Identification of Monopterus cuchia (Hamilton) and...

58

Chapter-VI

Identification of Monopterus cuchia

(Hamilton) and Monopterus albus (Zuiew) by

mitochondrial DNA-based markers

6.1 INTRODUCTION

The development of DNA-based genetic markers has a revolutionary impact on animal

genetics. It is theoretically possible to observe and exploit genetic variation in the entire

genome of organisms with DNA markers. In recent years, mtDNA, because of its fast

evolution i.e. 5 to 10 times faster than single copy nuclear genes (Avise, 1994), has been

widely applied in systematics, population genetics and conservation biology of animals

(Wang et al., 2000). The present study has used mitochondrial Cytochrome c Oxidase

subunit I (COI / cox1) gene and control region (CR or D–loop) as genetic marker for

identification of Monopterus cuchia and Monopterus albus.

A. Cytochrome C oxidase (COI)

Cytochrome c Oxidase subunit I (COI / cox1) gene suppose to be evolving faster than

16S rDNA has widely been used in molecular taxonomy to resolve the phylogenetic

relationships within the family and also to study intraspecific population genetic structure

in several groups of eukaryotes including fishes.

B. Mitochondrial control region marker (CR or D–loop)

The D–loop segment exhibits a comparatively higher level of variation than protein–

coding sequences due to reduced functional constraints and relaxed selection pressure

(Saccone et al., 1987).

An attempt has been made in the present investigation to study the molecular

variation between two Synbranchid eel species- Monopterus cuchia and Monopterus

albus. The study represents the first hand information generation, includes field data

59

collection, sample collection and analyses including DNA isolation, PCR, sequencing of

selected genes of mtDNA (D-loop region and COI) followed by sequence-based

molecular genetic variation between Monopterus cuchia and Monopterus albus.

The existing nucleotide and protein sequence of COI (from GenBank and

UniProtKB) of five other eel shaped fishes belonging to the families Anguillidae

(Anguilla bengalensis) and Mastacembelidae (Mastacembelus armatus, Macrognathus

pancalus, Macrognathus aral, Macrognathus aculeatus) also included in the present

study, to establish the evolutionary relationships of Monopterus albus and Monopterus

cuchia with other eel species. The goal of the present study is to identify potential

molecular markers that is linked to different Monopterus species and thus could

specifically differentiate M. cuchia and M. albus, which are valuable in aqua agriculture.

6.2 MATERIALS AND METHODS

Materials and methods followed in this study are included in Chapter-III.

6.3 RESULTS

6.3.1 PCR amplification and sequencing

The DNA samples of purified PCR product gave ~650 bp COI gene fragment and ~ 600

bp D-loop regions, amplified in separate reaction set for each gene by using the specific

primer pairs. The representative gel images for the PCR amplification profile for

mitochondrial and nuclear genes are shown in Figure 6.1(A-B).

6.1(A). PCR amplification profile of COI (~650 bp)

[product size ~650 bp; Lane 1-6: M. cuchia, Lane 7-12: M. albus, 13. 100 base pair

ladder (1kb)]

60

6.1 (B). PCR amplification profile of D-loop region (~600 bp).

[product size ~600 bp; Lane 1-6: M. cuchia, Lane 7-12: M. albus, 13. 100 base pair

ladder (1kb)]

Figure 6.1 (A-B). Representative gel images for PCR amplicons of two genes.

6.3.2 Data mining and Sequence analysis

6.3.2.1 Comparative sequence analysis COI gene

The COI genes of the present study ranged from 605(COI of Anguilla bengalensis) to 655

(COI of Macrognathus pancalus and Macrognathus aral) nucleotide long with molecular

weight of 185.602 kDa (in A. bengalensis) to 200.758 kDa (in Macrognathus aral)

respectively. The melting temperature ranged from 83.40 (COI of A. bengalensis) to

84.49 (COI of M. albus) at 0.1M salt concentration (Table 6.1). The frequency of AT in

COI mRNA (cDNA) sequence in different fishes of the present study ranged between

0.522 (in COI of M. albus) to 0.615 (in COI of Monopterus cuchia). On the other hand

frequency of GC ranged from 0.385 (in COI gene of Monopterus cuchia) to 0.478 (in

COI gene of Monopterus albus) (Table 6.1). The COI gene sequences were found to be

A:T rich (Table 6.1; Figure 6.2). The transition/ transversion frequency for the

nucleotides of the COI gene are- A=>T = 0.05, A=>C = 0.04, A=>G= 0.1, T=>A = 0.04,

T=>C= 0.19, T=>G= 0.2, C=>A= 0.04, C=>T= 0.2, C=>G=0.02, G=>A= 0.19,

G=>T=0.05, G=>C=0.04. Multiple sequence alignment of COI gene in M. cuchia and M.

albus has been presented in Figure 6.3.

61

Table 6.1. Statistics of COI cDNA sequence in seven eel species.

Statistical parameter M. cuchia M. albus Anguilla

bengalensis

Mastacembelus

armatus

Macrognathus

pancalus

Macrognathus

aral

Macrognathus

aculeatus

Sequence

source/GenBank

Accession numbers

KR705867

(This study)

KR705873

(This study)

*JX887590

*JX983365

*FJ459512

*HQ219137

*JX260905

Length (bp) 652 655 605 652 655 655 651

MW in single

stranded condition

(kDa)

200.473 200.571 185.602 199.973 200.715 200.758 199.515

Melting temperature

(0C) [salt] = 0.1M

80.68 84.49 83.40 83.83 83.80 82.86 82.79

Frequency of A + T 0.615 0.522 0.549 0.538 0.539 0.562 0.564

Frequency of G + C 0.385 0.478 0.451 0.462 0.461 0.438 0.436

*Sequence sources: GenBank of NCBI

Figure 6.2. Nucleotide sequence composition in the COI cDNA sequence of M. cuchia

and M. albus.

62

Figure 6.3. Multiple sequence alignment of COI gene in M. cuchia and M. albus. The

sizes of the bar diagram represent the degree of conservation of respective amino acid in

each alignment position. ‘-’ represent no conserved sequence.

6.3.2.2 Comparative sequence analysis of D-loop region

The D-Loop region sequence of the present study ranged from 610 (in both the

Monopterus species) to 950 (in Danio rerio) nucleotide long with molecular weights of

195.84 kDa (in M. albus), 195.95 kDa (in M. cuchia) and 304.272 kDa (in Danio rerio)

respectively. The melting temperature was found to be 81.70 (in M. cuchia) and 82.04 (in

M. albus) at 0.1M salt concentration against Danio rerio (78.06) (Table 6.2). The

63

frequency of AT in D-loop region (cDNA) sequence ranged from 0.582 (in M. albus) to

0.679 (in Danio rerio). On the other hand frequency of GC ranged from 0.321 (in Danio

rerio) to 0.418 (in M. albus) (Table 6.2). The D-loop region was found to A: T rich (Table

6.2; Figure 6.4). The transition/ transversion frequency for the nucleotides of the D-loop

region are- A=>T = 0.06, A=>C = 0.04, A=>G= 0.11, T=>A = 0.06, T=>C= 0.11,

T=>G= 0.04, C=>A= 0.06, C=>T= 0.17, C=>G=0.04, G=>A= 0.17, G=>T=0.06,

G=>C=0.04. Multiple sequence alignment of D-loop region in M. cuchia and M. albus

has been depicted in Figure 6.5.

Table 6.2. Nucleotide sequence statistics of the D-loop region cDNA sequence in M.

cuchia and M. albus.

Statistical parameter M. cuchia M. albus

Sequence source/GenBank Accession numbers KR705891 (Present study) KR705897 (Present study)

Length (bp) 610 610

MW in single stranded condition (kDa) 195.95 195.84

Melting temperature (0C) [salt] = 0.1M 81.70 82.04

Frequency of A + T 0.590 0.582

Frequency of C + G 0.410 0.418

Figure 6.4. Nucleotide composition of mitochondrial D-loop region cDNA sequence of

M. cuchia and M. albus.

64

Figure 6.5. Multiple sequence alignment of D-loop region in M. cuchia and M. albus.

The sizes of the bar diagram represent the degree of conservation of respective amino

acid in each alignment position. ‘-’ represent no conserved sequence.

65

6.3.2.3 Comparative sequence analysis of COI protein

The sizes of protein sequences of COI enzyme in the present study ranged between 216

(COI of Anguilla bengalensis) to 518 (COI of M. albus) amino acids. The amino acids

Leucine (frequency=0.118 in M. albus to 0.157 in M. cuchia) followed by Alanine

(frequency=0.089 in M. albus to 0.111 in M. cuchia) have been found predominantly rich

in the COI of these two eel species (Figure 6.6). Sequence analysis of COI protein

revealed +ve hydropathy on average (0.722 in M. albus and 0.913 in M. cuchia) (Table

6.3; Figure 6.7). The molecular weight of COI in the present study ranged from 23.151

kDa (in COI of Monopterus cuchia) to 57.519 kDa (in COI of M. albus). The Isoelectric

point of the COI ranged between 4.94 to 6.23 (Table 6.3; Figure 6.8). The Instability

index of COI of the present study ranged from 23.92 to 27.49 (Table 6.3). The pair-wise

sequence alignment of amino acid sequence revealed following differences in M. albus

from M. cuchia - T=>M (at position 31), S=>C (at position 42), V=>I (at position 64) ,

S=>A and V=>I (at position 116 and 118 respectively), S=>G (at position 134), A=>S

and I=>V (at position 187 and 188 respectively) and V=>I (at position 193) (Figure 6.9).

Table 6.3. Physico-chemical analysis of COI protein

Statistical

parameter

M. cuchia M. albus Anguilla

bengalensis

Mastacembelus

armatus

Macrognathus

pancalus

Macrognathus

aral

Macrognathus

aculeatus

Sequence Source/

UniProtKB Accession number

This study This study *S4SNW0

*S4SMK7

*B1A2X4

*G3DQ16

*I1TM91

No. of amino acids (aa)

217 518

216 217 218 218 217

MW (kDa)

23.151 57.519 23.157 23.163 23.34 23.354 23.207

pI 4.94 6.23 6.02 4.94 4.94 4.94 4.94

-ve charged

residues

9

25

8

9

9

9

9

+ve charged residues

4

17

6

4

4

4

4

Formula C1084H1680N2

54O281S12 C2728H4056

N638O673S30 C1083H1654N256

O280S13 C1086H1684N254O28

2S1 C1096H1695N255O

284S11 C1097H1697N255O

284S11 C1088H1688N254O

283S11

II 24.45 26.78 27.49 23.92 24.74 24.25 24.31

AI 123.23 106.02 105.23 125.02 124.45 124.45 125.02

GRAVY 0.913 0.722 0.702 0.917 0.915 0.916 0.907

MW: Molecular weight; pI: Isoelectric point; II: Instability index; AI: Aliphatic index; GRAVY: Grand average of

hydropathicity. *Sequence Source: UniProtKB.

66

Figure 6.6. Amino acid distribution histogram of for COI protein of M. cuchia and M.

albus.

A. B.

Figure 6.7. Hydropathicity plot for COI (Kyte-Doolittle scale: Kyte and Doolittle, 1982).

A. M. cuchia, B. M. albus.

67

Figure 6.8. Electrical vs pH graph for COI between two eel species.

Figure 6.9. Multiple sequence alignment of COI protein in M. cuchia and M. albus. The

sizes of the bar diagram represent the degree of conservation of respective amino acid in

each alignment position. ‘-’ represent no conserved sequence.

6.3.3 Molecular evolution

The Maximum-likelihood model parameters for data sets as estimated in Modeltest

(Posada and Crandall, 1998) are listed in Table 6.4. Pair-wise distances (P-distance) of

COI gene has been presented in Table 6.5a&b. P-distance of D-loop region gene and COI

protein have been depicted in the Tables 6.6 & 6.7 respectively. The bootstrap consensus

tree inferred from 1000 replicates was taken to represent the evolutionary history of the

taxa analyzed (Felsenstein, 1985).

68

Table 6.4. Maximum-likelihood model considered for phylogeny as estimated in

Modeltest (Posada and Crandall, 1998)

Parameter COI gene D-loop

region

COI

protein

Model HKY+G+I T92 mtREV24

Bayesian Information Criterion (BIC) scores 5581.044 3728.1 1698.75

Akaike Information Criterion, corrected (AICc) value 5249.71 3553.9 1501.84

Maximum Likelihood value (lnL) -2579.71 -1751.9 -717.53

Gamma distribution (G) 1.4366 n/a n/a

invariable (I) 0.4832 n/a n/a

Transition/Transversion bias (R) 2.132 1.254 0.019

Total positions in the final dataset 559 608 164

6.3.3.1 COI gene phylogeny

The Pair-wise distance of COI gene sequences among the different eel species of the

present study revealed shortest genetic distance (0.003) between Macrognathus aral and

Macrognathus aculeatus. The longest genetic distance (0.375) exists between

Monopterus albus and Monopterus cuchia followed by second longest genetic distance

(0.356) between Monopterus albus and Macrognathus aculeatus (Table 6.5a). The COI

gene phylogenic analysis involved 21 nucleotide sequences for, where there were a total

of 559 positions in the final dataset. Estimates of Evolutionary Divergence in Sequences

(COI gene) of M. cuchia and M. albus with Anguilla bengalensis has been shown in

Table 6.5 b.

Table 6.5a. Pairwise distance COI gene among seven eel species

1 2 3 4 5 6 7

1 Monopterus cuchia -

2 Monopterus albus 0.375 -

3 Anguilla bengalensis 0.277 0.320 -

4 Macrognathus aculeatus 0.282 0.356 0.300 -

5 Macrognathus aral 0.285 0.352 0.297 0.003 -

6 Macrognathus pancalus 0.295 0.355 0.292 0.178 0.183 -

7 Mastacembelus armatus 0.273 0.340 0.317 0.175 0.180 0.171 -

69

Table 6.5b. Evolutionary Divergence in COI gene sequences of M. cuchia and M. albus

with Anguilla bengalensis.

1 2 3 4 5 6 7 8 9 10 11 12 13

1 Monopterus cuchia 1

2 Monopterus cuchia 2 0.000

3 Monopterus cuchia 3 0.004 0.004

4 Monopterus cuchia 4 0.004 0.004 0.000

5 Monopterus cuchia 5 0.005 0.005 0.002 0.002

6 Monopterus cuchia 6 0.005 0.005 0.002 0.002 0.000

7 Monopterus albus 1 0.405 0.405 0.406 0.406 0.406 0.406

8 Monopterus albus 2 0.405 0.405 0.406 0.406 0.406 0.406 0.000

9 Monopterus albus 3 0.405 0.405 0.406 0.406 0.406 0.406 0.000 0.000

10 Monopterus albus 4 0.405 0.405 0.406 0.406 0.406 0.406 0.000 0.000 0.000

11 Monopterus albus 5 0.405 0.405 0.406 0.406 0.406 0.406 0.000 0.000 0.000 0.000

12 Monopterus albus 6 0.400 0.400 0.400 0.400 0.400 0.400 0.002 0.002 0.002 0.002 0.002

13 Anguilla bengalensis

JX887590

0.285 0.285 0.281 0.281 0.285 0.285 0.359 0.359 0.359 0.359 0.359 0.354 -

A. The evolutionary history of COI gene was inferred using the Maximum Parsimony

method. Tree-1 out of 9 most parsimonious trees (length = 419) is shown. The

consistency index is (0.702128), the retention index is (0.891892), and the composite

index is 0.653486 (0.626222) for all sites and parsimony-informative sites. The MP tree

was obtained using the Sub-tree-Pruning-Regrafting (SPR) algorithm (Nei and Kumar,

2000) (Figure 6.10-A).

B. The evolutionary history was inferred by using the Maximum Likelihood method

based on the Hasegawa-Kishino-Yano model (Hasegawa et al., 1985). The tree with the

highest log likelihood (-2588.6145) is shown. Initial tree(s) for the heuristic search were

obtained by applying the Neighbor-Joining method to a matrix of pairwise distances

estimated using the Maximum Composite Likelihood (MCL) approach. A discrete

Gamma distribution was used to model evolutionary rate differences among sites [5

categories (+G, parameter = 0.6077)]. The rate variation model allowed for some sites to

be evolutionarily invariable ([+I], 48.2788% sites) (Figure 6.10-B).

70

A.

B.

Figure 6.10. COI gene-based phylogenetic profile of M. cuchia and M. albus among seven eel

species. A. Maximum Parsimony tree, B. Maximum Likelihood tree. The percentage of replicate

trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985).

FJ459508 Monopterus cuchia




KF742427 Monopterus cuchia

AP002945 Monopterus albus

JX260828 Anguilla bengalensis bengalensis



EU417776 Macrognathus pancalus

FJ459512 Macrognathus pancalus


JX260905 Macrognathus aculeatus

JQ667548 Macrognathus aculeatus

HQ219137 Macrognathus aral




JX983365 Mastacembelus armatus



99

93

86

100

100

94

100

79

76

78

93

100

100

99

100

78

100

100



JQ667548 Macrognathus aculeatus




FJ459512 Macrognathus pancalus






KF742427 Monopterus cuchia








AP002945 Monopterus albus

92

100

87

71

81

100

100

96

100

100

69

99

100

59

71

The COI gene MP tree formed two distinct clades and revealed that M. cuchia is a

sister taxa of A. bengalensis plus M. albus with 100% bootstrap support. Macrognatus

pancalus in their successive sister taxa for ming a distinct clade. The second clade is

formed by Magrognathus aculeatus and Macrognathus aral (boot strap support 100%)

plus Mastacembelus armatus as their successive sister taxa (100% bootstrap support)

(Figure 6.10-A).

The COI gene ML tree also formed two distinct clades with bootstrap support

99%. The first clade is formed by Macrognathus aral plus M. aculeatus followed by

Macrognathus pancalus plus Mastacembelus armatus (bootstrap support 100%) as their

successive sister taxa. The second calde is formed by Monopterus cuchia (bootstrap

support 100%) followed by A. bengalensis plus Monopterus albus as its successive sister

taxa. (Figure 6.10-B).

Figure 6.11 showed the evolutionary history based on COI gene of 12

Monopterus taxon sampled from Assam and Manipur, inferred using the Maximum

Parsimony method. Tree-1 out of 104 most parsimonious trees (length = 224) is shown.

The consistency index is (0.961905), the retention index is (0.989757), and the composite

index is 0.954408 (0.952052) for all sites and parsimony-informative sites (in

parentheses). The percentage of replicate trees in which the associated taxa clustered

together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein,

1985). The analysis involved 15 nucleotide sequences. There were a total of 559

positions in the final dataset. The analysis included three Anguilla bengalensis sequences

of GenBank as outgroup.

The analysis COI gene phylogeny showed that Monopterus cuchia and

Monopterus albus are two evolutionarily distinct species having common ancestor. They

formed two distinct clades in the phylogenetic tree with bootstrap percentage 99 (Figure

6.11). This two species are having evolutionary distinctness with Anguilla bengalensis

(bootstrap value 96). Within the Monopterus cuchia populations, the samples of

population-4 (M. cuchia 1-3) formed distinct clades from population-2 (M. cuchia 4-6)

with bootstrap separation 84%, which indicates the possibility of existence of two

subspecies in Monopterus cuchia. Within the clades of the genus M. albus differences

exists in evolutionary distance (Figure 6.11).

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Figure 6.11. Maximum Parsimony-based phylogenetic tree of COI gene in the

Monopterus taxa collected from Assam and Manipur.

6. 3.3.2 D-loop phylogeny

The Pair-wise distance of D-loop region sequences among the different eel species of the

present study revealed the presence of genetic distance (0.013) within the Monopterus

cuchia samples collected from population-4 and population-2 (Table 6.6). Between

Monopterus cuchia and Monopterus albus samples, highest genetic distance (0.062)

exists between M. cuchia 4, 5, 6 (population-2) and M. albus-10, 11, 12 (Manipur)

populations. The longest genetic distance (0.810) exists between the outgroup sequence

Danio rerio and M. cuchia 4, 5, 6 (population-2 and 3) (Table 6.6).

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Table 6.6. Evolutionary Divergence in sequences of D-loop region

Sl.

No.

1 2 3 4 5 6 7 8 9 10 11 12 13

1 Monopterus cuchia 1 -

2 Monopterus cuchia 2 0.000

3 Monopterus cuchia 3 0.000 0.000

4 Monopterus cuchia 4 0.013 0.013 0.013

5 Monopterus cuchia 5 0.013 0.013 0.013 0.000

6 Monopterus cuchia 6 0.013 0.013 0.013 0.000 0.000

7 Monopterus albus 1 0.032 0.032 0.032 0.046 0.046 0.046

8 Monopterus albus 2 0.032 0.032 0.032 0.046 0.046 0.046 0.000

9 Monopterus albus 3 0.032 0.032 0.032 0.046 0.046 0.046 0.000 0.000

10 Monopterus albus 4 0.048 0.048 0.048 0.062 0.062 0.062 0.015 0.015 0.015

11 Monopterus albus 5 0.048 0.048 0.048 0.062 0.062 0.062 0.015 0.015 0.015 0.000

12 Monopterus albus 6 0.048 0.048 0.048 0.062 0.062 0.062 0.015 0.015 0.015 0.000 0.000

13 Danio rerio 0.789 0.789 0.789 0.810 0.810 0.810 0.790 0.790 0.790 0.804 0.804 0.804 -

The evolutionary history of Monopterus cuchia and Monopterus albus based on D-loop

region was inferred using the Maximum Parsimony method. Tree-1 out of 116 most

parsimonious trees (length = 321) is shown (Figure 6.12). The consistency index is

(0.980392), the retention index is (0.992308), and the composite index is 0.989216

(0.972851) for all sites and parsimony-informative sites (in parentheses). The percentage

of replicate trees in which the associated taxa clustered together in the bootstrap test

(1000 replicates) is shown next to the branches (Felsenstein, 1985). The MP tree was

obtained using the Close-Neighbor-Interchange algorithm (Nei and Kumar, 2000) with

search level 1 in which the initial trees were obtained with the random addition of

sequences (10 replicates). The analysis involved 13 nucleotide sequences. All positions

containing gaps and missing data were eliminated. There were a total of 608 positions in

the final dataset.

74

Figure 6.12. Maximum Parsimony tree of D-loop region in the Monopterus taxa

collected from Assam and Manipur.

The D-loop region based phylogenetic tree reveled that M. cuchia and M. albus are two

separate sister groups (bootstrap support 100%). Both in M. cuchia and M. albus taxon in

the tree showed sub-clades within the genus, indicating the possibility of existence of

sub-species in each of the species (Figure 6.12). Within the Monopterus cuchia

populations, the samples of population-4 (M. cuchia 1-3) formed a distinct clade

(bootstrap support 99%) from population-2 (M. cuchia 5-6) with bootstrap separation

100%, which indicates the possibility of existence of two subspecies in Monopterus

cuchia. Within the clade of the genus M. albus differences exists in evolutionary distance

and two distinct sub-clades are observed in bootstrap value 100% (Figure 6.12).

6.3.3.3 COI protein phylogeny

The Pairwise distance of COI protein sequences among the different eel species of the

present study revealed shortest genetic distance (0.001) between Macrognathus aral and

Macrognathus aculeatus. The longest genetic distance (0.177) exists between

Monopterus albus and Anguilla bengalensis (Table 6.7).

75

Table 6.7. Pairwise distance of COI protein among seven eel species

Sl. No. 1 2 3 4 5 6 7

1 Monopterus cuchia -

2 Monopterus albus 0.047 -

3 Anguilla bengalensis 0.150 0.177 -

4 Macrognathus aculeatus 0.028 0.072 0.166 -

5 Macrognathus aral 0.028 0.072 0.166 0.001 -

6 Macrognathus pancalus 0.038 0.082 0.166 0.014 0.014 -

7 Mastacembelus armatus 0.028 0.072 0.166 0.009 0.009 0.019 -

The phylogenetic analysis of COI protein involved 18 amino acid sequences. All

positions containing gaps and missing data were eliminated. There were a total of 164

positions in the final dataset.

A. The evolutionary history was inferred using the Maximum Parsimony method. Tree #1

out of 10 most parsimonious trees (length = 41) is shown. The consistency index is

(0.888889), the retention index is (0.965517), and the composite index is 0.918419

(0.858238) for all sites and parsimony-informative sites (in parentheses). The MP tree

was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (Nei and Kumar,

2000) with search level 1 in which the initial trees were obtained by the random addition

of sequences (10 replicates) (Figure 6.13-A).

B. The evolutionary history was inferred by using the Maximum Likelihood method

based on the General Reversible Mitochondrial + Freq. model (Adachi and Hasegawa,

1996). The tree with the highest log likelihood (-706.9901) is shown. The percentage of

trees in which the associated taxa clustered together is shown next to the branches. Initial

tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to

a matrix of pairwise distances estimated using a JTT (Jones-Taylor-Thornton) model

(Figure 6.13-B).

76

A.

B.

Figure 6.13. Molecular phylogenetic profile of M. cuchia and M. albus among different

bony fishes based on COI protein sequence. A. Maximum Parsimony tree, B. Maximum

Likelihood tree.

The COI protein MP tree formed two distinct clades and revaled that M. cuchia is

a successive sister taxa of A. bengalensis and M. albus. The second clade is formed by

Macrognathus aculeatus and Macrognathus aral plus Magrognathus aculeatus and

Q5N6D8 Anguilla bengalensis labiata

S4SNW0 Anguilla bengalensis bengalensis

U3LDB6 Anguilla bengalensis bengalensis

S4SMF3 Anguilla bengalensis bengalensis

Q94SG6 Monopterus albus

G8XSD9 Monopterus cuchia

W0I5X7 Monopterus cuchia


S4SMK4 Macrognathus aculeatus

G3DQ16 Macrognathus aral

J7JZZ0 Macrognathus aral

I1TM91 Macrognathus aculeatus

B1A2X4 Macrognathus pancalus

G8XSE0 Macrognathus pancalus


S4SMK7 Mastacembelus armatus

M4QDG0 Mastacembelus armatus

B1A304 Mastacembelus armatus

100

91

84

93

87

I1TM91 Macrognathus aculeatus

G3DQ16 Macrognathus aral

J7JZZ0 Macrognathus aral

S4SMK4 Macrognathus aculeatus

B1A2X4 Macrognathus pancalus



S4SMK7 Mastacembelus armatus

B1A304 Mastacembelus armatus

M4QDG0 Mastacembelus armatus



W0I5X7 Monopterus cuchia

Q94SG6 Monopterus albus

U3LDB6 Anguilla bengalensis bengalensis

Q5N6D8 Anguilla bengalensis labiata

S4SMF3 Anguilla bengalensis bengalensis

S4SNW0 Anguilla bengalensis bengalensis66

100

79

98

57

87

82

91

51

65

50

77

Macrognathus aral (boot strap support 893%) followed by Mastacembalus armatus as

their successive sister taxa (87% bootstrap support) (Figure 6.13-A).

The COI protein ML tree also formed two major clades and revaled that

Macrognathus aculeatus and Macrognathus aral are having close evolutionary

relationship, Macrognathus puncalus is their sister taxa and these three taxa formed a

clade with their successive sister taxon Mastacembelus armatus (bootstap support 91%).

The second clade is formed by Monopterus cuchia followed by its successive sister taxa

of M. albus and A. bengalensis (Figure 6.13-B).

6.3.4 Tertiary structures of COI from M. albus and M. cuchia

The tertiary structure of COI for M. cuchia has 9 helices, 15 helix-helix interacs, 10 beta

turns, 1 gamma turns, whereas the tertiary structure of COI for M. albus has 22 helices,

60 helix-helix interacs, 28 beta turns, 2 gamma turns(Figure 6.14 A-B). The ERRAT

verification revealed that the overall quality factor for the predicted tertiary structures of

COI is around 95%. ProCheck verification revealed structural accuracy as more than

90% of the aminoacid residues in the predicted 3D structures of COI are in the range of

most favoured region, which confirms the validity of the predicted structures (Figure

6.15). The functions of COI protein with respective ProFunc score are listed in Table 6.8.

A. B.

Figure 6.14. The tertiary structure of COI proteins from (A) M. cuchia, (B) M. albus displayed in

UCSF Chimera (Pettersen et al., 2004).

78

Figure 6.15: Ramachandran plot for backbone dihedral angles PSI (y) and PHI (f) for the

final structure of COI from A. Monopterus cuchia, B. Monopterus albus. The red region

represents the most favored region, yellow = allowed region, light yellow = generously

allowed region, white = disallowed region [ProCheck].

79

.

Table 6.8. Predicted functions of COI with respective ProFunc score (shown within

parenthesis)

Name

of the

Protei

n

Taxon

Protein name terms Gene Ontology (GO) terms

Cellular

component Biological

process

Biochemical

function

COI

M. cuchia

Oxidase (21.70), cytochrome (21.22),

cytochrome oxidase

(20.56), fragment

(8.12), state (5.28), reduced (3.60),

oxidized (2.87),

reduced state (2.39)

cell (29.01), cell part

(29.01),

membrane

(25.76), integral to

membrane

(24.67)

metabolic process (33.78), cellular

process (31.04),

cellular metabolic

process (29.05), oxidation

reduction (17.96)

catalytic activity (25.61),

transporter

activity (25.55),

transmembrane transporter

activity (25.55),

binding (24.74)

M. albus

Cytochrome (55.49),

oxidase (53.93),

cytochrome oxidase

(31.76), reduced (6.40), recombinant

(5.25), binding (4.00),

carbon (3.52), state (3.17)

membrane

(55.13), cell

(55.13) cell

part (55.13), integral to

membrane

(51.51)

metabolic process

(63.52), cellular

process (62.47),

cellular metabolic process (61.70),

oxidation

reduction (51.92)

catalytic activity

(62.74),

oxidoreductase

activity (51.92), metal ion binding

(51.61), binding

(51.61)

The sequences of D-loop region and COI genes have been successfully deposited to

GenBank Database of NCBI and Accession number has been obtained for each submitted

sequence (Table 6.9 of Annexure-I). The protein structure of COI of M. cuchia and M.

albus have been deposited to the Protein Model Database (PMDB) and PMDB-ID has

been assigned to each submitted structures (Table 6.10 of Annexure-I).

6.4 DISCUSSION

Efficient identification of the two Synbranchid eel species of the present study is critical

for aquaculture management as well as for eel conservation (Dudu et al., 2010). Thus,

identification of M. cuchia and M. albus has been supported by molecular

characterization in the present study instead of conventional methods (Huang, et al.,

2001). The present study has revealed an interesting point of difference for identification

of the two Synbranchid species that the cDNA sequence COI-gene of M. cuchia is more

A: T rich than that of M. albus (Figure 6.1). Moreover the COI protein of M. cuchia has

more Lucine (L) and Alananine (A) residues frequency than that of M. albus (Figure 6.3).

80

The Instability index in COI is in the range of stable molecule. The hydropathicity plot

(Figure 6.7) revealed that the COI protein is hydrophobic in nature. The COI gene of

present study has showed higher transversion frequency than the transition frequency for

the nucleotides of the eel species (A=>G= 0.1, G=>A= 0.19). The graph of electrical

charge as a function of pH for COI (Figure 6.8) revealed significant differences in the

Isoelectric points of M. cuchia (pI =4.94) and M. albus (pI = 6.23). The pair-wise

sequence alignment of amino acid sequence revealed 10 specific positions where amino

acid sequence differences exist between M. albus from M. cuchia (Figure 6.10).

Both Monopterus albus and Monopterus cuchia are regarded as species complex

and demands taxonomic revision (Dahanukar, 2010; IUCN, 2013). However, in the

present study, the genetic distance of COI gene (0.375) revealed that the two Monopterus

albus and Monopterus cuchia are not within a species complex but they are two well

established distantly related species. The COI gene MP tree revealed that M. albus has

more close evolutionary relationship with A. bengalensis than that of M. cuchia and M.

albus (Figure 6.11-A).The COI gene ML tree also revealed more evolutionary close

relatedness between A. bengalensis and Monopterus albus than that of M. albus and M.

cuchia (Figure 6.11-B).

The analysis COI gene phylogeny showed that Monopterus cuchia and

Monopterus albus are two evolutionarily distinct species rather than a species complex.

The high bootstrap value (84%) separation of population-4 (M. cuchia 1-3) and

population-2 (M. cuchia 4-6) in the phylogenetic tree strongly indicates the possibility of

existence of two subspecies within Monopterus cuchia. (Figure 6.12).

The high bootstrap value in the D-loop region based phylogenetic tree also

supports that M. cuchia and M. albus are two separate species (bootstrap support 100%)

(Figure 6.13). Within the Monopterus cuchia populations, population-4 (M. cuchia 1-3)

and population-2 (M. cuchia 5-6) showed evolutionary distinctness by forming two

distinct sub-clades (bootstrap separation 100%. This clearly supports the finding of COI

gene based phylogeny that there is higher possibility of existence of two subspecies in

Monopterus cuchia (Figure 6.13).The M. albus also shows evolutionary distance by

forming two distinct sub-clades and the bootstrap value 100% indicates the possibility of

existence of two subspecies within this species (Figure 6.13).

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The MP tree of COI protein revealed distinct evolutionary profile of M. cuchia

and M. albus and out of this two species M. albus has a very close evolutionary

relationship with Anguilla bengalensis) (Figure 6.14-A).The ML tree of COI protein

revealed that M. albus has a very close evolutionary relationship with Anguilla

bengalensis than that of M. cuchia of the same genus. All the phylogenetic trees revealed

an interesting point that Monopterus cuchia (family- Synbranchidae) is intermediate taxa

of the taxa belonging to family Mastacembelidae and ‘Anguillidae plus Synbranchidae’

(Figure 6.14B). The tertiary structures of COI are found to be structurally conserved and

revealed that COI is an alpha-domain protein.

6.5 CONCLUSION

Understanding of genetic relationships between the two species of eels under the genus

Monopterus is listed as high priority in the IUCN record (IUCN, 2013). The present study

based on mitochondrial D-loop and COI clearly revealed that M. cuchia and M. albus are

two distinct species. However the D-loop and COI gene based phylogenetic tree revealed

that both the species M. cuchia and M. albus might have two sub-species within each

species. Further, phylogeographic study based on sampling in large geographic area

along their distribution ranges will help to establish such sub-speciation. The present

study will certainly be helpful in understanding genetic variation between M. cuchia and

M. albus and will clarify taxonomic uncertainties (Dahanukar, 2010) mentioned by

earlier workers. Further, a microsatellite based studies on northeast Indian population of

Monopterus species complex by the present authors (unpublished data) also showed

genetic distinctness of M. cuchia and M. albus rather than a species complex.

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