Iraqi Journal of Agricultural Sciences –2021:52(1):249-258 Abdulrahman & et al. PHENETIC AND UNSUPERVISED MULTIVARIATE ANALYSIS POLYANTHUM (WIGHT) WALP Mahmoud Dogara Abdulrahman1, 2† Hasan Nudin Nur Fatihah1 Mohammed Abdul Manaf Ali1 Nashriyah Mat1 Moneruzzaman Khandaker1† 1Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Tembila Campus, 22200 Besut, Terengganu, Malaysia 2Department of Biological Sciences, Faculty of Science, Kaduna State University, Kaduna. Nigeria *Corresponding author: [email protected]; [email protected] ABSTRACT family is widely distributed in Asia has been the largest group of ; mainly trees and few shrubs. Distributed all over the world in tropical and subtropical areas. Syzygium is the largest genus with economical value found all over the Malaysian Peninsular. Evolutionary relationships within the Syzygium is unclear and there are currently no reliable criteria to divide the genus into manageable entities for systematic study. Species of Syzygium is the richest genus of woody in South East Asia with approximately 1000 or more species but little is known about the genus. Syzygium polyanthum Wight is one of the favourites Ulam that have been consumed for ages in Peninsular Malaysia and also as herbal medicine. The species is widely misunderstood due to extreme morphological variability, similarity in aroma and flavor. The species is substituted or adulterated with several other species. The study was aimed to construct phenetic tree and unsupervised multivariate analysis from morphological and anatomical the data matrix. Phenetic analysis, Principal component and hierarchical cluster analysis revealed they are two different cultivars species but inter variation exited among cultivars of same species. The above documented information has added new taxonomic information with regard to the identification of the cultivars in Peninsular Malaysia. The study recommends further study on de novo sequence of Serai kayu and Serai kayu hutan. Keywords: Phylogenetic, Morphology, Anatomy, Phenetic

مجلة العلوم الزراعية العراقية -2021 :52 (1(:249-258 عبد الرحمن وآخرون تحليل متعدد التخصصات غير الخاضع للرقابة SYZYGIUM POLYANTHUM (WIGHT) WALP محمود دوغارا عبد الرحمن حسن نودين نور فاتح محمد عبد المناف علي ناشرّية مات ، مناف علي نشريا مات منير الزمان خنداكر المستخلص تنتشر عائلة Myrtaceae على نطاق واسع في آسيا وقد كانت أكبر مجموعة من النباتات ؛ بشكل رئيسي األشجار والقليل من الشجيرات. موزعة في جميع أنحاء العالم في المناطق االستوائية وشبه االستوائية. Syzygium هو أكبر جنس ذو قيمة اقتصادية موجود في جميع أنحاء شبه الجزيرة الماليزية. العالقات التطورية داخل Syzygium غير واضحة وال توجد حالًيا معايير موثوقة لتقسيم الجنس إلى كيانات يمكن إدارتها للدراسة المنهجية. أنواع نباتات السيزيجيوم هي أغنى جنس من النباتات الخشبية في جنوب شرق آسيا مع ما يقرب من 1000 نوع أو أكثر ولكن ال ُيعرف سوى القليل عن الجنس. يعد Syzygium polyanthum Wight واحًدا من Ulam المفضلة التي تم تناولها على مر العصور في شبه الجزيرة الماليزية وأي ًضا كدواء عشبي. ُيساء فهم النوع على نطاق واسع بسبب التباين المورفولوجي الشديد والتشابه في الرائحة والنكهة. يتم استبدال أو غش األنواع بعدة أنواع أخرى. هدفت الدراسة إلى بناء شجرة ظاهرية وتحليل متعدد المتغيرات بدون إشراف من مصفوفة البيانات المورفولوجية والتشريحية. أظهر التحليل الوراثي والمكون الرئيسي والتحليل الهرمي العنقودي أنهما نوعان مختلفان من األصناف ولكن االختالف المتبادل بين األصناف من نفس النوع. أضافت المعلومات الموثقة أعاله معلومات تصنيفية جديدة فيما يتعلق بتحديد األصناف في شبه جزيرة ماليزيا. توصي الدراسة بمزيد من الدراسة حول تسلسل de novo لـ Serai kayu و Serai kayu hutan. الكلمات المفتاحية: علم الوراثة ، علم التشريح ، علم التشريح الوراثي

Received:2/2/2020, Accepted:22/5/2020

249 Iraqi Journal of Agricultural Sciences –2021:52(1):249-258 Abdulrahman & et al. INTRODUCTION millimeters long but differ in size and colour Myrtaceae family is a tropical trees and shrubs based on the species and enviromental factor with nearly about 55,000 species, grouped into (Gamage et al., 2003). Most members of the two sub-family, 17 tribes and 142 genera. genus their fruits are oblong ranging from 0.5 They are mainly found in large amount of to 3.5 centimeters long which are black or dark number in Central America, South America, purple, edible and flesh and almost all species Australia and southern hemisphere (Harrington bear seed within the fruits (Gamage et al., & Gadek, 2004; Craven & Biffin, 2010). 2003; Retamales et al., 2015). Most members Generally, the leaves of Myrtaceae family are their fruits are sweet, flavour and mildly sour in opposite direction, phloem internal, ever (Retamales et al., 2015). Syzygium polyanthum green and woody and abundance of oil glands W., is a tree or shrub ranging between 22m tall in most of the members (Abdulrahman et al., with glabrous or greenish brown bark. 2018a). They are bisexual, poly stemonous, Opposite leaves with eliptic apex angle and fully inferior or partial inferior ovaries mostly cuneate base shape. S polyanthum grow low with nectariferous hypanthium and land areas, widely distributed in tropical, actinomorphic flower (Wilson et al., 2001; subtropical region of the world. Syzygium Gamage et al., 2003). The fruit of the family polyanthum native to Malaysia, china, are normally divided as either fleshy (berry) or Thailand and . Known by Malay dry (capsular or nut-like), but numerous other ethnic group as Serai kayu and Serai kayu fruit types, such as drupes, and various hutan and locally identified by Indonesian as intermediates, also occur within the group laurel Indonesia or bay leaf, widely used in (Wilson 201). Syzygium is a large genus of their daily diet as Ulam in order to enhance Myrtaceae, occurring from Africa eastwards to their appetite (Abdulrahman et al., 2018b). the Hawaiian Islands and from India and Traditionally, the plants are known for the southern China southwards to south eastern treatment of diabetes, malarial fever, Australia and New Zealand (Van Wyk, 1985). gastrointestinal infection, ulcer, endometriosis In terms of species richness, the genus is and also health improvement through centered in Malaysia but in terms of its basic postpartum (Abdulrahman et al., 2018b). evolutionary diversity it appears to be centered Despite the medicinal history or record of in the Melanesian-Australian region (Hussin et Syzygium polyanthum in Peninsular Malaysia al., 1992). Syzygium are densely foliaceous the cultivars are widely misidentified or and large ever green plants with thick bark and misnamed in ecological inventories or surveys grayish brown utilised in woody industry which cause serious problems for biodiversity (Abdulrahman et al., 2018a). Wood from management. The taxonomy of Syzygium Syzygium is durable, close grained and whitish polyanthum is still controversial due to the (Soh & Parnell 2011). They have leathery extreme morphological variability. leaves, obovate elliptic or oblong ovate shape Multivariate data was utilised (qualitative and with six to twelve centimeters (Retamales et quantitative) to determine the relationship of al., 2015). There is wide variation in shape, closely related species. The present study aims presence of primary, secondary and tertiary at adding taxonomic information to the genus vein, shining, smooth and the tips of the leaves Syzygium; through phenetic and cluster are less acuminate and broad (Abdulrahman et analysis of Syzygium polyanthum cultivar al., 2018a). From the branch lets below the (Serai kayu and Serai kayu hutan) based on leaves panicles arise which are four to six morphology and anatomical data set. centimeters and being terminal or axillary MATERIALS AND METHODS (Retamales et al., 2015). The flower from Plant materials Syzygium is in cluster oblong or round shape, Samples of Serai kayu were collected from greenish white and scented. The calyx is Terengganu, Kelantan, Johor (Batu Pahat area) about four millimeters long with funnel shape and Penang; while Serai kayu hutan was and toothed (Abdulrahman et al., 2018b). The collected from Terengganu, Peninsular petals cohere looks like a small disk (Gamage Malaysia, and were taken to the Forest et al., 2003). Numerous stamens about four Research Institute of Malaysia (KEP),

250 Iraqi Journal of Agricultural Sciences –2021:52(1):249-258 Abdulrahman & et al. Universiti Kebangsaan Malaysia (UKMB) and importance (Table 1). The following plants University of Malaya (KLU) for identification. features can be observed and coded from The plant samples of Serai kayu and Serai herbarium specimen or live plants (Fatihah et kayu hutan were mounted, pressed and al., 2012; Monteiro et al., 2015). Table 2 deposited at the Herbarium of Faculty of present fourteen anatomical characteristics Bioresources and Food Industry, Universiti were observed, measured and coded (Fatihah Sultan Zainal Abidin (UniSZA) with voucher et al., 2012). Syzygium mytifolium was used as numbers of 395 and 396, respectively. outgroup to determine the relationship and Morpho anatomical measurement and closeness of the cultivars of Syzygium character coding polyanthum (Serai kayu and Serai kayu hutan) A total of twenty morphological characteristics in Peninsular Malaysia. Phenetic analysis was were observed, measured and coded , the carried out using Phylogenetic Analysis Using characteristics were all derived from the leaf Parsimony (PAUP) software (V. 4.0b1; parts of the plants (leaf length, width and Swofford) was used to generate trees (Fatihah shape) because they are taxonomically et al., 2012). Table 1. Morphological Characters and Character States Used S/N Characters Character coding 1 Leaf length 0=less or equal to 9 cm; 1= less or equal to 18 cm 2 Leaf width 0= less or equal to 2 cm; 1= less or equal to 4 cm 3 Petiole length 0= less or equal to 0.5 cm; 1= less or equal to 1 cm 4 Petiole morphology 0= base swollen; 1= pulvinate 5 Petiole attachment 0=marginal; 1=peltate central 6 Leaf shape 0= eliptic; 1= obovate 7 Leaf symmetry 0=symmetrical; 1=base asymmetrical 8 Leaf base angle 0=acute; 1= obtuse 9 Leaf apex angle 0=acute;1=acute to acuminate 10 Leaf apex shape 0=straight; 1=convex 11 Leaf base shape 0= cuneate; 1=convex 12 Leaf margin 0=entire; 1=serrate 13 Leaf lobation 0=unlobed; 1=bilobed 14 Leaf colour 0=evergreen; 1=partial 15 Leaf odour 0= nil; 1= aromatic 16 Vein type 0=pinnate; 1=basal 17 Vein agrophic 0=simple agrophic; 1=compound agrophic 18 Vein spacing 0=uniform; 1=irregular 19 Vein category 0=opposite percurrent; 1=random reticulate 20 Tertiary vein 0= present; 1= absent Table 2. Anatomical Characters and Character States Used S/N Anatomical characters Character state 1 Type of stomata 0=anisocytic; 1=anomocytic 2 No of palisade mesophyll layer 0=1 layer; 1= 3 layers 3 Spongy mesophyll layer 0=unequal; 1=equal 4 Oil gland 0=absent; 1=present 5 Cell inclusions in lamina 0=absent; 1=present 6 Location of stomata 0=abaxial; 1=adaxial 7 Density of stomata 0=moderate; 1=high 8 Trichome on lamina 0=absent; 1=present 9 Trichome on midrib 0=absent; 1=present 10 Trichome on petiole 0=absent; 1=present 11 Vascular tissue at midrib 0=close; 1=open 12 Presence of arm in vascular tissue 0= no; 1= yes 13 Vascular tissue at petiole 0=close; 1=open 14 Vascular tissue at stem 0=hexagonal 1= circle Unsupervised pattern establish relationship exist between Serai kayu Unsupervised pattern of multivariate analyses and Serai kayu hutan. were carried out through Principal RESULTS AND DISCUSSION Components Analysis (PCA) and Hierarchical Tree of phenetic was constructed in order to Cluster Analysis (HCA) using SIMCA-P establish the evolutionary relationship that (V.14.1 Umetrics Sweden) software to exists between Syzygium polyanthum cultivars (Serai kayu and Serai kayu hutan) based on the

251 Iraqi Journal of Agricultural Sciences –2021:52(1):249-258 Abdulrahman & et al. data matrix derived from their morphological with bootstrap value 90 %, the first clade and anatomical features respectively. Syzygium contain only the outgroup species Syzygium mytifolium was used as outgroup through out mytifolium (Figure 1). The second major the study. Phenetic construction using the clades divide into three subclades which morphological and anatomical data matrix contain all the five species of Serai kayu (Figure 1) was found to have Consistency accounting to bootstrap value of 64, 61 % and index (CI) of 0.8500, Retention index (RI) of the last clades collapse. The last clade with a 0.9524 and homoplasy index (HI) 0.1500; the bootstrap value of 100% contain all the five phylogenetic tree constructed from the species of Serai kayu hutan bases on the combine data divided in to three major clades combined data matrix (Figure 1).

Figure 1. Phenetic Tree of Morphological and Anatomical Features of Serai kayu and Serai kayu hutan. Under line numbers are percentage of bootstrap value. Branches without values have less than 50% bootstrap value

252 Iraqi Journal of Agricultural Sciences –2021:52(1):249-258 Abdulrahman & et al. Discrimination and classification of the plants main clusters in Serai kayu hutan. Similarly, in species based on multivariate analysis was PC2 three main clusters were formed which carried out using unsupervised analysis. This comprises of both the Serai kayu and Serai is due to the fact of unbiasness of the methods kayu hutan along the negative score plots PCA and HCA . The following study adopted (Figure 2). While in the positive score plots of the unsupervised pattern to discriminate and the PC2 two main clusters were formed of classified Syzygium polyanthum cultivars. both Serai kayu and Serai kayu hutan. The Spectra filter model was obtained from the PCA established relationship existed between combination of morphological and anatomical the two cultivars as no discrimination was data with X matrix with highest variation (R2X found in the PC2. Variation also existed (cum). 0.974) and the highest predictive power within the cultivar of same species as it can be (fitness of the model) (Q2 (cum).0.913). The seen in Serai kayu and Serai kayu hutan PCA constructed from combination of (Figure 2). Loading plots display the morphological and anatomical data; correlation structure of the variables. Loading established the relationship (similarities and plots revealed the variables responsible for the discrimination) that existed between the two discrimination between the two cultivars. The cultivars Serai kayu and Serai kayu hutan score and loading plots are complementing (Figure 2). The PCA clearly discriminate the each other. The position of objects in a given two species into two cultivar along the PC1 direction in a score plot is influenced by but no clear discrimination was found along variables lying in the same direction in the the PC2 (Figure 2). Along the PC1 two main loading plot. clusters were formed in Serai kayu and three

Figure 2. PCA Score Plot of morphological and anatomical features of Syzygium polyanthum (SK; Serai kayu and SKH; Serai kayu hutan)

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Figure 3. Loading line of PC1 morphological and anatomical features of Syzygium polyanthum (SK; Serai kayu and SKH; Serai kayu hutan)

Figure 4. Loading line of PC1 morphological and anatomical features of Syzygium polyanthum (SK; Serai kayu and SKH; Serai kayu hutan) Biplots was constructed augments and allow display clearly the variables responsible for the us to further understand the score plots and relationship among the examined cultivars loading line relationship and discrimination to (Figure 5).

Figure 5. Biplots of the morphological and anatomical features of Syzygium polyanthum (Serai kayu and Serai kayu hutan)

254 Iraqi Journal of Agricultural Sciences –2021:52(1):249-258 Abdulrahman & et al. Hierarchical cluster analysis (HCA) was was also divided into two subclade, the first constructed from the combination of subclade contain species of Serai kayu hutan morphological and anatomical data matrix; (Figure 6). Second subclade further divided resulted in to two major clades, with first into two mini clades. The first mini clade major clade divided into two subclades, the contain only one species of Serai kayu hutan first subclade contain two species of Serai and the second mini clade contain two species kayu and the second subclade contain three of Serai kayu hutan. species of Serai kayu. The second major clade

Serai kayu Serai kayu hutan Figure 6. Dendrogram of the HCA on the morphology and anatomy of Syzygium polyanthum cultivars (Serai kayu and Serai kayu hutan) As a result of diverse challenges attached to taxonomic identification of the plants (Peters the molecular identification of plants, wrong et al., 2014). In studying taxonomic identification happened with consequences in relationship in plants, morphological the wrong deposit of sequence in the NCBI characters are still consider valuable (Brazeau (gene bank). In the process of extracting DNA et al., 2017). Though molecular sequence data from plant sample, you might end of extracting is trying to outpace it but it remains bacterial or fungal DNA attached to the plant indispensable in order to established ancestral sample (Klingenberg & Gidaszewski, 2010). phenotypic conditions. The study first Looking at this numerous challenges; there is documents the phenetic analysis of the genus quite a number of reasons why phenetic Syzygium based on the morphological and analysis of morphological data is of paramount anatomical data matrix; with the case study of importance (Baum & Smith, 2011). There are Syzygium polyanthum cultivars as most of the some taxa that are presently extinct, which previous study carried out on the genus are make it very difficult for molecular studies purely on qualitative morphological and (Lawing & Polly, 2010). For extinct species, anatomical features. Analogous situation was you can only establish or traced their fossil previously reported by Fatihah et al. (2012) on record from phenetic analysis of their studies phylogenetic analysis of seven morphological data which can be obtained varieties of Ficus deltoidea Jack in Peninsular from the literatures (Lawing & Polly, 2010). Malaysia. previously genus Syzygium were Quite a number of studies have been carried classified in peninsular Malaysia based on out in order to identify plants material to physical observation of morphological data overcome adulteration and this add to the (Hussain et al., 1992). Hussain et al. (1992)

255 Iraqi Journal of Agricultural Sciences –2021:52(1):249-258 Abdulrahman & et al. also reported the species in (1901) Malaysia samples (Okada et al., 2010). HCA has been Syzygium divided into two species known as widely employed in other to discriminate Eugenia Syzygium and Jambosa group based plants according to their closest allies in terms on observable features of floral. Further divide metabolomic (Gad et al., 2013; N. Kumar et them in to three groups Syzygium, Jamboselle al., 2014). HCA employed in the present study and Jambosa. That is how it continues until found there is a strong relationship between late 90s where the genus was classified based the study cultivars. But variation existed even on the shape of vascular bundle (Hussain et al., within the same species of same cultivars. 1991). The following study used numerical Previously Yaradua et al. (2018) has also analysis to further investigation on the utilised numerical data for descrimination of taxonomic description of the genus Syzygium. genus Crotalaria. Chemical and bioactive The phenetic trees constructed from the descrimination of Ficus deltoidea was carried combination of morphology and anatomy were out in Penisular Malaysia (Yunusa et al., found to have lower consistency index and 2018). Raj et al. (2001) reported numerical higher retention index. The CI and RI show analysis were believed to be robust in plant how good the constructed phnetic tree was. It species identification toward similarity and has been previously reported by Fatihah et al. differences they exhibited. The present study (2012) and Monteiro et al. (2015) that; lower finds the two cultivars of Syzygium values of CI and higher in RI revealed how polyanthum based on morphological and good and robust the data matrix is in revealing anatomical data matrix; the cultivars were the relationship that existed between the monophyletic, exhibited a very strong examined species. In order to further validate relationship, but nevertheless, there is the results of phenetic analysis unsupervised variation between and within the cultivars. The recognition pattern is used, from which relationship and variation documented in the resulted in with how a particular plants sample present study might be as a result of mutation, differed from the others. The outcome of the cross pollination and sexual recombination. analysis would bring the idea on what features Therefore, this nictitated the morphometric they are sharing or what makes them different analysis of medicinal plants with respect to from each other (Gad et al., 2013). PCA and their environment to avoid adulteration of the HCA are the two most popular and widely plants either intentionally or unintentionally. used unsupervised pattern analysis (Lagunin et The study agrees with Fatihah et al. (2012) on al., 2014; Skov et al., 2014). Principal the phylogenetic analysis of Ficus in component analysis (PCA) is a technique that Terengganu state, Peninsular Malaysia. is unsupervised and provides useful visual CONCLUSSION result classification of the multivariate analysis The phenetic analysis of the two cultivars (Gad et al., 2013; Lagunin et al., 2014; Matera based on morphometric data have delimited et al., 2014; Song et al., 2014; Ballabio, 2015). the two group into two distinct cultivars with The technique (PCA) has pull out the variation intra variation within species of same cultivar. within the data set and displayed them The study has added taxonomic information to according to their relationships or similarities, the genus Syzygium. The study recommend and at the same time making the data set very further studies should be carried out on de readable. Results from the PCA revealed the to novo sequence of the two cultivars; as the two cultivars exhibit relationship but still were no complete genome sequence of the differences existed between the two cultivar species in National Centre for Biotechnology species. Where variation can clearly be seen in Information NCBI data base. cultivar of same species. Hierarchical cluster ACKNOWLEDGEMENTS analysis is used in multivariate data analysis The authors wish to acknowledge Universiti (Okada et al., 2010). The technique presents its Sultan Zainal Abidin (Unisza) Malaysia and results in a form of tree (dendrogram) that Kaduna State University (KASU) Nigeria. explains the relationships between the study

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Cultivars Morphological and Anatomical data matrix Sta 1 2 3 4 4 6 7 8 9 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 te 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 S 0 0 1 1 1 1 1 0 0 0 1 1 0 0 ? 0 0 0 0 1 1 1 1 0 1 0 1 0 0 0 0 0 0 0 M SK 1 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 1 0 0 0 1 1 1 0 1 0 1 0 0 0 0 0 0 0 1 SK 1 1 0 0 0 0 1 0 0 0 0 1 0 0 1 0 0 0 1 0 1 1 1 0 1 0 1 0 0 0 0 0 0 0 2 SK 1 1 0 0 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 0 1 1 1 0 1 0 1 0 0 0 0 0 0 0 3 SK 1 1 0 0 1 0 0 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 1 0 1 0 1 0 0 0 0 0 0 0 4 SK 1 1 0 0 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 1 1 0 1 0 0 1 1 1 1 0 1 1 1 5 SK 1 0 0 0 1 0 1 0 1 1 1 0 0 0 1 0 0 0 1 1 1 1 0 1 0 0 1 1 1 1 0 1 1 1 H1 SK 1 0 1 0 0 0 1 0 1 1 1 0 0 0 1 0 ? 0 1 1 1 1 0 1 0 0 0 1 1 1 0 1 1 1 H2 SK 1 0 0 0 1 0 1 0 1 1 1 0 0 0 1 0 0 0 1 1 1 1 0 1 0 0 1 1 1 1 0 1 1 1 H3 SK 1 0 0 0 1 0 1 1 1 0 0 0 0 0 1 0 0 0 1 1 1 1 0 1 0 0 0 1 1 1 0 1 1 1 H4 SK 1 0 1 0 1 0 1 1 1 0 0 0 1 0 0 0 0 0 ? 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 H5 REFERENCES 8.Gamage, H. K., M. S., Ashton, and B. 1.Abdulrahman, M. D., A. M. ALI, H. N. N., Singhakumara, 2003. Leaf structure of Fatihah, M. M., Khandaker, and N. Mat, 2018. Syzygium spp.(Myrtaceae) in relation to site Morphological and anatomical Studies of affinity within a tropical rain forest. Botanical Syzygium polyanthum (Wight) Walp. Journal of the Linnean Society, 141(3), 365- (Myrtaceae). Malayan Nature Journal, 70(3), 377. 309-322. 9.Harrington, M., and P. Gadek, 2004). 2.Abdulrahman, M. D., A. M., Ali, H. N. N., Molecular systematics of the Acmena alliance Fatihah, M. M., Khandaker, and N. Mat, (Myrtaceae): phylogenetic analyses and 2018. Traditional medicinal knowledge of evolutionary implications with reference to Malays in Terengganu, Peninsular Malaysia. Australian taxa. Australian Systematic Botany, Malayan Nature Journal, 70(3), 349-364. 17(1), 63-72. 3.Baum, D. A., and S. D. Smith, 2013. Tree 10.Hussin, K. H., D. F., Cutler, and D. M. thinking: an introduction to phylogenetic Moore, 1992. Leaf anatomical studies of biology: Roberts Greenwood Village, CO. Eugenia L.(Myrtaceae) species from the Malay 4.Brazeau, M. D., T., Guillerme, and M. R. Peninsula. Botanical Journal of the Linnean Smith, 2017. Morphological phylogenetic Society, 110(2), 137-156. analysis with inapplicable data. bioRxiv, 11.Klingenberg, C. P., and N. A. Gidaszewski, 209775: 1-34. 2010. Testing and quantifying phylogenetic 5. Craven, L. A., & E. Biffin, 2010. An signals and homoplasy in morphometric data. infrageneric classification of Syzygium Systematic Biology, 59(3), 245-261. (Myrtaceae). Blumea-Biodiversity, Evolution 12.Kumar, N., A., Bansal, G., Sarma, and R. and Biogeography of Plants, 55(1), 94-99. K. Rawal, 2014. Chemometrics tools used in 6. Fatihah, H. N., N., Mat, A. R., Zaimah, M. analytical chemistry: An overview. Talanta, N., Zuhailah, H., Norhaslinda, M., . Khairil, A. 123, 186-199. M. Ali, 2012. Morphological phylogenetic 13.Lawing, A. M., and P. D. Polly, 2010. analysis of seven varieties of Ficus deltoidea Geometric morphometrics: recent applications Jack from the Malay Peninsula of Malaysia. to the study of evolution and development. PloS one, 7(12), 52-64. Journal of Zoology, 280(1), 1-7. 7.Gad, H. A., S. H., El‐Ahmady, M. I., Abou‐ 14.Monteiro, R. F., A., Mantovani, and R. C. Shoer, and M. M. Al‐Azizi, 2013. Application Forzza, 2015. Morphological phylogenetic of chemometrics in authentication of herbal analysis of two early-diverging genera of medicines: a review. Phytochemical Analysis, Bromelioideae (Bromeliaceae). Rodriguésia, 24(1), 1-24. 66(2), 505-521.

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