Objects of research: Appraising of the species diversity and the variation according to space of Chrysomelidae in Nui Chua national park. Determining the food of Chrysomelidae and appraising the change of their food according to space.
Trang 1GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
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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
Trang 2This 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
Trang 3INTRODUCTION
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
Trang 4Determining 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)
Trang 5Measuring 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
Trang 6determining 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
Trang 71.1.3 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
Trang 8Studied 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
Trang 91.2.3 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
Trang 10Specimens 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
Trang 11parameters 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"
(http://species.h-its.org)
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
Trang 122.5 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.
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