International Journal of Zoology

and Research (IJZR)

ISSN(P): 2278-8816; ISSN(E): 2278-8824
Vol. 6, Issue 6, Dec 2016, 1-12
TJPRC Pvt. Ltd

HEMICHORDATE EVOLUTIONARY
RELATIONSHIPS BASED ON CODING GENES
BIBHUTI PRASAD BARIK
Assistant Professor, Department of Zoology, Khallikote University, Brahmapur, Odisha, India
ABSTRACT
Evolutionary relationship within hemichordates is very interesting. The objectives of the current study were to
infer evolutionary relationship among hemichordates based on coding genes (ATPase alpha, cytochrome b and histone)
along with in silico proteomic analysis. Evolutionary relationships were inferred showing similar species clustered
together but did not form distinct clades as per their lineage and morphological similarities. It was noticed that some
species appeared to be polyphyletic and this could be assumed by possible mutations and adaptive radiations.
The variation in nucleic acid composition is observed and may be attributed to mutational pressure. GC content of the
genes was predicted and significantly varied. This variation might have played a crucial role in lineage based on patterns
of base composition within and among species. The amino acid composition of the translated proteins, atomic

trend among hemichordates. The species have evolved and expected to have adapted to different ecological environment.
KEYWORDS: Hemichordate, Coding Genes, Evolution, Translation, In Silico & Physiochemical Properties

Received: Oct 03, 2016; Accepted: Oct 24, 2016; Published: Oct 28, 2016; Paper Id.: IJZRDEC20161

INTRODUCTION

Original Article

composition of corresponding amino acids and physiochemical parameters has been used to determine the evolutionary

Evolutionary studies of hemichordates are significant in understanding chordate evolution. Molecular

phylogenetic analyses only have not yet provided robust support for the hemichordate evolution. The objectives of
the current study were to infer phylogenetic relationship among hemichordates based on ATPase alpha,
cytochrome b and histone genes along with in silico proteomic analysis of the translated proteins.

MATERIALS AND METHODS

Retrieval of Sequences and Taxon Sampling
The selected coding gene sequences of hemichordates available in Gen Bank (Benson et al., 2013)
database were retrieved using a PERL script. The sequences were filter searched and were selected based on gene
types using Bioedit software version 7.0.5.3 (Hall, 1999). These gene sequences were considered for phylogenetic
analysis and their corresponding proteomic analysis were also carried out.
Multiple Sequence Alignment and Phylogenetic Analysis
The retrieved gene sequences were saved and fasta formatted for multiple sequence alignment.
The sequences were aligned using CLUSTAL W (Thomption et al., 1994). For pair wise sequence alignment the
gap opening penalty and extension penalties were 15 and 6.66 respectively. The aligned file was exported for
phylogenetic analysis. Five different methods (ML, NJ, ME, UPGMA and MP) were adopted to perform
phylogenetic analysis using MEGA 7 software (Kumar et al., 2016). The branch length and consistency, retention
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Bibhuti Prasad Barik

and composites indices are shown in table 1.

Table 1: Branch length and indices of CI, RI and CI
Sl.No.

Gene

Sum of Branch Length

Consistency Retention Composite
Index
Index
Index
ML
NJ
ME UPGMA
1.
ATPase alpha
-501 2.166 2.166
2.211
0.837
0.806
0.781
2.
Cytochrome b
-156 1.076 1.076
1.081
0.685
0.453
0.340
3.
Histone
-360 9.455 9.455
9.098
0.458
0.887
0.411
ML: Maximum Likelihood, NJ: Neighbour Joining, ME: Minimum Evolution, UPGMA: Unweighted Pair Group
Method with Arithmetic Mean, MP: Maximum Parsimony, CI: Consistency Index, RI: Retention Index and CI:
Composite Index.
All characters were equally weighted and unordered. Alignment gaps were treated as missing data. The
percentage of replicate trees in which the associated taxa clustered together in the bootstrap was 500 replicates. The
evolutionary distances were computed.
Nucleic Acid Composition
Nucleotide composition of the gene sequences were computed using Bioedit (Hall, 1999).
Translation and In silico Physiochemical Characterization
The gene sequences were translated to their corresponding protein sequences and the compositions of amino acids
were predicted using Bioedit. The physicochemical properties such as atomic composition, molecular weight, theoretical
pI, instability indices, aliphatic indices and grand average of hydropathicity etc. were computed using ExPASys
ProtParam tool (Gasteiger et al., 2005).

RESULTS & DISCUSSIONS

Phylogenetic Analysis
Maximum Likelihood Trees
The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model
(Tamura and Nei, 1993). The trees were with the highest log-likelihood. Initial tree(s) for the heuristic search were
obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pair wise distances estimated using
the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.
The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included
were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated (Figure 1-3).
100

S.k owalevsk ii AY580280.1

P.flava AY580292.1

S.bromophenolosus DQ994686.1
H.sp. MR-2009 FJ555327.1

0.2

Figure 1: ML Tree Based on ATPase Alpha Gene

Impact Factor (JCC): 2.1739

NAAS Rating: 2.59

Hemichordate Evolutionary Relationships Based on Coding Gene

P.koehleri EU728442.1

64
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Figure 3: ML Tree based on Histone Gene

Neighbor Joining Trees
The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal
trees were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the
phylogenetic trees (Figure 4-6). The evolutionary distances were computed using the Maximum Composite Likelihood
method (Tamura et al., 2004) and are in the units of the number of base substitutions per site.
100

S.k owalevsk ii AY580280.1

P.flava AY580292.1

S.bromophenolosus DQ994686.1
H.sp. MR-2009 FJ555327.1

0.1

Figure 4: NJ Tree based on ATPase Alpha Gene

www.tjprc.org

editor@tjprc.org

Bibhuti Prasad Barik

P.koehleri EU728442.1

62
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Minimum Evolution Trees
The evolutionary history was inferred using the Minimum Evolution method (Rzhetsky and Nei, 1992). The trees
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phylogenetic trees (Figure 7-9). The evolutionary distances were computed using the Maximum Composite Likelihood
method (Tamura et al., 2004) and were in the units of the number of base substitutions per site. The ME trees were
searched using the Close-Neighbor-Interchange (CNI) algorithm (Nei and Kumar, 2000).

Impact Factor (JCC): 2.1739

NAAS Rating: 2.59

Hemichordate Evolutionary Relationships Based on Coding Gene

S.k owalevsk ii AY580280.1

100

P.flava AY580292.1
S.bromophenolosus DQ994686.1
H.sp. MR-2009 FJ555327.1

0.1

Figure 7: ME Tree based on ATPase Alpha Gene

P.k oehleri EU728442.1

64
83

S.bromophenolosus EU728444.1
H.plank tophilus EU728443.1
Ptychoderidae.sp. EU728449.1
G.berk eleyi EU728447.1
P.flava EU728448.1

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B .c la v ig e ru s
1
6 49 .1
10
B .c la g er us F N 9 08
3
B .c la vi
15
FN 908 65 5.1
B.c lav ige rus
32
44
B.clavigerus FN908656.1
64

cl a

ige

10
10

40

27
15

B.

v
cla

ru

08

.1
ru
64 4
ige
908
FN
l av
c
s
.
01.1
B
ru
553
ige
FJ5
lav
009
B .c
2
1.1
R
0860
p. M
F N9
H.s
acta
p
m
603.1
R.co
FN 9 0 8
pacta
R.com
02.1
90
ta FN 86
73
R.compac

B.

18
13

B.clavigerus FN908633.1
31
20.1
us FN9086
14
B.claviger
55
.1
8
2
74
N9086
erus F
g
vi
.1
la
25
B.c
6
8
0
N9
.1
rus F
621
vige
9 08
B.cla
9
64
.1
s FN
4 9
u
r
63 5
e
8
ig
0
v
N9
1
F
B.cla
2
.1
r us
23
ige
86
la v
.1
90
B.c
19
FN
6
s
8
ru
1
90
ige
N
3.
v
F
1
la
6
r us
B.c
08
e
9
g
N
vi
cla
sF
B.
ru
ige
v
c la
B.

cla

e
v ig

9
FN

Figure 9: ME Tree based on Histone Gene

www.tjprc.org

editor@tjprc.org

Bibhuti Prasad Barik

UPGMA Trees
The evolutionary history was inferred using the UPGMA method (Sneath and Sokal, 1973). The optimal trees
were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the
phylogenetic trees (Figure 10-12). The evolutionary distances were computed using the Maximum Composite Likelihood
method and were in the units of the number of base substitutions per site.
S.k owalevsk ii AY580280.1

100

P.flava AY580292.1
S.bromophenolosus DQ994686.1
H.sp. MR-2009 FJ555327.1

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Figure 10: UPGMA Tree Based on ATPase Alpha Gene

P.k oehleri EU728442.1

66
79

S.bromophenolosus EU728444.1
H.plank tophilus EU728443.1
G.berk eleyi EU728447.1
P.flava EU728448.1

100

Ptychoderidae.sp. EU728449.1

B.clavig
erus FN
908640
.1
B.cla
vigeru
s FN
34
9086
B.cla
4
2
100
.1
vige
25
rus
FN9
B.c
086
lav
38.1
ige
94
rus
B.c
FN
lav
908
ige
647
H.
rus
sp
.1
FN
.M
90
B.
R8
cla
64
20
8 .1
vig
09
B.
er
cla
FJ
us
55
vig
53
FN
er
01
90
us
.1
86
FN
46
90
.1
86
45
.1
37

56

B.clavigerus FN908639.1
74

99

0.2

61

63.1
086
FN 9
cta
2.1
6
45
mpa
086
R.co
N9
58
.1
ta F
36
86
pac
34
90
om
.1
FN
R.c
24
86
rus
.1
90
ige
33
lav
FN
86
B.c
.1
us
90
er
28
vig
FN
86
cla
us
90
B.
er
FN
vig
us
cla
er
B.
vi g
cl a
B.

B.
cla
vig
er
B.
us
cla
vig
FN
e
90
r
B.c
us
86
lav
FN
25
ige
9
.1
08
rus
6
B.c
15
FN
2
lav
.1
90
77 0
ige
86
r us
34
FN
6
.
B.c
1
908
lavig
637
eru
.1
s FN
908
B.cla
614
436
vige
.1
rus F
N90
97
8631
B.clavi
.1
gerus
FN9086
16.1
B.clavigeru
35
s FN9086
18.1
10

B.clavigerus FN9
08641.1

Figure 11: UPGMA Tree Based on Cytochrome b Gene

56

38

1
3.
64
08
1
9
4.
FN
64
us
08
.1
er
N9
00
g
i
F
53
v
s
55
ru
cla
FJ
ige
B.
9
v
0
02.1
cla
-20
B.
086
MR
N9
44
p.
aF
99
E.s
04.1
act
p
086
om
85
FN 9
R.c
acta
omp
601.1
8
.c
R
N90
cta F
mpa
89 100
R.co
603.1
08
FN9
pacta
99
R.com
68.1
100
86
90
s FN
B.clavigeru

72

B.clavigerus FN908622.1

B.clavigerus FN908667.1

47

B.clavigerus FN908632.1

64

29
67

100

31

FN
90
86
19
08
cta
.1
R. c
6
F
09
om
N9
.1
pa
08
ct a
60
FN
R.c
6.1
90
om
99
86
pac
05
ta F
.1
N9
R.co
086
mpa
08.1
cta
10 7
0 6
FN 9
086
B.cla
99
07.1
vigeru
s FN
65
9086
66.1
100
B.clavig
erus FN
908665
.1
100
B.clavigerus FN9
08613.1

us

FN
9

cta

m
pa

er
B.clavig

B.cla

R.
co
mp
a

.1
60
86
1
90
5.
FN
65
08
us
9
er
6.1
FN
5
6
vig
us
08
cla
er
N9
.1
B.
vig
sF
49
ru
cla
86
90
ge
B.
i
v
FN
.1
cla
rus
B.
650
76 68
ige
908
lav
FN
49
B.c
rus
.1
4
0
5
ige
10
086
lav
B.c
FN9
rus
vige
57.1
B.cla
9086
s FN
vigeru
8652.1
us FN90
62
4443

41

B.clavigerus FN908653.1

vig
er

64

908651.1
B.clavigerus FN

B.
cla

99

34

R.
co

R.compacta
FN908612
.1
R.com
pacta
FN908
610.1
B.cla
vige
rus F
N90
B.c
8664
lavig
.1
eru
sF
B.c
N90
53
lav
865
ige
10 100
9.1
R.
rus
0
co
FN
mp
908
R.
ac
658
co
ta
.1
mp
FN
R.
90
ac
co
8
t
60
aF
m
0
pa
N9
.1
ct
0
86
a
FN
61
.1
90
85
99
.1
100

39

18

17.1
s FN9086
24
B.clavigeru
1
8621.
FN90
us
er
g
.1
B.clavi
92
0
862
N90
rus F
35.1
vige
6
la
8
.c
0
B
34
N9
.1
sF
39
eru
630
08
lavig
9
00
1
.c
1
B
FN
7.
62
r us
08
ige
6.1
N9
lav
2
c
F
.
B
86
rus
.1
90
ige
23
FN
lav
86
us
B.c
90
er
N
ig
F
v
us
cla
B.
er
vig
cla
.
B

R.compacta FN908611.1

Figure 12: UPGMA Tree based on Histone Gene

Impact Factor (JCC): 2.1739

NAAS Rating: 2.59

Hemichordate Evolutionary Relationships Based on Coding Gene

MP Trees
The evolutionary history was inferred using the Maximum Parsimony method (Figure 13-15). The MP trees were
obtained using the Subtree-Pruning-Regrafting (SPR) algorithm with search level 0 in which the initial trees were obtained
by the random addition of sequences (10 replicates). All positions containing gaps and missing data were eliminated.
H.sp. MR-2009 FJ555327.1

100

S.bromophenolosus DQ994686.1
S.k owalevskii AY580280.1
P.flava AY580292.1

Figure 13: MP Tree based on ATPase Alpha Gene

P.koehleri EU728442.1

76
80

S.bromophenolosus EU728444.1
H.planktophilus EU728443.1
G.berkeleyi EU728447.1
P.flava EU728448.1

95

Ptychoderidae.sp. EU728449.1

Figure 14: MP Tree based on Cytochrome b Gene

88
59
29
45

20

.1

B .c lav igerus FN908616.1

B.c lav ige rus FN
908 61 8.1
B .c la vi
ge ru s F
N 90 86 1
B .c la
7. 1
v ig e ru
s FN
B .c la
90 86
2
v ig e
3 2 .1
ru s
B .c
FN9
43
086
B .c lav ig e
2 2 .1
r us
78
la v
B.
FN
ig e
c
90 8
rus
B . la vi
631
ge
cl a
F
N9
.1
r
B.
08
c l a v ig e u s F
63
N9
4 .1
vig ru s
08
FN
er
6
14
us
90
.1
86
FN
37
90
.1
86
21
.1

2.1

99

45

.1

48

64

.1

86

48

9
FN

-200

57

E.sp. MR-2009 FJ555300.1

08

61

5.

0
N9

86

30

.1

6. 1
F
us
62
er
08
us
N9
1
v ig iger
F
a
l
27.
c
av
r us
086
B . B .cl
ige
N9
lav
sF
c
u
.
r
.1
B
ige
624
la v
40
908
B .c
0
s FN
34 7
eru
36.1
6
ig
8
v
9
0
la
N9
B.c
rus F
.1
vige
26
B.cla
908620
rus FN
ge
vi
la
35
B.c
.1
33
18
s FN9086
19
B.clavigeru
B.clavigerus FN908625.1
53

21

.1
64 1
90 8
.1
s FN
63 9
908
.1
FN
40

86

.1

. MR

e ru

90

47

38
86

86
ru s
44
FN
.1
908
0
643
9 FJ
.1
555
B.cla
26
3
01.1
vigeru
9
7
s FN
9086
B.clavi
46.1
31
gerus
FN908
668.1
B.clavigerus
48
FN908667
.1
21
H.sp

la v ig

FN

86

86
90

90

08

90

FN
90

N9

90

FN

FN

ru s

sF

FN

FN

us

us

ig e

e ru

us
us

r us

ige

er

r us

er
er

lav

ige

er

ig e

v ig

v ig
vig

B.c

l av

vi g

vig

lav

la v

cla

c la
cla

B .c

cl a

B .c

B .c

B .c

B.

B.
B.
B.

B.
cla

99

R.compacta FN908602.1
.1
98
FN908603
R.compacta
81
04.1
6
8
0
9
a FN
ct
pa
1.1
0
R.com
6
99
8
N9 0
cta F
4.1
mpa
866
0
R.co
9
9
1
9
10.
s FN
eru
086 2. 1
N9
lavig
1
B.c
ta F
86
1
c
.
0
a
9
p
11
FN
om
86
ta
R.c
90
ac
FN
mp
o
a
t
c
5
8
R.
ac
mp
co
R.

54

19

71

81

91

cla
B.

59

R .c om

N9

82

57

v ig

er

us

FN

90

86

56

.1

66

51.

0 .1

086

08

B.clavigerus FN908658.1

rus
F

N9

2.1

R.c om pac ta

ig e

rus
F

65

.1

08

49

B.c lav ige rus FN

908 654 .1
B .c la vige
ru s F N
90 86 57
.1
B .c la
v ig e ru
s FN
B .c la
9 0 86
5 0 .1
v
ig
e ru s
B .c
F N9
lav
086
ig e
5 3 .1

la v

s
FN
9

86

5.

N9
0

65

e ru

sF

B .c

68

ru

FN

la v
ig

v ig
e

B.

c la

ig

32
96

B.

la
v

s
90
8

.1
07
.1
86
05
90
86
.1
FN N 9 0
08
a
.1
3 .1
c t ta F 9 0 8 6
662
66
c
pa
08
N
9 08
9
9.1
N
m
5
pa ta F
FN
0 86
ta F
co m
c
cta
FN9
pac
R . R .co m p a
pa
om
e ru s
co
om
R .c
.1
la v ig
0
0
R . R .c
6
B .c
N 9 08
95 1
c ta F
9
m pa
R .c o
61 .1
N 90 86
pa cta F
FN 90 859 9.1

31

B.
c

er
u

6957

B.clavigerus FN908628.1
B.claviger
us FN9086
35.1
B.clavi
65
99
gerus
FN908
B.cla
619.1
vigeru
44
s FN
B.cla
87
9086
vige
23.1
99
B .c
rus
FN9
lav
R.
ige
086
B
r us
13.1
R . com .clav
FN
c o pa
ig e
908
c
m
665
p a ta F r u s F
.1
N9
N9
ct
a
08
F N 086
66
06
6. 1
90
.1
86
09
.1
73

29

Figure 15: MP Tree based on Histone Gene

www.tjprc.org

editor@tjprc.org

Bibhuti Prasad Barik

Recently Osborn and colleagues showed that despite their great morphological diversity, most deep-living
hemichordates form a single clade (Osborn et al., 2012). In the current study, ATPase alpha, cytochrome b and histone
genes sequences were used to infer for phylogenetic affiliations among species belonging to different species of
hemichordates. Phylogenetic trees were investigated by different methods including most parsimonious trees to infer
phylogeny. The tree showed more or less similar species clustered together but did not form distinct clades as per their
lifestyles. The result also indicated that several species appear to be polyphyletic and several unrelated species appear to
share the same clade.
Nucleic Acid Composition
Nucleotide composition of the gene sequences were predicted (Table 2) and the result showed that GC content of
ATPase alpha gene was below 50% i.e. 47.53% in case of P. flava and lowest (42.52%) in case of S. bromophenolosus. GC
content of cytochrome b gene was high i.e. 62.9% in case of Ptychoderidae. sp and lowest (53%) in case of P. flava.
Similarly in case of histone gene Enteropneusta.sp. contains 45.65% of GC with lowest (8.11%) in R. compacta. The
variation in nucleic acid composition is observed and may be attributed to mutational pressure. In the representative
species of hemichordata, i.e., Balanoglossus carnosus, possible influence of mutational pressure due to compositional
constraints in codon usage was noticed (Karumathil, 2016).
Table 2: Nucleic Acid Composition of Gene Sequences of Different Species
Sequence
Length

A+T
%

G+C
%

Hemichordata. sp.

1208

52.98

47.02

315

325

307

261

S. kowalevskii

1233

56.53

43.47

375

322

297

239

P. flava

1233

52.47

47.53

338

309

311

275

S. bromophenolosus

1103

57.48

42.52

321

313

261

208

P. koehleri

421

55.34

44.66

100

133

50

138

H. planktophilus

306

54.25

45.75

72

94

30

110

S. bromophenolosus

386

58.81

41.19

93

134

50

109

G. berkeleyi

392

54.08

45.9

101

111

55

125

P. flava

405

53.0

46.9

115

100

53

137

Gene

Species

ATPase alpha

Cytochrome b

Impact Factor (JCC): 2.1739

Molecular
Weight
26.08(A)
21.61(C)
25.41(G)
26.90(T)
30.41(A)
19.38(C)
24.09(G)
26.12(T)
27.41(A)
22.30(C)
25.22(G)
25.06(T)
29.10 (A)
18.86(C)
23.66(G)
28.38(T)
23.75(A)
32.78(C)
11.88(G)
31.59(T)
23.53(A)
35.95(C)
9.80(G)
30.72(T)
24.09(A)
28.24(C)
12.95(G)
34.72(T)
25.77(A)
31,89(C)
14.03(G)
28.32(T)
28.40 (A)
33.83 (C)
13.09(G)
NAAS Rating: 2.59

Hemichordate Evolutionary Relationships Based on Coding Gene

Ptychoderidae. sp.

386

62.9

37.05

116

127

40

103

B. clavigerus

1074

4.28

6.05

23

23

39

26

Enteropneusta.sp.

276

45.65

54.35

75

51

72

78

Hemichordata. sp.

276

44.2

53.26

61

61

72

75

R. compacta

456

8.11

14.91

15

22

36

32

Histone

24.69(T)
30.05(A)
26.68(C)
10.36(G)
32.90(T)
2.14%(A)
2.42%(C)
3.63%(G)
2.14%(T)
27.17%(A)
28.26%(C)
26.09%(G)
18.48%(T)
22.10%(A)
27.17%(C)
26.09%(G)
22.10%(T)
3.29%(A)
7.02%(C)
7.89%(G)
4.82%(T)

GC content of the genomic DNA is significantly varied and this variation might have played a significant role in
the evolution. Two main evolutionary processes have been raised to explain the patterns of variation of base composition
within and among species: biases in the process of mutation, such that the rates of change from G-C and A-T are not
constant in time or space (Sueoka, 1988); and natural selection, either on overall GC content or on specific patterns of
codon usage (Eyre-Walker, 1999). The most active neutralistselectionist debate has concern of GC evolution in
vertebrates (Mooers and Holmes, 2000). Such explicitly phylogenetic studies of GC dynamics are rare, perhaps because of
concerns that changes in base composition can affect the accuracy with which we reconstruct trees. The effects of GC
variation on phylogenetic inference need to be explored further. The phylogenetic effects of GC differences apply to
neighbouring nucleotide sites (Karlin and Mrazek, 1997).
Translation and In silico Physiochemical Characterization
The amino acid composition of proteins has been used to determine the trend among species (Bogatyreva, 2006)
more specifically hemichordates to understand its evolutionary perspective (Sorimachi, 1999) and to identify the
contrasting feature of proteins (Gaur, 2010). The species have evolved and adapted to different ecological environment.
Their common origin indicates that amino acid composition of proteins of different kinds of eukaryotes may be similar.
However, it is interesting to observe the contrasting features introduced due to their local ecological adjustment (Table 3).

www.tjprc.org

editor@tjprc.org

10

Bibhuti Prasad Barik

Table 3: Amino Acid Composition of Translated Proteins of Different Species

Protein

ATPase alpha

Cytochrome b

Histone

Species
Hemichordata.sp.
S.kowalevskii
P.flava
S.bromophenolosus
P.koehleri
H.planktophilus
S.bromophenolosus
G.berkeleyi
P.flava
Ptychoderidae.sp.
P.bahamensis
H.kupfferi
M.psammophilus
B.clavigerus
Enteropneusta.sp.
Hemichordata.sp.
R.compacta

Molecular
Weight
99696.46
100817.78
101385.63
90818.96
37657.14
27695.36
34269.69
34448.44
35454.90
34007.26
46433.81
45275.08
47181.50
118762.82
22656.89
23456.61
48711.65

Maximum no.
Amino Acids
Thr 325
Ala 375
Ala 338
Ala 321
Cys 138
Cys 110
Thr 134
Cys 125
Cys 137
Thr 127
Ala 153
Cys 161
Cys 174
Asn 963
Cys 78
Cys 75
Asn 351

The percentage of occurrence of the amino acids in proteins depends, at least for some of the residues, on the
protein dimension (Carugo, 2008). The amino acid composition along with atomic composition (Table 4) of conserved
residues in present-day proteins, i.e., those residues which are unchanged between an ancestral sequence and any given
descendant sequence, is determined by two factors the amino acid composition within ancestral sequences, and the relative
probability of conservation of each amino acid between an ancestral and an extant descendant sequence (Brooks et al.,
2002).
Table 4: Atomic Composition (No. of Carbon, Hydrogen, Nitrogen, Oxygen
and Sulphur) of Protein Sequences of Different Species
Protein
ATPase alpha

Cytochrome b

Histone

Species name
Hemichordata.sp.
P.flava
S.bromophenolosus
S.kowalevskii
G.berkeleyi
H.planktophilus
P.flava
P.koehleri
Ptychoderidae.sp
S.bromophenolosus
B.clavigerus
Enteropneusta sp
R.compacta

Carbon
1986
2024
1755
2009
690
550
701
746
683
678
190
459
177

Hydrogen
3261
3213
2802
3212
1014
827
1157
1103
1014
1000
300
766
328

Nitrogen
547
547
462
552
156
153
233
165
150
150
60
142
86

Oxygen
524
597
549
586
172
133
171
189
175
173
52
126
49

Sulphur
28
16
12
18
1
1
2
1
0
1
1
1
0

Physiochemical properties of the translated proteins were computed (Table 5). The molecular weight of the
proteins ranged from 45275 to 4288.9. The computed isoelectric point (pI value) of all species ranges from 12.48 to 4.96.
The instability indices indicated higher values in R.compacta and those of Ptychoderidae.sp was lower. The aliphatic index
refers to the relative volume of a protein that is occupied by aliphatic side chains and contributes to the increased
thermostability of protein. In the present study aliphatic indices of Ptychoderidae sp. (116.37) were found to be higher than
those of others. This indicates that proteins of this species are more stable than those of others over a wide temperature
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Hemichordate Evolutionary Relationships Based on Coding Gene

11

range. Thus it may be assumed that Ptychoderidae species are more likely to change and adapt to varied environments.
Grand average of hydropathicity (GRAVY) values indicates the solubility of proteins: negative GRAVY values of most
hemichordate species showed it to be hydrophilic in nature with few exceptions indicating a little surface accessibility of
the protein to interact with water.
Table 5: In silico Physiochemical Properties of Proteins of Different Species of Hemichordates
Protein

Species
MW
Tpl
II
AI
Hemichordata.sp.
44083.7
10.71
61.89
111.75
P.flava
45275.0
8.61
35.89
80.39
ATPase alpha
S.bromophenolosus 39543.0
4.96
26.09
93.32
S.kowalevskii
45052.0
9.04
29.78
82.34
G.berkeleyi
14278.6
6.12
28.28
111.26
H.planktophilus
11742.6
11.77
87.33
70.91
P.flava
15649.4
12.43
73.92
81.97
Cytochrome b
P.koehleri
15439.0
5.25
29.85
117.39
Ptychoderidae.sp
14126.4
5.27
25.49
116.37
S.bromophenolosus 14052.3
5.23
35.28
115.32
B.clavigerus
4288.9
10.95
73.12
73.78
Enteropneusta sp
10322.0
11.02
49.94
83.91
Histone
Hemichordata sp
10561.7
11.02
40.91
73.15
R.compacta
4445.1
12.48 192.55 55.71
M.W: Molecular Weight, T.pI: Theoretical pI, I.I: Instability Index, A.I: Aliphatic

Gravy
0.194
-0.353
-0.155
-0.291
0.709
-0.600
-0.966
0.746
0.600
0.698
-0.511
-0.551
-0.710
-2.151
Index, GRAVY: Grand

Average of Hydropathicity.

CONCLUSIONS
The accumulation of mutations can eventually lead to differences between ATPase alpha, cytochrome b and
histone proteins with respect amino acid composition (Table 5). Thus, over a very long evolutionary time, mutation and
drift (Hughes, 2010) appear to be able to overcome the conservative effect of stabilizing selection on physiochemical
properties of proteins and give rise to a certain degree of functional differentiation.
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Bibhuti Prasad Barik

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