Genetic studies on the swimming crab (portunus pelagicus linnaeus, 1758) with implications for fisheries management

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY ___________________________ MUHAMMAD ARIFUR RAHMAN GENETIC STUDIES ON THE SWIMMING CRAB Portunus pelagicus LINNAEUS, 1758 WITH I

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY _

MUHAMMAD ARIFUR RAHMAN

GENETIC STUDIES ON THE SWIMMING CRAB (Portunus pelagicus LINNAEUS, 1758) WITH IMPLICATIONS FOR

FISHERIES MANAGEMENT

MASTER THESIS

KHANH HOA – 2018

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY _

MUHAMMAD ARIFUR RAHMAN

GENETIC STUDIES ON THE SWIMMING CRAB (Portunus pelagicus LINNAEUS, 1758) WITH IMPLICATIONS FOR

Topic allocation Decision

Decision on establishing the

Committee:

Defense date: 6 th June, 2018

Supervisors:

Prof Dr Henrik Glenner

Dr Dang Thuy Binh

Chairman:

Assoc Prof Ngo Dang Nghia

Faculty of Graduate Studies:

KHANH HOA – 2018

UNDERTAKING

I undertake that the thesis entitled: “Genetic Studies on the Swimming Crab

(Portunus pelagicus Linnaeus, 1758) with Implications for Fisheries Management”

is my own work The work has not been presented elsewhere for assessment until the

time this thesis is submitted All the given information is true to best of my knowledge

14.06.2018

Muhammad Arifur Rahman

Last but not least, I would like to thank to all of my family members for supporting me spiritually throughout the program

Thank you!

14.06.2018

Muhammad Arifur Rahman

TABLE OF CONTENTS

UNDERTAKING iii

ACKNOWLEDGMENT iv

TABLE OF CONTENTS ……… ………v

LIST OF ABBREVIATIONS vii

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF APPENDICES x

ABSTRACT xi

CHAPTER 1: INTRODUCTION 1

1.1 Background 1

1.2 Environmental change and human impact on marine population 2

1.3 Applications of advance genomic techniques in fisheries management 3

1.4 Rationale of the research 4

1.5 Objectives of the study 4

1.6 Thesis contents 5

CHAPTER 2: LITERATURE REVIEW 6

2.1 Overview of the study area 6

2.1.1 Overview of Vietnam climate 6

2.1.2 Overview of Vietnamese coastline 6

2.2 Study on swimming crab (Portunus pelagicus Linnaeus, 1758) 8

2.2.1 Classification 8

2.2.2 Habitat and distribution 9

2.2.3 Life cycle 10

2.3 Overview of the research methodology 11

2.3.1 Molecular markers for studying population genetics 11

2.3.2 Restriction site-associated DNA sequencing (RADseq) 14

2.3.3 Next generation sequencing (NGS) 15

2.4 Overview on population genetics 16

2.4.1 Population genetics of marine organisms 16

2.4.2 Review on Portunus pelagicus 16

2.5 Population genetic study and fisheries management 18

CHAPTER 3: MATERIALS AND METHODS 20

3.1 Sampling sites and tissue collection 20

3.2 Research outline 23

3.3 Research methodology 24

3.3.1 DNA extraction and digestion 24

3.3.2 EzRAD library preparation 24

3.3.3 SNP discovery and filtering 25

3.3.4 Outlier loci detection 26

3.3.5 Genetic diversity and effective population size 26

3.3.6 Analyses of population structure 26

3.3.7 Historic migration pattern 27

CHAPTER 4: RESULTS AND DISCUSSION 28

4.1 Results 28

4.1.1 Reference genome and SNP detection 28

4.1.2 Outlier loci detection 28

4.1.3 Genetic diversity of Portunus pelagicus 28

4.1.4 Effective population size (Ne) 29

4.1.5 Genetic differentiation of Portunus pelagicus 29

4.1.6 Population structure 30

4.1.7 Historic migration pattern 32

4.2 Discussion 33

4.3 Implications for fisheries management 36

4.3.1 Record keeping 36

4.3.2 Gear regulation and habitat monitoring 37

4.3.3 Sanctuary establishment 37

4.3.4 Implementation of ban season 38

4.3.5 Control of pollution 38

4.3.6 Implementation of management action plan 39

4.3.7 Creating awareness 39

CONCLUSION AND RECOMMENDATIONS 40

REFERENCES 41 APPENDICES a

AFLP Amplified Fragment Length Polymorphisms

A, T, C, G Adenine, Thymine, Guanine, Cytosine

BI Bayesian inference

bp Base pairs

DNA Deoxyribonucleic acid

RAD Restriction site Associated DNA Sequencing RAPD Random Amplification of Polymorphic DNA RFLP Restriction Fragment Length Polymorphisms RNA Ribonucleic acid

SNPs Single nucleotide polymorphisms

16S rRNA 16S ribosomal Ribonucleic acid

PCR Polymerase Chain Reaction

MVP Minimum Viable Population

COI Cytochrome Oxidase I

Cm Centimeter

GENO Genotype

Km Kilometer

Table 4.3 Log probabilities of the data given the model (*marginal likelihood, based

on the Bezier approximation score) and Δ values (difference from largest 1Lm value) and rank according to largest likelihood value 33

LIST OF FIGURES

Figure 2.1 The economical zones and activities in Vietnamese coastal area (Thanh et

al., 2004) 7

Figure 2.2 Blue swimming crab 9

Figure 2.3 Ventral view of male and female P pelagicus 9

Figure 2.4 Distribution of P pelagicus 10

Figure 2.5 Life cycle of P pelagicus 11

Figure 2.6 SNPs 14

Figure 2.7 DNA library construction using ezRAD technique 15

Figure 3.1 Dorsal view of P pelagicus (A Male and B Female) 20

Figure 3.2 Map of sampling stations for genetic study of P pelagicus along the coastal line in Vietnam 22

Figure 3.3 Flow diagram of the research work 23

Figure 4.1 Population structure results for K=3 amongst P pelagicus populations obtained using STRUCTURE on a data set of 80 individuals and 721 polymorphic SNPs (A) Optimal K value, (B) Bar plot of P pelagicus populations, (C) Individual group distribution percentage (%) among sampling sitesError! Bookmark not defined Figure 4.2 (A) Principal Component Analysis (PCA) of P pelagicus based on neutral polymorphic SNP markers (B) Scatter plot from PCoA of P pelagicus 31

Figure 4.3 Principal Component Analysis (PCA) of P pelagicus based on under selection (divergent) loci 32

Figure 4.4 Berried female blue swimming crab in the landing center 38

LIST OF APPENDICES

Appendix No.1 Purification of total DNA a Appendix No.2 Calculation of concentration of the samples a Appendix No.3 Collection of DNA Libraries b Appendix No.4 Quality scores and base calling accuracy (Phred scale) d Appendix No.5 Individual and SNPs filtering step by step e Appendix No.6 Detection of outlier SNPs using multiple algorithms f

Genetic Studies on the Swimming Crab (Portunus pelagicus Linnaeus,

1758) with Implications for Fisheries Management

ABSTRACT

The blue swimming crab (Portunus pelagicus Linnaeus, 1758) is distributed

throughout the coastal areas from north to south of Vietnam It is one of the commercially exploited crab fishery resources in the country This is the first study to provide a broad survey of genetic variation, population structure and historic

migration pattern of P pelagicus along the Vietnamese coastline The crab samples

were collected from the north (Hai Phong and Quang Ninh), central (Khanh Hoa and Phu Yen) and south (Phu Quoc and Rach Gia) of Vietnam The study used a panel of single nucleotide polymorphisms (SNPs) generated from restriction site-associated DNA (RAD) sequencing Here, 731 polymorphic SNP markers including 10 outlier loci were identified from 80 individuals After removing outlier loci, 721 putatively neutral SNPs were used to assess fine-scale population structure in the Vietnamese swimming crab The average number of observed alleles (Na) was 1.975±0.004 and effective number of alleles (Ne) was 1.318±0.005 The average observed heterozygosity (Ho) per locus was 0.275±0.004 while the average expected

heterozygosity (He) was 0.221±0.003 Pairwise Fst values among the locations ranged

from 0.00582 to 0.07314 (P<0.05) indicating significant genetic differentiation among

the populations Effective population size (Ne) was found small (Ne<500) Population structure analyses indicated that P pelagicus in the south (Phu Quoc and Rach Gia) is

isolated from the remaining population from the north and center, and should be managed as a different stock Historic migration pattern based on Bezier approximation score supported Panmixia model The findings suggested that immediate action plan should be implemented for the conservation and management

of P pleagicus populations along the Vietnamese coastline The results might be used for understanding current status of natural population of P pelagicus under changing

environment due to the natural and anthropogenic activities with implications for fisheries management

CHAPTER 1: INTRODUCTION

1.1 Background

Vietnam is a tropical to subtropical country located in the eastern most of the Indochina Peninsula and occupies about 331,210 km2 The 200 nautical miles exclusive economic zone (EEZ) covers about 1 million km2 and 3260 km long coastline along the East Sea (Vietnamese name of South China Sea) is contributing largely to the country‟s economy The coastal zone is divided into four natural parts: the Gulf of Tonkin in the North, the central coast, the southeast coast and the Gulf of

Thailand in the South (Thanh et al., 2004) There are approximately 11000 marine

species of them more than 2000 fish species inhabiting in 20 typical marine

ecosystems (Vietnamnet, 2016a & Thanh et al., 2004) The predominant coastal

habitats are bays, estuaries, coral reefs, mangroves, lagoons, wetlands and seagrass beds The local ocean currents and currents coming from the North or from the Pacific through the East Sea Straits are major currents depending on the monsoons In winter, current flows in the North East-South West direction coming from the North while in summer ocean current flows in the South West-North East direction coming from the central coast However, Vietnam is one of the top five severely affected countries by climate change due to its long coastlines and rapid unregulated economic activity in coastal areas (IPCC, 2001 & GFDRR, 2011) Thanh (2006) stated that climate change and human activities are responsible to change the highly resourceful coastal ecosystems in Vietnam Human activities includes unregulated coastal tourism and development, upstream forest and mangrove deforestation, mining, building sea and river dikes, construction of big dams in river mouths, dredging channels, agriculture, aquaculture, overexploitation, illegal fishing methods, cage culture, pollution from industry, agriculture and domestics (Vietnamnet, 2016b; World Bank, 2011 & Thanh

et al., 2004) These have led to serious damage of coastal habitats with accompanying

decrease in marine biodiversity and fishery production

Swimming crab (P pelagicus) are distributed in the wild throughout the long coastal waters from north to south of Vietnam (Thuy, 2000 & Ha et al., 2014) Locally this crab is known as “Ghẹ xanh” It is present in large numbers with great value for

commercial fisheries in Vietnam through exporting in USA, Europe and Japan (WWF, 2016) In 2013, Vietnam became the leading supplier of fresh blue swimming crab in Japan (VASEP, 2013) Although the crab has huge consumption locally but data was not found regarding this A study showed that in 2008 the catch was 11300 tonnes in Kien Giang waters but in 2013 it was declined to 7800 tonnes due to overharvesting

(Ha et al., 2016) Increasing demand and overexploitation due to the growing coastal

population, export trade, tourism, pollution and industrialization are threatening the sustainability of the wild stocks of swimming crab in Vietnam

1.2 Environmental change and human impact on marine population

Change in sea water parameters due to climate change is a growing concerns for fishery managers that causing substantial extinction of marine vertebrates and invertebrates (Waples and Naish, 2009) potentially altering the genetic structure, reproduction and culture of the species (Hoegh-Guldberg and Bruno, 2010 &

Wernberg et al., 2014) Climate niche modeling predicts that presently a highly

occupied area by species and communities will no longer be suitable for them (Dunlop

et al., 2012) that could be the greatest global threat to marine biodiversity over the

next several decades (Leadley et al., 2010) For instance, Munguia-Vega et al (2015)

reported climatic bottleneck effect on pink abalone population genetic structure in Baja California

Besides climatic variability, change in genetic variation and gene flow alteration in marine organisms was also reported due to overfishing (Hutchings and Reynolds,

2004; Smith, 1994; Aho et al., 2005 & Kenchington, 2003), mangrove degradation (Nehemia and Kochzius, 2017) and pollution (Nevo et al., 1987; Hamilton et al., 2016; Cimmaruta et al., 2003 & Puritz and Toonen, 2011) Lind and Grahn (2011) found that Gasterosteus aculeatus populations are genetically distinct at polluted sites

by pulp mill effluent Yu et al (2016) discovered low levels of genetic diversity and

no evidence of population structure in Hyporhamphus sajori that might be due to

fairly high gene flow via transport of eggs and larvae by the Kuroshio and Tsushima warm current

1.3 Applications of advance genomic techniques in fisheries management

A number of recent reviews have addressed the prevailing lack of integration of genetic information into fisheries management schemes of the particular species (Kochzius, 2009 & Hauser and Carvalho, 2008) Currently, genetic studies have become indispensable for sustainable fishery management because it helps to understand the stock size, gene flow, distribution and migration pattern of subpopulations in mixed fisheries (Utter, 1991) For example, in Baja California

marine reserve, Munguia-Vega et al (2015) found high allelic diversity, a large effective population size (Ne) and a lack of a recent genetic bottlenecks in pink

abalones compared to other fishing sites after a climatic bottleneck

Using restriction site-associated DNA sequencing (RADseq) and molecular markers can identify and score thousands of genetic markers which are randomly distributed across the target genome of a population Nowadays, a new type of marker, single nucleotide polymorphism (SNP) is considered as the best molecular tool for studying population genetics because it is less error-prone in comparison to other markers when

considering the assessment of thousands (Vignal et al., 2002) SNPs have been isolated for several species such as Litopenaeus vannamei (Du et al., 2010), Scylla

paramamosain (Ma et al., 2011 & Feng et al., 2014) and P pelagicus (Miao et al.,

2017)

Molecular population genetic studies of the swimming crab (P pelagicus) provide necessary information for effective management of wild stocks (Klinbunga et al.,

2007 & Klinbunga et al., 2010a) Genetic diversity and population differentiation of

P pelagicus was analyzed by using different methods and markers including allozyme

analysis, AFLP, RAPD and mtDNA (Bryars and Adams, 1999; Klinbunga et al., 2007; Klinbunga et al., 2010a; Klinbunga et al., 2010b; Sienes et al., 2014; Ma et al., 2016; Ren et al., 2016 & Fujaya et al., 2016) in Thailand, China, Indonesia, Australia

and so on For the first time, using traditional ABI solid sequencing method 91 SNP

markers for P pelagicus were isolated and characterized from 8426 bp long quality DNA sequences in China (Miao et al., 2017) Despite these studies found high

high-genetic variation in swimming crab but the information is still inadequate to

understand the management and conservation of this species using genetic data Absence of available SNP data has limited the studies on molecular population

genetics and fisheries management of P pelagicus in Vietnam Therefore, deeper

insights on genetic study of swimming crab in Vietnam are needed

1.4 Rationale of the research

For the sustainable management of the commercially exploited P pelagicus, the

primary step could be a clear understanding of its genetic diversity that can be used to understand the stock structure, evaluation of the levels of gene flow and historic migration pattern In this research, environmental change and anthropogenic stresses are predicted to impact the swimming crab population in Vietnam by altering genetic structure and creating stress to shift them from their current habitat The purpose of

this study is to understand the connectivity and substructure of P pelagicus

populations along the Vietnamese coastline that will allow an evaluation of the influence of changing environmental conditions To apply this to the north-south temperature variation, this research was designed to examine fine-scale population structure and historic migration pattern of swimming crab using RAD sequencing method and SNPs marker Moreover, the research is also planned to understand the historic migration pattern and distribution in the selected stocks of swimming crab This will enhance management of this valuable resource and provide a basis for extending this to a wider array of species

1.5 Objectives of the study

To meet the above goal, the following objectives are going to be pursued:

1 To describe and understand the genetic diversity and population structure of

swimming crab (P pelagicus) along the Vietnamese coastline

2 To detect historic migration pattern of P pelagicus stocks in Vietnam

1.6 Thesis contents

To complete the work, at the very beginning I learned the laboratory techniques such

as DNA Extraction, Gel electrophoresis, DNA concentration, EzRAD in the Laboratory for the Molecular Biology Studies lead by Dr Dang Thuy Binh, Senior lecturer, Institute for Biotechnology and Environment Meanwhile, I reviewed the current literatures on the species, global impact of climate change and human intervention on genetic structure, use of genetic information for fishery management, biology of swimming crab, ecological and economic importance of swimming crab in Vietnam and so on to form ideas and logic in solving problems I tried to find out the gaps in knowledge regarding the genetic structure and migration pattern of swimming crab in Vietnam and abroad I found a little difference in the literatures, which may be due to species geography and research methods However, I summarized the information related my research work to-date in Chapter 2 (Literature Review) Further, I presented the methods I used as a revolutionary approach, such as, EzRAD, NGS, SNPs etc., for completing my thesis on swimming crab in Chapter 3 In Chapter

4, I tried my best to interpret the results and discuss it clearly Finally, I made conclusion and recommendations based on my research findings

CHAPTER 2: LITERATURE REVIEW

2.1 Overview of the study area

2.1.1 Overview of Vietnam climate

Vietnam is located on the eastern margin of the Indochina Peninsula at 14.05°N and 108.27°E and occupies about 331,211.6 km2 It has a diverse climate across the country, with southern parts that are near the equator experiencing a tropical climate and northern parts in the humid subtropics showing greater seasonal variation In northern regions temperature ranges from 22-27.5ºC in summer to 15-20ºC in winter, while southern areas temperature range from 28-29ºC in summer and 26-27ºC in winter Annual rainfall ranges from 700-5000 mm, with northern area generally receiving more than the south Average annual temperature has increased by 0.4ºC since 1960 (projected to increase by 1ºC by 2050), with the rate of increase more rapid

in the dry seasons and more intense in the southern parts of the country Observations showed that mean sea level rise (SRL) is increasing and projected to rise by between

28 cm (low-emission scenario) to 33 cm (High-emission scenario) by 2050 All the information in this paragraph is cited from the report published by GFDRR (2011)

2.1.2 Overview of Vietnamese coastline

The Vietnamese coast is located in a tropical monsoon zone with a total of 114 river mouths and estuaries The area experiences northeast (NE) wind from October to April and southwest (SW) wind from May to September The mean wind velocity is 2.5-5 m/s The mean temperature ranges from 22.6-27.2ºC, increasing southward The mean annual rainfall ranges between 1000 mm and 2400 mm with the least precipitation occurring along the central coast The coastal tides include diurnal,

semidiurnal and mixed types with range of 0.5 to 4.0 m (Thanh et al., 2004) The

coastal current varies in velocity and direction according to season and location Major upwelling areas in Vietnamese coastal zone are along the central coast, offshore

of the Mekong deltas and in the Gulf of Tonkin Out of 20 typical coastal ecosystems estuaries, bays, lagoons, mangroves, coral reefs, sea grass beds etc are predominant and have high biological diversity The most important economic activities such as

water way and ports, agriculture, fishery, industry, mining and rapid tourism etc are

concentrated along the coastal line (Figure 2.1)

Figure 2.1 The economical zones and activities in Vietnamese coastal area (Thanh et

al., 2004)

Along with climate change human activities have caused the loss of coastal habitats such as mangroves, corals reefs, seagrass beds etc The mangroves declined to 155000

ha in 2000 from 400000 ha in 1943 (Thanh et al., 2004) Most of the coastal

aquaculture ponds are built in the areas occupied by mangroves Coral reefs and sea grasses are destroyed by changing in sea water parameters, strong waves and storms, and pollution as well as other destructive practices such as sediment excavation, illegal fishing gears and the dynamite fishing Population increase, rapid unregulated

tourism, agriculture, aquaculture, industrialization and transportation activities have released a large amount of pollutants into the coastal zone including heavy metals, chemical fertilizers and pesticides, dyes, untreated waters etc directly or indirectly by

river flow (Thanh et al., 2004 & World Bank, 2011) Along the coastline, there are up

to 12 large urban centers, 90 ports and harbors, hundreds of fishing docks and around 238,600 industrial facilities (Thanh, 2006) Every year, Vietnam‟s coastline receives a river water discharge of around 880 billion cubic meters Oil pollution is the most serious in near shore areas Calculations show that in 1995, there was 47 tons of oil released into Vietnamese sea from different sources It has been known that there have been 40 oil spills within coastal areas in Vietnam (Thanh, 2006) The extract of coastal minerals from around 100 mineral mines has altered inshore landscapes The rivers have been dammed to create many reservoirs in the catchments and coastal plains for irrigation and hydroelectric power has obstructed the migration for the spawning of fish species The fishery stock showed a clear declining tendency due to overexploitation and use of destructive methods; loss of habitat, and pollution For example, in the last decades the Vietnam‟s fish stock in the Gulf of Tonkin has been reduced to some 185,500 tonnes equaling only 24.6% of the original stock observed during the 1960s-70s At the important fishing grounds, fish stocks have decreased by

up to 25–30%, and even 50% in some sites (Thanh, 2006)

2.2 Study on swimming crab (Portunus pelagicus Linnaeus, 1758)

2.2.1 Classification

Portunus pelagicus (Figure 2.2) is known as the blue swimming crab found in the

intertidal estuaries The species is ideal for aquaculture because of its rapid growth, easy to raise, quick delivery, resistance to both nitrate (Romano and Zeng, 2007a) and ammonia (Romano and Zeng, 2007b) The males are bright blue in colour with white spots, while the females are brown with green staining and are more rounded carapace

as shown in Figure 2.3 (Marshall et al., 2005) Male crab has a long, pointy apron

while the female have a rounded (V-shaped) apron at the underside The classification

of P pelagicus is as follows:

Species: Portunus pelagicus

2.2.2 Habitat and distribution

P pelagicus (Figure 2.4) is a scavenging tropical marine species and widely

distributed in Australia, Indian and Pacific oceans, including the east coast of Africa

and southern Japan as well (Stephenson, 1962; Pazooki et al., 2012 & Olgunoglu and

Olgunoglu, 2016) It is also reported from the Mediterranean Sea, having entered via

the Suez Canal and therefore considered as lessepsian migrant (Foka et al., 2004 & Lai et al., 2010) It is distributed from the intertidal zone to approximately 50 m depth

(Kangas, 2000) The species abundance is high in shallow bays including corals, sandy, muddy or sea grass habitats This species is in very high demand for commercial and food purposes in South and Southeast Asia In Vietnam the species is

distributed throughout the long coastlines and islands (Thuy, 2000 & Ha et al., 2014)

Figure 2.3 Ventral view of male and female P pelagicus

(Source: http://museum.wa.gov.au/explore/articles/blue-swimmer-crab)

Figure 2.2 Blue swimming crab (Source: https://en.wikipedia.org/wiki/File:Portun

us_pelagicus_male.jpg)

Figure 2.4 Distribution of P pelagicus (Source: DFWA, 2011)

2.2.3 Life cycle

The life cycle of P pelagicus consists of mainly 4 stages to become an adult crab

These are 1 Zoea (Zoea-I, Zoea-II, Zoea-III and Zoea-IV), 2 Megalopae, 3 Juvenile

and 4 Adult stage (Figure 2.5) (Josileen and Menon, 2004) Estuarine crabs tend to

mate in autumn during the female moults although the female spawn in late summer The fecundity could vary from 180000 to 2 million The eggs are fertilized by the

stored sperm and attach to the female abdomen when laid (Innocenti et al., 2003 & DFWA, 2011) The eggs take 8 days at 25ºC while at 20ºC take 18 days for fully developed and ready to release Hatching time depends on water temperature such as

at 24ºC it takes 15 days (Smith, 1982) At normal condition, the female incubate the eggs for 18 days and leave the eggs as they hatch into Zoea (DFWA, 2011)

Figure 2.5 Life cycle of P pelagicus (Source: DFWA, 2011; Josileen and Menon,

2004 & Wikipedia, Last edited: 23 March 2018)

2.3 Overview of the research methodology

2.3.1 Molecular markers for studying population genetics

In population genetics, molecular markers have been widely used in analysis of

genetic diversity and genetic relatedness (Martinez et al., 1999 & Roa et al., 1997)

Markers are used in molecular fisheries biology to identify a particular sequence of DNA in a pool of unknown DNA for the purpose of studying genetic variation and population structure in marine organisms According to Khan (2015) there are 11 molecular markers However, the most common markers are described below:

2.3.1.1 Restriction fragment length polymorphisms (RFLPs)

Restriction fragment length polymorphisms (RFLPs) are differences among individuals in the lengths of short DNA fragments cut by restriction endonuclease enzymes to separate the fragments according to their size through gel electrophoresis

(Botstein et al., 1980) Each restriction endonuclease targets different nucleotide

sequences in a DNA strand and therefore cuts at different restriction sites RFLP requires high quality large DNA sample for analysis that is time consuming and labour intensive

2.3.1.2 Random amplified polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) is the random amplification of polymorphic DNA segments from short synthetic oligonucleotide sequences (5-10

nucleotides) (Williams et al., 1990) It can analyse the genetic diversity of an

individual by using random primers from polymerase chain reaction (PCR) The very simple genotyping technology makes RAPD popular in many laboratories However, RAPD cannot distinguish between heterozygous and homozygous locus (dominant mode of inheritance), produce low level of polymorphism and difficult to interpret due

to mismatch between the primer and the template Due to problems in experiment reproducibility and low reliability, many journals don‟t accept experiments merely based on RAPDs anymore

2.3.1.3 Amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) is a PCR-based tool uses a

combination of restriction enzymes to digest genomic DNA and PCR (Vos et al.,

1995) It is capable to detect various polymorphisms in different genomic regions simultaneously Therefore, it has become the superior genetic marker with high levels

of diversity However, the method is highly complex that need to use different kits making it expensive

2.3.1.4 Microsatellites/ Simple sequence repeats (SSRs)

Microsatellites are the outstanding, most popular and extensively employed markers

in population genetic study and evolutionary biology (Miah et al., 2013; Muneer, 2014 & Vieira et al., 2016) Microsatellites are special repeat sequences

Abdul-(Simple sequence repeats, SSRs) containing repeated nucleotide sequences typically from 2 to 7 nucleotides per unit (Tautz and Renz, 1989) The primer design for PCR amplifies these repeat sequences using specific sequences at the ends of the

microsatellite locus However, this technique requires of prior characterization of sequences containing microsatellite loci to allow primers design for PCR, making it an

experimentally long, labour intensive and costly process (Zalapa et al., 2012 & Liu et

al., 2016)

2.3.1.5 Single nucleotide polymorphisms (SNPs)

Single nucleotide polymorphisms (SNPs) are polymorphic DNA markers that can be identified by single nucleotide differences By using limiting enzyme to break down genomic DNA into short segments and attach adapters to both ends, large number of

genetic variants such as SNPs can be identified from the sequence data (Baird et al.,

2008) SNP is defined as a genomic locus where two or more alternative bases occur with appreciable frequency (>1%) SNPs have gained high popularity for genetic study because they are the most abundant genetic variations distributed in the genome SNPs reflect the DNA sequence differences of two individuals of the same species at the base pair level, when a single nucleotide (A, T, G, C) in the genome is altered

(Figure 2.6) A variant is considered to be a SNP when differentiation occurs by

1/1000 nucleotides or more (Pierce, 2003) If a SNP occurs within a gene, then the gene is described as having more than one allele With the SNPs, we can compare the genetic diversity of different species, even with broad categories, to examine evolution as well as to determine the classification relationship between species

Carreras et al (2017) used molecular SNPs in the study of genetic variation of

Mediterranean fish populations, and comparison of results with other traditional markers showed that the use of SNPs more effective SNPs are able to be identified with high confidence more recently because of much deeper sequencing coverage provided by the next generation sequencing (NGS) technologies

Figure 2.6 SNPs (The upper DNA molecule differs from the lower DNA molecule at a

single base-pair location Source: http://knowgenetics.org/snps/)

2.3.2 Restriction site-associated DNA sequencing (RADseq)

Restriction site-associated DNA sequencing (RADseq) can identify and score thousands of genetic markers, randomly distributed across the genome, from a group

of individuals using Illumina technology RAD methods are being used and developed with many techniques such as mbRAD (Miller & Cribb, 2007 & Baird et al., 2008), 2b-RAD (Wang et al., 2012), ddRAD (Peterson et al., 2012), and newly developed ezRAD (Toonen et al., 2013) Puritz et al (2014a) studied the strengths and

weaknesses of different RAD protocols to help the researchers when selecting a RAD protocol Here, ezRAD method has selected as it differs from others in its flexibility to use any restriction enzyme

ezRAD is a novel strategy based on RAD sequencing developed at Texas A&M University Corpus Christi, USA to prepare genome libraries that uses the Illumina

TruSeq Library Preparation Kit (Toonen et al., 2013) The technique requires little

technical expertise and investment in laboratory equipment This technique uses two restriction enzymes (RE) namely Sau3AI and MboI suitable for sequencing from 300

to 500 bp Toonen et al., (2013) applied ezRAD method for genomic genotyping in

non-model organisms The process of creating ezRAD library is shown in Figure 2.7

Figure 2.7 DNA library construction using ezRAD technique (Source:

https://www.slideshare.net/LutzFr/bioinformatics-workshop-sept-2014)

2.3.3 Next generation sequencing (NGS)

Sequencing is defined as orderly nucleotides identification process in a polymer of

nucleic acids of a given DNA or RNA fragment (Kumar et al., 2012) to encodes the

necessary information for organisms to survive and reproduce DNA sequencing is very much effective in core research into why and how organisms live as well as in practically all branch of biological research NGS is a type of DNA sequencing method which can be used to sequence the whole genome in a day (Bhejati and Tarpey, 2013) This technology has revolutionized genomic research including the analysis population genetic diversity Although Sanger sequencing, Maxam-Gilbert sequencing ABI/Solid sequencing was available but we used NGS as it offers novel and rapid ways for genome characterization Commercially available technologies are Roche/454 (Pyrosequencing), Illumina/Solexa, Helicos BioSciences, Life/APG-SOLiD system etc In this project, Illumina HiSeq sequencing was chosen to discover SNPs Illumina sequencing technology works in three basic steps: amplify, sequence, and analyze The process begins with purified DNA The DNA gets chopped up into smaller pieces and given adapters, indices, and other kinds of molecular modifications that act as reference points during amplification, sequencing, and analysis (Wikipedia, Last edited: 3 March 2018)

2.4 Overview on population genetics

2.4.1 Population genetics of marine organisms

Rumisha et al (2017) studied the genetic diversity and connectivity in East African Giant Crab (Scylla serrata) for the effective management purposes The study

analysed eight microsatellites loci from 230 tissue samples of the crab collected from Kenya, Tanzania, Mozambique, Madagascar and South Africa The study found lower current nucleotide diversity than the historic nucleotide diversity due to either overexploitation or historic bottlenecks in recent years In their study, microsatellite

loci also did not show significant genetic variation (p>0.05) Tracey et al (2011) observed low level of genetic variability in eight populations of Homarus americanus

The species were geographically isolated but found genetically similar Gold and

Richardson (1991) identified normal genetic variability in red drum (Sciaenops

ocellatus) populations from the Atlantic coast of the southeastern USA and the

northern Gulf of Mexico Gold et al (2013) compared the population genetic among Cobia (Rachycentron canadum) from the Northern Gulf of Mexico, U.S Western

Atlantic and Taiwan using nuclear-encoded microsatellites and mitochondrial DNA (mtDNA) sequences The study didn‟t recommend using of Cobia broodstock from Taiwan in U.S aquaculture facilities because both genetic markers from Taiwan showed significant differences with Cobias in U.S waters

2.4.2 Review on Portunus pelagicus

A number of literatures studied the effect of different environmental factorss on

different activities and functions of P pelagicus (Lesatng et al., 2003; Bryars and Havenhand, 2006; Hajisamae et al., 2015 & Sugumar and Vasu, 2013) These study

suggested need for effective management of the stocks of swimming crab According

to Edgar (1990) and Kangas (2000) high gene flow levels are expected in this species

as it exhibits moderately long planktonic larval stages (26-45 days) and high mobility

during the crab phase Bryar and Adams (1999) studied population genetics of P

pelagicus using allozyme analysis at seven polymorphic loci in Australia Samples

were collected from 11 sites covering three regions Four discrete subpopulations and

no evidence of population sub-structuring within each population were recognized

Sezmis (2004) in his PhD research, found significant genetic difference as well as

genetic heterogeneity in P pelagicus populations in Australian waters This study was

based on six microsatellite loci and polymorphism of a 342-bp fragment of cytochrome oxidase subunit I (COI)

Ren et al (2016) studied swimming crab populations in four regions from

southeastern sea of China based on mitochondrial COI gene The results showed high haplotype diversity (0.6833-0.8142) and low nucleotide diversity (0.0021 - 0.0034) In

addition, pairwise Fst values ranged from 0.02346 to 0.03655, suggested limited

population genetic structure and high levels of gene flow among populations along the distribution areas of China

Klinbunga et al (2010a) studied the genetic differentiation of P pelagicus in Thai

waters using RAPD analysis The study found significant genetic heterogeneity and strong genetic differentiation (P < 0.01 for θ and P < 0.0001 for the exact test) and limited gene flow among populations The study concluded that all the populations are

separate and should treat as a different exploited stock Chai et al (2017) published the population structure of P pelagicus in coastal areas of Malaysia inferred from

microsatellites Their results revealed low levels of genetic differentiation among the

populations and the existence of inbreeding among different populations of P

with P pelagicus available in GenBank The study also indicated hybridization and

population differentiation among the populations

Miao et al (2017) used traditional ABI solid sequencing method for P pelagicus in

China The study identified and characterized 91 SNP markers from 8426 bp long high-quality DNA sequences Of these 91 SNP loci, each had bi-alleles with the minor allele frequency ranging from 0.0167 to 0.5000 The observed heterozygosity per locus ranged from 0.0333 to 1.0000, while the expected heterozygosity per locus was

from 0.0333 to 0.5085 Forty-four loci showed significant deviation from Hardy–Weinberg equilibrium (HWE)

2.5 Population genetic study and fisheries management

Without acknowledging the population genetics structure and variation, climate

change effects on marine organisms are drastically underestimated (Balint et al.,

2011) Samuiloviene and Kontautas (2012) warned that ignoring the population genetic structure may result in loss of genetic diversity, reduced productivity and ecological damage Waples and Naish (2009) stated that any changes to marine ecosystems alter the selective regimes that component species experience and hence can be expected to produce and evolutionary approach They pointed out that harvest, artificial propagation and climate change are important factor in this regard The authors also mentioned that quantitative genetic and molecular genetics approaches can accomplish this Finally, the authors suggested that studies of marine and anadromous populations have provided a great deal of information about natural

levels of genetic variability Casey et al (2015) stated that modern genetic and

genomic approaches are likely to help address some of the most pressing fisheries

management challenges Review by Ovenden et al (2015) guided that genetic

analytical approaches are important for species identification, stock structure, resolving mixed-stock fisheries, ecosystem monitoring, genetic diversity and resilience and so on The authors summarized that genetic data can offer a versatile and useful tools for informing fisheries managers about issues that have a biological basis As for example, Spies and Punt (2015) studied the utility of genetics in marine fisheries management Their results showed that managed fishing can reduce stock sizes below target reference points when distinction populations are not managed based on the results of genetic testing Samuiloviene and Kontautas (2012) also considered genetic management as an important component of strategies that ensure the conservation and recovery of Atlantic salmon and brown trout populations in the wild

Ciftci and Okumus (2002) stated that molecular genetic studies of natural population are dependent on the polymorphic neutral markers and offer the possibility of investigation of population structure Genetic studies also provide scientific data for regulation of harvest to protect weaker populations and finally long term management

of fisheries resources Wenne et al (2007) also reviewed the current development of

genomic technologies and pinpoint their potential beneficial applications as well as implications for fisheries management and aquaculture

Ungfors et al (2009) found lower genetic variation in edible crab (Cancer pagurus) in

Swedish waters using microsatellite DNA They suggested that the stakeholders can take precautionary approach such as implementing landing size restrictions, no fishing below a certain local biomass or above a defined fishing mortality for the management

of the edible crab populations In order to prevent the extinction of wild Baltic salmon and a further decrease of naturally produced populations, the International Baltic Sea Fisheries Commission (IBSFC) in 1997 adopted the Salmon Action Plan 1997-2010

by releasing salmon fry, parr and smolt with extinct populations (Samuiloviene and Kontautas, 2012)

Marine reserve is one of the major ways for rebuilding depleted populations and

conserving biodiversity in marine ecosystems (Green et al., 2014) For instance, this

technique showed positive result for population genetics in abalone fisheries in

California (Munguia-Vega et al., 2015) Based on the study result on fiddler crab

Austruca occidentalis, Nehemia and Kochzius (2017) suggested that mangrove

restoration might be one of the most effective ways to conserve genetic diversity in the fiddler crab with environmental change

CHAPTER 3: MATERIALS AND METHODS

3.1 Sampling sites and tissue collection

Blue swimming crab Portunus pelagicus (Figure 3.1) was collected along the

north-south geographical temperature gradient (Cat Ba Island - Hai Phong City; Ha Long Bay - Quang Ninh Province in the North, Nha Trang Bay and Van Phong Bay - Khanh Hoa province; Song Cau and Tuy Hoa - Phu Yen province in the Center, and Phu

Quoc Island, Rach Gia City - Kien Giang Province in the South) (Figure 3.2 and

Table 1) The crabs were collected following simple random sampling at the

exploitation site, transported alive in aerated sea water to the laboratory where they were kept in aquaria until dissected All tissue samples were taken from chelipeds of fresh crab and preserved in 98% ethanol immediately after collection

Figure 3.1 Dorsal view of P pelagicus (A Male and B Female)

Table 3.1 Portunus pelagicus sample site information and genetic diversity based on neutral SNPs, including number of samples

collected (N), observed number of alleles (Na), effective number of alleles (Ne), observed and expected heterozygosity (Ho/He) and percentage of polymorphic loci (%P)

Figure 3.2 Map of sampling stations for genetic study of P pelagicus along the coastal

line in Vietnam

3.2 Research outline

The study was consisted of several steps designed in the following flow chart

Step 1 Sample collection and preservation

(98% ethanol)

Step 2 DNA extraction (QIAGEN), Gel electrophoresis and DNA concentration (Qubit)

Step 3 DNA library preparation by ezRAD protocol

Cut genomic DNA (Sau3AI

Adaptor ligation PCR amplification

Step 4 Next Generation Sequencing

Illumina HiSeq 2500/4000

Step 5 dDocent, SNP calling and SNP filtering

Step 6 Advanced genomic analysis (STRUCTURE, PCoA, MIGRATE-N)

Figure 3.3 Flow diagram of the research work

3.3 Research methodology

3.3.1 DNA extraction and digestion

Genomic DNA was extracted from preserved tissue samples using the DNeasy Blood

& Tissue Kit (Qiagen) following to the manufacturer's instructions (Appendix 1), and

treated with RNase (100 mg/mL) to remove residual RNA Instead of getting max 300

µl elution, extracted DNA was continuously eluted three times (100 µl elution/time) to get better DNA quality All elution were assessed using gel electrophoresis (1% agarose gel) The best elution (sharp, high molecular bands, no smear) was selected to

determine the concentration by Qubit® 2.0 Fluorometer (Invitrogen) (Appendix 2)

Selection criteria was concentration ≥ 3 ng/µl Selected DNA template (100 ng) were then purified with AmpureXP (Agentcourt) SPRI beads using a 2:1 template to bead volume ratio

Purified DNA from each crab individual was simultaneously digested with two

restriction enzymes: MboI and Sau3AI (NEB) (Appendix 3) Each digestion was

performed in 25 µl reactions: 2.5 µl SmartCut Buffer (10X), 0.5 µl MboI and 0.5 µl Sau3AI (5 unit/µl), and 21.5 µl of DNA template (elute from the bead) Digestions were incubated at 37 ºC for 3 h to overnight, and then 65 ºC for 20 mins, cleaned with PEG solution (10 g PEG, 7.3 g NaCl, water fill up to 49 ml), and elute with 20.1 µl Illumina Resuspension Buffer

3.3.2 EzRAD library preparation

Cleaned digestions were inserted directly into the Illumina TruSeq nano DNA library Prep kit following the Sample Preparation v2 Guide starting with the “Perform End Repair” step Digested libraries were end repaired, 350 bp size-selected by SP bead, 3ʹ ends were adenylated and Illumina adapters were ligated to the digested genomic DNA samples

PCR reactions were performed using a total volume of 15 µl including 1.5 µl Illumina PCR Primer Cocktail, 6 µl Illumina Enhanced PCR Mix, 1.875 µl ddH2O and 5.625 µl DNA libraries Biorad thermocyclers (Icycler) were used under the following temperature program: initial denaturation at 95 ºC for 3 min, followed by 8 cycles of

98 ºC for 20s, 60 ºC for 15s and 72 ºC for 30s Final extension was done at 72 ºC for 5 min and the soaking temperature was set to 4 ºC

PCR products (The 400–500 bp fragments (of which 120 bp are the ligated adapters) were inspected using a 1.5% agarose gel with ethidium bromide run at 90 V for 20 min and bands were visualized under UV transilluminator PCR products were purified using SP Beads (1:1), and quantified using qPCR DNA libraries were sequenced as paired-end 100 bp runs on HiSeq 2500/4000 system (Illumina) in Texas A&M University Corpus Christi Genomics Core Laboratory, USA

3.3.3 SNP discovery and filtering

The SNPs detection is implemented by dDocent v2.0 (Puritz et al., 2014b) First, raw FastQ files were trimmed using Trimmomatic v0.3 (Bolger et al., 2014) to

simultaneously remove Illumina adapter sequences, and any base that have a quality

score (Q-score Appendix 4) of less than 10 (Toonen et al., 2013) These reads were

saved for de novo assembly Rainbow v2.0.2 program (Chong et al., 2012) was

applied to clustered and assembled forward and reverse read files into a final assembly

of reference contigs These contigs were then clustered to reference genomes by

CD-hit v4.6.1 (Fu et al., 2012 & Li and Godzik, 2006) based on overall sequence

similarity (90% by default) Quality trimmed reads were mapped to the reference genomes using BWA v0.7.12 (Li and Durbin, 2009 & Li and Durbin, 2010) with the MEM algorithm (Li, 2013) SAM files were converted to BAM files using

SAMTOOLS (Li et al., 2009) and output was further restricted to reads with mapping

quality above 10

SNPs calling was performed using Freebayes v0.9.21(Garrison and Marth, 2012) with default parameters Raw SNPs files are concatenated into a single variant call format

(VCF) file using VCFtools v0.1.11 (Danecek et al., 2011) The raw SNPs calls are

then filtered with some instances of VCFtools The first instance filters out minor allele frequency (MAF) (0.01 - 0.5), minimum mean depth (mean DP ≤ 10), INDEL loci, this decomposed insertion and deletion genotypes The second round of filtering removes HWE (Hardy-Weinberg Equilibrium with p < 0.001, and then genotyped

0.95 (This applied a genotype call rate (95%) across all individual) Finally, filter out last minimum quality score (Q) < 30, and allele balance (AB) (0.25 - 0.75)

3.3.4 Outlier loci detection

We used BayeScan v2.1 (Foll and Gaggiotti, 2008) and Lositan (Antao et al., 2008)

methods with default settings to detect loci with greater than expected levels of divergence among geographic populations Bayesian approach was used that allows the estimation of the posterior probability of a given locus being under the effect of selection (Foll and Gaggiotti, 2008) Loci putatively under selection were defined as those with false discovery rate (FDR) < 5%, alpha-values significantly >0 (i.e with Q-values smaller than 0.05), while loci putatively under balancing selection had alpha-values significantly smaller than 0 All other loci were considered neutral were further

used for studying population genetics

3.3.5 Genetic diversity and effective population size

Allele frequencies, expected (He) and observed heterozygosity (Ho) were estimated using the package GenAlEx v6.5 (Peakall and Smouse, 2012) We used GENODIVE 2.0b25 (Meirmans and van Tienderen, 2004) to calculate equilibrium estimates of the

level of allelic fixation via pairwise Fst The significance of pairwise Fst values was assessed with an AMOVA test and 1000 permutations in GENODIVE Pairwise Fst

value was confirmed by Genepop v4.51 (Rousset, 2008)

Estimates of genetic effective population size (Ne) were generated with NEESTIMATOR v2b (Do et al., 2014) with a minor allele frequency cutoff of 0.05

Effective population size was calculated for all sampled sites individually, for the north (HP-QN), center (KH-PY) and south (KG) coastal water, and for all the individuals combined into a single population (along the north – south coast of Vietnam)

3.3.6 Analyses of population structure

We used the model-based Bayesian clustering method implemented in STRUCTURE

v2.3.4 (Pritchard et al., 2000) to infer the number of K (K=1-5) with 5,000 iterations

of burn-in followed by 5,000 iterations of Markov Chain Monte Carlo (MCMC), using

correlated allele frequencies admixture model The optimal value of K is evaluated using the Evanno method (Evanno et al., 2005) by Structure Harvester v0.6.94 (Earl

and vonHoldt, 2012)

We tested the population connectivity along Vietnamese coastal line using two different data sets: neutral loci, and loci suppose under selection (divergent loci) We used information from two data sets to identify the hierarchical pattern of genetic

structure in this species: (1) a STRUCTURE analysis (Pritchard et al., 2000), and (2) a

principle coordinates analysis (PCoA) of genetic distances between individuals (Smouse and Peakall, 1999) using GenAlEx v6.5 (Peakall and Smouse, 2012)

We performed the STRUCTURE analysis using two dataset of 80 individuals with

optimal K=3 For optimal K=3 we carried out 5,000 iterations of burn-in followed by

5,000 iterations of MCMC, under the correlated allele frequencies model allowing admixture PCA (for neutral and divergent loci) was performed in GenAlEx v6.5

using a covariance-standardized genetic distance of Fst, and allows for a graphical

description of the genetic divergence among populations in multivariate space

3.3.7 Historic migration pattern

Historic gene flow between populations was estimated using the Bayesian inference implemented in MIGRATE-n v3.6.11 (Beerli and Felsenstein, 2001) The run was performed using 500,000 recorded genealogies sampled every 100 steps, preceded by

a burn-in of 20,000 Four hot chains were used with temperatures: T1 = 1.0, T2 = 1.5, T3 = 3.0 and T4 = 1.0x106 After optimization, the maximum mutation-scaled

effective population size (θ) prior was set at 0.1 while the maximum mutation-scaled migration (M) prior was set at 20,000 Five hypotheses of migration among

populations were tested: (1) symmetric migration rates between all sites (Panmixia Model), (2) non-symmetric migration rates between all sites (Full Model) (3) migration between all sites only from the north to the south (North-South Model), (4) migration between all sites only from the south to the north (South-North Model), (5) migration occurring only between neighboring, north-center sites but no migration between south population (South Separate Model) The most likely model was chosen using the Bezier ln produced by Migrate-N according to Beerli (2009)

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Results

4.1.1 Reference genome and SNP detection

Results of sequencing of swimming crab specimens from three locations generated

604123297 reads with a reading length of 101 bp Reads with low quality base (Q

<20) and adapters were removed The optimal reference genome of 3280843 bp is constructed from 9583 high-quality reads SNPs were called using Freebayes v0.9.21 (Garrison and Marth, 2012) with BAM input and optimal reference genome Initially,

107115 raw SNPs were detected from 86 individuals those were filtered using

VCFtools v0.1.11 (Danecek et al., 2011) VCFfilter has reduced number of

individuals and SNPs with each filter index Finally, the data set consisted of 731 SNPs from 80 individuals Information on individuals removed and SNPs filtration

after each step of analysis is presented in Appendix 5

4.1.2 Outlier loci detection

A total of 731 polymorphic SNPs in P pelagicus stocks across all sample sites were

successfully discovered BayeScan identified no SNPs as outliers (FDR≥0.05) The Lositan Selection Workbench (Lositan) identified 10 outlier loci After removing outlier loci, the remaining 721 polymorphic SNPs were considered as neutral and 61 putatively under selection with FDR<5% and 95% confidence interval were used for

further data analysis Detection of outliers is presented in Appendix 6

4.1.3 Genetic diversity of Portunus pelagicus

Genetic diversity of P pelagicus based on neutral SNPs was presented in Table 3.1

Observed number of alleles per locus (Na) in KG, KH-PY and HP-QN was 1.935±0.009, 1.992±0.003 and 1.945±0.009, respectively (mean 1.957±0.004) Number of effective alleles per locus (Ne) was 1.335±0.009, 1.327±0.007 and 1.293±0.008 in KG, KH-PY and HP-QN, respectively Observed heterozygosity (Ho) range: 0.253-0.288 and expected heterozygosity (He) range: 0.206-0.231 among populations Highest percentage of polymorphic loci (%P) was observed in KH-PY (99.17%) and lowest was in KG population (93.48%) Similar values for the indices of