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 early Miocene, and throughout Africa and East Asia in the
late Tortian (8 Mya). These murrels were probably origi-
There are over 34,000 valid fish species globally (Eschmeyer nated from south Himalayan region in India during early
and Fricke 2016). The teleost being the largest infraclass in Eocene epoch (Bohme 2004).
ray-finned fishes covers more than 95% of all fish and half of The snakeheads are important food fishes (Chattopadhyay
the extant vertebrate species. In teleost, the perciformes is 1975; Jhingran 1982) as well as medicinal and pharmaceu-
the largest order with around 156 families, including tical values (Michelle et al. 2004). They are carnivorous, air-
Channidae family. This family is commercially important breathing fish (Froese and Pauly 2018), and can survive for a
and are of two genera Channa and Para channa with 29 and longer time without water. They are found in swamps,
three species, distributed in Asia and Africa, respectively reservoirs, drains, ponds, canals, rivers, small streams, rice
(Gold et al. 1990). Fossil records of the family Channidae fields, mining pools, roadside ditches and lakes across
are reported by Roe (1991) from Indian subcontinent, in southern Asia, southern China, Indo-China and the Sunda
early Eocene, about 50 million years ago (Mya). These Islands (Hossain et al. 2008). These channid species are
species were spread in western Eurasia about 17 Mya in 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 using Lieca CW4000 Karyo software. Average length of the
list (2016), the status of channid fishes mainly falls under homologous chromosome pair was used for estimating the
least concern v3.1, a few species come under data deficient length of short arm (p), long arm (q), arm ratio (q/p), cen-
v3.1, C. diplogramme is under vulnerable B1 ab(iii) ? 2 tromeric index and relative length (%). The arm ratios were
ab(iii) v3.1, and C. bleheri and C. harcourtbutleri are under used to classify the chromosomes as metacentric (m), sub-
near threatened v3.1. metacentric (sm), subtelocentric (st) and telocentric (t) as
Till date, the cytogenetic profiles of many channid species suggested by Levan et al. (1964). The ideograms were built
(table 1) have been investigated. Dhar and Chatterjee (1984) according to the relative length and centromeric index.
explained the karyotype analysis and chromosomes evolution of Silver nitrate (AgNO3) and chromomycin A3 (CMA3)
five Channa species, namely C. barca, C. orientalis, C. punc- staining of chromosomes was done as described by Howell
tata, C. stewartii and C. striata. The karyomorphological studies and Black (1980) and Ueda et al. (1987) to localize tran-
by various workers in channid species reported a different scriptionally active region and GC-rich nucleolar organizer
diploid chromosome number (2n) in the same species (Nayyar region (NOR) respectively. Giemsa stained karyotype anal-
1966a; Manna and Prasad 1973). The 2n in C. punctata was yses, AgNO3 and CMA3 positive sites were determined from
reported as 34 (Nayyar 1966a) and 32 (Manna and Prasad 1973; more than 50 metaphase complements of each specimen
Rishi 1973). Similarly in C. orientalis, the 2n has been reported (over 150 metaphase complements per species) using 100 9
as 42 (Klinkhardt et al. 1995), 52 (Banerjee et al. 1988), 76 objective lens with oil immersion in light microscope.
(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 Analysis of chromosome evolution
using Giemsa, silver nitrate (AgNO3) and chromomycin A3
(CMA3) staining. Since the DNA sequences in channid species The direction of chromosome evolution (Robertsonian
are available in NCBI GenBank, a comparative analysis of mechanism) in Channa species was found by the combined
chromosomes and DNA sequences were also included in this study of cytogenetic and molecular techniques using Ore-
study. To find the evolutionary correlation between the karyol- chromis potani as an out group in both analyses. Whereas,
ogy and DNA sequences, the karyological information was two different types of methods has been used for analysis of
superimposed on the phylogenetic tree constructed from DNA hypothesis of chromosome evolution. The software Chro-
sequences of mitochondrial gene 16S rRNA. 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
Materials and methods rRNA gene sequence downloaded from NCBI and aligned
using molecular evolutionary genetics analysis (MEGA
The Indian species cytogenetically investigated in this study 5.05) software (Tamura et al. 2011). The best-fit nucleotide
were orange-spotted snakehead (C. aurantimaculata; Musi- substitution model for the 16S rRNA dataset was selected
kasinthorn 2000), dwarf snakehead (C. gachua; Hamilton based on the Akaike information criterion (AIC) in J-model
1822), great snakehead (C. marulius; Hamilton 1822), test (Darriba et al. 2012). The ML- chromosome evolution
Ceylon snakehead (C. orientalis; Bloch 1801), spotted tree was constructed on 16S rRNA tree using ChromEvol
snakehead (C. punctata; Bloch 1793) and striped snakehead 1.3 (Mayrose et al. 2010). The chromosome evolution model
(C. striata; Bloch 1793). The live individuals (n = 3 of each gave information about loss or gain of chromosome, dupli-
species) of each of the six Channa species were collected by cations or semiduplications mechanism. For understanding
netting from northeastern region of India. Since all the the time based chromosome evolution, the node age of
specimens were in their juvenile stage, their sex could not be available 16S rRNA gene of Channa species were estimated,
identified by visual examination. which determined the approximate time of split between
For chromosome preparations, the live specimens were lineages. This was used for estimating the time when chro-
injected with 0.05% colchicine intramuscularly at the rate of mosomal changes occurred in context of geological time
1.0 mL/100 g body weight of fish to arrest chromosomes at scale. The 16S rRNA gene matrix of genus Channa were
metaphase stage. After 90 min of injection, the specimens analysed under Bayesian framework with uncorrelated log-
were immersed in benzocaine solution and sacrificed. The normal–relaxed clock model in BEAST v. 1.6.1 (Drummond
kidney tissues were dissected out for the preparation of and Rambaut 2007).
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 Results
chromosome slides were stained with 6% Giemsa in phos-
phate buffer (KH2PO4 and Na2HPO4 with pH 6.8) for 20 The karyotype formula (with 2n and fundamental arm
min at room temperature. The homologous chromosomes number (NF)) of the studied species were observed as
were paired and arranged in decreasing order of their size by 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, from one pair in C. aurantimaculata and C. orientalis to four
4m ? 10sm ? 30t (44 and 58) in C. marulius, pairs in C. punctata (figures 1c–6c; table 2). The nonse-
12m ? 10sm ? 14st ? 16t (52 and 74) in C. orientalis, quential staining of other metaphase chromosome spreads
18m ? 10sm ? 4st (32 and 60) in C. punctata and with CMA3 positive sites determined one to four pairs of
6m ? 2sm ? 10st ? 22t (40 and 48) in C. striata (figur- NORs as bright zones against black background (figures 1d–
es 1a–6a; table 2). Minimum 2n was found in C. punctata 6d; table 2), which were in agreement with AgNORs. In four
and maximum in C. gachua, whereas NF ranged from 48 in species (figures 1, 2, 3, 6) the location of AgNO3 and CMA3
C. striata to 98 in C. aurantimaculata. The C. auranti- signals were at telomeric position of the chromosomes,
maculata possessed maximum 28 metacentric chromosomes whereas in C. orientalis (figure 4) and C. punctata (fig-
with 2n = 52, whereas the metaphase spreads of C. marulius ure 5), the signal positions were intercalary at the homolo-
(2n = 44) showed maximum 30 telocentric chromosome, gous pair of the chromosomes.
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- Chromosome evolution
sented in figures 1b–6b. The species-wise chromosome
statistics are given in table 3. The analysis suggested the best model for chromosome
The AgNO3 stained NOR (AgNOR) signals in metaphase evolution with chromosome gain or loss and constant
chromosome complements of six Channa species ranged 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 chromosome) and fission (gain), which showed events
ChromEvol software. The rate parameter estimated in the inferred with an exp [0.5 (figure 7). Therefore, this study
depicted model are: 103.23 for chromosome losses, 31.07 has focussed on the haploid chromosome numbers estimated
for chromosome gains and 3.02 for chromosome demidu- by the Bayesian method. The Bayesian time-calibrated tree
plication. The results also showed the occurrence of poly- allowed us to infer that the Channa species diverged from
ploidization events and suggested that duplication of Oreoleuciscus potanini in early Miocene around 20.81 Mya
chromosome occurred during the chromosome evolution. (figure 8), probably due to the change occurred in chromo-
The main events were fusion (loss in number of 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)

showed that in all the cases the 2n was 44 except by Nayyar
The centromere based description of karyomorphology to (1966a) who reported 40. The presence of high number (15
group chromosomes as metacentric, submetacentric, subte- pairs) of telocentric chromosomes in Indian populations may
locentric and telocentric/acrocentric and their size-wise be considered more close to primitive species (Campos and
arrangement have been used for prediction of inter-rela- Cuevas 1997).
tionship among the families and closeness of species (Lopez In C. striata, the 2n = 40 was first reported by Nayyar
and Fenocchio 1994). On the contrary, the karyomorpho- (1966a) and later several workers reported different diploid
logical investigations in fishes are strenuous due to their chromosome number, karyomorphology and NF, but most of
small size and high number of chromosomes. The present the authors reported; 2n = 40 as given by Kumar et al.
work aimed to cytogenetically reinvestigate some species of (2013). Further, the numbers of telocentric/subtelocentric
the genus Channa, of the evolutionary advance fish family, chromosomes were higher than that of the metacentric/sub-
Channidae of the freshwater teleost fishes (Dhar and Chat- metacentric chromosomes in all the studies. The number of
terjee 1984; Bhatti et al. 2013). 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 Page 11 of 14 112

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 reduce further with more number of metacentric chromo-
absence of telocentric chromosomes. some and intercalary NORs.
The nonsequential, silver-staining bearing NOR is gen- The establishment of different karyotypes in Channa spe-
erally visualized in transcriptically active site of rDNA. The cies during evolution occurred by three mechanisms (Gold
GC-rich active DNA of NORs in many vertebrates, includ- 1979), namely aneuploidy, polyploidy and Robertsonian
ing fishes (Gold et al. 1990) has been identified by CMA3 rearrangements (i.e. change in diploid number of chromo-
positive site staining which not only provide the number and some without change in NF value, Booke 1974). This is also
localization of NORs but also indicate GC-rich content of responsible for the karyotype establishment and change in
transcriptically active sites of rRNA genes in other fish chromosome number in many other fish family (Legrande
species (Jankum et al. 2003). 1980). Whereas, the Robertsonian mechanism was not only
The AgNO3 stained those NORs sites (table 1; figur- responsible for establishment of different karyotypes
es 1c–6c), which expressed themselves during the preceding (2n = 32 to 56) in genus Channa, but also changed NF values
interphase by attaching to a composite of acidic proteins (NF = 48 to 98). It is a centromeric shift with pericentric
associated with nucleolus and pre-RNA (Jorden 1987). The inversion that has been emphasized by several workers (Le-
nonsequential CMA3 stained the GC-rich active sites of grande 1981). Therefore, it gave the unambiguous explana-
DNA and confirmed the same position as AgNOR (table 2; tion of species wise change in chromosome number with
figures 1d–6d). The presence of intercalary NOR (C. ori- fundamental values, otherwise explanation of different kary-
entalis and C. punctata) were due to centromeric fusion of otypes and polyploidy would have been very difficult.
two chromosomes. The study of Manna and Prasad in 1973 and Dhar and
The analysis of karyomorphology and CMA3 signals Chatarjee in 1984 did not indicate polyploidy in Channidae
among the Channa species suggests that they evolved species, because the study of total and relative chromosomal
through a complicated chromosome evolution process with length (table 3) in meiotic division did not indicate polyploidy.
the occurrence of polyploidization, duplication, rearrange- The hypothesis that could explain the observed differ-
ments, fusion and loss of chromosomes. However, there is ences in karyotypes, suggests that changes in chromosome
no evidence of a fixed pattern of these type of events among number occurs gradually over the time by multiple events of
these Channa species. Moreover, C. punctata and C. ori- chromosomal rearrangements. The most explanatory mech-
entalis are from same origin as evidenced by presence of one anism for chromosome evolution in Channa species is
pair intercalary NOR/CMA3 signals. Further, the probable ‘Robertsonian mechanism with pericentric inversion or
hypothesis of the chromosome evolution in Channa species centromere shift’ proposed by Dhar and Chatterjee (1984).
in this study is shown in figure 9. It is hypothesized that the The presence of asymmetrical karyotype in all species
chromosome number in the new evolving species will 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 Banerjee S. K., Misra K. K., Banerjee S. and Ray-Chaudhuri S.
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evolution of snake-head fish (family Channidae). J. Fish Biol.
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to Denton 1973, the present species are formed by the for- Bertollo L. A. C., Takahashi C. S. and Moreira F. O. 1978
mation of biarmed chromosomes from rod-shaped telocen- Cytotaxonomic consideration on Hoplias lacrdae (Pisces, Ery-
thrinidae). Braz. J. Genet. 1, 103–120.
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second includes Parachanna obscura, C. lusius, C. miro- the cyprinodontid fish, Fundulusnotatus from the Upper Tombig-
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Corresponding editor: H. A. RANGANATH