Study on diversity of leaf beetle and relationship with plant in environmental condition at núi Chúa national park, Vietnam by molecular bological tool

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ---------------------------- Nguyen Thi Dinh STUDY ON DIVERSITY OF LEAF BEETLE (CHRYSOMELIDAE) AND RELATIONSHIP WITH PLANT IN ENVIRONMENTAL CONDITION AT NÚI CHÚA NATIONAL PARK, NINH THUAN PROVINCE, VIETNAM BY MOLECULAR BOLOGICAL TOOL Major: Ecology Code: 9 42 01 20 SUMMARY OF BIOLOGICAL DOCTORAL THESIS Ha Noi - 2018 This dissertation was completed at Graduate University Science and Technology – Vietnam Academy of Science and Technology The first supervisor : Asso. Prof. Dr. Nguyen Van Sinh The second supervisor: Dr. Jesús Gómez-Zurita The first reviewer: The second reviewer: The thirt reviewer: . This dissertation will be defended before the academic evaluation board of the doctoral thesis, at meeting room in Graduate University Science and Technology – Vietnam Academy of Science and Technology, in ...h’, date 2018. Please reference in: - Library of Graduate University Science and Technology - Vietnam national library 1 INTRODUCTION 1. Reason for choosing the topic Biomes are interdependent. Plant communities relate to physical properties of environment as: temperature, humidity, solar radiation Those physical properties change with altitude and vary the structure of plant communities. Plant are food of insects and have an important role in the diversity and distribution of insects. Study on responding of herbivore with the change of plant communities in altitude will help us over the understand about relationship interdependent each other in the ecosystem. Chrysomelidae is the biggest family in Coleoptera order. Their food are plant, therefore Chrysomelidae relates to plant in all their life and sampling them is easy. For those reasons, Chrysomelidae is a suitable object to study the interaction of biomes in the ecosystem. Nui Chua national park includes the area of semi-driest forest in Vietnam. Plant communities change in altitude from dry forest on low land through semi-humid forest green humid forest on a high mountain. For those reasons, Nui Chua national park is the ideal site to carry out researching. For above all reason, I choose the topic “ Study on the diversity of leaf beetle (Chrysomelidae) and relationship with plant in environmental condition at Nui Chua national park, Ninh Thuan province, Vietnam by molecular biological tool ” 2. Objects of research Appraising of the species diversity and the variation according to space of Chrysomelidae in Nui Chua national park. 2 Determining the food of Chrysomelidae and appraising the change of their food according to space. 3. The main contents of the dissertation Using ADN Barcoding to appraise the species diversity of Chrysomelidae in Nui Chua national park. Using AND Barcoding to determine the food of Chrysomelidae in Nui Chua national park. Determine the component variables of Chrysomelidae and their food according to space in Nui Chua national park. CHAPTER 1. OVERVIEW 1.1. The topic relates to the dissertation 1.1.1. Using the molecular biological tool to measure biodiversity 1.1.1.1. Biodiversity and problems related Definement biodiversity: Biodiversity is the diversity in three levels: gene, species and ecosystem. In there, the diversity of species is used in studying biology, conservation and ecology. Mora et al. (2011) predicted that there are about 8,7 million (± 1,3 million) of eukaryote on the earth; among there, about 2,2 million (± 0,18 million) live under the ocean. And they think that about 86% of living species on the land and 91% of living species under ocean are waiting for describing. So far, total known species in the world are approximately 1.900.000 species and every year there are 18.000 new species to be described (Chapman 2009). In there, tropical forest supports 50% of biodiversity on the earth (Myers 1988, Mittermeier et al. 1998) and 80% of known insect species (WWF), 1998) although the tropical forest only covers <10% of areas of the earth (Achard et al. 2002). 3 Measuring Biodiversity: Biologist usually measures the biodiversity by measuring species richness. There are two main methods to measure species richness, that are quality (the number of species) and quantity (the number of individual or living mass per unit of area) method. The best method for measuring biodiversity is to measure the abundance of all organism following space and time, using a classification (the number of species), function and interactions between species. Or measuring a changing of biodiversity in space and time. But, today we could not to exactly do this highly because of the lack of data. Crisis of biodiversity: is lost of gene, species and ecosystem. Vos et al. (2015) estimated that the lost in nature about with ratio 0,1 species per million species per year and now aday, the ratio of extinct is higher 1000 times when compare with natural extinct and in the future could be higher 10.000 times. Levin (2002) shown that average of 20 minutes there is one species be lost. Impediment of classification in studying biodiversity: The lack of name and difficulty to recognize species in ecological research is impediment of classification (New, 1984). This influence on conservational research. Because of there are many species to be extinct before described and we will not know how many species are on the earth. This impediment makes us misunderstand about biodiversity on the earth. 1.1.1.2. Using molecular biological tools to measure rapid biodiversity Using molecular biological tool to measure the diversity of species: ADN barcode in Mitochromosome (COI gene) is the useful tool to determine animal species. ADN barcode is a good tool for 4 determining known tspecies and detecting new species (Hebert et al. 2003, 2010, Savolainen et al. 2005). ADN barcode support data for classification more exactly (Thompson et al. 2012). ADN barcode help determining species in the case species only recognized in family or genus level. In the plant, the ADN in chromosome are used for determining species. Gene rbcLa and matK are barcode for plant land (CBOL Plant Working Group 2009). Add this, two sites trnH-psbA and nrITS are puted forward as supplemental ADN barcode for plant (Hollingsworth et al. 2011, Li et al. 2011). Using molecular biological tool to study ecological interaction: By this method, plant ADN is extracted from the insect's gut. A part of Chloroplast ADN are amplified, sequences of plant ADN are compared with Genbank. This method permits us to determine host plant of hebivove with no observing field. 1.1.2. Heterogeneity of the environment and how it influences biodiversity. Haller et al. (2013) said that, there are two space pattern on the earth: superpopulation pattern in their organism live in a two or many fragmentary and environmental gradient pattern. The heterogeneity of environment by two factors: the first because of the change of environment and continuously because of fragment in space. They conclude that the heterogeneity of environment was created regime of choices differently motivate diversity that is an important motive for species formation. 5 1.1.3. Chrysomelidae is suitable object to apply the molecular biological tool to measure biodiversity and study interaction in the ecosystem. Chrysomelidae is a big family in Coleoptera order. Chrysomelidae is great diversity (the number species and their host plant) and easy to collect Therefore Chrysomelidae is suitable object to apply the molecular biological tool to study their diversity and interaction between Chrysomelidae and their host plant (Price 2002). 1.1.3.1. Overview about studying Chrysomelidae in Vietnam. Chrysomelidae in Vietnam was studied about classification, biology, ecology from 1975 to 2008. Specially in North of Vietnam (Tam Đảo, Hòa Bình, Hà Nam, Ninh Bình..), and midle of Vietnam (Quảng Bình, Quảng Trị and Thừa Thiên Huế) and Highland of Vietnam (Đặng Thị Đáp & Medvedev, 1982- 1989; Đặng Thị Đáp et al. 2005-2008). Classification of Chrysomelidae in Vietnam is studied by many foreign scientists as Medvedev (1983- 2015), Kimoto (1997, 1998, 2000). There are about 700 species of Chrysomelidae are recorded and published in Vietnam. Predicting the number of species Chrysomelidae can be over 1000 species. This show that Chrysomelidae in Vietnam are great diversity and many species are waiting to describe new for science. But information about host plant of Chrysomelidae in Vietnam are limited. Đặng Thị Đáp (1983) studied about the influence of landscape to distribution of Cassidinae subfamily, but haven’t researched about responding of Chrysomelidae with environmental gradient yet. 2005 year, Tạ Huy Thịnh et al. 6 Studied about insects in Nui Chua national park, but he did not study about Chrysomelidae community. . 1.1.3.2. Overview about studying Chrysomelidae on the world. Chrysomelidae on the world were deeply studied about host plant (Jolivet và Hawkeswood 1995), biology, classification, evolution, ecology. (Jolivet, và Cox , 1996). Now aday, scientist used molecular biological tool to study phylogeny of Chrysomelidae (Montelongo và Gómez-Zurita, 2014; ), measuring their diversity of Chrysomelidae (Thormann et al., 2016) and seek their host plant (De la Cadena et al. 2016; Kishimoto-Yamada et al. 2013; Jurado- Rivera et al.2009). There are research about the change of species by altitude, almost research are in South and Central America and on coleoptera and butterfly (24,5%), only have several research on Chrysomelidae. 1.2. Research site 1.2.1. Locality Research was carried out in the Núi Chúa National Park, Ninh Thuan province, South of Vietnam. The Núi Chúa national park was located from 11°35'25" to 11°48'38" of North latitude and from 109°4'5" to 109°14'15" of East longitude. Núi Chúa includes 29.865 ha, in there the land are 22.513 ha and the ocean is 7.352 ha, and the buffer zone is 7.350 ha in area 1.2.2. Terrain The shape of Núi Chúa national park as Tortoise with a head in South, tail in Xốp peak. Núi Chúa includes many peaks, the highest peak is Cô Tuy mountain where is 1039m in high. 7 1.2.3. Climate and hydrography Climate in Núi Chúa national park is dry, rain season is later and end earlier than the other area in Vietnam, starting from September in October and ending in December. 1.2.4. Plant ecology Ecosystem in Núi Chúa national park is a semi-dry plant in low mountain and evergreen plant in high mountain. 1.2.5. Fauna and flora Flora in Núi Chúa national park have recorded 1.504 vascular plant species belong to 85 orders, 147 families, 596 genera in 7 phyla Fauna in Núi Chúa national park have recorded 330 vertebrate species, in there 84 animal species, 163 bird species and 83 amphibians – reptiles species, 46 species in there are in Vietnam red books. In research 2004 year, Tạ Huy Thịnh et al., have recorded 10 orders, 95 families and 361 insect species in Núi Chúa. Summary, the Núi Chúa national park is unique and driest forest in Việt Nam and plant communities change with altitude from dry forest on the lowland, through semi-humid to humid evergreen forest on a high mountain, therefore Núi Chúa is the ideal site to carry out this research. Chapter 2: RESEARCH METHOD 2.1. Sampling and delimitation biomes in Núi Chúa national park. 2.1.1. Sampling 8 Specimens were collected 10 times in two years (2012 and 2013 years) fixed and along 5 paths in the Núi Chúa national park: Mái Nhà, Đá Đỏ, Ao Hồ, Núi Ông và Suối Trục. In every path, sampling from the altitude at sea level (corresponding to the dry ecoregion) go to up at 449m in altitude (corresponding to the transition ecoregion and a part of the humid ecoregion) and the Ðá Hang path was only collected one time in the first fieldworking (5/2012) (Figure 2.2). Chrysomelidae are collected by beating around fixed sites along the paths in each sampling. Beating is carried out during 10 minutes from shrub, low trees up to high trees until it is impossible to stop (about 2,5 m). The collected beetles were immediately stored in vials containing absolute ethanol (96%) for preservation of DNA, vials labeled for geographical location and sampling path. Figure 2.2: The sampling map of Chrysomelidae in Núi Chúa national park (a) The site of the Núi Chúa national park in Việt Nam map (b) Outline the shape of the Núi Chúa national park and sampling paths (c) Sampling sites in sampling paths with grey area are assumed transitional area. 2.1.2. Delimitation biomes in Núi Chúa national park. Delimitation biomes in the Núi Chúa national park are based on previous studies of Núi Chúa, combining with analyzing some 9 parameters of the Chrysomelidae population as Sorensen – Dice index by using the sliding window method (Barton et al. 2013). 2.2. Molecular biological method ADN of all specimens of Chrysomelidae collecting in the Núi Chúa national park are extracted by DNeasy Blood and Tissue (Qiagen Iberia) kit by laboratory protocol. We used whole specimens, which were recovered after DNA extraction, mounted dry and labelled with a voucher number for the future. We amplified cytochrome c oxidase 1 (cox1) gene to delimit Chrysomelidae and amplified locus cpDNA PsbA-TrnH to determine host plant of Chrysomelidae. 2.3. Method for delimitation Chrysomelidae species Delimitation Chrysomelidae species by molecular data: We used phylogenetic methods: Generalized Mixed Yule-Coalescent (GMYC) pattern with single and multi threshold (Pons J. et al. 2006) and và sự Poisson Tree Processes (bPTP) pattern (Zhangj-J et al. 2013). We used some softwares as r8s, PADTHd8, BEAST 1.8.1 to get ultrametric tree. Using "splits " package (Ezard et al. 2009) in R 3.1.1 software to delimit by GMYC pattern with single and multi threshold. bPTP pattern is ran online on the web "bPTP server" ( Classification species by morphology. Specimens are classified by key of Kimoto (2000, 1989, 1982, 1981). The result is compared with result of molecular biological methods. 2.4. Method determination host plant of Chrysomelidae in Núi Chúa national park: Using BAGpipe program. 10 2.5. Estimate expected species richness of Chrysomelidae in Núi Chúa national park. Using EstimateS 9.1 software. 2.6. Method research interaction between Chrysomelidae and environment. 2.6.1. Analyzing the change of Chrysomelidae with spaces: Using EstimateS 9.1 software (Colwell 2013) to calculate Jacard and Sorensen indexes. Measuring dissimilarity by “betapart” package R. 2.6.2. Canonical Correspondence Analysis (CCA) to detect factor imfluent on interaction between Chrysomelidae and their host plant: Using “vegan” 2.0-10 package in R. 2.6.3. Mesruring Beta diversity of interaction between Chrysomelidae and their host plant by biome (altitude): Using “BAT” and “betalink” 2.1.0 packages in R software. 2.6.4. Analysing topology of interaction between Chrysomelidae and their host plant. Using “Bipartite” 2.05 package in R software. Chapter 3: RESULTS OF RESEARCH 3.1. Delimitation of biomes We recommend elevational intervals from 160 m to 320 m as a transition zone between dry biome and moist biome in the sampling area. The midpoint (300 m) is considered as the strong boundary of the transition area for further analysis of the Chrysomelidae community and their host plant according to elevation. 3.2. Species diversity of Chrysomelidae in Núi Chúa national park. 3.2.1. Species diversity of Chrysomelidae of Chrysomelidae in Núi Chúa national park basing on morphology Total 520 specimens of Chrysomelidae collected in the Núi Chúa national park. Using classificatinal keys of some authors, we identified all specimens are in 141 morphospecies. Almost specimens 11 are identified at the genus level, some specimens are to family level, a few specimens are at species level. In specimens are identified to species, there are 13 new species for science, 11 species in there belong to Monolepta Chevrolat genus: M. decreta, M. demimuta, M. densopunctata, M. dubia, M. fluctuans, M. fuscicorne, M. interruptomarginata, M. ochracea, M. quotidiana, M. semicostata, M. thomaswagneri and 2 species belong to Paleosepharia Laboissiere genus: P. frontis và P. nuichua. Almost specimens belong to Eumolpinae, Galerucinae and Alticinae subfamilies. Remaining 7 subfamilies are few specimens, in there two subfamilies Cryptocephaninae and Hispinae there are no collected specimens in some paths. Diversity of species is from 22 species to 58 species in sampling paths and from 85 species to 95 species in two elevations (300m). (Table 3.1). Table 3.1: Species diversity of Chrysomelidae in sampling paths and according to altitude in the Núi Chúa national park (Note: Morphospecies are above mark “/” and bPTP species“/”) Subfamily The number of specimens The number of species Paths Biomes AH ĐĐ MN NÔ ST ĐH 300m Alticinae 49 23/25 4/4 5/5 6/6 8/8 6/7 6/6 17/18 12/12 Bruchinae 7 3/3 - - 2/2 2/2 - - 3/3 - Chlamysinae 3 2/2 - - - 1/1 1/1 - - 2/2 Chrysomelinae 2 2/2 1/1 - - - - 1/1 1/1 1/1 Clytrinae 12 7/7 1/1 - 3/3 - 4/4 3/3 5/5 4/4 Criocerinae 2 2/2 - - - - 2/2 - - 2/2 Cryptocephalinae 14 9/9 4/4 1/1 1/1 1/1 5/5 1/1 7/7 4/4 Eumolpinae 221 41/51 12/13 7/8 20/22 18/21 19/20 18/19 26/32 30/33 Galerucinae 146 43/45 8/8 6/6 17/17 17/17 19/20 9/9 28/29 28/29 Hispinae 38 9/9 1/1 3/3 8/8 2/2 2/2 1/1 8/8 3/3 Total 494 141/155 31/32 22/23 57/59 49/52 58/61 39/40 95/103 86/90 12 3.2.2. Species diversity of Chrysomelidae in Núi Chúa national park based on ADN data The number of species is from 155 species to 186 species depending delimitational method (Table 3.2). Exception the result of GYMC pattern with multiple threshold, reminding results match highly with morphospecies (90 %), only 8 species by bPTP method are split with single threshold method and two species combined in one form (Table 3.3). The number of species only has one individual win 49,7% (bPTP species) Table 3.2: The result of delimitation species based on ADN of cox1 gên of Chrysomelidae in Núi Chúa national park according to different algorithm and pattern Tree Algor ithm Thresh old The number of species The number of clade Probabiliy in GMYC The number of species matches with morphospeci es The number of species split with morpho species The number of species mix togethe r compar ing with morpho species ML R8s single 178 [175-181] 63 [62-65] 500.063 122 18 0 Multiple 186 [183-186] 62 [61-62] 505.887 119 21 0 Pd8 Single 160 [155-163] 65 [64-66] 522.670 124 16 0 Multiple 161 [152-166] 76 [73-76] 527.336 39 20 81 BI SC Single 162 [157-166] 67 [65-67] 2480.504 124 16 0 Multiple 165 [153-165] 94 [91-96] 2491.387 39 21 80 ULN Single 164 [158-166] 66 [66-68] 2463.070 122 18 0 Multiple 173 [170-174] 67 [66-67] 2466.495 119 21 0 ML - bPTP 155 - - 126 14 0 3.2.3. Expected species diversity of Chrysomelidae in Núi Chúa national park The result of estimating expected species diversity of Chrysomelidae shown that the species richness of Ao Hồ, Suối Trục, 13 Đá Đỏ, Mái Nhà and Núi Ông paths are higher from twice (exception Jack 1 estimator) to 4,3 times (Chao 2 estimator Mái Nhà) (Table: 3.4). When split data according to altitude, the result shown that the specimens collected