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Ministry of Education Vietnam Academy of and Training Science and Technology MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ---------------------------- VAN HONG THIEN BUILDING PHYLOGENETIC TREES FOR THE ARACEAE IN SOUTHERN VIETNAM BASED ON MORPHOLOGICAL AND MOLECULAR MARKERS Major: Ecology Code: 62 42 01 20 SUMMARY OF BIOLOGY DOCTORAL THESIS Ho Chi Minh City – 2017 The work was realized in Graduate University of Science and Technology, Vietnam Academy of Science and Technology. Advisor 1: Dr. Lưu Hong Truong Advisor 2: Dr. Nguyen Phi Nga Examiner 1: Assoc. Dr. Nguyen Van Ket Examiner 2: Assoc. Dr. Truong Thi Dep Examiner 3: Dr. Vu Ngoc Long The thesis will be evaluated by doctoral committee at Graduate University of Science and Technology, Vietnam Academy of Science and Technology on …… 2017. The thesis is available at: - Library of Graduate University of Science and Technology - National Library of Vietnam LIST OF ABBREVIATIONS CI : Consistency index DNA : Deoxyribonucleic acid HN : Herbarium of Institute of Ecology and Ecological Resources HNU : Herbarium of University of Science, Vietnam National University, Ha Noi IGS : Intergenic spacer ISI : International Scientific Indexing K : Herbarium of Kew MEGA : Molecular Evolutionary Genetics Analysis P : Herbarium of Paris PAUP : Phylogenetic Analysis Using Parsimony PCR : Polymerase Chain Reaction PHH : Herbarium of University of Science, Vietnam National University Ho Chi Minh City RC : Rescaled consistency index RI : Retention index SGN : Herbarium of Southern Institute of Ecology VNM : Herbarium of Institute of Tropical Biology Sect. : Section 1 INTRODUCTION 1. The necesstiy of the thesis Araceae in Vietnam were first systemerized by the French botanist Gagnepain in 1942 (Gagnepain, 1942). Pham-Hoang (2000) in the book "An Illustrated Flora of Vietnam" (Volume III) as well as Nguyen (2005) in the "Checklist of Plant Species of Vietnam" (Volume III, edited by Nguyen Tien Ban) provided the brief information on the taxonomy of species in Araceae. Most recently, the monograph work on Araceae in Vietnam was conducted by Nguyen (2006), according to which the author made a list of 116 species belonging to 23 genera in Vietnam. However, much of the research samples in Nguyen Van Du's work (2006) were collected in the northern regions of Vietnam. In addition, since 2006, there have been 13 new species and 4 new records of Araceae in Vietnam (mostly in the South) which were published by many authors, thereby, increasing the total number of species of Araceae in Vietnam up to 133 (Nguyen 2007 & 2008; Hetterscheid, 2006 & 2013; Nguyen & Croat, 2010; Gong et al., 2012; Bruggeman et al., 2013; Nguyen & Bui, 2013; Nguyen et al., 2013; Luu et al., 2013 & 2014; Hoang et al., 2016; Van et al., 2016). Moreover, many species and genera in Araceae have currently changed and have not been unified in the classification (Nguyen, 2006; Gusman & Gusman, 2006; Cusimano et al., 2011; Hetterscheid & Claudel 2012; Nauheimer et al., 2011; Nauheimer & Boyce, 2013). Thus, the knowledge of Araceae in Vietnam, especially in the South of Vietnam, is still incomplete, many species are propably unknown or unclear in terms of classification. Moreover, in Vietnam, no reseach has been conducted on the use of morphological characteristics and especially molecular markers for the purpose of constructing the phylogenetic tree for systematizing and providing taxonomic information of Araceae. For the above reason, we decided to implement the project: “Building phylogenetic trees for the Araceae 2 in southern Vietnam based on morphological and molecular markers”. 2. Goal of the subject Building phylogenetic trees for the Araceae in southern Vietnam based on morphological and molecular markers, thereby (1) indicating the evolutionary relationship between taxa in Araceae; (2) re-classifying and/or confirming the exact identification for ambiguous species; (3) recording the new species of Araceae for the flora in Vietnam. 3. The main contents of the study Collecting plant materials; observation of specimens in hebariums; identification and decription of materials; amplification and sequencing of trnL-trnF IGS and matK chloroplast DNA regions; data processing methods. CHAPTER 1: LITERATURE REVIEW 1.1. Introduction to Aracea Araceae is a family of Monocotyledon of the Magnoliophyta with about 2824 species of 107 genera (Govaert & Frodin, 2002) and was first described in 1789 by Jussieu (Jussieu, 1789). Araceae has very featured morphological characteristics, which there is a spathe varied in shape, size and color. Spathe is responsible for protecting spadix and is usually divided into two parts, limb and tube. Another characteristic of the Araceae is spadix; spadix is divided into two forms: bisexual and unisexual, unisexual spadix is the one that carries only famale or male flowers, while bisexual spadix is a type that carries both male and female flowers. Spadix of many species in Araceae in general and most of the species in southern Vietnam in particular have a sterile portion located between the male and female part or at the end. In addition, flowers of Araceae are divided into two types, including bisexual and unisexual flowers. According to many studies, bisexual flowers often appear in subfamilies of ancient classifications such as Pothoideae, 3 Monsteroideae and Lasioideae; while the unisexual flowers are more common in the subfamily of higher evolutionary levels, such as Aroideae (Mayo et al., 1997; Nguyen, 2006; Boyce et al., 2012). 1.2. Research status with taxanomic Araceae 1.2.1. The studies on taxanomic Aracea in the world Araceae was first described in 1789 by Jussieu, in which he categorized all climbing species into a group called Pothos L., while terrestrial species were classified as Arum L. and Dracontium L. (Jussieu, 1789). Schott (1860) set up the first classification system for the Araceae. This system was then modified and developed by many researchers (Hooker, 1883; Engler, 1876b & 1820b; Grayum, 1990; Bogner & Nicolson, 1991; Mayo et al., 1997). In the field of molecular marker application research in Araceae, the first work was done by French et al. (1995). Later, further research into the construction of the phylogenetic tree for Araceae were also conducted by several authors, for example, Barabe et al. (2002) and Cabrera et al. (2008) built the phylogenetic tree for Araceae based on a combination of two non-coding regions of the chloroplast DNA. Cusimano et al. (2011) and Nauheimer et al. (2012) used phylogenetic tree of Cabrera et al. (2008) and some more genera to form a phylogenetic tree for Araceae based on chloroplast markers. 1.2.1. Studies on the taxonomic Aracea in Vietnam Gagnepain (1942) first described Araceae in Vietnam in the "Flore Général de L'Indochine". Many of the later studies of Araceae in Vietnam were carried out by a number of authors (Pham-Hoang, 1993 & 2000, Nguyen, 2005). Nguyen (2006) reported that in Vietnam there were 116 species belonging to 23 genera. From 2006 to date, there have been 13 new species and 4 new records of Araceae published in Vietnam. Among them, genus Arisaema have four new species and three new records, followed by genus Amorphophallus with four new species and one new records, 4 Typhonium and Alocasia with two new species and one new species for Rhaphidophora (Hetterscheid, 2006; Nguyen, 2008; Nguyen & croat, 2010; Gong & Li, 2012; Bruggeman et al., 2013; Hetterscheid & Claudel, 2013; Luu et al., 2013 & 2014; Nguyen et al., 2013; Nguyen et al., 2015; Nguyen & Quang, 2015; Nguyen et al., 2016; Van et al., 2016). CHAPTER 2. CONDITION AND METHOD 2.1. Research subject The species of Araceae is distributed in southern Vietnam, from Da Nang City. 2.2. Research Method 2.2.1. Field survey method The field survey method is implemented in the following steps: (1) Listing the number of species available in southern Vietnam; (2) selecting sample location; (3) recording the distribution of species; (4) documentary photography; (5) collecting samples for dried specimens; (6) DNA sample collection. 2.2.2. Method in the laboratory 2.2.2.1. Method of morphological characterization includes steps: (1) observation of specimens in hebariums (HN, VNM, HNU, SGN, PHH, P and K); (2) species identification by morphological comparison method; (3) building morphological characteristics of species in southern Vietnam. 2.2.2.2. Molecular method includes the steps: (1) selection of molecular markers in research of building phylogenetic tree; (2) total DNA extraction; (3) PCR reaction; (4) Purification of PCR products and sequencing. 2.2.3. Data processing methods 2.2.3.1.Phylogenetic tree based on morphological characteristics: building phylogenetic tree for 103 taxa, including 101 species, 1 subspecies and 1 form in southern Vietnam based on 33 morphological characteristics by PAUP software (Swofford, 5 2002), according to the Maximum Parsimony method with 1000 bootstrap replicates (Felsenstein, 1985). 2.2.3.2. Phylogenetic tree based on molecular markers: building phylogenetic tree for 70 taxa, including 64 species, 1 subspecies and 1 form which were collected in southern Vietnam and 4 taxa from Genbank data by PAUP * software (Swofford, 2002) and MEGA6 (Tamura et al., 2013) by Maximum parsimony, Neighbor joining and Maximum likelihood methods. CHAPTER 3. RESULT AND DISCUSSION 3.1. Results of the construction of the Phylogenetic tree based on morphological characteristics The results on the phylogenetic tree in Figure 3.1 showed that the resolution was not high because the branches tended to start from the same root. The morphological relationships of 101 species, 1 subspecies and 1 form were basically divided into 4 subfamilies: Pothoideae (I), Monsteroideae (II), Lasioideae (III) and Aroideae (IV). 3.1.1. Subfamily Pothoideae and Monsteroideae The phylogenetic tree in Fig. 3.1 showed that the two subfamily of Pothoideae and Monsteroideae with bisexual flower (3-tribe Potheae, Anadendreae and Monstereae) grouped together with bootstrap as 83% and were separated from subfamily Aroideae with unisexual flower . This result was consistent with the view of the classification system established by Mayo et al.
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