GENES & GENOMICS 30 (3) : 215-222 (June 2008) ©The Genetics Society of Korea Genetic Variation and Divergence in Two Critically Endangered Mahseer Species Tor tor and Tor putitora (Pisces : Cypriniformes) Assessed by RAPD Markers Debashish Ghosh and Md. Samsul Alam* Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh Received January 17, 2008; accepted April 7, 2008 ABSTRACT Tor tor and Tor putitora are two important cyprinid species distributed in many countries of the South and South East Asia. The species are critically endangered in Bangladesh. Therefore information on genetic structure of the species would be useful to take management plan for conservation and stock enhancement of these two species. Random Amplified Polymorphic DNA (RAPD) markers were used to assess the genetic variation in these species. Upon screening of 27 random decamer primers six primers giving consistent banding patterns with good resolution were selected for the analysis of twelve fish of each species. The primers generated 44 loci of which 41 were polymorphic. The frequency of polymorphic loci was 86.36% in T. tor and 31.83% in T. putitora. The band-sharing based genetic similarity of T. tor was lower than that in T. putitora. The gene diversity in T. tor was 0.270 while that in T. putitora was 0.106. The genetic identity and genetic distance values between the two species were 0.863 and 0.147 respectively. The population differentiation value (PhiPT) between the two species was highly significant (P <0.001). No species diagnostic bands were detected in either of the species though fifteen private bands, 13 for T. tor and two for T. putitora were obtained. The present study detected higher levels of genetic variability in T. tor compared to T. putitora. Key words: genetic variation, mahseer, RAPD, Tor tor, Tor putitora. INTRODUCTION China (Rahman, 1989; Menon, 1992; Roberts, 1999; Chen and Yang, 2004). Mahseers are commercially Mahseer, a group of medium to large sized fishes important game fish as well as highly esteemed table of the family Cyprinidae are distributed in many fish fetch a high market price and are potential South and South East Asian countries including candidate for aquaculture (Ogale, 2002). Two mahseer Indonesia, Java, Malaysia, Laos, Myanmar, India, species T. tor and T. putitora are found in the hilly Bangladesh, Nepal, Pakistan, Afganistan and South streams of Sylhet, Mymensingh, Netrokona, Dinajpur and Kaptai reservoir of Chittagong Hill Tracts in *To whom correspondence should be addressed. Bangladesh (Rahman, 1989) but at present, their Email: [email protected]. availability is limited mostly to the Kangsha and 216 GENES & GENOMICS (2008) 30 : 215-222 Someshwari River in Netrokona district. These species diversity of two indigenous mahseer species, Tor inhabit rivers and lakes, but grow better in rivers tambroides and T. douronensis (Cyprinidae) cultured with a rocky bottom. They travel toward headwaters in Sarawak, Malaysia. Very recently, Mohindra et al. at the start of the rainy season and downstream at (2007) discriminated five mahseer species from the end of the rainy season. T. tor is considered as Indian peninsula using RAPD markers. the most beautiful of all endemic fish of Bangladesh. The Randomly Amplified Polymorhic DNA (RAPD) T. putitora is important as a principal game as well fingerprinting technique allows detection of DNA as food fish and is reported to grow up to 54 kg polymorphisms by randomly amplifying multiple (Froese and Pauly, 2003). Mahseer can be used in regions of the genome through PCR using single polyculture, cage culture and for river ranching without arbitrary primers designed independent to target any negative effect on mrigal (Cirrhinus mrigala ) and DNA sequence (Williams et al., 1990; Welsh and the common carp (Tripathi, 1995 cited by Ogale, 2002). McClelland, 1990). RAPD fingerprinting technique is The abundance of mahseers fish has been declining simple, fast, sensitive and allows the examination of in the natural distribution ranges. T. tor and T. genomic variation without prior knowledge of DNA putitora have been declared as critically endangered sequences (Williams et al., 1990; Welsh and McClelland, species in Bangladesh (Ameen et al., 2000; Hussain 1990). The present study was the first attempt to reveal and Mazid, 2001). The major possible factors attributed nuclear DNA level variation within and between the for the depletion of mahseer stocks include degradation two critically endangered cyprinid species T. tor and of aquatic ecological conditions, indiscriminate fishing T. putitora in Bangladesh using RAPD markers. of broodstock and juveniles, pollution and population pressures on resources. The declining trend in the MATERIALS AND METHODS populations of mahseers needs immediate attention for their in situ conservation and rejuvenation in Sample collection and DNA extraction natural waters by protecting their habitats as well as Twelve specimens of T. tor were collected from two through propagation-assisted rehabilitation strategies. amateur fish farmers who stocked the fingerlings A contingent of T. putitora was introduced in Bangladesh collected from the Swamesshari and the Kangsha from Nepal in 1991 for stock enhancement (Hussain River under Netrokona district in July-August, 2005. and Mazid, 2001). Artificial breeding technique of T. Even after repeated attempts, no more samples of putitora has been established but culture of this this species could be collected from the fishermen species has not been disseminated throughout the indicating the scarcity of this fish. T. putitora fingerlings country due to unavailability of fish seed. On the produced through induced breeding were collected other hand, however, the artificial breeding technique from the Freshwater Station of Bangladesh Fisheries of T. tor could not be established yet. Research Institute (BFRI), Mymensingh and reared Information on stock structure and genetic variation in the ponds for two months before being used for is prerequisite of any species requiring conservation collection of fin samples. and stock improvement. Identification of polymorphic In order to carry out the RAPD analysis, approx- markers with consistent scorable alleles is a crucial imately 250 mg of fin tissue was clipped from each of step to generating population genetic data (Ferguson the 24 fish anesthetized with 2-Phenoxy ethanol using et al., 1995). There is very limited information on any scissors and preserved in 95% ethanol in separate class of genetic markers or stock structure in T. tor microfuge tube. The samples were kept in -20ﾟC until and T. putitora. Mohindra et al. (2004) examined extraction of genomic DNA. Genomic DNA was prepared amplification of Cyprinus carpio, Catla catla and from the fin tissue following sodium dodecyl sulfate Barbus barbus microsatellite markers in T. putitora (SDS)-Proteinase-K digestion, phenol: chloroform: and reported the genetic variability in samples isoamyl alcohol (25:24:1) extraction and alcohol collected from three rivers and one lake in India. precipitation method as described by Islam and Alam Nguyen et al. (2006) studied the mitochondrial DNA (2004). .