Journal of Genetics (2019)98:112 Ó Indian Academy of Sciences

https://doi.org/10.1007/s12041-019-1156-4 (0123456789().,-volV)
(0123456789().,-volV)

RESEARCH ARTICLE

Evolutionary analysis of genus Channa based on karyological

and 16S rRNA sequence data

RAVINDRA KUMAR1, VISHWAMITRA SINGH BAISVAR1, BASDEO KUSHWAHA1, GUSHEINZED WAIKHOM2

and MAHENDER SINGH1*

1Molecular Biology and Biotechnology Division, ICAR-National Bureau of Fish Genetic Resources, Lucknow
226 002, India
2Institute of Bioresources and Sustainable Development, Imphal 795 001, India

*For correspondence. E-mail: dr.mahendersingh29@gmail.com.

Received 16 May 2019; revised 20 August 2019; accepted 9 September 2019

Abstract. A wide range of diploid number of chromosomes and the body size of Channa congeners are useful combination of characters
for studying the factors controlling the body size. In this study, the karyological information was superimposed on the evolutionary tree
generated by 16S rRNA mitochondrial gene sequences. Here, the metaphase chromosome complements stained with Giemsa, AgNO3 and
CMA3 were prepared from six snakehead murrel fish species collected from northeast India. The diploid chromosome numbers and the
fundamental arms of C. aurantimaculata (2n = 52, NF = 98), C. gachua (2n = 56, NF = 84), C. marulius (2n = 44, NF = 58),
C. orientalis (2n = 52, NF = 74), C. punctata (2n = 32, NF = 60) and C. striata (2n = 40, NF = 48) were calculated by the analysis of
metaphase chromosome complements. Both methods of nucleolar organizer region (NOR) localization, silver nitrate and chromomycin A3,
revealed NOR pairs of 1, 2, 3, 1, 4 and 3 in C. aurantimaculata, C. gachua, C. marulius, C. orientalis, C. punctata and C. striata,
respectively. The subject species showed primitive type of asymmetrical chromosomes, except the C. punctata. The variation in 2n for
C. orientalis (2n = 52, 78) and C. gachua (2n = 52, 78, 104) of a complete haploid set indicates the possibility of either ploidy change in
C. orientalis and C. gachua, if we consider 2n = 52 or the Robertsonian rearrangements in different populations of these two species. The
chromosome evolution tree was constructed on 16S rRNA ML-phylogenetic tree using ChromEvol 1.3. The analysis of chromosome
evolution explained the loss or gain of chromosome, duplications or semiduplications mechanism. For time scaling the chromosome
evolution, the node age of available 16S rRNA gene of Channa species were estimated, which was also used for estimating the time when
chromosomal changes occurred in context of geological time-scale.

Keywords. AgNO3; nucleolar organizer region; Channa genus; chromomycin A3; giemsa; karyotype; IUCN red list.

Introduction
There are over 34,000 valid fish species globally (Eschmeyer
and Fricke 2016). The teleost being the largest infraclass in
ray-finned fishes covers more than 95% of all fish and half of
the extant vertebrate species. In teleost, the perciformes is
the largest order with around 156 families, including
Channidae family. This family is commercially important
and are of two genera Channa and Para channa with 29 and
three species, distributed in Asia and Africa, respectively
(Gold et al. 1990). Fossil records of the family Channidae
are reported by Roe (1991) from Indian subcontinent, in
early Eocene, about 50 million years ago (Mya). These
species were spread in western Eurasia about 17 Mya in
early Miocene, and throughout Africa and East Asia in the
late Tortian (8 Mya). These murrels were probably origi-
nated from south Himalayan region in India during early
Eocene epoch (Bohme 2004).
The snakeheads are important food fishes (Chattopadhyay
1975; Jhingran 1982) as well as medicinal and pharmaceu-
tical values (Michelle et al. 2004). They are carnivorous, air-
breathing fish (Froese and Pauly 2018), and can survive for a
longer time without water. They are found in swamps,
reservoirs, drains, ponds, canals, rivers, small streams, rice
fields, mining pools, roadside ditches and lakes across
southern Asia, southern China, Indo-China and the Sunda
Islands (Hossain et al. 2008). These channid species are
considered as a potential aquaculture species in addition to
112 Page 2 of 14 Ravindra Kumar et al.
the important capture fishery resource. As per the IUCN red
list (2016), the status of channid fishes mainly falls under
least concern v3.1, a few species come under data deficient
v3.1, C. diplogramme is under vulnerable B1 ab(iii) ? 2
ab(iii) v3.1, and C. bleheri and C. harcourtbutleri are under
near threatened v3.1.
Till date, the cytogenetic profiles of many channid species
(table 1) have been investigated. Dhar and Chatterjee (1984)
explained the karyotype analysis and chromosomes evolution of
five Channa species, namely C. barca, C. orientalis, C. punc-
tata, C. stewartii and C. striata. The karyomorphological studies
by various workers in channid species reported a different
diploid chromosome number (2n) in the same species (Nayyar
1966a; Manna and Prasad 1973). The 2n in C. punctata was
reported as 34 (Nayyar 1966a) and 32 (Manna and Prasad 1973;
Rishi 1973). Similarly in C. orientalis, the 2n has been reported
as 42 (Klinkhardt et al. 1995), 52 (Banerjee et al. 1988), 76
(Dhar and Chatterjee 1984) and 78 (Manna and Prasad 1973;
Arkhipchuk 1999; Ruma et al. 2006). In this study to confirm the
cytogenetic profile, six channid species were investigated by
using Giemsa, silver nitrate (AgNO3) and chromomycin A3
(CMA3) staining. Since the DNA sequences in channid species
are available in NCBI GenBank, a comparative analysis of
chromosomes and DNA sequences were also included in this
study. To find the evolutionary correlation between the karyol-
ogy and DNA sequences, the karyological information was
superimposed on the phylogenetic tree constructed from DNA
sequences of mitochondrial gene 16S rRNA.
Materials and methods
The Indian species cytogenetically investigated in this study
were orange-spotted snakehead (C. aurantimaculata; Musi-
kasinthorn 2000), dwarf snakehead (C. gachua; Hamilton
1822), great snakehead (C. marulius; Hamilton 1822),
Ceylon snakehead (C. orientalis; Bloch 1801), spotted
snakehead (C. punctata; Bloch 1793) and striped snakehead
(C. striata; Bloch 1793). The live individuals (n = 3 of each
species) of each of the six Channa species were collected by
netting from northeastern region of India. Since all the
specimens were in their juvenile stage, their sex could not be
identified by visual examination.
For chromosome preparations, the live specimens were
injected with 0.05% colchicine intramuscularly at the rate of
1.0 mL/100 g body weight of fish to arrest chromosomes at
metaphase stage. After 90 min of injection, the specimens
were immersed in benzocaine solution and sacrificed. The
kidney tissues were dissected out for the preparation of
metaphase chromosomes using hypotonic treatment, Car-
noy’s fixative (3:1 of methanol and acetic acid) and flame-
drying technique (Bertollo et al. 1978). The metaphase
chromosome slides were stained with 6% Giemsa in phos-
phate buffer (KH2PO4 and Na2HPO4 with pH 6.8) for 20
min at room temperature. The homologous chromosomes
were paired and arranged in decreasing order of their size by
using Lieca CW4000 Karyo software. Average length of the
homologous chromosome pair was used for estimating the
length of short arm (p), long arm (q), arm ratio (q/p), cen-
tromeric index and relative length (%). The arm ratios were
used to classify the chromosomes as metacentric (m), sub-
metacentric (sm), subtelocentric (st) and telocentric (t) as
suggested by Levan et al. (1964). The ideograms were built
according to the relative length and centromeric index.
Silver nitrate (AgNO3) and chromomycin A3 (CMA3)
staining of chromosomes was done as described by Howell
and Black (1980) and Ueda et al. (1987) to localize tran-
scriptionally active region and GC-rich nucleolar organizer
region (NOR) respectively. Giemsa stained karyotype anal-
yses, AgNO3 and CMA3 positive sites were determined from
more than 50 metaphase complements of each specimen
(over 150 metaphase complements per species) using 100 9
objective lens with oil immersion in light microscope.
Analysis of chromosome evolution
The direction of chromosome evolution (Robertsonian
mechanism) in Channa species was found by the combined
study of cytogenetic and molecular techniques using Ore-
chromis potani as an out group in both analyses. Whereas,
two different types of methods has been used for analysis of
hypothesis of chromosome evolution. The software Chro-
mEvol 1.3 was used to model the chromosome evolution in
Channa with input of morphology and number of haploid
chromosomes. The ML phylogenetic tree was based on 16S
rRNA gene sequence downloaded from NCBI and aligned
using molecular evolutionary genetics analysis (MEGA
5.05) software (Tamura et al. 2011). The best-fit nucleotide
substitution model for the 16S rRNA dataset was selected
based on the Akaike information criterion (AIC) in J-model
test (Darriba et al. 2012). The ML- chromosome evolution
tree was constructed on 16S rRNA tree using ChromEvol
1.3 (Mayrose et al. 2010). The chromosome evolution model
gave information about loss or gain of chromosome, dupli-
cations or semiduplications mechanism. For understanding
the time based chromosome evolution, the node age of
available 16S rRNA gene of Channa species were estimated,
which determined the approximate time of split between
lineages. This was used for estimating the time when chro-
mosomal changes occurred in context of geological time
scale. The 16S rRNA gene matrix of genus Channa were
analysed under Bayesian framework with uncorrelated log-
normal–relaxed clock model in BEAST v. 1.6.1 (Drummond
and Rambaut 2007).
Results
The karyotype formula (with 2n and fundamental arm
number (NF)) of the studied species were observed as
28m ? 18sm ? 6t (52 and 98) in C. aurantimaculata,
 Karyological evolution of genus Channa Page 3 of 14 112

Table 1. Comparative karyomorphological variations in the species of genus Channa.

Species 2n Chromosome formula NF Reference(s)

C. punctata 34 16m ? 8a 50 Nayyar (1966a)

32 18m ? 12sm ? 2st 62 Manna and Prasad (1973)
32 10m ? 18sm ? 4t/a 60 Rishi (1973)
34 16m ? 14sm ? 4t/a 64 Dhar and Chatterjee (1984)
32 16m ? 16sm 64 Dhar and Chatterjee (1984)
32 24m ? 8sm 64 Banerjee et al. (1988)
32 10m ? 18sm ? 4t/a 60 Arkhipchuk (1999)
32 24m ? 2sm ? 6t – Ruma et al. (2006)
32 18m ? 12sm ? 2st 62 Kumar et al. (2013)
32 18m ? 10sm ? 4st 60 Present study
C. striata 40 10m ? 30t/a 50 Nayyar (1966a)
40 8m ? 2sm ? 30t/a 54 Nayyar (1966b)
40 8m ? 2sm ? 16st ? 14t 50 Manna and Prasad (1973)
40 8m ? 6st ? 26t/a 54 Dhar and Chatterjee (1984)
40 8m ? 2sm ? 2st ? 28t 54 Banerjee et al. (1988)
44 4m ? 2sm ? 38t 50 Wattanodorn et al. (1985)
40 8m ? 6st ? 26t 54 Chatterjee (1989)
44 2m ? 2sm ? 40t 48 Donsakul and Magtoon (1991)
42 6m ? 36t/a 50 Supiwong et al. (2009)
40 6m ? 2sm ? 10st ? 22t 48 Kumar et al. (2013)
40 6m ? 2sm ? 10st ? 22t 48 Present study
C. gachua 78 12m ? 12sm ? 54t/a 102 Nayyar (1966b)
78 16m ? 10sm ? 52t 104 Banerjee et al. (1988)
112 4m ? 2sm ? 106t 116 Donsakul and Magtoon (1991)
78 12m ? 12sm ? 4st ? 50t 102 Manna and Prasad (1973)
52 12m ? 10sm ? 14st ? 16t 86 Kumar et al. (2013)
56 24m ? 16sm ? 6st ? 10t 96 Singh et al. (2013)
104 (4n) 6sm ? 98t (autotetraploid) 112 Tanomtong et al. (2014)
56 12m ? 16sm ? 18st ? 10t 84 Present study
C. marulius 40 10m ? 30a 50 Nayyar (1966b)
44 4m ? 40t 48 Barat (1985)
44 4m ? 4st ? 36a 48 Donsakul and Magtoon (1991)
44 8m ? 36t 52 Bhatti et al. (2013)
44 4m ? 40t/a 48 Khuda-Bukhsh et al. (1986)
44 4m ? 10sm ? 30t 58 Present study
C. orientalis 76 2m ? 6sm ? 68t/a 84 Dhar and Chatterjee (1984)
78 12m ? 12sm ? 4st ? 50t/a 102 Manna and Prasad (1973)
52 16m ? 10sm ? 52t/a 104 Banerjee et al. (1988)
78 – – Arkhipchuk (1999)
42 2m ? 2sm ? 38t/a 46 Klinkhardt et al. (1995)
78 34m ? 2sm ? 42t – Ruma et al. (2006)
52 12m ? 10sm ? 14st ? 16t 74 Present study
C. aurantimaculata 52 28m ? 18sm ? 6t 98 Present study
C. argus 48 – – Arkhipchuk (1999)
48 – – Cui et al. (1991)
48 4sm ? 44t/a 74 Lingyun (1982)
48 4sm ? 44st – Kang et al. (1985)
C. asiatica 44 – – Cui et al. (1991)
44 4m ? 8sm ? 32st – Kang et al. (1985)
46 2m ? 8sm ? 36st – Kang et al. (1985)
C. barca 38 6m ? 6sm ? 4st ? 22t/a 54 Dhar and Chatterjee (1984)
38 6m ? 6sm ? 4st ? 22t/a 54 Chatterjee (1989)
C. lucius 48 2m ? 2sm ? 2st ? 42t 52 Donsakul and Magtoon (1991)
88 2m ? 86t 90 Chavananikul et al. (1993)
48 2m ? 46t/a 54 Khakhong et al. (2014)
C. maculata 42 4m ? 2sm ? 36st – Kang et al. (1985)
C. micropeltes 44 2sm ? 42t 46 Donsakul and Magtoon (1991)
44 2m ? 42t 46 Supiwong and Jearranaiprepame (2009)
C. marulioides 38 Magtoon et al. (2006)
C. stewartii 66 12m ? 6sm ? 6st ? 42t/a 90 Dhar and Chatterjee (1984)
104 2m ? 102t/a 106 Rishi and Haobam (1984)
112 Page 4 of 14 Ravindra Kumar et al.

Table 1 (contd)
Species 2n Chromosome formula NF Reference(s)

P. obscura 42 16m ? 18t/a 50 Hinegardner and Rosen (1972)

34 16m ? 18t/a 50 Nayyar (1966b)
O. potanini 48 – – Arkhipchuk (1999)

Acrocentric chromosomes, reported by some workers, have been shown under telocentric as t/a.

Figure 1. (a) Karyotype, (b) idiogram, (c) AgNOR signal on upper row, (d) CMA3 signal on lower row in homologous chromosomes in C.
aurnimaculata.

12m ? 16sm ? 18st ? 10t (56 and 84) in C. gachua,
4m ? 10sm ? 30t (44 and 58) in C. marulius,
12m ? 10sm ? 14st ? 16t (52 and 74) in C. orientalis,
18m ? 10sm ? 4st (32 and 60) in C. punctata and
6m ? 2sm ? 10st ? 22t (40 and 48) in C. striata (figur-
es 1a–6a; table 2). Minimum 2n was found in C. punctata
and maximum in C. gachua, whereas NF ranged from 48 in
C. striata to 98 in C. aurantimaculata. The C. auranti-
maculata possessed maximum 28 metacentric chromosomes
with 2n = 52, whereas the metaphase spreads of C. marulius
(2n = 44) showed maximum 30 telocentric chromosome,
which is near to primitive species (2n = 48; showing telo-
centric chromosome) than other studied species (Dhar and
Chatterjee 1984). The ideograms of each species are repre-
sented in figures 1b–6b. The species-wise chromosome
statistics are given in table 3.
The AgNO3 stained NOR (AgNOR) signals in metaphase
chromosome complements of six Channa species ranged
from one pair in C. aurantimaculata and C. orientalis to four
pairs in C. punctata (figures 1c–6c; table 2). The nonse-
quential staining of other metaphase chromosome spreads
with CMA3 positive sites determined one to four pairs of
NORs as bright zones against black background (figures 1d–
6d; table 2), which were in agreement with AgNORs. In four
species (figures 1, 2, 3, 6) the location of AgNO3 and CMA3
signals were at telomeric position of the chromosomes,
whereas in C. orientalis (figure 4) and C. punctata (fig-
ure 5), the signal positions were intercalary at the homolo-
gous pair of the chromosomes.
Chromosome evolution
The analysis suggested the best model for chromosome
evolution with chromosome gain or loss and constant
duplication (figure 7), describe the evolutionary scenario
 Karyological evolution of genus Channa Page 5 of 14 112

Figure 2. (a) Karyotype, (b) idiogram, (c) AgNOR signal on upper row, (d) CMA3 signal on lower row in homologous chromosomes in C.
gachua.

Figure 3. (a) Karyotype, (b) idiogram, (c) AgNOR signal on upper row, (d) CMA3 signal on lower row in homologous chromosomes in C.
marulius.

best in genus Channa (forming two lineages) inferred by
ChromEvol software. The rate parameter estimated in the
depicted model are: 103.23 for chromosome losses, 31.07
for chromosome gains and 3.02 for chromosome demidu-
plication. The results also showed the occurrence of poly-
ploidization events and suggested that duplication of
chromosome occurred during the chromosome evolution.
The main events were fusion (loss in number of
chromosome) and fission (gain), which showed events
inferred with an exp [0.5 (figure 7). Therefore, this study
has focussed on the haploid chromosome numbers estimated
by the Bayesian method. The Bayesian time-calibrated tree
allowed us to infer that the Channa species diverged from
Oreoleuciscus potanini in early Miocene around 20.81 Mya
(figure 8), probably due to the change occurred in chromo-
some number during evolution.
112 Page 6 of 14 Ravindra Kumar et al.

Figure 4. (a) Karyotype, (b) idiogram, (c) AgNOR signal on upper row, (d) CMA3 signal on lower row in homologous chromosomes in
C. orientalis.

Figure 5. (a) Karyotype, (b) idiogram, (c) AgNOR signal on upper row, (d) CMA3 signal on lower row in homologous chromosomes in
C. punctata.
 Karyological evolution of genus Channa Page 7 of 14 112

Figure 6. (a) Karyotype, (b) idiogram, (c) AgNOR signal on upper row, (d) CMA3 signal on lower row in homologous chromosomes in
C. striata.

Table 2. Cytogenetic profiles of six species of genus Channa.

Chromosome types
AgNOR CMA3
Species 2n NF m sm st t (pair) (pair) AgNOR/ CMA3 position (chromosome type)

1 C. aurantimaculata 52 98 28 18 – 06 1 1 Terminal of p arm (SM)

2 C. gachua 56 84 12 16 18 10 2 2 Terminal of p arm (ST)
3 C. marulius 44 58 4 10 – 30 3 3 Terminal of p arm (2SM) ? terminal of p arm (1T)
4 C. orientalis 52 74 12 10 14 16 1 1 Intercalary (1M)
5 C. punctata 32 60 18 10 4 – 4 4 Intercalary (1M) ? terminal of p arm (2M)
? terminal of p arm (1SM)
6 C. striata 40 48 6 2 10 22 3 3 Terminal of p arm (1SM) ? terminal of p arm (2ST)

NF, fundamental arm number.

Discussion The studies of many workers on C. marulius (table 1)

The centromere based description of karyomorphology to
group chromosomes as metacentric, submetacentric, subte-
locentric and telocentric/acrocentric and their size-wise
arrangement have been used for prediction of inter-rela-
tionship among the families and closeness of species (Lopez
and Fenocchio 1994). On the contrary, the karyomorpho-
logical investigations in fishes are strenuous due to their
small size and high number of chromosomes. The present
work aimed to cytogenetically reinvestigate some species of
the genus Channa, of the evolutionary advance fish family,
Channidae of the freshwater teleost fishes (Dhar and Chat-
terjee 1984; Bhatti et al. 2013).
showed that in all the cases the 2n was 44 except by Nayyar
(1966a) who reported 40. The presence of high number (15
pairs) of telocentric chromosomes in Indian populations may
be considered more close to primitive species (Campos and
Cuevas 1997).
In C. striata, the 2n = 40 was first reported by Nayyar
(1966a) and later several workers reported different diploid
chromosome number, karyomorphology and NF, but most of
the authors reported; 2n = 40 as given by Kumar et al.
(2013). Further, the numbers of telocentric/subtelocentric
chromosomes were higher than that of the metacentric/sub-
metacentric chromosomes in all the studies. The number of
telocentric chromosomes were more than subtelocentric
Table 3. Chromosomes statistics of six undertaken species of genus Channa.
112

C. marulius C. striatus C. orientalis C. gachua C. punctatus C. aurantimaculata

Mean Mean Mean Mean Mean Mean Mean Mean

Mean p/q RL CM Mean p/q RL CM p/q RL CM p/q RL CM Mean p/q RL CM Mean p/q RL CM
Page 8 of 14

1 0.555/0.958 3.091 m 0.707/1.01 3.590 m 0.706/ 2.911 m 0.505/ 2.329 m 0.974/1.135 4.347 m 0.681/0.959 2.93 m
0.858 0.757
2 0.631/0.908 2.956 m 0.707/0.959 3.483 m 0.696/ 2.825 m 0.449/ 1.863 m 0.833/1.011 3.800 m 0.550/0.857 2.51 m
0.822 0.560
3 0.378/1.024 2.567 sm 0.707/0.909 3.377 m 0.454/ 2.114 m 0.433/ 1.827 m 0.857/1.011 4.056 m 0.555/0.752 2.33 m
0.681 0.556
4 0.404/0.988 2.475 sm 0.454/0.863 2.753 sm 0.444/ 2.140 m 0.398/ 1.767 m 0.832/1.010 3.692 m 0.545/0.706 2.24 m
0.706 0.560
5 0.353/1.063 2.591 sm 0.343/1.135 3.091 st 0.429/ 1.879 m 0.379/ 1.632 m 0.832/0.858 3.225 m 0.551/0.758 2.34 m
0.580 0.505
6 0.454/0.849 2.233 sm 0.294/0.943 2.587 st 0.348/ 1.461 m 0.393/ 1.565 m 0.831/0.984 3.328 m 0.540/0.757 2.32 m
0.438 0.454
7 0.378/0.912 2.256 sm 0.283/0.929 2.535 st 0.376/ 2.157 sm 0.449/ 2.495 sm 0.631/0.883 3.121 m 0.510/0.651 2.07 m
0.782 0.903
8 0/1.489 2.686 t 0.298/0.935 2.577 st 0.364/ 2.057 sm 0.443/ 2.398 sm 0.555/0.833 2.862 m 0.579/0.782 2.43 m
0.741 0.856
9 0/1.439 2.595 t 0.227/0.831 2.212 st 0.359/ 1.963 sm 0.364/ 2.162 sm 0.580/0.823 2.912 m 0.459/0.555 1.81 m
0.696 0.808
10 0/1.237 2.231 t 0/1.151 3.166 t 0.348/ 1.934 sm 0.343/ 2.213 sm 0.480/1.043 3.138 sm 0.464/0.556 1.82 m
0.690 0.856
11 0/1.363 2.458 t 0/1.186 2.480 t 0.353/ 1.876 sm 0.318/ 1.977 sm 0.454/1.010 3.016 sm 0.454/0.555 1.80 m
Ravindra Kumar et al.

0.655 0.753
12 0/1.212 2.185 t 0/1.136 2.375 t 0.294/ 2.327 st 0.300/ 1.932 sm 0.419/0.959 2.84 sm 0.454/0.630 1.94 m
0.957 0.747
13 0/1.161 2.094 t 0/1.313 2.744 t 0.278/ 2.287 st 0.293/ 1.785 sm 0.350/0.808 2.386 sm 0.454/0.505 1.71 m
0.950 0.674
14 0/1.136 2.049 t 0/1.111 2.322 t 0.298/ 2.249 st 0.299/ 1.651 sm 0.349/0.822 2.413 sm 0.378/0.480 1.53 m
0.910 0.596
15 0/1.186 2.140 t 0/1.060 2.216 t 0.263/ 2.119 st 0.252/ 2.047 st 0.309/0.984 2.665 st 0.349/0.727 1.92 sm
0.875 0.857
16 0/1.212 2.185 t 0/1.083 2.264 t 0.254/ 2.0844 st 0.254/ 2.042 st 0.258/0.808 2.197 st 0.339/0.686 1.83 sm
0.866 0.853
17 0/1.161 2.094 t 0/0.909 1.901 t 0.219/ 1.926 st 0.193/ 1.733 st – – – 0.354/0.686 1.86 sm
0.816 0.746
18 0/1.136 2.049 t 0/0.849 1.774 t 0.208/ 1.849 st 0.153/ 1.522 st – – – 0.348/0.686 1.85 sm
0.785 0.671
19 0/1.035 1.866 t 0/0.813 1.701 t 0/1.010 1.879 t 0.158/ 1.548 st – – – 0.334/0.645 1.75 sm
0.681
20 0/1.085 1.957 t 0/0.403 0.842 t 0/0.909 1.691 t 0.157/ 1.361 st – – – 0.354/0.713 1.91 sm
0.580
21 0/0.934 1.684 t – – – 0/0.858 1.597 t 0.139/ 1.590 st – – – 0.304/0.605 1.63 sm
0.722
 Karyological evolution of genus Channa Page 9 of 14 112

CM

p, short arm; q, long arm; RL, relative length; m, metacentric; sm, submetacentric; st, subtelocentric; t, telocentric; CM, chromosome morphology; mean, average of two homologous
chromosomes in this species. The study also revealed three

sm

sm

–
–
t
t
t
pairs of silver NOR and CMA3 positive sites at subtelo-
centric chromosomes.

1.62

1.44

1.80
1.35
1.26
Mean
C. aurantimaculata

In C. gachua, different karyomorphology was reported by

–
–
RL

different workers. The findings in terms of 2n = 78 is sim-

ilar to that of Nayyar (1966a), Manna and Prasad (1973),
0.274/0.530
0.294/0.611
CM Mean p/q

Banerjee et al. (1988) and Kumar et al. (2013). In most of

0/1.010
0/0.757
0/0.707
the earlier findings, there was high number of telocentric
chromosomes in this species. Two pairs of AgNO3 stained

–
–
NOR and CMA3 positive sites at homologous subtelocentric
chromosomes were observed in this species. In C. orientalis,
–

–
–
–
–
– the 2n = 52 was also reported by Banerjee et al. (1988),
whereas in the same species 2n = 78 was reported by Manna
Mean
RL

and Prasad (1973), Arkhipchuk (1999) and Ruma et al.

–

–
–
–
–
–

(2006). The interesting fact is the huge variation in 2n of C.

C. punctatus

orientalis (2n = 52 vs 78) and C. gachua (2n = 52, 78,

CM Mean p/q

104). The difference of 26 chromosomes (equals to a com-

plete haploid set n, if we consider 2n = 52) in 2n as reported
–

–
–
–
–
–

by different authors in the same species, indicates the pos-

sibility of ploidy change in different populations of C. ori-
st

st

t
t
t
t
t

entalis and C. gachua (Gold 1979) has happened in different

populations of these two species. The presence of one pair of
1.476

1.354

2.142
1.537
1.444
1.397
1.211
Mean

intercalary AgNO3 stained NOR and CMA3 positive site is

RL

probably a fusion of two homologous chromosomes at

C. gachua

metacentric position.
0/1.161
0/0.833
0/0.782
0/0.757
0/0.656
0.123/

0.128/
CM Mean

0.677

0.606

The karyomorphological information of C. punctata has

p/q

also been ambiguous as Nayyar in 1966 reported the diploid

number of chromosome (2n = 34 with karyotype,
–
–

16m ? 18t), that were not supported by Rishi in 1973 with

t

t
t
t

the diploid number and chromosome morphology (2n = 32

1.597

1.503

1.315
1.268
1.174

with karyotype, 10m ? 18sm ? 4t). Whereas, Dhar and

Mean
RL

Chatterjee in (1984), reported two form of diploid number

–
–
C. orientalis

(2n = 32 and 34, with NF = 64). This type of variation of

0/0.858

0/0.808

0/0.707
0/0.681
0/0.631

2n and NF were also reported in C. punctata (Manna and

CM Mean

Prasad 1974; Black and Howell 1978; Legrande and

p/q

–
–

Cavander 1980). The present study shows the diploid

number of chromosome in C. punctata, 2n = 32 with
–

–
–
–
–
–

karyotype 18m ? 10sm ? 4st and NF = 60, without any

telocentric chromosomes. This may be assigned to the
Mean

karyotype evolutions due to Robertsonian rearrangements of

RL

–
–
–
–
–

chromosome (Gold 1979) by the centric fusion (most com-

mon) or dissociation of chromosome (Denton 1973). In
C. striatus
CM Mean p/q

C. punctata and C. orientalis, the presence of intercalary

silver NOR and CMA3 positive site at metacentric homol-
ogous chromosome pair are confirmed as centric fusion, the
–

–
–
–
–
–

widely observed event in fishes (Galleti et al. 2000).

–

–
–
–
–
–

The presence of higher number of chromosome in teleost

t

fishes is more advanced characteristic than the primitive

1.548
Mean

2n as 48 (Hinegardner and Rosen 1972; Stingo 1979). The

RL

–
–
–
–
–

bi-armed chromosomes were used for making the karyotype,

if chromosomes differ in size and shape, it is marked as
C. marulius
Table 3 (contd)

asymmetrical, whereas if they differ only in size then it is

Mean p/q

chromosomes.
0/0.858

marked as symmetrical. It has been generated by absence or

presence of microsome/telocentric chromosome (Stebbins
23. 0

0
0
0
0
0

1971 and Morescalchi 1975). The asymmetrical species

22

24
25
26
27
28

were termed as unimodel, as in this study, except C.

112 Page 10 of 14 Ravindra Kumar et al.

Figure 7. Karyotype evolution and generalized ancestral chromosome state of the genus Channa under maximum likelihood optimization
phylogenetic tree.
 Karyological evolution of genus Channa
Figure 8. Bayesian time-calibrated maximum clade-credibility tree using a molecular-clock lognormal chronogram of 21 Channa species
and out group as O. potanini with node ages in MYA.
punctata which has been considered as symmetrical due to
absence of telocentric chromosomes.
The nonsequential, silver-staining bearing NOR is gen-
erally visualized in transcriptically active site of rDNA. The
GC-rich active DNA of NORs in many vertebrates, includ-
ing fishes (Gold et al. 1990) has been identified by CMA3
positive site staining which not only provide the number and
localization of NORs but also indicate GC-rich content of
transcriptically active sites of rRNA genes in other fish
species (Jankum et al. 2003).
The AgNO3 stained those NORs sites (table 1; figur-
es 1c–6c), which expressed themselves during the preceding
interphase by attaching to a composite of acidic proteins
associated with nucleolus and pre-RNA (Jorden 1987). The
nonsequential CMA3 stained the GC-rich active sites of
DNA and confirmed the same position as AgNOR (table 2;
figures 1d–6d). The presence of intercalary NOR (C. ori-
entalis and C. punctata) were due to centromeric fusion of
two chromosomes.
The analysis of karyomorphology and CMA3 signals
among the Channa species suggests that they evolved
through a complicated chromosome evolution process with
the occurrence of polyploidization, duplication, rearrange-
ments, fusion and loss of chromosomes. However, there is
no evidence of a fixed pattern of these type of events among
these Channa species. Moreover, C. punctata and C. ori-
entalis are from same origin as evidenced by presence of one
pair intercalary NOR/CMA3 signals. Further, the probable
hypothesis of the chromosome evolution in Channa species
in this study is shown in figure 9. It is hypothesized that the
chromosome number in the new evolving species will
Page 11 of 14 112
reduce further with more number of metacentric chromo-
some and intercalary NORs.
The establishment of different karyotypes in Channa spe-
cies during evolution occurred by three mechanisms (Gold
1979), namely aneuploidy, polyploidy and Robertsonian
rearrangements (i.e. change in diploid number of chromo-
some without change in NF value, Booke 1974). This is also
responsible for the karyotype establishment and change in
chromosome number in many other fish family (Legrande
1980). Whereas, the Robertsonian mechanism was not only
responsible for establishment of different karyotypes
(2n = 32 to 56) in genus Channa, but also changed NF values
(NF = 48 to 98). It is a centromeric shift with pericentric
inversion that has been emphasized by several workers (Le-
grande 1981). Therefore, it gave the unambiguous explana-
tion of species wise change in chromosome number with
fundamental values, otherwise explanation of different kary-
otypes and polyploidy would have been very difficult.
The study of Manna and Prasad in 1973 and Dhar and
Chatarjee in 1984 did not indicate polyploidy in Channidae
species, because the study of total and relative chromosomal
length (table 3) in meiotic division did not indicate polyploidy.
The hypothesis that could explain the observed differ-
ences in karyotypes, suggests that changes in chromosome
number occurs gradually over the time by multiple events of
chromosomal rearrangements. The most explanatory mech-
anism for chromosome evolution in Channa species is
‘Robertsonian mechanism with pericentric inversion or
centromere shift’ proposed by Dhar and Chatterjee (1984).
The presence of asymmetrical karyotype in all species
indicate that all the species are comparatively primitive
112 Page 12 of 14 Ravindra Kumar et al.
Figure 9. Hypothesis of chromosome evolution in genus Channa.
except C. punctata that showed symmetrical advanced type
of karyotype (Dhar and Chatarjee 1984), without telocentric
chromosomes. It is widely accepted that all the karyotypes of
present day species were established from primitive kary-
otype of 48 telocentric chromosomes (Post 1965). According
to Denton 1973, the present species are formed by the for-
mation of biarmed chromosomes from rod-shaped telocen-
tric chromosomes. According to molecular phylogenetic
analysis, the Channidae species are divided in to two major
linages: the first includes C. stewartii, C. aurantimaculata,
C. barca, C. gachua, C. orientalis, C. punctata and the
second includes Parachanna obscura, C. lusius, C. miro-
peltis, C. maculate, C. argus, C. asiatica, C. striata,
C. maruloides and C. marulius. These results fill the infor-
mation gap in evolutionary relationship of genus Channa
(Dhar and Chatarjee 1984) and are also useful in phyloge-
netics, conservation and characterization of these species.
These findings also update the karyotype information of genus
Channa with nonsequential staining of AgNOR and CMA3,
which can be used for characterization of Channa species.
Acknowledgments
Author gratefully thank the Department of Biotechnology for their
financial support and ICAR-National Bureau of Fish Genetic
Resources, Lucknow for providing work place facilities and
encouragement.
References
Arkhipchuk V. V. 1999 Chromosome database. Database of Dr.
Victor Arkhipchuk.
Banerjee S. K., Misra K. K., Banerjee S. and Ray-Chaudhuri S.
P. 1988 Chromosome numbers, genome sizes, cell volumes and
evolution of snake-head fish (family Channidae). J. Fish Biol.
33, 781–789.
Barat A. 1985 A study of chromosomes in some Indian teleost
(Pisces). Ph.D. thesis, Kalyani University, Kalyani, India.
Bertollo L. A. C., Takahashi C. S. and Moreira F. O. 1978
Cytotaxonomic consideration on Hoplias lacrdae (Pisces, Ery-
thrinidae). Braz. J. Genet. 1, 103–120.
Bhatti M. Z., Rafiq M. and Mian A. 2013 Kayrotype of sol
(Channa marulius) from Indus river, Pakistan. J. Anim. Plant
Sci. 23, 475–479.
Black A. and Howell W. M. 1978 A distinctive chromosomal race of
the cyprinodontid fish, Fundulusnotatus from the Upper Tombig-
bee river system of Alabama and Mississippi. Copeia 2, 280–288.
Bohme M. 2004 Migration history of air-breathing fishes reveals
neogene atmospheric circulation patterns. Geology 32, 393–396.
Booke H. E. 1974 A cytotaxonomic study of the round white-
fishes, genus Prospolium. Copeia 1,115–119.
Campos H. H. and Cuevas C. 1997 Karyotypes of the most
primitive catfishes (Teleostei: Siluriformas: Diplomystidae). J.
Zool. Syst. Evol. Res. 35, 113–119.
Chavananikul V., Wattanodorn S. and Wattanavijarn W. 1993 A
study of chromosome number and morphology in 7 species of
Thai freshwater fish. J. Aquat. Anim. Diseases 14, 19–34.
Chatterjee K. 1989 Cytotaxonomic and electrophoretic investiga-
tions on Indian air-breathing fishes. In Fish genetics in India (ed.
P. Das and A. G. Jhingran), pp. 83–99. Printers and Publishers,
New Delhi.
Chattopadhyay S. K. 1975 Studies on freshwater fishes of Belighata
Bheries, Calcutta. Rec. Zool. Surv. India 357, 359–360.
Cui J., Ren X. and Yu Q. 1991 Nuclear DNA content variation in
fishes. Cytologia 56, 425–429.
Darriba D., Taboada G. L., Doallo R. and Posada D. 2012 J Model
Test 2: more models, new heuristics and parallel computing. Nat.
Methods 9, 772.
Denton T. E. 1973 Fish chromosome methodology, (ed. C. Charles),
pp. 166. Tomas Springfield, Illinois.
 Karyological evolution of genus Channa
Dhar N. J. and Chatterjee K. 1984 Chromosomal evolution in
Indian murrels (Channiformes: Channidae). Caryologia 37,
359–371.
Donsakul T. and Magtoon W. 1991 A chromosome study on five
species of fishes (Channa, family Channidae), from Thailand,
Bangkok. In The Proceedings of the 29th Kasetsart University
Annual Conference (Fisheries Section), pp. 561–574.
Drummond A. J. and Rambaut A. 2007 BEAST: Bayesian
evolutionary analysis by sampling trees, BMC Evol. Biol. 7, 214.
Eschmeyer W. N. and Fricke R. 2016 Catalog of fishes (http://
researcharchive.calacademy.org). Electronic version Accessed 1
Nov. 2016.
Froese R. and Pauly D. (ed) 2018 Fish base, www.fishbase.org (12/
2018).
Galleti P. M., Aguilar C. T. and Molina W. 2000 An overview of
marine fish cytogenetics. Hydrobiologia 420, 55–62.
Gold J. R. 1979 Cytogenetics. In Fish physiology (ed. W. S. Hoar,
D. T. Randall and J. R. Brett), vol. 8, pp. 353–405. Academic
Press, New York.
Gold J. R., Li Y. C., Shipley N. S. and Powers P. K. 1990 Improved
methods for working with fish chromosomes with a review of
metaphase chromosome banding. J. Fish Biol. 37, 563–575.
Hamilton F. 1822 An account of the fishes found in the river
Ganges and its branches. Archibald Constable, Edinburgh.
Hinegardner R. and Rosen D. E. 1972 Cellular DNA content and
the evolution of teleostean fishes. Am. Nat. 106, 621–644.
Hossain M. K., Latifa G. A. and Rahman M. M. 2008 Observations
on induced breeding of snakehead murrel, Channa striata
(Bloch, 1793). Int. J. Sustain. Crop Prod. 3, 65–68.
Howell W. M. and Black D. A. 1980 Controlled silver-staining of
nucleolus organizer regions with a protective colloidal developer:
a 1-step method. Experientia 36, 1014.
IUCN 2016 The IUCN red list of threatened species. Version
2016-3. \www.iucnredlist.org[. Downloaded on 08 March
2017.
Jankum M., Oclaewicz K., Pardo B. G., Martinez P., Woznicki P.
and Sanchez L. 2003 Localization of 5SrRNA loci in three
coregonid species (Samonidae). Genetica 119, 183–186.
Jhingran V. G. 1982 Fish and fisheries of India, pp. 467–470.
Hindustan Publishing, Delhi.
Kang L., Yucheng L. and Dun Z. 1985 Studies on the karyotypes
and C-banding patterns of three species in Channidae (Pisces).
Acta. Genet. Sin. 12, 470–477.
Khakhong S., Supiwong W., Tanomtong A., Sriuttha M., Jear-
ranaiprepame P., Soemphol W. et al. 2014 A first chromosomal
characterization of NORs in splendid snakehead fish, Channalu-
cius (Perciformes, Channidae). Cytologia 79, 133–139.
Khuda-Bukhsh A. R., Chanda T. and Barat A. 1986 Karyomor-
phology and evolution in some Indian hill stream fishes with
particular reference to polyploidy in some species. In Indo-
Pacific Fish Biology: Proceedings of the Second International
Conference on Indo-Pacific fishes (ed. Uyeno T., Arai R.,
Taniuchi T. and K. Matsuura), pp. 886–898. Ichthyological
Society of Japan, Tokyo.
Klinkhardt M., Tesche M. and Greven H. 1995 Database of fish
chromosomes. Westarp Wissenschaften, Hohe Börde.
Kumar R., Kushwaha B., Nagpure N. S., Behera B. K. and Lakra
W. S. 2013 Karyological and molecular diversity in three
freshwater species of the genus Channa (Teleostei, Perciformes)
from India. Caryologia 66, 109–119.
Legrande W. H. 1980 The chromosome complement of Arius felis
(Siluriformes: Ariidae). Jpn. J. Ichthyol. 27, 82–84.
Legrande W. H. 1981 Chromosomal evolution in North American
catfishes (Siluriformes: Ictaluridae) with particular emphasis on
the Madtoms, Noturus. Copeia 1, 33–52.
Legrande W. H. and Cavander T. M. 1980 The chromosome
complement of the stonecat Madtoms, Noturus flavus
Page 13 of 14 112
(Siluriformes: Ictaluridae) with evidence for the existence of a
possible chromosomal race. Copeia 2, 341–344.
Levan A., Fredga K. and Sandberg A. 1964 Nomenclature for
centromeric position on Chromosomes. Hereditas 52, 201–220.
Lingyun Z. J. L. 1982 Karyotype analysis of the snake-head
Ophiocephalus argus. J. Beijing Normal Univ. (Nat. Sci.) 3.
Lopez P. A. and Fenocchio A. S. 1994 Confirmation of two
different cryptotypes for the neotropical fish Hoplias malabricus
Gill 1903 (Characiformes). Cytobios 80, 217–221.
Magtoon W., Donsakul T. and Rangsiruji A. 2006 Karyotypes of
two Channid fishes (family Channidae): Channa maruloides and
C. asiatica. In 32nd Congress on Science and Technology of
Thailand, Bangkok.
Manna G. K. and Prasad R. 1973 Chromosomes in three species of
fish (Channa). Nucleus 16, 150–157.
Manna G. K. and Prasad R. 1974 Cytological evidence for two
forms of Mystus vittatus as two species. Nucleus 17, 4–8.
Mayrose I., Barker M. S. and Otto S. P. 2010 Probabilistic models
of chromosome number evolution and the inference of poly-
ploidy. Syst. Biol. 59, 132–144.
Michelle N. Y. T., Shanti G. and Loqman M. Y. 2004 Effect of
orally administered Channa striata extract against experimen-
tally-induced osteoarthritis in rabbits. Int. J. Appl. Res. Vet. Med.
2, 171–175.
Morescalchi A. 1975 Chromosome evolution in the caudate
amphibians. Evol. Biol. 8, 338–387.
Musikasinthorn P. 2000 Channa aurantimaculata, a new channid
fish from Assam (Brahmaputra River basin), India, with
designation of a neotype for C. amphibeus (McClelland, 1845).
Ichthyol. Res. 47, 27–37.
Nayyar R. P. 1966a Karyotype studies in seven species of
Cyprinidae. Genetica 35, 95–104.
Nayyar R. P. 1966b Karyotype studies in thirteen species of fishes.
Genetica 37, 78–92.
Post V. A. 1965 Vergleichende Untersuchungen der Chromo-
somenzahlen hei Süßwasser-Teleosteern. J. Zool. Syst. Evol. Res.
3, 47–93.
Rishi K. K. 1973 Somatic karyotypes of three teleosts. Genen.
Phaenen. 16, 101–107.
Rishi K. K. and Haobam M. S. 1984 Karyotypes of three forms of
fishes having high chromosome number. Int. J. Acad. Ichthyol. 5,
1–2.
Roe L. J. 1991 Phylogenetic and ecological significance of
Channidae (Osteichichthys, Teleostei) from the early Eocene
Kuldana formation of Kohat, Pakistan. Contributions from
the Museum of Paleontology, University of Michigan. 28,
93–100.
Ruma F., Ahmed A. T. A. and Alam S. S. 2006 Karyotype analysis
of Channa punctata Bloch and Channa orientalis Schneider with
Giemsa, CMA3 and DAPI. Cytologia 71, 425–430.
Singh S. S., Singh C. B., Thoidingjam L. and Waikhom G. 2013 A
new report of karyotype in the freshwater snakehead fish,
Channa gachua (Channidae: Perciformes) from Northeast India,
Manipur. Int. J. Res. Fish. Aquac. 3, 7–12.
Stebbins G. L. 1971 Chromosomal evolution in higher plants.
Edward Arnold Ltd., London.
Stingo V. 1979 New developments in vertebrate cytotaxonomy II.
The chromosomes of the cartilagenous fishes. Genetica 50,
227–239.
Supiwong W. and Jearranaiprepame P. 2009 Cytogenetics of giant
snakehead fish (Channa micropeltes) from the Northeast of
Thailand. In The Proceedings of 12th National Graduate
Research Conference, Khon Kaen. BMP6, 1584–1590.
Supiwong W., Jearranaiprepame P. and Tanomtong A. 2009 A new
report of karyotype in the chevron snakehead fish, Channa
striata (Channidae, Pisces) from Northeast Thailand. Cytologia
74, 317–322.
112 Page 14 of 14 Ravindra Kumar et al.
Tamura K., Peterson D., Peterson N., Stecher G., Nei M. and
Kumar S. 2011 MEGA5: molecular evolutionary genetics
analysis using maximum likelihood, evolutionary distance, and
maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739.
Tanomtong A., Supiwong W., Jearranaiprepame P., Khakhong S.,
Kongpironchuen C. and Getlekha N. 2014 A new natural
autotetraploid and chromosomal characteristics of dwarf snake-
head fish, Channa gachua (Perciformes, Channidae) in Thailand.
Cytologia 79, 15–27.
Corresponding editor: H. A. RANGANATH
Ueda T., Irie S. and Kato Y. 1987 Longitudinal differentiation of
metaphase chromosomes of Indian muntjac as studies by
restriction enzyme digestion, in situ hybridization with cloned
DNA probes and Distamycin A plus DAPI fluorescence staining.
Chromosoma 95, 251–257.
Wattanodorn S., Chavananikul V. and Wattanavijarn W. 1985 Cell
culture derived from Snakehead fish (Channa striata). Thai J.
Vet. Med. 15, 305–312.