Int. J. Biosci.

2012

International Journal of Biosciences (IJB)

ISSN: 2220-6655 (Print) 2222-5234 (Online)
Vol. 2, No. 3, p. 88-96, 2012
http://www.innspub.net

RESEARCH PAPER OPEN ACCESS

Comparative analysis of genetic diversity among three species of

the freshwater fish genus Garra (Osteichthys: Cyprinidae) using
restriction fragment length polymorphism
M Raja, P Malaiammal, S Nandagopal, M Muralidharan, M Arunachalam*
Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University,
Alwarkurichi- 627 412, Tamil Nadu State, India
Received: 04 August 2011
Revised: 14 September 2011
Accepted: 16 September 2011

Key words: Garra, morphometric, meristic, RFLP, genetic diversity, Western Ghats.
Abstract
The genetic variation in three species of the freshwater cyprinid Garra was studied using the traditional
morphometric, meristic and Restriction fragment length polymorphism as molecular tool analysis. Samples were
collected from their respective geographic locations of southern Western Ghats. Based on the 46 morphometric and
18 meristic characters employed during this study 23 characters showed variation among the three species and hence
were utilized for the PCA ordination. The principal component analysis was performed using 15 morphometric and 8
meristic characters of which 12 components were extracted and the first three axes showed eigenvalues >1 and they
explained the variance about 81.46 % of the total variance. The genome size of the species Garra mullya ranged from
3.8-6.15 µg/mg, Garra kalakadensis ranged from 3.25- 6.3 µg/mg and Garra gotyla stenorhynchus ranged from 3.9-
6.15 µg/mg. Based on the electrophorogram, different bands of fragments in each lane and band volume were
analyzed, According to Hind III enzyme the electrophorogram analysis showed maximum fragment length
polymorphism in Garra mullya which had four fragments and the total volume of bands was 12.582 nmoles. Based
on the Eco R1 enzyme digestion the electrophorogram analysis revealed that the maximum fragment length
polymorphism in Garra mullya composed of four fragments and the total volume of bands in the entire lane was
10.5965 nmoles. Based on the Hind III and Eco R1 restriction enzymes, the cluster analysis clearly showed that the
Garra mullya and Garra kalakadensis grouped together while Garra gotyla stenorhynchus with distinct genetic
distance did not cluster with the other two species. Garra gotyla stenorhynchus can also be distinguished
morphologically from Garra mullya and Garra kalakadensis.
*Corresponding Author: M Arunachalam  [email protected]

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Introduction DNA sequence of a genome that can be detected by

Fishes are the most diverse group of living vertebrates, breaking the DNA into pieces with restriction enzymes
with more than 24,600 extant species currently known. and analyzing the size of the resulting fragments by gel
The Order Cypriniformes is the planet’s largest electrophoresis.
monophyletic group of freshwater fishes, with over
400 genera and 3000 species native to Asia, Europe, DNA-based techniques for species identification have
Africa, and North America (Nelson, 2006). recently started to be applied towards a wide variety of
Cypriniformes in peninsular India is represented by 4 fish species, including closely related species belonging
families, 8 sub families, 40 genera and 166 species. The to the same family or genus (Davidson 1998; Bossier
family Cyprinidae has 4 subfamilies, 27 genera and 135 1999; Lockley and Bardsley 2000). The information on
species (Arunachalam et al., 2008). Historically the genetic resource of Indian fish fauna is very limited,
morphology of fishes is the main source of information especially native cyprinids. The genetic variation in
for taxonomic and evolutionary studies. Systematic different populations of the freshwater cyprinid species
ichthyologists still rely on morphology for taxonomic Puntius filamentosus was studied using Restriction
characters as species have characteristics shape, sizes, Fragment Length Polymorphism (RFLP) analysis. The
pigmentation patterns, disposition of fins and other genomic size of the different populations of P.
external features which help in identification and filamentosus was found between 3.45 and 3.80ng/mg.
classification. Meristic traits are often considered to be The study could prove one population as distinct.
the most reliable taxonomic characteristics, because (Johnson et al., 2007).
most are easy to determine.
The cyprinid fish genus Garra includes bottom
All the organisms are subjected to mutations as a result dwelling fishes usually found in fast flowing streams
of normal cellular operations or interactions with the where they cling to rocks using the highly modified
environment, leading to genetic variation. In mouth which acts as a sucker. Garra is a common
conjunction with selection and genetic drift, there cyprinid and are mostly inhabitants of rapid running
arises genetic variation within and among individuals, waters, adapted with suctorial disc to attach
species and higher order taxonomic groups. This themselves to the substratum in swift currents and
variation are useful to geneticists, but it must be horizontally placed paired fins, especially the pectorals.
heritable and discernable to the researcher, whether as The flattening of the head and anterior part is as a
a recognizable phenotypic variation or as a genetic result of efforts to utilize more and more of the
mutation distinguishable through molecular anterior part of the body for adhesion. The proboscis
techniques. Several marker types are highly popular in and the tubercles help to lessen the velocity and impact
genetics. of the rushing torrents. Adaptive features in all the
species being similar most of the morphological
The development of molecular techniques has helped features show overlap and hence the taxonomic
to investigate fish systematics. The realm of methods characters commonly used are not helpful in clear cut
developed for molecular studies offers new suites of diagnosis of the species and hence taxonomic
characters for analyzing relationships among fish ambiguities exist.
systematics (Hillis, et al., 1996; Ferraris and Palumbi,
1996) and hence have been effectively applied from the The genus is widely distributed from southern China,
level of populations to orders. A restriction fragment across South East Asia, India and the Middle East to
length polymorphism or RFLP, is a variation in the northern and central Africa. Because of the extreme

89 Raja et al.
 Int. J. Biosci. 2012

morphological divergence of the genus Garra, the present in each sample were determined using UV
sister group relationships of this genus is of extreme spectrophotometer. Isolated DNA samples were
interest to systematic ichthyologists, fish biologists, subjected to restriction enzyme digestion at 37º C for 2
and evolutionary biologists alike. h using EcoR1 and Hind III enzyme. After incubation
each samples were loaded in to 1% agarose gel and
In the present study, the aim is to analyze the overall electrophoresed at 50 – 100 V for one and half hours.
features of morphometric, meristic characters, and After electrophoresis, gel was placed in the gel
Restriction pattern of nuclear DNA using the document unit (BIORAD) and bands were visualized
restriction enzymes Hind III, and Eco RI regions in and were photographed. The DNA fragment in each
order to evaluate patterns of genetic variation between lane was viewed and number of band and band volume
species of Garra and to determine the phylogenetic were documented using TOTAL LAB gel analyzing
relationship of three species of the endemic genus software. The band volume data were used for
Garra in selected streams and rivers of the peninsular construction of similarity cluster using PAST software
India. (Free ware: version. 1.83).

Materials and Methods

Fish samples were collected from three geographically
isolated east flowing river systems of the Western
Ghats. Garra mullya was collected from the upstream
of Hanumannadhi, which forms a sub basin of
Tamiraparani river and G. kalakadensis was collected
from Pachiyar in Kalakad Mundandhurai Tiger
Reserve and G. gotyla stenorhynchus collected from
Nellithurai (Bhavani River) near Mettupalayam (Fig.
1.) These native cyprinids were collected using mono
filamentous, multi filamentous gill nets of different
mesh sizes from 8-28 mm and cast nets. Fishes were
identified in the field and a portion of gill and muscle
tissues were fixed in 95% ethanol and were kept in the
ice cubes for molecular analysis. Few individuals were
preserved in 10% formalin for morphological studies.
Large specimens were injected with 15% formalin
carefully through the vent prior to preservation.
Specimens were kept preserved in Manonmaniam Fig. 1. The Experimental fishes of the genus garra.
Sundaranar University Museum of Natural History
(MSUMNH). Measurements were made point to point Results
using digital caliper. The meristic and morphometric In the present study the Principal component analysis
measurements are based on the method by Hubbs and based on the 46 morphometric and 18 meristic
Lagler (1956). characters were employed. As 23 characters showed
variation among the three species and hence these
The genomic DNA was isolated by phenol-chloroform characters were used for PCA ordination of which 15
method (Sambrook et al., 1989). Amount of DNA are morphometric and 8 were meristic characters

90 Raja et al.
 Int. J. Biosci. 2012

(Table 1). Of these 12 components were extracted and characters but none of them was significant in factor
the first three axes showed the eigen value >1 and also loadings.
variation is 81.46 % of the total variance (Table 2). The
first component alone explained about 53.7 % variance Characters such as head length and predorsal scales
in characters such as: Snout length, Pre nasal length, varied between Garra gotyla stenorhynchus and
Head width, Transverse breast rows, Pre anal scales, Garra kalakadensis (Head length 20.56 – 25.48 vs.
Pelvic insertion to anal origin, Lower transverse rows, 26.37 - 28.32; predorsal scales 10 vs.10 - 12) as shown
Circumpeduncular scales, Head depth at occiput and by the PCA (Fig. 2) Disc width and pelvic fin rays of
Peduncle depth showed higher factor loadings (> 0.8). Garra mullya were lower in values compared to the
The second axis explained 18.38 % variance and higher other two species and hence it has been plotted
loadings were noted in two characters Disc width and between the G. kalakadensis and G. gotyla
branched pelvic fin rays. The 3rd and 4th components stenorhynchus in the ordination.
together explained 9.28 % variance among the

Table 1. Morphometric and meristic measurements of Garra mullya, Garra kalakadensis and Garra gotyla
stenorhynchus.
Characters Garra mullya Garra kalakadensis Garra gotyla
(n=10) (n=10) stenorhynchus
(n=10)
Range Mean ± SD Range Mean ± SD Range Mean ± SD
Snout length (SL) 45.54 - 52.39 48.89 ± 1.98 41.38 - 46.98 44.85 ± 1.61 49.26 - 58.43 54.88 ± 2.77
Pre nasal length (PNL) 32.1 - 38.09 35.57 ± 2.13 25.89 - 33.8 29.48 ± 2.73 34.29 - 42.28 38.31 ± 2.22
Head width (HW) 68.79 - 76.58 72.95 ± 2.73 64.54 - 69.12 66.82 ± 1.66 69.78 - 78.75 74.96 ± 2.85
Head depth at nostril (HDN) 34.56 - 45.32 39.45 ± 3.52 33.1 - 40.23 37.68 ± 2.36 45.39 - 53.00 47.36 ± 2.69
head depth at pupil (HDP) 45.08 - 57.41 52.07 ± 3.66 48.22 -56.13 51.81 ± 2.96 54.79 - 62.93 59.11 ± 2.58
Head depth at occiput (HDO) 63.21 - 71.73 66.66 ± 3.45 55.84 - 65.4 61.24 ± 2.97 70.73 - 79.14 74.65 ± 2.64
Peduncle depth (PD) 9.82 - 13.1 12.18 ± 0.88 9.82 - 10.59 10.24 ± 0.27 12.70 -18.07 14.76 ± 1.41
Dorsal origin to anal origin (DA) 31.22 - 40.89 37.89 ± 2.58 31.3 - 35.6 33.5 ± 1.06 35.28 - 47.12 39.5 ± 2.97
Dorsal fin base (DFB) 15.03 - 20.06 17.03 ± 1.54 11.76 -15.71 13.44 ± 1.12 16.73 - 23.10 18.3 ± 1.77
Pectoral insertion to anal origin (PECA) 48.78 - 57.43 54.62 ± 2.36 48.53- 56.22 52.16 ± 2.40 54.25 - 61.90 58.01 ± 2.92
Pelvic insertion to anal origin (PELA) 21.61 - 26.12 24.39 ± 1.18 19.41- 24.38 22.13 ± 1.49 23.75 - 34.33 27.25 ± 2.89
Head length (HL) 20.39 - 25.93 24.52 ± 1.63 26.37- 28.31 27.48 ± 0.68 20.56 - 32.60 24.05 ± 2.93
Body depth (BD) 18.23 - 25.3 22.56 ± 1.96 16.22- 21.16 19.24 ± 1.60 19.21 - 29.70 22.88 ± 2.74
Disc length (DL) 22.93 - 29.91 26.11 ± 2.76 26.52- 34.39 30.52 ± 2.68 28.27- 35.27 33.12 ± 2.19
Disc width (DW) 37.16 - 46.27 41.75 ± 3.06 48.49 - 55.39 51.01 ± 2.07 46.47- 54.31 50.26 ± 2.80
Branched pelvic fin rays (pelvic) 7-8 7.3 ± 0.45 8 8± 0.00 8 8 ± 0.00
Branched pectoral fin Rays (pectoral) 11-13 12± 0.43 12 - 14 12.90 ± 0.57 12 - 15 13.92 ± 0.79
Pre dorsal scales (predor) 10-11 10.9 ± 0.29 10 - 12 11.10± 0.88 10 10 ± 0.00
Lower transverse rows (Lowtr) 3-4 3.8 ± 0.45 3 3± 0.00 4 4 ± 0.00
Circumpeduncular scales (circp) 15-16 15.8± 0.39 12 - 14 12.60 ± 0.70 16 16 ± 0.00
Circumferential scales (circf) 22 22 ± 0.00 16 - 20 19.60 ± 1.26 22 22 ± 0.00
Transverse breast rows (breast) 5 5 ± 0.00 5 5± 0.00 6 6 ± 0.00
Pre anal scale (pre anal) 16-21 17.6 ± 2.02 14 - 17 15.70± 1.06 21-23 21.75 ± 0.62

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genome size of Garra mullya ranged from 3.8-6.15

µg/mg, while Garra kalakadensis showed the range
from 3.25- 6.3 µg/mg and Garra gotyla stenorhynchus
showed the range from 3.9-6.15 µg/mg. The result of
the restriction analysis showed that there was clear
separate DNA banding pattern for the three species
and the fragment migration were ranged from 5000-
2000bp. Based on the electrophorogram, different
bands of fragments in each lane and band volume were
analyzed (Table 4, 5). According to Hind III enzyme
the electrophorogram analysis showed maximum
fragment length polymorphism in Garra mullya which
had four fragments 1.056 nmoles, 5.842 nmoles, 3.216
nmoles and 2.468 nmoles respectively and the total
volume of bands along the entire lane was 12.582
nmoles. Garra kalakadensis had three restriction
fragments 4.236 nmoles, 3.924 nmoles and 3.124
nmoles and the total volume of bands in the entire lane
was 11.284. Garra gotyla stenorhynchus had four
restriction fragments with corresponding band
volumes of 1.649 nmoles, 0.926 nmoles, 3.418 nmoles
Fig. 2. Agarose gel image and restriction pattern of the and 2.236 nmoles and the total volume of bands in the
genus garra. entire lane was 8.229 nmoles.

The DNA content of each species and the

corresponding O.D. values are given in Table 3. The

Table 2. Component matrix of four axes from PCA of morphological data.

Characters Axis 1 Axis 2 Axis 3 Axis 4
Snout length (SL) 0.874* -0.139 0.194 -0.162
Pre nasal length (PNL) 0.864* 0.206 0.088 0.191
Head width (HW) 0.813* 0.198 0.350 0.020
Head depth at nostril (HDN) 0.787 -0.281 -0.100 0.232
head depth at pupil (HDP) 0.709 -0.401 0.110 0.021
Head depth at occiput (HDO) 0.900* -0.057 0.032 -0.098
Peduncle depth (PD) 0.935* 0.041 -0.190 0.031
Dorsal origin to anal origin (DA) 0.772 0.359 -0.247 0.000
Dorsal fin base (DFB) 0.766 0.335 0.026 0.221
Pectoral insertion to anal origin (PECA) 0.755 -0.097 -0.424 0.070
Pelvic insertion to anal origin (PELA) 0.827* -0.004 -0.314 -0.095
Head length (HL) -0.729 -0.057 -0.576 0.122

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Body depth (BD) 0.607 0.447 -0.290 -0.395

Disc length (DL) 0.288 -0.676 -0.293 -0.140
Disc width (DW) 0.006 -0.924* -0.097 0.107
Branched pelvic fin rays (pelvic) 0.018 -0.844* 0.250 -0.236
Branched pectoral fin Rays (pectoral) 0.424 -0.761 -0.081 -0.053
Pre dorsal scales (predor) -0.673 0.277 -0.012 -0.565
Lower transverse rows (Lowtr) 0.834* 0.204 -0.108 -0.323
Circumpeduncular scales (circp) 0.835* 0.460 0.049 0.079
Circumferential scales (circf) 0.747 0.431 0.078 0.050
Transverse breast rows (breast) 0.827* -0.502 0.018 -0.019
Pre anal scale (preanal) 0.842* -0.244 0.250 -0.149
Percent of variance explained 53.79 18.38 5.35 3.93

Table 3. DNA Purity and Genome size of the three species of genus Garra.

Species No. Absorbance at Purity of DNA Total amount

260nm 280nm µg/mg
1 0.084 0.1377 1.64 4.2
2 0.076 0.1284 1.69 3.8
Garra mullya 3 0.092 0.1490 1.62 4.6
4 0.102 0.1693 1.66 5.1
5 0.123 0.2115 1.72 6.15
1 0.126 0.2179 1.73 6.3
2 0.108 0.1825 1.69 5.4
Garra kalakadensis 3 0.089 0.1441 1.62 4.45
4 0.085 0.1385 1.63 4.25
5 0.065 0.1040 1.60 3.25
Garra gotyla 1 0.078 0.1263 1.62 3.9
stenorhynchus 2 0.089 0.1450 1.63 4.45
3 0.096 0.1622 1.69 4.8
4 0.123 0.2152 1.75 6.15
5 0.110 0.1870 1.70 5.5

Table 4. Number of fragments and band volume of Electrophorogram for the three species of genus Garra based on
the Hind III restriction pattern.
Fish species Number of Fragments Band volume Total band volume
(n moles) (n moles)
1 1.056
Garra mullya 2 5.842 12.582
3 3.216
4 2.468
Garra kalakadensis 1 4.236
2 3.924 11.284
3 3.124
Garra gotyla stenorhynchys 1 1.649
2 0.926 8.229
3 3.418
4 2.236

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Table 5. Number of fragments and band volume of Electrophorogram of the genus Garra based on the Eco RI
restriction pattern.
Fish species Number of Fragments Band volume Total Band volume
(n moles) (n moles)
1 2.3645
Garra mullya 2 3.1004 10.5965
3 4.1252
4 1.0064
Garra 1 4.5692
kalakatensis 2 3.6541 11.4773
3 3.2540
Garra gotyla stenorhynchys 1 1.2301
2 0.6359 7.1364
3 3.1540
4 2.1164

1.2301 nmoles, 0.6359 nmoles, 3.1540 nmoles, and

2.1164 nmoles and the total volume of bands in the
entire lane was 7.1364 nmoles.

Fig. 3. Principal Component Ordination for three

Fig. 4. Genetic distance among the three species of
garrine species Garra mullya ( ), Garra gotyla
Garra based on the RFLP band volume.
stenorhynchus ( ) and Garra kalakadensis ( ) based
on morphological characters.
Based on the Hind III and Eco R1 restriction enzymes,
the cluster analysis clearly showed that Garra mullya
Based on the electrophorogram, different bands of
and Garra kalakadensis grouped together while Garra
fragments in each lane and band volume were analyzed
gotyla stenorhynchus with distinct genetic distance
(Table 5). Based on the Eco R1 enzyme digestion, the
did not cluster with the other two species (Fig. 3)
electrophorogram analysis revealed that the maximum
Garra gotyla stenorhynchus can also be distinguished
fragment length polymorphism in Garra mullya
morphologically from Garra mullya and Garra
composed of four fragments with each band volume of
kalakadensis by the presence of proboscis.
2.3645 nmoles, 3.1004 nmoles, 4.1252 nmoles and
1.0064 nmoles respectively and the total volume of
Discussion
bands in the entire lane was 10.5965 nmoles. Garra
The genus Garra known for its higher degree of
kalakadensis possessed three restriction fragments
plasticity ascribed to its adaptive features comprises a
4.5692 nmoles, 3.6541 nmoles and 3.254 nmoles and
number of species some with distinct characters and
the total volume of bands in the entire lane was 11.4773
few others with close resemblances owing to the more
nmoles. Garra gotyla stenorhynchus had three
similar morphological characters. Most of the
restriction fragments each with a band volume of
morphological traits of fishes are similar and often

94 Raja et al.
 Int. J. Biosci. 2012

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