Multiple population structure of the giant eel Anguilla marmorata in Thua Thien Hue, Vietnam base on COI sequences

DOI: https://doi.org/10.21203/rs.3.rs-40345/v1

Abstract

The giant mottled eel Anguilla marmorata is a species of great economic, ecological, and conservative values in the Southeast Asian region. The research aims to conservation and evaluation of the genetic diversity of A. marmorata populations living in Thua Thien Hue, Vietnam. The sequencing of the barcode region of the cytochrome c oxidase subunit 1 gene was carried out for 48 individuals of A. marmorata , which were collected from five different ecosystem regions. The sequences were analyzed using various genetic, phylogenetic, and population analyses to assess their variability. A total of 20 polymorphic sites and 17 haplotypes were identified. Very low fixation (Fst = -0.073 – 0.003; < 0.05) was found for A. marmorata populations in the region. The populations showed signs of recent populations’ expansion, besides negative Tajima’s D test, Fu’s Fs, Fu and Li’s D* and F* test values. The genetic evolution of eel occurs in a randomized pattern over a large population, with the likelihood of rare alleles appearing in the population. In Thua Thien Hue (Viet Nam) territories, we indicated the five areas for conservation units with moderate eels’ populations’ diversity.

Introduction

In Viet Nam, there were 5 out of 19 species/ subspecies belong to the genus Anguilla (Watanabe 2003), (Watanabe et al. 2004), (Watanabe et al. 2005), namely: A. nebulosa (McClelland, 1844), A. japonica (Temminck & Schlegel, 1984), A. marmorata (Quoy & Gaimard, 1824), A. celebensis (Kaup, 1856), and A. bicolor pacifica (Schmidt, 1928) (Phung 2001). A. marmorata was identified to appear in the central region of Vietnam from Nghe An to Khanh Hoa (Nguyen et al. 2018). In Thua Thien Hue, Anguilla marmorata is widely distributed, exploited and cultivated with high economic value (Huyen and Linh 2019). They are used as breeding stock in farm and food in the golden-colored and silver-colored life stages (Watanabe et al. 2009).

A. marmorata has a catadromous life-history strategy (Arai 2016) with distances from several hundred to thousands of kilometers (Arai 2014). During migration between oceans and freshwater during special stages of the life cycle, strong environmental changes have shaped not only their physiological characteristics (Wang et al. 2014), (Le et al. 2009) but also the genetic structure of eels (Ishikawa et al. 2004); (Fahmi et al. 2015); (Li et al. 2015); (Pavey et al. 2015); (Laporte et al. 2016), (El-Nabi et al. 2017). Besides, upheaval conditions of river management, water containment, overfishing, and pollution may have influenced the movement of upstream eels (Wasserman et al. 2011), (Le et al. 2009) and downstream (Linh et al. 2010), (Huyen et al. 2012), (Huyen and Phu 2015) led to the increase of population decline risk of the giant mottled eel in the wild. Although in the world A. marmorata is ranked at LC level (least concern) (Pike et al. 2019), but it has been listed in the Viet Nam Red Data Book as VU (Vulnerable) lever since 2007 (Ministry of Science Technology and Environment 2007).

DNA barcoding provides an efficient method for species-level identifications (Hubert et al. 2008), to address questions relating to the ecology and evolution of natural systems (Kress et al. 2015) based on short, standardized gene regions (Hebert and Barett 2005), (Hebert et al. 2003). For animals, markers located on the mitochondrial genome, such as cytochrom b (cytb) gene, cytochrom c oxyase 1 (COI) gene, and D-loop region…. often have been recommended using by scientists (Kress et al. 2015). In which, a short DNA sequence in the mitochondrial gene encoding COI with 600 base pairs (bp) (Hebert and Barett 2005), (Hebert et al. 2003) has been accepted as a practical, standardized, species-level DNA barcode for many groups of animals (Hebert et al. 2003), (Kress et al. 2015). To assess the genetic diversity of the A. marmorata population distributed in Thua Thien Hue (TTHue), Vietnam regarding the distribution environment and examining the evolution and development of populations, identifying conservation units for A. marmorata in TTHue, we designed this study basing on the amplification and sequencing of polymerase chain reaction (PCR) for the COI subunit of A. marmorata collected in 5 different ecological regions.

Materials And Methods

Ethics statement

All animal protocols were approved by the Committee on the Ethics of Animal Experiments of Hue University, Vietnam (permit No. DHH2019-02-113), and were performed strictly with the Guide for fishing capture and animals of Institute of Biotechnology. Animals were fishing capture by fishermen and allowed the Provincial Department of Fisheries.

Collection of specimens

Wild adult specimens of A. marmorata were collected from 5 localities in Thua Thien Hue, Viet Nam, including Phong Dien (PD), Thao Long dam (DTL), Truoi dam (DTR), Nam Dong (ND), Bu Lu and Lang Co (PL) from October 2017 to October 2018 (Fig. 1). A. marmorata species were identified using the keys developed by Watanabe (2004). Tissues from the adductor muscle were dissected from fresh specimens, preserved in 95% ethanol, and frozen at -80 °C until DNA extraction.

Genomic DNA isolation and amplification

Total genomic DNA was extracted by a description of Kumar et al. (2007) with a modification for A. marmorata. The primer used to amplify the COI gene segment was designed based on the nucleotide sequence of the two gene bank codes: A. marmorata - AP007242.1 and A. marmorata - HQ141374.1. The nucleotide sequences of the primers are A. marFw-1: 5’-GCACTAAGCTTCTAATCCG- 3’ and A. marRv-1: 5’-GATGATTATTGTGGCAGAAG-3’. The PCR amplifications were performed in 35 µL reaction volumes: 1 µL DNA, 1 µL F-primer (10 mM), 1 µL R-primer (10 mM), 7 µL PCR buffer (10X), 0.5 µL dNTP (10 mM), 0.2 µL Enzyme Taq (5 UI/ µL) and sterile distilled water addition to full volume 35 µL. PCR amplification was performed on the machine (MJ-Mini™ Persanol Thermal Cycle, Bio-Rad) according to the following thermal cycling: 95 ° C / 5 minutes; followed by 30 cycles: 95 °C / 45 seconds, 51 °C / 30 seconds, and 72 °C / 1 minute; The last is 72oC / 7 minutes. PCR product quality was checked by running electrophoresis on 1% agarose gel with buffers used as TAE 1X (Tris - acetate 40 mM + EDTA 1 mM) and stained with ethidium bromide fluorescent dye (EtBr 0, 5 µg / L) while washing the gel with distilled water for 10 minutes. The gel is observed by the Gel Documentation image analysis system.

Sequence alignment and molecular phylogenetic analysis

For all sequence analyses, COI genetic similarities were evaluated using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST) to identify A. marmorata sequences. The raw DNA sequences were edited using BioEdit (Hall 1999) and the pairwise, as well as multiple alignments of sequences, which was performed using ClustalW (Larkin et al. 2007) alignment editor. Multiple sequence alignment was also checked manually, and the consensus sequences were obtained. Molecular phylogenetic analyses were performed in MEGA. X software (Kumar et al. 2018). Sequence data were subsequently analyzed for Neighbour Joining methods with bootstraps of 1000 replicates (Felsenstein 1985). Nucleotide composition analysis was carried out using BioEdit. Transition and transversions were equally weighted. The final consensus sequences were submitted to the National Center for Biotechnology Information (NCBI) database with accession number - MN067923 to MN067970. Standard genetic diversity indices, such as the number of haplotypes, polymorphic sites (S), haplotype numbers (h), haplotype diversity (Hd), nucleotide diversity (π), population mutation rates based on the number of segregation sites (θω) and mean the number of pairwise differences (θπ) was calculated with DnaSP 5.0 (Librado and Rozas 2009). Statistical analysis to distinguish DNA sequences evolving randomly (neutrality) with those evolving under a non-random process was done using Tajima’s D (Tajima 1989); Fu’s Fs tests; Fu & Li's Fs test and Fu & Li's D test (Tamura and Nei 1993). The Network ver. 5.0 software (http://www.fluxus-engineering.com/) was implemented to estimate phylogenetic relationships among the unique haplotypes. Genetic differentiation, genetic distance, and migration rate among the populations were estimated by calculating the F statistic (Fst) between the populations and testing their significance with 1000 permutations.

Results

Genetic variation

Sequences of the 843 bp COI gene were determined in 48 specimens, 20 polymorphic sites, and 17 haplotypes were detected. 12 haplotypes were found in only one population, 2 (H1 and H9) were found equally between two populations, only 1 (H7) was found in three populations, and 2 (H4 and H5) were found in all five populations (Table 1 and Fig. 2). Haplotype positions were showed in Table 1.

Table 1

Variable position of 17 haplotypes of COI of A. marmorata in Thua Thien Hue, Vietnam

Haplo-type

GenBank accession number

Nucleotide position beginning from 5´ end

39

75

171

174

189

210

237

270

342

387

420

582

585

611

657

690

747

804

808

812

H1

MN067923, MN067925,

MN067938

A

C

C

T

T

C

T

G

G

A

G

T

A

T

A

C

C

A

A

G

H2

MN067924

*

*

*

*

*

T

C

*

*

*

A

*

*

*

*

*

*

*

*

*

H3

MN067926

*

*

*

*

*

*

C

*

A

*

A

*

*

C

*

T

*

*

*

*

H4

MN067927, MN067928,

MN067929, MN067930,

MN067932, MN067934,

MN067937, MN067939,

MN067943, MN067946,

MN067947, MN067956,

MN067958, MN067959,

MN067960, MN067963,

MN067964, MN067966,

MN067967, MN067970

*

*

*

*

*

*

C

*

*

*

*

*

*

*

*

*

*

*

*

*

H5

MN067931, MN067935,

MN067940, MN067942,

MN067950, MN067954,

MN067962, MN067965

*

*

*

*

*

*

C

*

*

*

*

*

*

*

*

*

*

G

*

*

H6

MN067933

*

*

*

 

C

 

C

*

*

*

*

*

*

*

G

*

*

*

*

*

H7

MN067936, MN067945,

MN067957

*

*

*

*

*

*

C

*

*

 

A

*

*

*

*

*

*

*

*

*

H8

MN067941

*

*

T

*

*

*

C

A

*

*

*

*

*

*

*

*

*

*

*

*

H9

MN067944, MN067969

*

*

*

*

*

*

C

*

*

G

*

*

*

*

*

*

*

 

*

*

H10

MN067948

C

         

C

*

*

*

*

*

*

*

G

*

*

*

*

*

H11

MN067949

*

*

*

*

*

*

C

*

*

*

*

G

G

*

G

*

*

G

*

*

H12

MN067951

C

*

*

*

*

*

C

*

*

*

*

*

*

*

*

*

*

*

*

*

H13

MN067952

C

*

*

*

*

*

C

*

*

*

A

*

*

*

*

*

*

*

*

*

H14

MN067953

C

T

T

*

*

*

C

A

*

*

*

*

*

*

*

*

*

*

*

*

H15

MN067955

*

*

*

*

*

*

C

*

*

*

*

*

*

*

G

*

*

*

*

*

H16

MN067961

*

*

*

C

   

C

*

*

*

*

*

*

*

*

*

T

*

*

T

H17

MN067968

*

*

*

*

*

*

C

*

*

*

*

*

*

*

*

*

*

*

C

*

Haplotype diversity (h) and nucleotide diversity (π) based on COI genes for the 5 values are listed in Table 2. Haplotype diversity (h) and nucleotide diversity (π) values for the whole population in TTHue are 0.801 and 0.00196, respectively. Haplotype diversity of 5 populations in different eco-regions ranges from 0.7556 to 0.9000. In particular, the DTR population has the highest haplotype diversity value. The lowest belongs to DTL and PL. Nucleotide diversity values in these populations ranged from 0.00161 to 0.00242 with the highest in the PD population.

Concerning demographic estimators, methods of Tajima’s D test, Fu’s Fs, Fu, and Li’s D* and F* test were used to test population neutrality (Tajima 1989), (Tamura and Nei 1993). The results in Table 2 show that all exposed exhibited significant negative values ​​for all neutrality tests with p < 0.05. These indicators support standing 'expansion in these areas. The population of eel in TTHue evolved by a random selection expanded population, and rare alleles appear in the population with high frequency. Also, the value of Fu and Li’s D * = -2.03402 (p < 0.05) indicated that in the study population, many individuals were showing a big difference compared to other individuals in the population. Besides, when considering the values ​​of Tajima's D test, Fu's Fs, Fu and Li's D * and F * test in 5 individual populations according to the sampling area, the trend was similar but the value p > 0.05 except for Fu's Fs results for the COI gene in the PL. This shows that the difference in the individual eel is only significant when selected with large populations (14 individuals or more).

Table 2

Neutrality test results for COI data obtained for A. marmorata in Thua Thien Hue, Viet Nam

Population

DTL

DTR

ND

PD

PL

TTHue

No. of sample

10

5

9

14

10

48

h

5

4

6

9

5

17 (35.42%)

S

7

4

6

9

6

20

Hd

0.75556 ± 0.0168

0.9 ± 0.0259

0.88889 ± 0.00828

0.87912 ± 0.00621

0.75556 ± 0.01678

0.801 ± 0.00267

π

0.00185

0.00190

0.00178

0.00242

0.00161

0.00196

θɷ

2.47

1.92

2.21

2.83

2.12

4.51

θπ

1.56

1.60

1.50

2.04

1.36

1.65

Tajima’s D

-1.573**

-1.0938

-1.3984

-1.06599

-1.4929

-2.03402*

Fu's Fs

-1.181*

-1.405*

-2.978*

-4.742*

-1.507

-12.228192*

Fu and Li's Fs

-1.818

-1.0938

-1.5509

-1.07521

-1.6893

-2.93225*

Fu and Li's D

-1.634

-1.0938

-1.3904

-0.8946

-1.51

-2.70391*

Abbreviations: h haplotype numbers, S number of segregation sites, Hd haplotype diversity, π nucleotide diversity, θɷ Watterson’s theta based on S, θπ theta based on π. *P < 0.05

Population genetic structure

Significant Fst values were observed in all pairwise comparisons between populations for the COI gene (p < 0.001; Table 3). Pairwise FST values ranged from -0.073 to 0.048, indicating weak genetic differentiation between populations (Fst<0.05). The biggest difference occurred in two populations DTL and PD (Fst =0.04558), while Pairwise Nm value ranged from -0,01961 to 0.011456 (< 1) indicating extremely low gene flow among the populations.

Table 3. Fst value and gene flow among 5 populations of A. marmorata in Thua Thien Hue, Viet Nam

Population

DTL

DTR

ND

PD

PL

DTL

--

0.012

-0.012

-0.011

0.001

DTR

-0.038*

--

0.003

-0.012

0.011

ND

-0.042*

-0.073*

--

-0.020

-0.011

PD

0.046*

-0.045*

0.012*

--

-0.010

PL

0.003*

-0.041*

-0.045*

0.048*

--

Data allow the diagonal is Fst; Nm values are data above the diagonal.

* indicates the significance level of Fst value at p < 0.001.

Haplotype network analysis

The median-joining network (Figure 2) illustrates the polymorphic sites, including the number and frequency of the haplotypes for COI sequences. The COI network was radial-like with a high number of unique haplotypes closely related to 1 central haplotype (H4). Dominant haplotype H4 accounted for 27.87% (20/48) of all 48 specimens. This also indicated that H4 is an ancestral haplotype. H5 also showed the presence of all sub-populations in the study area but with little correlation with other haplotypes, which indicated the specificity of it. The haplotypes tended to be dispersed in comparison to the central haplotype, revealing a tendency to generate discrepancies among independent individuals. The giant mottled eel A. marmorata populations in TTHue are strongly correlated with each other, and there seems no separation of an individual population.

The evolutionary history was inferred using the Unweighted pair group method with arithmetic mean (UPGMA) (Sneath and Sokal 1973) in figure 3 with 1000 replicates of the bootstrap test (Felsenstein 1985). The sum of the branch length is 0.07028378. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Nei-Gojobori method (Nei and Gojobori 1986) in MEGA X (Kumar et al. 2018). The UPGMA tree showed that most of the haplotypes were weakly associated (less than 50% bootstrap support) or unresolved, the differences among which were possibly due to low nucleotide. Haplotypes of COI of A. marmorata in TTHue were clustered into 2 obvious branches. H8 and H14 stand in the same independent branches with the remaining 14 haplotypes with bootstrap support of 56%. H5 and H15 together with H12 and H13 were grouped into 2 subgroups in branch 2 with the highest correlation (bootstrap> 60%). H4, H7, and H17 also form a group but have an extremely low bootstrap support rate of only 17%. The remaining haplotypes have a discrete distribution in the branch.

Discussion

A. marmorata with long life and long migrate distance could have a strong population genetic structure. Genetic diversity within and between populations provides a potential genetic resource for future adaptation and can be vital for the fitness of a population (Xu et al. 2012). It is mainly explained by several historical and contemporary processes, such as genetic drift, effective migration, natural selection, fragmentation, and range expansion (Slatkin 1985). Earlier works have analyzed eel population genetics-based mainly on allozymes (Pantelouris et al. 1970, 1971) and demonstrated genetic differentiation between geographical locations. The genetic structure of A. marmorata was later reassessed based on the region of the Cyt b (Fahmi et al. 2015), mitochondrial (mt) DNA (Ishikawa et al. 2004), mitochondria control region (mtCR) (Donovan et al. 2012) and 16S rRNA genes (Gagnaire et al. 2009). COI gene barcode region exhibited a credible population-based diversity that exceeded that obtained with sequencing the mitochondrial Cytb gene, showing a percentage of haplotypes from the total sequences equaling 45% for COI, versus 15% for Cytb (El-Nabi et al. 2017). For the first time in Viet Nam, we could identify the level of genetic diversity of A. marmorata based on COI markers basic. Results of the COI gene showed high levels of haplotype diversity (h = 0.7556–0.9000) and low levels of nucleotide diversity (π = 0.00161–0.00242). 17 haplotypes were identified in the 48 samples analyzed (35.42%). It is a similar trend with some other researchers on Anguilla species. In A. marmorata, Famil et al were found 44 haplotypes, h = 0.937 ± 0.013 and π = 0.861 ± 0.002 (%) in Indonesia base on analyzed the Cyt b gene (Fahmi et al. 2015) and 14 haplotypes, nucleotide diversity (p = 2.156%) and haplotype diversity (h = 0.780) from mtCR sequences in Pohnpei and Kosrae (Donovan et al. 2012). On another hand, 129 haplotypes were also identified with h = 0.92–1.00 and π = 0.13–1.06% for 6 other species of tropical eels (A. interior, A. nebulosi nebulosi, A. bicolor pacific, A. bicolor bicolor, A. celebesensis, A. borneensis) in Indonesia determined by Famil et al. (2015). El-Nabi et al (2017) found 44 segregating nucleotide sites, 33 haplotypes, haplotype diversity (h = 0.94), and nucleotide diversity (π = 0.008) when analyzed the COI of A. anguilla with 525 nucleotides in length (El-Nabi et al. 2017).

Researchers often use Fst to assess gene flow, a higher Fst value indicates a lower level of gene flow (Nm) and higher genetic differentiation among populations (Hedrick 2005). Fst reflects the level of inbreeding within populations (Wright 1984) or the extent to which populations are differentiated (Hartl and Clark 2007). The presence of genetic structure is an outcome of limited gene flow and a high level of genetic drift within each reproductively isolated group. Fst values below 0.05 indicate negligible genetic differentiation, whereas values greater than 0.25 indicate high genetic differentiation within the analyzed population (Weir 1996). Fst values of A. marmorata in TTHue, Viet Nam were significant but weak (Fst = -0.073–0.003; p < 0.05) lower than that was found for A. anguilla populations in Egypt and worldwide (Fst = 0–0.04) (El-Nabi et al. 2017) and high Nm values were detected among the populations. This may be because primitive and highly conservative of the COI gene in this species. However, DTR and ND showed high and significant Fst values (Fst = 0.073, p > 0.05); this indicated a high degree of genetic differentiation among 2 populations, which endorsed a signal of isolation due to distance. Populations may be divided by major geographic barriers such as land barriers and oceanographic patterns.

The negative Tajima’s d test and Fu’s Fs neutrality test values were obtained for all tested populations, thus rejecting the null hypothesis of neutral evolution of the COI marker. This indicated that most populations of A. marmorata in TTHue, Vietnam have been in expansion. These results also showed a similar trend of variation when compared with Famil et al. (2015) and El-Nabi et al. (2017) studies.

Results of the median-joining network of the COI gene showed several ambiguous connections among the 5 populations. The demographic analyses using UPGMA for all haplotypes show 2 similar branches one branch contains two types of haplotypes (COI) from two populations ND and PD and the other branch contains most of haplotypes from the remaining 5 populations. The ramification of the haplotypes has a low Boostrasp's support value. This can be explained by several reasons. The first, Anguilid have a catadromous life-history strategy, spawning in remote tropical seas with larvae that are transported back by currents to their nursery grounds in freshwater or coastal areas (Arai 2016). The golden eel growth stage may be as short as two to three years in warm productive habitats, but about six to 20 years or more in more northerly latitudes, e.g. in the Pearl River, China (Williamson and Boëtius 1993). Intense migration and long-lived habitats in the inland ecosystems have driven the formation of haplotypes independently of individual species. Secondly, A. marmorata, a catadromous eel, migrates upstream on nights, following the lunar cycle. The dramatic environmental changes between ocean and freshwater during particular phases of their life cycle shape their physiological features, e.g. visual sensitivity, olfactory ability, and salinity tolerance (Wang et al. 2014). This can also be proposed as a cause of the occurrence of rare genes and single haplotypes. This induces then isolation by time (IBT) of spawning groups. IBT causes a restriction in gene flow, taking place between early and late spawners (Hendry and Day 2005). And the last, based on analyzed three different phylogenetic trees of A. marmorata population in Thua Thien Hue, Vietnam, Huyen and Linh (2020) showed that there was the high genetic similarity of individuals in eel populations in Thua Thien Hue and it was divided into two separate groups that are guided by the migration process and specific ecological (Huyen and Linh 2020).

Declarations

Availability of data and materials

The datasets of COI sequences analyzed during the current study are available on the GenBank with accession number from MN067923 to MN067970. The data were simultaneously made available to ENA in Europe and the DNA Data Bank of Japan.

Ethics approval and consent to participate

All animals and samples applied to international, national, and regional and Institutional guidelines for animal care and rules in Vietnam.

Consent for publication

Not applicable

Competing interests

The authors report no conflict of interest.

Funding

This work was supported by Hue University (No. DHH2019-02-113) − 03 Le Loi str., Hue city, Viet Nam; The Domestic Master/Ph.D. Scholarship Programme of Vingroup Innovation Foundation (VINIF. 2019. TS. 32); and The Ph.D. scholarship 2018 of Southeast Asian Regional Center for Graduate Study and Research in Agriculture (SEARCA) - College, Los Baños, Laguna 4031 Philippines.

Authors' contributions

Both authors discussed and designed the experiments. Kieu Thi Huyen conducted the main experiments and data analysis. All authors wrote, read, and approved the final manuscript.

Acknowledgments

We thank Mr. Dang Thanh Long with his technician's assistance in laboratories.

References

Arai T (2014) Evidence of local short-distance spawning migration of tropical freshwater eels, and implications for the evolution of freshwater eel migration. Ecol Evol 4:3812–3819. https://doi.org/10.1002/ece3.1245

Arai T (2016) Biology and Ecology of Anguillid Eels (ed. Arai T.): Taxonomy and Distribution. CRC Press: 1–20 [Accessed on 6 April 2018]. https://www.crcpress.com/Biology-and-Ecology-of-Anguillid-Eels/Arai/p/book/9781482255157

Donovan S, Pezold F, Chen Y, Lynch B (2012) Phylogeography of Anguilla marmorata (Teleostei: Anguilliformes) from the eastern Caroline Islands. Ichthyol Res 59:70–76. https://doi.org/10.1007/s10228-011-0245-z

El-Nabi SESH, El-Desoky MS, Mohammed-Geba K (2017) Genetic population diversity of European eel Anguilla anguilla elvers in two Egyptian water bodies, Rosetta (Rachid) estuary and Burullus lake. Genes Genom 39:1035–1045. https://doi.org/10.1007/s13258-017-0572-1

Fahmi MR, Solihin DD, Soewwardi K, Pouyaud I, Berrebi P (2015) Molecular phylogeny and genetic diversity of freshwater Anguilla eels in Indonesian waters based on mitochondrial sequences. Life environment 65(3):139–150. https://www.documentation.ird.fr/hor/fdi:010066025 [Accessed on 12 May 2019]

Felsenstein J (1985) Confidence-Limits on Phylogenies – an approach using the bootstrap. Evolution 39:783–791. DOI:10.1111/j.1558-5646.1985.tb00420.x

Gagnaire PA, Minegishi Y, Aoyama J, Réveillac E, Robinet T, Bosc P, Tsukamoto K, Feunteun E, Berrebi P (2009) Ocean currents drive secondary contact between Anguilla marmorata populations in the Indian Ocean. Marine Ecology Progress Series 379:267–278. DOI:10.3354/meps07895

Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT." In Nucleic acids symposium series, vol. 41, no. 41, pp. 95–98. [London]: Information Retrieval Ltd., c1979-c2000

Hartl DL, Clark AG (2007) Principles of population genetics, 4th edn. Sinauer Associates, Sunderland

Hebert PDN, Barrett RDH (2005) Identifying spiders through DNA barcodes. Can J Zool 83:481–491. https://doi.org/10.1139/z05-024

 

10.1098/rspb.2002.2218

Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes, Proc. Biol. Sci. 207 (1512): 313–321. https://doi.org/10.1098/rspb.2002.2218

Hedrick PW (2005) A standardized genetic differentiation measure. Evolution 59(8):1633–1638. https://doi.org/10.1111/j.0014-3820.2005.tb01814.x

Hendry AP, Day T (2005) Population structure attributable to reproductive time: isolation by time and adaptation to time. Mol Ecol 14:901–916. https://doi.org/10.1111/j.1365-294X.2005.02480.x

Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, Burridge M, Watkinson D, Dumont P, Curry A, Bentzen P, Zhang J, April J, Bernatchez L (2008) Identifying Canadian Freshwater Fishes through DNA Barcodes. PLoS ONE 3(6):e2490. https://doi.org/10.1371/journal.pone.0002490

Huyen KT, Linh NQ, Hue HT (2012) Resources of grass eel (Anguilla marmorata) in Quang Binh estuaries and protected techniques. Journal of Agriculture Rural Development 2/2012:115–122. (in Vietnamese with English abstract)

Huyen KT, Linh NQ (2019) Characterization of giant mottled eel (Anguilla marmorata) gastrointestinal tract that origin from Thua Thien Hue, Vietnam. J Fish Res 3(2):4–9. 10.35841/fisheries-research.3.2.4-9 ) ( :. DOI

Huyen KT, Phu VV (2015) Status of exploitation and management measures and cosnevation development for eel (Anguilla marmorata) resources in Huong river system. Hue University Journal of Science 104(5). (in Vietnamese with English abstract)

Huyen KT, Linh NQ (2020) Phylogenetic analysis of Anguilla marmorata population in Thua Thien Hue, Vietnam based on the cytochrome C oxidase I (COI) gene fragments. AMB Expr 10:122. https://doi.org/10.1186/s13568-020-01059-7

Ishikawa S, Tsukamoto K, Nishida M (2004) Genetic evidence for multiple geographic populations of the giant mottled eel Anguilla marmorata in the Pacific and Indian oceans. Ichthyol Res 51:343–353. https://doi.org/10.1007/s10228-004-0241-7

Kress WJ, Garcia-Robledo C, Uriarte M, Erickson DL (2015) DNA barcodes for ecology, evolution, and conservation. Trends Ecol Evol 30:25–35. https://doi.org/10.1016/j.tree.2014.10.008

Kumar R, Singh PJ, Nagpure NS, Kushwaha B, Srivastava SK, Lakra WS (2007) A non- invasive technique for rapid extraction of DNA from fish scales. Indian Journal of Experimental Biology 45: 992–997. [Download on 6 March 2018] https://pdfs.semanticscholar.org/2d3f/616c0f78098c25b74c5e42446d7e20cc586d.pdf

Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35(6):1547–1549. https://doi.org/10.1093/molbev/msy096

Laporte M, Pavey SA, Rougeux C, Pierron F, Lauzent M, Budzinski H, Labadie P, Geneste E, Couture P, Baudrimont M, Bernatchez L (2016) RAD-Sequencing reveals within-generation polygenic selection in Response to Anthropogenic organic and metal…. Mol Ecol 25(1):219–237. https://doi.org/10.1111/mec.13466

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. https://doi.org/10.1093/bioinformatics/btm404

Le QD, Shirai QD, Nguyen K, Miyazaki DC, Arai N T (2009) Heavy metals in a tropical eel Anguilla marmorata from the Central part of Vietnam. Water Air Soil Pollut 204:69–78. https://doi.org/10.1007/s11270-009-0027-7

Li L, Jia YH, Li P, Yin SW, Zhang GS, Wang XL, Wang YY, Wang XJ, Zang X, Ding YD (2015) Molecular cloning, characterization, and expression of vacuolar-type-H+-ATPase B1 (VHAB1) gene in the gill of Anguilla marmorata. Genet Mol Res 14(3):8008–8020. DOI:10.4238/2015.july.17.9

Librado P, Rozas J (2009) DnaSP v5: A Software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25(11):1451–1452. https://doi.org/10.1093/bioinformatics/btp187

Linh NQ, Nghia VD, Minh TD, Thanh ND, Lam HV, Tram NDQ, Hue HT (2010) Study of the season, time appears the eel fingerlings in Quang Binh provine. Journal of Agriculture Rural Development 12/2010:195–200. (in Vietnamese with English abstract)

Ministry of Science Technology and Environment (2007) Viet Nam Red Data Book. Part 1. Animals. Natural Science and Technology Publishing House, Ha Noi

Nei M, Gojobori T (1986) Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 3(5):418–426. DOI:10.1093/oxfordjournals.molbev.a040410

Nguyen AT, Tsukamoto K, Lokman PM (2018) Composition and distribution of freshwater eels Anguilla spp. in Vietnam Fish Sci 84(8):987–994. https://doi.org/10.1007/s12562-018-1239-9

Pantelouris EM, Árnason A, Tesch FW (1970) Genetic variation in the eel. II. Transferrins, haemoglobins and esterases in the eastern North Atlantic. Possible interpretations of phenotypic frequency differences. Genet Res 16:277–284. https://doi.org/10.1017/S0016672300002536

Pantelouris EM, Árnason A, Tesch FW (1971) Genetic variation in the eel. III. Comparisons of Rhode Island and Icelandic populations. Implications for the Atlantic eel problem. Mar Biol 9:242–249. https://doi.org/10.1007/BF00351386

Pavey SA, Gaudin J, Normandeau E, Dionne M, Castonguay M, Audet C, Bernatchez L (2015) RAD sequencing highlights polygenic discrimination of habitat ecotypes in the panmictic American eel. Curr Biol 25:1666–1671. https://doi.org/10.1016/j.cub.2015.04.062

Phung NH (2001) Vietnamese Animals - Episode X. Publishing scientific and technical, Hanoi (in Vietnamese)

Pike C, Crook V, Gollock M, Jacoby D (2019) Anguilla marmorata (errata version published in 2020). The IUCN Red List of Threatened Species 2019: e.T166189A167699312. [Downloaded on 08 May 2020]. https://dx.doi.org/10.2305/IUCN.UK.2019-3.RLTS.T166189A167699312.en

Slatkin M (1985) Gene flow in natural populations. Annu Rev Ecol Syst 16:393–430. https://www.annualreviews.org/doi/pdf/10.1146/annurev.es.16.110185.002141

Sneath PHA, Sokal RR (1973) Numerical Taxonomy. Freeman, San Francisco. https://www.cabdirect.org/cabdirect/abstract/19730310919

Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123(3):585–595. https://www.genetics.org/content/123/3/585.short

Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526. https://doi.org/10.1093/oxfordjournals.molbev.a040023

Wang FY, Fu WC, Wang IL, Yan HY, Wang TY (2014) The giant mottled eel, Anguilla marmorata, uses Blue-shifted rod photoreceptors during upstream migration. PLoS ONE 9(8):1–11. https://doi.org/10.1371/journal.pone.0103953

Wasserman RJ, Weyl OLF, Strydom NA (2011) The effects of instream barriers on the distribution of migratory marine-spawned fishes in the lower reaches of the Sundays River, South Africa. Water South Africa 37(4):495–504. DOI:10.4314/wsa.v37i4.7

Watanabe S (2003) Taxonomy of the Freshwater Eels, Genus Anguilla Schrank, 1798. In: Aida K., Tsukamoto K., Yamauchi K. (eds) Eel Biology. Springer, Tokyo. https://link.springer.com/chapter/10.1007%2F978-4-431-65907-5_1

Watanabe S, Aoyama J, Tsukamoto K (2004) Reexamination of Ege’s (1939) Use of taxonomic characters of the genus Anguilla. ulletin of Marine Science 74:337–351. https://www.ingentaconnect.com/content/umrsmas/bullmar/2004/00000074/00000002/art00006 [Download on 5 March 2018].

Watanabe S, Aoyama J, Nishida M, Tsukamoto K (2005) A molecular genetic evaluation of the taxonomy of eels of the genus Anguilla (Pisces: Anguilliformes). Bull Mar Sci 76(3):675–690. https://www.ingentaconnect.com/content/umrsmas/bullmar/2005/00000076/00000003/art00006 [Download on 5 March 2018].

Watanabe S, Miller MJ, Aoyama J, Tsukamoto K (2009) Morphological and meristic evaluation of the population structure of Anguilla marmorata across its range. J Fish Biol 74:2069–2093. https://doi.org/10.1111/j.1095-8649.2009.02297.x

Weir BS (1996) Genetic Data Analysis II: Methods for discrete population genetic data. Sinauer Associates, Inc., Sunderland

Williamson GR, Boëtius J (1993) The eels Anguilla marmorata and A. japonica in the Pearl River, China, and Hong Kong. Asian Fish Sci 6:129–138

Wright S (1984) Evolution and the genetics of populations, volume 4: variability within and among natural populations (Vol. 4). University of Chicago press

Xu X, Wu X, Yu Z (2012) Comparative studies of the complete mitochondrial genomes of four Paphia clams and reconsideration of subgenus Neotapes (Bivalvia: Veneridae). Gene 494:17–23. https://doi.org/10.1016/j.gene.2011.12.002