Assessment of Morphometric and Genetic Variation in Three Freshwater Fish Species of the Genus Garra ( Osteichthyes: Cyprinidae )
, Nabeel Mannalamkunnath ALIKUNHI1
, Selvaraj CHINNATHAMBI2
, Ramaiya SORNAM2
, Murugaiyan KALAISELVAM1
, Subramanian MANIVANNAN3
1Annamalai University, Centre of Advanced Study in Marine Biology, Parangipettai, 608502, Tamil Nadu, India; [email protected]
2Manonmaniam Sundaranar University, Department of Biotechnology, Sri Paramaayani Centre for Environmental Sciences, Awarkurichi, 627412, Tamil Nadu, India
3Sathak College of Arts and Science, Department of Biotechnology, Mohammed, Sholinganallur, Chennai-119, Tamil Nadu, India
The present study evaluated the patterns of morphometric and genetic variation using RAPD-PCR techniques for the first time on three species of Garra, viz. G. mullya, G. kalakadensis and G. gotyla stenorhynchus, collected from various river basins of South-India.
The results of morphological analysis revealed that G. mullya and G. kalakadensis hold many similar characters compared to the other congener, G. gotyla stenorhynchus. However, the G. gotyla stenorhynchus fish species exhibited distinct variation in the morphological characters such as snout length, pre-nasal length, inter-nasal width, gap width, lower jaw to isthmus, head depth at pupil, dorsal fin length and disc width from the other two species of Garra. However, certain morphometric characters overlapped. Hence the RAPD finger printing was used to assess the levels of genetic variation in Garra spp. using RAPD-PCR technique. A total of 72 reliable fragments were detected using 10 Operon primers, ranging from 2600 molecular weight to 3100. The shared RAPD fragments found in both G. mullya and G. kalakadensis with fixed frequencies were observed with all the investigated primers, implying their genetically closer relationship.
However, the similarity index observed for G. gotyla stenorhynchus was less with the other two species specifying a genetically distant link.
The present investigation thus contribute to the knowledge on morphological and genetic variation in these three Garra species.
Keywords: fresh water fishes, genetic variations, morphological changes, PCR , RAPD
India is a land of ‘biological paradise’ which supports a great variety of flora and fauna with its diverse topography, climate and natural resource. The fresh water resources of Indian subcontinent are very rich but largely unexplored.
An estimate states that there are 12 major rivers and 48 lesser rivers with a total catchment area of 277.6 million hectares (Ghosh and Ponniah, 2001). Out of the 2500 species of fishes, recognized in the Indian subcontinent, 930 are categorized as freshwater species (Jayaram, 1999) and account for 9.3% of global inland fish production (Ghosh and Ponniah, 2001). However, studies on fresh- water fishes in the Indian subcontinent have been limited to scattered works on commercial fisheries and have been largely restricted to some of the major river systems. The fin fish population in many fresh water resources of the country still remains unexplored for their taxonomical de- lineation.
Taxonomy is the pioneering exploration of life on earth, which lays the foundation for the phylogenetic tree of life (Wilson, 2004). It provides requisite database for ecology and conservation science and makes access to the vast and still largely unexplored biodiversity to humanity (Wilson,
2004). For ichthyo-taxonomical studies, morphometric characters are generally being used in discriminating many fish species (Anyanwu and Ugwumba, 2003; Eyo 2002, 2003; Teugels, 1992). They are measurable features which are helpful for separating closely related genera, species and even populations within them (Cadrin, 2000).
The development of molecular techniques has received considerable attention for investigating the genetic diver- sity of the fishes. Advances in molecular techniques in- creased the availability of different DNA-based markers, which has become efficient tools in conservation genetic studies (Haig, 1998; Avise, 2004). Random amplified poly- morphic DNA (RAPD) is a simple and straightforward PCR-based technique that enabled dramatic improve- ments in genetic diversity analysis within the past decade.
This technique uses arbitrary primers for amplification of discrete regions of genome (Williams et al., 1990). RAPD markers have been used to evaluate the genetic diversity in numerous organisms and on fish populations belonging to the same family or genus (Ali et al., 2004; Cooper, 2000;
Lockley and Bardsley, 2000).
The Garra Hamilton-Buchanan genus, belonging to the Garrinae subfamily, is widely distributed in fresh water basins of the world.
Received 13 January 2011; accepted 28 February 2011
length, pelvic axillary scale length, maxillary barbel length, rostral barbel length, distance b/w pect fin and vent, dis- tance b/w pelc fin and vent and disc width distance b/w vent and anal fin were taken. They were examined using correlation matrix in PCA and cluster analysis by the PAST software.
The total genomic DNA was extracted from muscle tissue following standard Phenol-Chloroform proto- col (Sambrook et al., 1989). The quantification of DNA was done by UV spectrophotometer analysis. The quan- tity of DNA was measured by obtaining the absorbance reading at 260 nm and the purity of DNA was checked by calculating the ratio of absorbance readings at 260 nm and 280 nm. After isolation, the DNA samples were taken out and mix with 7 µl Bromophenol blue (sample loading dye) and a 15 µl of mixed DNA product was loaded in 1.5
% Agarose gel (50 ml) containing Ethidium bromide at the concentration of 20 µl per 50 ml of gel. The electro- phoresis was carried out for 1 to 2 hours at 50 volts. After electrophoresis gel was placed in the UV transilluminator and bands were visualized and were photographed in gel documentation system.
For the RAPD analysis, the DNA extracts were sub- jected to PCR amplification with primers purchased from Operon Technologies, U.S.A. Ten different primers were tested on fish samples and four better responding were selected for further studies. The RAPD profile generated by each set of primer was scored for presence or absence of an amplification product using agarose gel. The pres- ence of a band was scored as 1 and the absence of it as 0.
Cluster analysis were performed and dendrograms plotted based on pair wise genetic distance estimated using the un- weighed pair group method with arithmetic mean (UP- GMA) based on Nei (1978).
Results and discussion
Morphometric measurement values obtained for the three species of Garra are shown in the Tab. 1. Garra mul- lya and G. kalakadensis are similar regarding morpho- logical characters comparing to other congener, G. gotyla stenorhynchus. G. gotyla stenorhynchus exhibited distinct variation in the morphological character such as snout length, pre-nasal length, inter-nasal width, gap width, low- er jaw to isthmus, head depth at pupil, dorsal fin length and disc width. The principale components analysis revealed that G. mullya and G. kalakadensis are more similar species than G. gotyla stenorhynchus (Fig. 2). The cluster analysis also exhibited a similar trend with G. gotyla stenorhynchus, distinguishable from the other two Garra species, G. mul- lya and G. kalakadensis. (Fig. 3). The morphometric re- sults are insignificant to support the established genetic structure of the population that often leads to taxonomic uncertainty (Daniel, 1997; Ponnian and Gopalakrishnana, 2000; Garg et al., 2009b). The investigation was further extended to analyze the genetic variation.
According to Talwar and Jhingran (1991), this genus is represented by 21 species in the Indian subcontinent.
Among this three closely related species, Garra mullya (Sykes, 1839), Garra kalakadensis (Rema Devi, 1993) and Garra gotyla stenorhynchus (Jerdon, 1849) are abundantly present in various fresh water basin of Tamil Nadu, South India. They slightly differ in their morphological features but certain characters overlap each other. The purpose of this study was to evaluate patterns of morphological and genetic variation in the primary range of the species, by examining banding pattern in several mitochondrial and nuclear genes, using RAPD-PCR techniques.
Materials and methods
Fish samples were collected from three geographically isolated river systems of the Western Ghats (Fig. 1). The Garra kalakadensis was collected from Kalakkad Mun- dandhurai Tiger Reserve (site 1); G. mullya from upstream of Hanumannadhi, one of the sub-basins of river Tamira- parani (site 2); G. gotyla stenorhynchus from Bhavani River near Mettupalayam (site 3). Fifteen individuals of each fish species were collected and preserved in 10% formalin (i.e., 4% formaldehyde solution) for morphometric stud- ies and fresh fish samples were preserved in 90% ethanol for isolation of genomic DNA. The morphometric mea- surements viz. standard length, preanal length, predorsal length, prepelvic length, prepectoral length, preoccipital
length, snout to opercle distance, upper jaw length, snout length, prenasal length, orbit width, interorbital width, in- ternasal width, head width, gape width, peduncle length, anal fin height, head depth at nostril, head depth at pu- pil, head depth at occiput, caudal peduncle depth, caudal fin length, dorsal fin height, pectoral fin length, pelvic fin Fig. 1. The map showing various sites of fish collection for the experiment
The isolation of high quality DNA is essential for many molecular biology applications using polymerase chain re- action (Chakraborty et al., 2008). This systematics realm of methods offers new suites of characters for analyzing the relationship among the fishes (Hillis, et al., 1996; Car- valho and Pitcher, 1995). In the present study, an optimum quantity of DNA was found in all the extracts of the fish Tab. 1. The proportional values of morphometric
measurements of the three species No. Morphometric measurements G.
kalakadensis G. gotyla stenorhynchus 1 Standard length 527.245 666.54 878.455 2 Snout to urocentrum 499.593 643.08 817.7092 3 Pre-anal length 413.107 532.59 673.5358 4 Pre-dorsal length 267.896 533.235 426.8375 5 Pre-pelvic length 278.871 520.56 464.5125 6 Pre-pectoral length 117.579 158.22 204.8717 7 Pre-occipital length 129.907 152.205 205.195 8 Snout to opercle 99.325 94.5 169.2817 9 Upper jaw length 66 64.515 90.6 10 Snout length 81.392 94.56 129.0333 11 Pre-nasal length 53.368 64.845 75.79667
12 Orbit width 39.348 43.035 53.21
13 Inter orbital width 72.788 82.365 112.4533 14 Inter nasal width 47.608 56.22 78.13667 15 Head width 116.088 144.72 173.38
16 Gape width 75.2 91.44 115.1833
17 Peduncle length 74.134 82.125 134.6658 18 Anal fin height 115.392 137.235 189 19 Head depth at nostril 65.972 76.71 126.2533 20 Head depth at pupil 84.472 95.085 144.0167 21 Head depth at occiput 96.912 112.74 157.71 22 Peduncle depth 69.396 82.515 127.1275 23 Caudal fin length 169.384 194.445 329.7167 24 Dorsal fin height 141.612 162.675 241.06 25 Pectoral fin length 151.052 173.955 223.7933 26 Pelvic fin length 129.472 148.2 196.4667 27 Pelvic axillary scale length 42.984 36 82.21 28 Maxillary barbell length 25.084 15.12 28.74333 29 Rostral barbel length 36.088 36 42.70667 30 Distance b/w pect fin/vent 270.648 325.545 428.71 31 Distance b/w pelc fin/vent 111.704 124.905 156.3933 32 Disc width 61.024 57.195 70.85667 33 Distance b/w
vent/anal fin 42.708 47.055 70.88
Tab. 2. Nucleotide sequences of RAPD primers (OPA-01-10) showing amplification status with the three fish species (best performing primers are in bold)
Primer code Primer sequence (5’to 3’) Molecular Weight (Da) Amplification Polymorphism
OPA-01 CAGGCCCTTC 2985 ND ND
OPA-02 TGCCGAGCTG 3035 + ++
OPA-03 AGTCAGCCAC 2987 + ++
OPA-04 AATCGGGCTG 2964 ND ND
OPA-05 AGGGGTCTTG 3048 ND ND
OPA-06 GGTCCCTGAC 3056 + ++
OPA-07 GAAACGGGTG 3108 + ++
OPA-08 GTGACGTAGG 3012 ND ND
OPA-09 GGGTAACGCC 2978 ND ND
OPA-10 GTGATCGCAG 2992 ND ND
ND: Not Detected; +: Amplification present; ++: Polymorphic Bands Present; -: No Polymorphic Bands Present; **:Selected primers for this study
Tab. 3. The pair wise comparison of Nei’s genetic identity of three species of Garra
Species G. mullya G.
kalakadensis G. gotyla stenorhynchus
G. mullya **** 0.2369 0.3544
G. kalakadensis 0.2369 **** 0.5912
G. gotyla stenorhynchus 0.3544 0.5912 ****
-2.4 -1.6 -0.8 0.8 1.6 2.4 3.2 4
Component 1 -4
-3.2 -2.4 -1.6 -0.8 0.8 1.6 2.4 3.2
Fig. 2. The PCA scatter diagram showing the variation of the three Garra species (red for Garra mullya, green for Garra ka- lakadensis and pink for Garra gotyla stenorhynchus)
OPA-6 primers produced the maximum number of ampli- fied products. Reproducible polymorphic bands from the RAPD analysis were screened qualitatively for presence or absence in each sample. The shared RAPD fragments found in both G. mullya and G. kalakadensis with fixed frequencies were also observed in all investigated prim- ers, implying their genetically close relationships. The pair wise comparison of genetic distance of three species revealed that G. mullya are more similar, considering the G. kalakadensis index, with less genetic distance (Tab. 4).
However, G. gotyla stenorhynchus was showing higher ge- netic distances with two species, which was higher with G.
kalakadensis. A similar observation was found with cluster analysis, in which G. gotyla stenorhynchus was separated from the other two species of Garra (Fig. 5). Hence, the present investigation revealed the taxonomical relation be- tween three Garra species, which will contribute much to the least studied fresh water fishes of India.
Genetic approaches offer powerful tools for examin- ing the current status of populations, for understanding the population changes for its conservation (Belfiore and Anderson, 2001). RAPD technique is one of the most fre- quently used molecular methods for taxonomic and sys- tematic analyses of various organisms (Garg et al., 2009a;
2010). The present study evaluated morphometric charac- ters and patterns of genetic variation in three Garra spe- cies. Both the morphological and genetic analysis revealed that G. mullya and G. kalakadensis had many similar char- species with various primers. A total of 72 reliable frag-
ments were detected and observed using 10 Operon prim- ers ranging from 2600 molecular weight to 3100. Thus 4 of the better responded primers viz. OPA-02, OPA-03, OPA-06 and OPA-7 were used for further studies (Tab.
2). Each of the random primers produced distinct poly- morphic banding patterns at all of the fishes (Fig. 4). The Fig. 3. Cluster analysis of three Garra species
Fig. 4. RAPD pattern with various primers tested for the three species of Garra. Lane no. (1-4) different individuals of Garra mul- lya, Lane no. 5-8 different individuals of Garra Kalakadensis and Lane no. 9-12 different individuals of Garra gotyla stenorhynchus.
Arrows indicate the species specific markers
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acters, whereas G. gotyla stenorhynchus exhibited distinct variations. The present structure of genetic diversity is the invisible dimension of biological diversity, being the re- sult of the evolutionary history of the species exposed to natural selection pressures in variable environmental con- ditions. Natural selection at the local level is an evolution- ary force opposed to gene flow. The combination of the two forces creates a powerful mechanism for maintaining within-species diversity (Edward et al., 2002).
The present investigation revealed the morphometric and genetic variation of three Garra species. The results of morphological approach revealed that G. mullya and G.
kalakadensis are more similar in comparison to the other congener, G. gotyla stenorhynchus. The latter exhibits dis- tinct variation both in the morphological character and genetic fragments. The present investigation contribution to the knowledge on morphological and genetic variation to the Garra species. However, much specific molecular biomarkers are required for understanding the taxonomi- cal relations of many other species of this group, which are widely distributed in various fresh water basins of India.
The authors are thankful to the authorities of Anna- malai University, Manonmaniam Sundaranar University and Mohammed, Sathak College of Arts and Science for providing facilities.
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