras do DNA de Artemesia vulgaris L.

Abstract

Artemisia vulgaris L. belongs to Asteraceae, is a herbal plant that has various benefits in the medical field, so that its use in the medical field can be explored optimally, the plant must be thoroughly identified. This study aims to identify A. vulgaris both in terms of descriptive morpho-anatomy and DNA barcoding using BLAST and phylogenetic tree reconstruction. The morpho-anatomical character was observed on root, stem, and leaf. DNA barcoding analysis was carried out through amplification and alignment of the rbcL and matK genes. All studies were conducted on three samples from Taman Husada (Medicinal Plant Garden) Graha Famili Surabaya, Indonesia. The anatomical slide was prepared by the paraffin method. Morphological studies revealed that the leaves of A. vulgaris both on the lower-middle part and on the upper part of the stem have differences, especially in the character of the stipules, petioles, and incisions they have. Meanwhile, from the study of anatomy, A. vulgaris has an anomocytic type of stomata and its distribution is mostly on the ventral part of the leaves. Through the BLAST process and phylogenetic tree reconstruction, the plant sequences being studied are closely related to several species of the genus Artemisia as indicated by a percentage identity above 98% and branch proximity between taxa in the reconstructed phylogenetic tree.

Keywords:
Artemisia vulgaris; DNA barcode; morpho-anatomy; matK; phylogenetic tree; rbcL

Resumo

Artemisia vulgaris L., pertencente à família Asteraceae, é uma planta herbácea com diversos benefícios à área médica. E para que seu uso na área médica possa ser explorado de forma otimizada, a planta deve ser minuciosamente identificada. Este estudo tem como objetivo identificar A. vulgaris por meio de análise morfoanatomia descritiva, quanto de código de barras do DNA utilizando BLAST e reconstrução de árvore filogenética. As características morfoanatômica foram observadas na raiz, caule e folha. A análise do código de barras do DNA foi realizada através da amplificação e alinhamento dos genes rbcL e matK. Todos os estudos foram feitos em três amostras de Taman Husada (Jardim de Plantas Medicinais) Graha Famili, Surabaya, Indonésia. A lâmina anatômica foi preparada pelo método da parafina. Estudos morfológicos revelaram que as folhas de A. vulgaris, tanto na parte média inferior quanto na parte superior do caule, apresentam diferenças, principalmente nos caracteres das estípulas, pecíolos e incisões. Já o estudo da anatomia revelou que A. vulgaris possui estômatos do tipo anomocítico e sua distribuição é principalmente na parte ventral das folhas. Através do processo BLAST e da reconstrução da árvore filogenética, as sequências de plantas em estudo apresentam estreira relação com várias espécies do gênero Artemisia, conforme indicado por uma identidade percentual acima de 98% e proximidade de ramos entre táxons na árvore filogenética reconstruída.

Palavras-chave:
Artemisia vulgaris; código de barras de DNA; morfoanatomia; matK; árvore filogenética; rbcL

1. Introduction

Artemisia vulgaris L. is a member of the genus Artemisia which belongs to the Asteraceae family, this family has significant economic and medical value. A. vulgaris has an important record in the history of medicine and is known as the “mother of herbs” in the Middle Ages (Ekiert et al., 2020). This plant is commonly used in traditional medicine practices, because of its therapeutic benefits, especially in urology (Zubair et al., 2020). Phytochemical studies of A. vulgaris resulted in the isolation of sterols, triterpenes, and flavonoids (Farrag et al., 2015). According to Barney and Ditommaso (2003), A. vulgaris displays morphological and physiological heterogeneity at extreme levels in various ecologies, including branch properties, branching degree, leaf morphology, and rhizome diameter (Barney and Ditommaso, 2003). Therefore, in addition to identifying morpho-anatomically to prevent misrecognition, identification at the molecular level such as DNA barcoding is also required. Apart from the morphological diversity, molecular identification is required to confirm the identity of the plant before the plant undergoes further extractions and the extracts are used as ingredients in herbal or pharmaceutical products.

In general, species of the genus Artemisia are found in temperate regions, especially in the northern hemisphere and with limited numbers in the southern hemisphere (Oberprieler et al., 2009). This genus has many morphological and phytochemical characteristics, both of which are associated with the geographical origin of the plant habitat (Abuhadra et al., 2017). For example, various studies on the plant trichomes indicate the content of secondary metabolites stored by trichomes. Several Asteraceae species have trichomes containing terpenoids, alkaloids, phenols, and oils (Dorly et al., 2015). Mercado et al. (2021) reported that Artemisia copa has schizogenous secretory ducts distributed in the leaf midvein and cortex in the stem (Mercado et al., 2021). The use of anatomical markers alone will not be sufficient to produce maximum taxonomic data, because these markers also depend on environmental factors (Zagoto and Violita, 2019). To determine the content of secondary metabolites for pharmaceutical purposes, anatomical markers can also be used. A molecular approach, such as DNA barcoding develops into conclusive authentication and taxonomic assignment for phytopreparation quality assurance (Ulrich-Merzenich et al., 2007; Patwardhan et al., 2014).

DNA barcoding is a taxonomic method to identify DNA of organisms or a particular species (Sarvananda, 2018). DNA barcoding is a powerful and efficient tool for the identification of poorly studied species using short and standardized DNA fragments (Genievskaya et al., 2017; Kress, 2017; Ariyanti et al., 2021). There are 7 candidate DNA plastid markers, namely the atpF-atpH, matK, rbcL, rpoB, rpoC1, psbK-psbI, and trnH-psbA. However, based on various assessment criteria, the recommended DNA barcode for plants is a combination of rbcL and matK loci (CBOL Plant Working Group, 2009). One of the weaknesses of DNA barcodes is the non-universality of markers, therefore the selection of DNA barcode types in taxonomic studies is critical. Each taxa groups have its standard barcode. For example, in animals, the mitochondrial cytochrome oxidase I (COI) gene is generally used for phylogenetic study. However; the same gene cannot be employed in plants, as it lacks sufficient variations due to a low mutation rate (Ho et al., 2021). To select universal barcodes for plants, various molecular markers have been identified, including cpDNA regions such as matK (maturase-K) and rbcL (ribulose-biphosphate carboxylase large subunit) (Hilu and Liang, 1997; CBOL Plant Working Group, 2009). These areas were selected for three reasons: universality, sequence quality, and discriminatory (CBOL Plant Working Group, 2009; Hollingsworth, 2011). There is a close correlation between plant identification activities to obtain chemical compounds for medical purposes. Hence, this study aims to identify A. vulgaris based on morpho-anatomic and DNA barcode markers to reveal a more in-depth medical potential of this plant. This investigation is expected to add important insights since A. vulgaris has become a commodity for herbal medicine and medicinal products.

2. Materials and Methods

2.1. Materials

Three individuals of adult A. vulgaris plant samples were taken in September 2019 at Taman Husada Graha Famili, Surabaya, East Java, Indonesia. The plant species identification was confirmed by comparison with the herbarium collection at the Purwodadi Botanical Garden, Indonesian Institute of Sciences, Pasuruan, Indonesia. Three voucher specimens (No. AV0101112021, No. AV0201112021, and No. AV0301112021) were deposited in Plant Systematic Laboratory, Department of Biology, Universitas Airlangga, Surabaya, Indonesia.

2.2. Morpho-anatomical characterization

The morphologically observed organs of Artemisia vulgaris were based on the research by El-Sahhar et al. (2010), which is limited to vegetative organs (root, stem, and leaves) (El-Sahhar et al., 2010). Anatomical analysis were based on plant organs (rhizomes, stem, and leaves) used by Janaćković et al. (2021), except for generative organs. Anatomical preparations were prepared using the paraffin method. Briefly, plant tissue were immersed into paraffin blocks to produce thin preparations (Ilham et al., 2022). Sample preparation using this method includes the following steps: cutting, fixation, aspiration, dehydration, dealcoholization, infiltration, embedding, trimming, staining, and mounting (Sari et al., 2016). The process of making anatomical preparations began with cutting the tissue with a thickness of 0.5-1 cm, then the pieces of plant tissue were fixed in a solution of FAA (Formalin, Glacial Acetic Acid, and 70% Alcohol) and simultaneously aspirated (Santos et al., 2016; Susetyarini et al., 2020). The fixation stage is the most important part of histology and cytology techniques because it prevents protein denaturation (Jamie, 2010). Dehydration is a process to remove all the fluid contained in the tissue (Rina, 2013). Dehydration process was carried out using graded alcohol (50%, 70%, 95%, and 100%) and dealcoholization using clearing agents such as xylol (Santos et al., 2016; Susetyarini et al., 2020). Clearing using xylol is useful for removing alcohol from the tissue and replacing it with a certain solution (Waheed, 2012). Then, the purification medium was replaced using paraffin (as a planting medium) that previously had thawed with an incubator. Plant tissue was embedded into paraffin blocks to solidifies, then the paraffin block will be carved into a trapezium and will be cut to produce a thin ban using microtome (Santos et al., 2016; Susetyarini et al., 2020). According to Rina (2013), embedding of paraffin is done three times in a certain period of time. Paraffin band was attached to a glass object that had coated with albumin. The last procedure was to color a thin paraffin band with safranin and fast green compounds, then cover it at second time with a cover glass (Santos et al., 2016; Susetyarini et al., 2020). Anatomical preparations will be observed under a light microscope to observe the tissues with magnificent 200-400 ×. All chemical materials were produced by Merck (Germany) for analytical analysis. Morpho-anatomical data were taken once for representative characters.

2.3. DNA barcoding

2.3.1. DNA extraction

DNA extraction was performed using the Tiangen Plant Genomic Kit (Tiangen, Kit). The DNA was extracted from three individuals young leaves of Artemisia vulgaris and each replicate has a mass of 0.1 g. The concentration and purity of DNA was measured using a Thermo Scientific Multiskan Go with absorbances of λ 260 nm and λ 280 nm.

2.4. PCR amplification and sequencing

DNA amplification using 2 cpDNA primers, rbcL (Forward: 5'-AAG TTC CTC CAC CGA ACT GTA-3'; Reverse: 5'-TAC TGC GGG TAC ATG CAA G-3') and matK (Forward: 5'- TGG TTC AGG CTC TTC GCT ATT G-3'; Reverse: 5'-CTG ATA AAT CGG CCC AAA TCG C-3'). These two primers had previously used on Soncus arvensis (Wahyuni et al., 2019), Achillea millefolium (Ilham et al., 2022), Pluchea indica (Wahyuni et al., 2022), and Cosmos caudatus (Purnobasuki et al., 2022). These primers had been specially designed for the Asteraceae family, so that the primers attachment to the target gene has a high chance. The PCR reaction mixture was 35 μL containing: 7.5 μL GoTaq®Green Master Mix; both rbcL and matK primers with the volume of forward and reverse primers is 1.5 μL (concentration 350-500 nM), 5 μL of DNA template (50 ng-1) and nuclease-free water was added until the volume reached 35 μL. Amplification using PCR was carried out by setting it into 5 stages with different temperatures and times: predenaturation 95°C for 5 minutes which occurred for 1 cycle; 35 cycles for denaturation 94°C for 30 seconds, annealing 56°C for 45 seconds, extension 72 °C for 45 seconds; and one cycle for final extension 72°C for 5 minutes. Determination of the PCR product and DNA quality was using gel electrophoresis with 1% agarose gel (Murtiyaningsih, 2017) and visualized with a UV transluminator. Subsequently, the PCR product were sent for sequencing at 1st Base Sequencing Service (Axil Scientific Pte.Ltd., Singapore).

2.5. Data analysis

The data from the morpho-anatomical analysis were analyzed descriptively, while the DNA sequenced from the 1st Base Sequencing Service was further processed using Bioedit 7.4 and Mega X software. Geneious 2021.3.4 software was also used to determine the percentage of nitrogen base composition (Purnobasuki et al., 2022). Furthermore, the consensus data was used for the DNA alignment or matching stage through the online software BLAST (Basic Local Alignment Search Tool). In the final stage, the sample plant sequence data and sequence data from the GenBank database were subjected to phylogenetic tree reconstruction using Neighbor-Joining Method. The data used to form the phylogenetic trees were derived from DNA sequences of the 3 replications of A. vulgaris and 15 plant DNA sequences from the GenBank database that were closely related to the sample plants. Table 1 shows a list of 15 plants from the GenBank database with additional information.

Table 1
Plant data downloaded from GenBank.

Plant (rbcL)	Acession Number	Plant (matK)	Accesion number
Artemisia sieversiana	MG951499.1	Artemisia vulgaris	KR231888.1
Artemisia selengensis	MG951498.1	Artemisia argyi	KM386991.1
Artemisia selengensis	MG951497.1	Artemisia montana	KF887960.1
Artemisia nakaii	MG951494.1	Artemisia sp.	KF697692.1
Artemisia japonica	MG951491.1	Artemisia montana	LC635379.1
Artemisia hallaisanensis	MG951490.1	Artemisia stolonifera	NC_049572.1
Artemisia gmelinii	MG951489.1	Artemisia vulgaris	KX581939.1
Artemisia fukudo	MG951488.1	Artemisia vulgaris	KX581938.1
Artemisia freyniana	MG951487.1	Artemisia vulgaris	KX581937.1
Artemisia capillaris	MG951485.1	Artemisia vulgaris	KX581936.1
Chrysanthemum x morifolium	MK986830.1	Chrysanthemum x morifolium	MK986830.1
Chrysanthemum boreale	MN913565.1	Chrysanthemum	KX522942.1
Chrysanthemum lucidum	NC_040920.1	Chrysanthemum	KX783651.1
Chrysanthemum lucidum	MH028788.1	Ajania fruticulosa	KX526529.1
Chrysanthemum	KX522942.1	Chrysanthemum indicum	MW633069.1

3. Result

The results of descriptive morpho-anatomical observations and processing of DNA sequence data from sample plants and Asteraceae plants are shown in the figures and tables below.

3.1. Morpho-anatomical characterization

The morphology of the vegetative organs including root, stem, and leaves are shown in Figure 1. The general anatomy of the vegetative organs are shown in Figures 2 and 3. Meanwhile, the appearance of the accessory organs of the epidermis including trichomes and stomata are shown in Figure 4.

Figure 1
The morphology of the vegetative organs of Artemisia vulgaris: A. Habitus, B. Root, C. Stem and leaves, D. Distribution of internodes at the base of the stem (BI: basal internodes), E. Adaxial side, F. Abaxial side, G. Distribution of leaves on the middle-bottom of the stem (MBS: distribution of leaves in the middle-bottom of the stem, PT: petiole, and ST: stipule), H. Distribution of leaves on the top of the stem (marked with a red circle).

Figure 2
Anatomy of A-B. Stem and C-D. Rhizomes. EP: epidermis, CL: collenchyma, CR: cortex, VB: vascular bundles, PT: pith, PL: phloem, CB: cambium, and XL: xylem.

Figure 3
A-B. Anatomy of leaves Artemisia vulgaris. ST: Stomata. EP: epidermis, PC: parenchymal cortex, MP: Mesophyll, VB: vascular bundles, PL: palisade, SP: sponge, XL: xylem, and PL: phloem.

Figures 4
Anatomy of stomata and trichomes of Artemisia vulgaris: A. Stomata, B. Glandular trichomes, C. T-shaped trichomes, D. Spiral trichomes, E. Dragger- shaped trichomes.

3.2. DNA barcoding

Quantitative measurement results of Artemisia vulgaris DNA are shown in Table 2 while qualitative measurement results are shown in Figure 5. Results of DNA processing in the form of nitrogen base percentages in Table 3, results of alignment using the BLAST NCBI program in Table 4, results of alignment with plants of close and distant relatives in Figure 6, and the phylogenetic trees in Figure 7.

3.2.1. Measurement of DNA purity and concentration

Table 2
Results of measurement of DNA purity and concentration.

Sample Name	Absorbance 1 (260 nm)	Absorbance 2 (280 nm)	Purity	Concentration (µg/µl)
Artemisia vulgaris 1	0.127	0.064	1.51	3.15
Artemisia vulgaris 2	0.103	0.071	1.45	1.95
Artemisia vulgaris 3	0.116	0.061	1.43	2.6

3.2.2. Visualization of electrophoresis results

Figure 5
Amplification results of A. rbcL gene and B. matK gene.

3.2.3. DNA sequence analysis

Table 3
Comparison of the number of nitrogenous bases in Artemisia vulgaris.

Plant	Percentage of nitrogen bases
Artemisia vulgaris rbcL 1	A: 29.4%, C: 25.9%, G: 19.2%, T: 25.5%
Artemisia vulgaris rbcL 2	A: 30.0%, C: 25.7%, G: 18.8%, T: 25.5%
Artemisia vulgaris rbcL 3	A: 29.9%, C: 25.7%, G: 19.0%, T: 25.5%
Artemisia vulgaris matK 1	A: 29.1%, C: 18.2%, G: 15.9%, T: 36.8%
Artemisia vulgaris matK 2	A: 28.6%, C: 18.1%, G: 16.9%, T: 36.4%
Artemisia vulgaris matK 3	A: 28.5%, C: 18.2%, G: 16.9%, T: 36.4%

3.2.4. Alignment with sequences registered in GenBank

Table 4
Summary of alignment results of rbcL using BLAST.

rbcL
Plant	Max. Score	Total Score	Query Cover	E-Value	Percentage Identity	Accession Number
Artemisia sieversiana	900	900	100%	0.0	99.80%	MG951499.1
Artemisia selengensis	900	900	100%	0.0	99.80%	MG951498.1
Artemisia selengensis	900	900	100%	0.0	99.80%	MG951497.1
Artemisia nakaii	900	900	100%	0.0	99.80%	MG951494.1
Artemisia japonica	900	900	100%	0.0	99.80%	MG951491.1
Artemisia hallaisanensis	900	900	100%	0.0	99.80%	MG951490.1
Artemisia gmelinii	900	900	100%	0.0	99.80%	MG951489.1
Artemisia fukudo	900	900	100%	0.0	99.80%	MG951488.1
Artemisia freyniana	900	900	100%	0.0	99.80%	MG951487.1
Artemisia capillaris	900	900	100%	0.0	99.80%	MG951485.1
Chrysanthemum x morifolium	894	894	100%	0.0	99.59%	MK986830.1
Chrysanthemum boreale	894	894	100%	0.0	99.59%	MN913565.1
Chrysanthemum lucidum	894	894	100%	0.0	99.59%	NC_040920.1
Chrysanthemum lucidum	894	894	100%	0.0	99.59%	MH028788.1
Chrysanthemum x morifolium	894	894	100%	0.0	99.59%	KX522942.1
matK
Plant	Max. Score	Total Score	Query Cover	E-Value	Percentage Identity	Accession Number
Artemisia vulgaris	1279	1279	99%	0.0	99.86%	KR231888.1
Artemisia argyi	1279	1279	99%	0.0	99.86%	KM386991.1
Artemisia montana	1279	1279	99%	0.0	99.86%	KF887960.1
Artemisia sp.	1279	1279	99%	0.0	99.86%	KF697692.1
Artemisia montana	1279	1279	99%	0.0	99.86%	LC635379.1
Artemisia stolonifera	1279	1279	99%	0.0	99.86%	NC_049572.1
Artemisia vulgaris	1279	1279	99%	0.0	99.86%	KX581939.1
Artemisia vulgaris	1279	1279	99%	0.0	99.86%	KX581938.1
Artemisia vulgaris	1279	1279	99%	0.0	99.86%	KX581937.1
Artemisia vulgaris	1279	1279	99%	0.0	99.86%	KX581936.1
Chrysanthemum x morifolium	1232	1232	99%	0.0	99.84%	MK986830.1
Chrysanthemum x morifolium	1232	1232	99%	0.0	99.84%	KX522942.1
Chrysanthemum x morifolium	1232	1232	99%	0.0	99.84%	KX783651.1
Ajania fruticulosa	1232	1232	99%	0.0	99.84%	KX526529.1
Chrysanthemum indicum	1232	1232	99%	0.0	99.84%	MW633069.1

Figure 6
Comparison of the studied Artemisa vulgaris gene sequences with plants from the GenBank database. A. rbcL, B. matK. AV: Artemisia vulgaris being studied, AS: Artemisia sieversiana accession MG951499.1, CXM: Chrysanthemum x morifolium accession KX522942.1, AVG: A. vulgaris accession KR231888.1, CI: Chrysanthemum indicum accession MW633069.1. Blue circles indicate differences in nitrogenous bases and red circles indicate gaps.

Figure 7
Phylogenetic tree of A. rbcL, B. matK.

4. Discussion

4.1. Morpho-anatomy characterization

Artemisia vulgaris has a shrub habitus, rarely seen in the form of perennial herbs, annual herbs or biennial herbs, this shrub habitus is common in most plants of the Artemisia genus (Abuhadra et al., 2017) (Figure 1A). A. vulgaris is a very common plant growing on nitrogenous soils, like weedy and uncultivated areas, such as waste places and roadsides (Setyawati et al., 2015). The plant has woody and brown root (Figure 1B) (Adams et al., 2012; Setiawati et al., 2008). The plant from the genus Artemisia has an erect stem, semiwoody, and the upper third of the stem is branched (Figure 1C). In terms of internodes, it generally has basal internode at the main stem, while in other areas of the stem, the internode is too short and surrounded by lush foliage (El-Sahhar et al., 2010) (Figure 1D). The young stem is terete and pubescent and older stem is somewhat purplish (Farrag et al., 2015; Wiart, 2003). The leaves of A. vulgaris are arranged densely and alternately, the position of the leaves are more inclined to the top of the stem (Figure 1C). The upper part of the lamina (dorsal) is dark green while the lower part (ventral) is light green (Barney and Ditommaso, 2003) (Figure 1E and 1F). Abaxial leaves also have tomentose hairs (Hayat et al., 2009b; Setyawati et al., 2015). This plant species has leaf with bifacial symmetry (Noorbakhsh et al., 2008). Most leaves of A. vulgaris are simplex, petiolate and stipulate. Leaf blades that located in the middle and bottom of the stem have edges with very deep notches (have highly dissected margins), pinnately lobed and the lobes are broadly lanceolate in shape with entire margins and apex acute, these primary lobes are often shallowly cleft by one or more secondary lobes (Figure 1G). The leaves at the top of the stem are also simplex, sessile (without petioles), exstipulate, and smaller in size compared to the leaves at the bottom, which are lanceolate in shape and the edges are not incised (Figure 1H). The leaves have a distinctive aroma when crushed (Weston et al., 2005; Farrag et al., 2015) and spicy taste (Borzabad et al., 2010).

The stem anatomy of A. vulgaris is almost the same as Artemisia absinthium var. absinthium and Artemisia absinthium var. calcigena (Konowalik and Kreitschitz, 2012), which consists of epidermal, collenchyma, cortex, transport bundles, and pith tissues (Figure 2A and 2B). The outermost tissue of the stem is called the epidermis, which is composed of a compact and dense layer of cells with a square to rectangular shape. There are no protective hairs or only a small number of broken bases of trichomes on the epidermis were observed (Ivanescu et al., 2015). Beneath the epidermis, there is a supporting tissue of young stem, namely collenchyma. The difference between epidermis and the collenchyma is related to the size of the cells and the number of cell layers. The collenchyma cells of A. vulgaris were larger and consisted of more than one layer of cells. According to Leroux (2012), in stem and petioles, collenchyma typically occurs in a peripheral position and can be found immediately beneath the epidermis (Leroux, 2012). The cortex which is composed of circular parenchyma cells, was not the largest tissue that composes the stem. The vascular bundles in this plant are collateral type, arranged in different sizes and have alternately circle patterns. Each bundle has a well-defined fibrous cap abutting the phloem (El-Sahhar et al., 2011). The arrangement of the xylem vessels in this plant is similar to Artemisa absinthium, the vessels are arranged in radial rows, continuously or discontinuously (Ivanescu et al., 2015). The cambium can be differentiated and represented by the presence of 3-4 cell rows (Farrag et al., 2015). The largest tissue that composes the stem is the pith. This tissue is formed by large parenchyma cells and is in the central region. The anatomy of the A. vulgaris rhizome in this study is similar to Artemisia umbelliformis rhizome (Janaćković et al., 2021) (Figure 2C and 2D). The rhizome is also known as a modified stem. From the observation, the size of the rhizomes’ cortex, is quite large when compared to the stem.

Histology of A. vulgaris leaves has the same structure with flowering plants, composing of epidermal, mesophyll, cortical parenchyma, and vascular tissue (Figure 3A and 3B). On the leaf lobe surface, a single-layered epidermis was observed on both sides of the leaf. On the frontal side, the leaf epidermis is composed of a single layer cells with irregular polygonal shapes. In some areas of the epidermis, there are several gaps indicating the presence of stomata. According to Noorbakhsh et al. (2008), the plant from genus Artemisia have stomata only on the abaxial or inferior side of the leaf, such as in Artemisia absinthium var. absinthium and var. calcigena (Noorbakhsh et al., 2008; Konowalik and Kreitschitz, 2012). Hussain et al. (2019) also reported that from 13 Artemisia species, the majority showed the presence of stomata on the abaxial side. However, according to El-Sahhar et al. (2011) and Farrag et al. (2015), A. vulgaris stomata are found on both sides of the leaf, but the most abundant is on the abaxial side. In Artemisia fragrans, the number of stomata on the adaxial side is larger than the abaxial side (Sadigova, 2022). In addition, the type of stomata of this plant is anomocytic (Hussain, 2020), similar to the stomata of Artemisia china (Ermayanti et al., 2004) (Figure 4A). In this type of stomata, the subsidiary cells are indiscernible from other epidermal cells (Karbalaei et al., 2021).

Besides stomata, 2 general types of trichomes, namely glandular trichomes and non-glandular trichomes were also present in the epidermis. Trichomes play an important role in plant taxonomy due to its variance in size, shape, morphology, cell number and composition (Schilmiller et al., 2008). Non-glandular trichomes helps to deter herbivores, disperse seeds, absorb water and so on. While glandular trichomes act as storage, and secretion of various types of plant secondary metabolites (Wu et al., 2012). Several secondary metabolites produced by glandular trichomes are utilized in pharmaceutical industry for health purposes (Liu et al., 2019). The anatomy of the glandular trichomes of A. vulgaris is similar to Artemisia annua. These trichomes are composed of two basal cells, two stalk cells and six secretory cells (Duke and Paul, 1993; Juliarni et al., 2007; Cui et al., 2020) (Figure 4B), which are similar to other plants in general (Feng et al., 2021). These six secretory cells are border to the secretory cavity and contribute to filling the apical subcuticular space with secondary metabolites (Anders et al., 2012). Glandular trichomes of A. vulgaris only have one shape, while non-glandular trichomes have several shapes, such as letter “T” shape, spiral and Dragger-shaped. T-shaped trichomes are composed of stalk and elongated cells (Soetaert et al., 2013) (Figure 4C). T-shaped trichomes not only found in the same species (Noorbakhsh et al., 2008), but also in other species such as Artemisia roxburghiana, Artemisia dubia, Artemisia moorcroftiana, Artemisia tangutica, Artemisia biennis, Artemisia macrocephala, Artemisia Japonica, Artemisia amygdalina, Artemisia absinthium, and Artemisia sieversiana (Hayat et al., 2009a). In some species, such as Artemisia annua, the T-shaped trichomes have the oddity of being able to synthesize the anti malarial drug Artemisinin (Judd et al., 2019). Spiral trichomes have a folding form along their entire length, being erect or appressed (Roudi et al., 2020) (Figure 4D). Whereas dragger-shaped hair is rarely present and consists of 1 to 4 short basal cells with a dragger-shaped apical end cell (Farrag et al., 2015) (Figure 4E). In complex and changeable environment, non-glandular trichomes on plant surface plays important role than glandular trichomes. The morphology and structure of non-glandular trichomes varies with plant species, so the characteristics of non-glandular trichomes are regarded as important characteristics for microscopic identification of plant medicinal materials (Cui et al., 2020). Unlike the plants studied having 1 form of glandular trichomes and 3 forms of non-glandular trichomes, other plants such as Artemisia argyi have 2 forms of glandular trichomes and 1 form of non-glandular trichomes (Cui et al., 2020).

Many traits can be identified from the epidermis. The compact arrangement of epidermal cells and presence of stomata and trichomes are the main features of A. vulgaris leaf epidermis (El-Sahhar et al., 2011). According to Dickison (2000), characteristics of the leaf epidermis play an important role in the identification of a species. These characters include the types of trichome glands, number of subsidiary cells, size of the guard cells, orientation and distribution of the stomata and sizes of the epidermal cells. The foliar epidermal cells of Artemisia are remarkable characters for the distinction of different species (Hayat et al., 2009a). The tissue after epidermis is the mesophyll tissue. Mesophyll of A. vulgaris differentiated into palisade and spongy tissue (Figure 3A and 3B). Half of the mesophyll thickness is occupied by palisade tissue. The palisade tissue is located on one side of the leaf namely the adaxial side (Juliarni et al., 2007; Noorbakhsh et al., 2008; Farrag et al., 2015). The palisade tissue cells are oval, tightly arranged in 1 row and discontinuous in the midrib³. The palisade cells contain red-brown colored content (Farrag et al., 2015). When compared with Artemisia china, the palisade tissue of A. china has a slightly rounded shape (Ermayanti et al., 2004). Sponge tissue is located on the abaxial side of the leaf, with irregularly shaped cells and very low intercellular density, causing large intercellular spaces. These intercellular spaces show aerenchyma (Farrag et al., 2015). The midrib is more prominent on the abaxial side, where it displays the parenchymal cortex and bundle vessels in a spheroidal shape (Noorbakhsh et al., 2008) (Figure 3A and 3B). The xylem is located at the top (El-Sahhar et al., 2011), characterized by the presence of large vessels arranged radially. The plants studied were cultivated plants, therefore they differed from wild plants in terms of leaf vascular tissue. In the wild plant, the vascular tissue consisted of one large central vascular bundle and a smaller one at each side, while the cultivated plant showed only one central vascular bundle (Farrag et al., 2015) (Figure 3A and 3B).

4.2. DNA barcoding

4.2.1. Measurement of DNA purity and concentration

The results of DNA purity and concentration using a spectrophotometer is shown in Table 2. The DNA purity of Artemisia vulgaris has a value of 1.43-1.51, while the concentration ranges from 1.95-3.15µg/µl. DNA purity is determined by comparing the wavelength absorbed by DNA (260 nm) and contaminants such as protein and phenol (280 nm). The formula is λ 260 nm/ λ 280 nm and good DNA purity values are 1.8-2.0 (Dewanata and Mushlih, 2021). If the comparison value of λ 260/λ 280 is less than 1.8, it indicates the presence of phenol or protein contaminants from the extraction process, whereas if the ratio is more than 2.0, the DNA contains RNA (Rizko et al., 2020). Contamination could be from incomplete lysis of cell components or due to the presence of phenol during the isolation process (Gross-Bellard et al., 1973).

4.2.2. Visualization of electrophoresis results

The primers used in the PCR process succeeded in amplifying the rbcL and matK genes in Artemisia vulgaris, indicated by a good intense DNA band (Figure 5A and 5B). The thickness of the rbcL and matK gene bands belonging to A. vulgaris has the same quantity as Achillea millefolium (Ilham et al., 2022) and Cosmos caudatus (Purnobasuki et al., 2022), because the primer used was same. According to the National Laboratory of Enteric Pathogens (1991) the key to the success of the PCR in diagnosis resides in its ability to amplify regions within a single molecule of DNA which may have etiologic significance (National Laboratory of Enteric Pathogens, 1991). There are several factors that influence the appearance of DNA during the PCR process: (1) triphosphate deoxyribonucleotide (dNTP), (2) primer design, (3) DNA template, (4) buffer composition, (5) number of cycles during PCR, 6) enzymes and (7) technical and nontechnical factorsy (Yuwono, 2007). Among the seven factors, primer design is the most important factors in determining the success of PCR. This is because the primer identifies the region of the DNA of interest. Primer design can be done based on a known DNA sequence or from a sequence related to the target protein (Triyani et al., 2016). From the results of sequencing by the 1st Base and processing using Bioedit 7.4 and Mega X, the rbcL gene had 490 bp, 494 bp, and 495 bp of nucleotides. Meanwhile, the matK gene were 666 bp, 697 bp, and 698 bp. Compared with studies using the same primers for Achillea millefolium by Ilham et al. (2022) and Cosmos caudatus by Purnobasuki et al. (2022), the number of nucleotides was almost similar, with rbcL gene, ranging from size 470 -500 bp while matK gene from 690-710 bp. Furthermore, A. vulgaris DNA sequences were subjected to BLAST (Basic Local Alignment Search Tool) process, to compare the sequences with other DNA sequences in BOLD (Barcode of Life Database) (BOLD, 2021) and GenBank NCBI (NCBI, 2021; Tindi et al., 2017).

4.2.3. DNA sequence analysis

DNA sequence analysis was performed to determine the percentage of each nitrogenous base that makes up a gene. The rbcL gene from the three replications of Artemisia vulgaris has a composition of 29.4-30.0% adenine, 18.8-19.2% guanine, 25.7-25.9% cytosine, and 25.5% thymine. Meanwhile, the matK gene consists of 28.5-29.1% adenine, 15.9-16.9% guanine, 18.1-18.2% cytosine, and 36.4-36.8% thymine (Table 3). From the results, adenine and thymine dominates the nucleotide composition of the two genes. Adenine-thymine dominance also occurs in other plants such as Cosmos caudatus (Purnobasuki et al., 2022), Sonchus arvensis (Wahyuni et al., 2019), Pinus (Sing et al., 2021), seagrass (Stevanus and Pharmawati, 2021), and Andrographis paniculate (Arif et al., 2019). High adenine-thymine content is found in chloroplast-based genes as well as mitochondria-based genes such as the COI gene with similar characteristics (Nugroho et al., 2017; Rahayu et al., 2019). The result is in line with Smith et al. (2011) that adenine and thymine comprise most of the mitochondrial and plastid genome sequences. The higher adenine-thymine content than the guanine-cytosine content is due to the high variability in nucleotide composition and the higher rate of nucleotide substitution in the amplified gene (Ismail et al., 2020).

4.2.4. Alignment with sequences registered in GenBank

The results of the sequences alignment using the BLAST program showed that rbcL and matK of Artemisia vulgaris from the Taman Husada Graha Famili Surabaya collection had a high similarity percentage (percentage identity) of more than 98% in both plants from the same genus or plants from different genus within the Asteraceae family (Table 4). According to Janda and Abbott (2007), if the percentage identity has a value of 100% or above 97%, it can be confirmed as the same species. The absence of A. vulgaris sequences from the GenBank database for the rbcL gene is probably due to the plant species has not been registered. Besides percentage identity, the similarity or closeness between plant species can also be determine using other parameters such as e-value, query cover, and score. E-value shows similarity between the studied A. vulgaris DNA sequences with plants registered in the GenBank database, if the value is close to or same as 0.0, the similarity is even higher (Sahaba et al., 2021). Query coverage is the percentage of the nucleotides length aligned with the existing database on BLAST (Fathiya et al., 2018). The homology level will be higher if the query coverage percentage value is also higher (Mukhopadhyay et al., 2018).

There were differences in the number of nitrogen bases and gaps when the studied plants were compared with other plants that were closely related and distantly related. In the case of the rbcL gene, comparisons were made with Artemisia sieversiana accession MG951499.1 and Chrysanthemum x morifolium accession KX522942.1, while the matK gene was compared with A. vulgaris accession KR231888.1 and Chrysanthemum indicum accession MW633069.1. The type of mutation that occurs is a point mutation. Comparison based on rbcL gene shows that the transversion occurs at nitrogen bases number 724, 816, and 1212, while the transition occurs at nitrogen base number 725 (Figure 6A). In addition, comparison based on matK gene also shows mutation with a more complex variety with transition, transversion, insertion and deletion activities. The transition occurs at nitrogen base number 339, transversion at 385 while insertion and deletion occur at 751,752, 753, 754, 755, 756, 757, 758, 759, and 1040. The three plant replicates studied had different positions of nitrogen bases and gaps that were 100% similar when compared to the plants from the GenBank database, indicating that the three replicates were indeed the same species. This is in accordance with the statement of Yang et al. (2018) that each individual has an SNP (Single Nucleotide Polymorphism) or commonly known as point mutation, and the same species will have SNP positions at the same range of base pairs. If the comparison between the sequences based on the rbcL gene is further analyzed, the difference in the composition of the nitrogen base between the studied plants and Artemisia sieversiana accession mg951499.1 has a small quantity if compared to the studied plants with chrysanthemum x morifolium Accession KX5222942.1 (Figure 6A). This is similar with the matK gene, the difference in nitrogen bases and the gaps between the plants under study with A. vulgaris accession KR231888.1 are smaller than plants under study with Chrysanthemum indicum accession MW633069.1. This indicates a close relationship between levels of taxon groups with more or fewer differences in nitrogenous bases.

Point mutation is a mutation that involve the replacement of one base pair of nucleotide (Julianti et al., 2015). Differences in nucleotide composition are generally caused by substitution events, substitution activities include transition and transversion (Huelsenbeck and Nielsen, 1999). Meanwhile, gaps in the DNA sequence are caused by insertion and deletion activities (Fan et al., 2007). According to Pandey et al. (2020), the barcoding gap is defined as the relation between the maximum intraspecific distances within each species, and the minimum interspecific distance with its nearest neighbor (Pandey et al., 2020). The consequences of point mutation are broadly divided into three. The first is a silent mutation or synonym mutation, which occurs when changes in the nitrogenous bases in the DNA sequence do not change the sequence of amino acids that make up the protein (Zheng et al., 2014). Next is a missense mutation, a change that causes the replacement of one amino acid with another in a protein-coding gene region (Cotton and Scriver, 1998). The last is nonsense mutation, which adds a premature stop signal that hinders any further translation of a protein-coding gene (Torella et al., 2020).

4.2.5. Phylogenetic tree

rbcL gene provides a good resolution in classifying species based on their taxonomic level (Figure 7A). The rbcL gene phylogenetic tree is split into 3 main clusters, except for Artemisia fukudo accession MG951488.1 which is not part of the three clusters. Cluster number one contains three replicated plants being studied with the same branch size, this indicates they are identical and came from the same parent or ancestor. According to Chen et al. (2015), rbcL is a gene sequence in the chloroplast genome that is derived directly from the parent. If there is a discovery of plant sequences based on the rbcL gene that has minimal variation and a highly conservative site, it can be concluded that these plants came from the same parent or the same ancestor. Cluster number two contains many different species in the genus Artemisia and the last cluster contains various species in the genus Chrysanthemum. It should be noted that A. vulgaris from the GenBank database is not yet available for the rbcL gene. This is not the case for matK gene because A. vulgaris data is already available in the GenBank database (Figure 7B). The phylogenetic tree is split into three main clusters, except for Artemisia montana accession KF887960.1. Unlike rbcL gene phylogenetic tree, matK gene phylogenetic tree offers resolution of placing the same species into different clusters. This is the case for A. vulgaris, which occupies clusters number one and two. The reason could be because matK has vast substitution rates in comparison to other chloroplast genes, and its gene sequence lies among the lowest conserved plastid genes (Zarei et al., 2020). However, in plants of different genus, there is no problem in separation for example genera Chrysanthemum and Ajania do not mix with plants from the genus Artemisia. Even so, the similarity between rbcL and matK based phylogenetic trees still position the three plant replications studied into the same group. This indicates that the same apomorphic/synapomorphic characters have been derived from the monophyletic characters in these samples forming a cluster (Pangestika et al., 2015). A similar case has been reported by Tallei and Kolondam (2015) that single locus of matK gene cannot be used to differentiate species in Myristica; it can only be used to differentiate the genus level within the Myristicaceae family. Ho et al. (2021) also reported that the discriminating abilities of the matK and rbcL genes in jewel orchid plants showed a different level of efficiency, the rbcL gene was considered to have better discriminating power than the matK gene alone or the rbcL+matK gene combination. According to Alasmari (2020) the separability of the matK gene to differentiate between species is not always reliable, because in their study, only 2 samples of the 4 sequences tested could be identified correctly at the species level. The ideal locus for DNA barcoding of plants remains debatable since some loci are efficient for some taxonomic groups and the species discrimination of these genes varies among plant species. Although the result of the matK gene phylogenetic tree is not sufficiently accurate to differentiate plants at species level, the rbcL gene-based data would help with this deficiency, this is in accordance with the study by Dong et al. (2012) that one DNA barcoding marker is not enough to obtain sufficiently accurate and specific identification results (Dong et al., 2012), so matK and rbcL must be used as dual DNA barcoding procedures (Alasmari, 2020).

In this study, it can be concluded that A. vulgaris has distinctive morphological characteristics on its leaves. The leaves distributed on the bottom-center are characterized by being large, petiolate, stipules, and having deep notches. While the leaves located at the top of the stem are smaller in size, do not have petioles and stipules and the incisions are not too deep or do not have incisions at all. The key feature that is used as an anatomical standard to distinguish A. vulgaris from other plants is the histology of the epidermis, because it retains various unique characteristics of the stomata and trichomes. Trichomes have 2 types, namely glandular with one shape and non-glandular with three shapes (T-shaped, spiral, and dragger). Through the BLAST process and phylogenetic tree reconstruction, the plant sequences being studied are closely related to several species of the genus Artemisia as indicated by a percentage identity of above 98% and the proximity of branches between taxa in the reconstructed phylogenetic.

5. Conclusion

In summary, Artemisia vulgaris exhibits a shrub-like appearance and is commonly found in nitrogen-rich uncultivated areas. Its distinct features include an upright stem, and densely arranged alternate leaves with varied sizes and shapes, emitting a unique aroma when crushed. Notably, the leaves possess different types of trichomes, influencing their taxonomy and secondary metabolite production. The stem anatomy consists of distinct tissues, including epidermis with anomocitic stomata, collenchyma, cortex, vascular bundles, and pith. Through DNA barcoding using the rbcL and matK genes, the study establishes the plant's close relationship with other Artemisia species within the Asteraceae family. This comprehensive exploration sheds light on both the physical and molecular characteristics of Artemisia vulgaris, contributing to its identification and classification in the plant realm.

Acknowledgements

The authors thank Plant Medicinal Garden “Taman Husada Graha Famili” Surabaya, East Java Indonesia for providing sample plants. This study was financially supported by Universitas Airlangga, Indonesia 2023 Fiscal Year, following The Decree of Rector of Universitas Airlangga Number: 1601/UN3.LPPM/PT.01.03/2023.

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Publication Dates

• Publication in this collection
  26 Feb 2024
• Date of issue
  2024

History

• Received
  11 Sept 2023
• Accepted
  06 Jan 2024