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3. Genetic diversity and molecular declining population of four Channid species from North India and possible strategies Prof. Iqbal Parwez Department of Zoology, Aligarh Muslim University Aligarh-202002, INDIA 2nd international conference on Integrative Biology Summit, Chicago, USA (August, 04-05, 2014)

4. Piscine Biodiversity • Among vertebrates, fishes occupy remarkable status due their highest species diversity totalling to nearly 25,000 species which is 3-6 times more compared to other vertebrate groups. • India is one of the mega-biodiversity countries in the world and occupies the 9th position in terms of freshwater mega-biodiversity (Mittermeier & Mitterrneier, 1997). • In India, there are 2,500 species of fishes; of which930 live in freshwater and 1570 in sea water (Kar, 2003). • According to Conservation Assessment & Management Plan (CAMP) workshop report (1998), 329 freshwater fishes in India are under the risk of decline or extinction.

5. Decline in fish Biodiversity • According to Conservation Assessment and Management Plan (CAMP) workshop held in 1997, 327 freshwater fishes in India are under the risk of decline or extinction. • Anthropogenic factors responsible for decline of fish species • Pollution load into aquatic ecosystem • Modification/ Destruction of aquatic habitat • Construction of Dam • Diversion or reclamation of river beds for urbanization & consequent reduction of water discharge in rivers. • Consequent reduction in natural habitat area • Introduction of exotic (non-native) fish species • Over fishing • Global climatic variations • END RESULT……. • Decrease in Ichthyofaunal Diversity

6. (CAPM Workshop Report 1998) Based on IUCN Red List Categories Status of assessed freshwater fishes of India

7. Why catalogue and conserve biodiversity • Biodiversity supports livelihood and sustainability development. • To secure the IPRs related to fish biodiversity to maintain our status on our biological resvoiurs and their potential benefits.

8. The way forward……… • Outline goals: • Sustainable utilization of these resviours. • Appropriate planning of biodiversity conservation & management strategies. • Identification, listing and understanding the threat level and formulating species specific conservative plan.

9. Response to New Challenges & Development • Legislation of Biological Diversity Act 2002 (BDA 2002) • Biological Diversity Rules (2004) • National Biodiversity Action Plan (NBAP 2008) • All aimed at launching viable Plans and Programmes & Policies towards “ Biodiversity Conservation”

10. Genetic factors • Apart from anthropogenic factors, genetic factorsare also responsible for decline of fish species due to: • Loss in genetic variability • Inbreeding • Improper breeding programmes • Genetic variability is important for survival of species because species having more genetic variation will be less susceptible to adverse environmental conditions.

11. Existing Molecular Methods for Genotyping • Even though there are several approaches to do genotyping such as • RFLP • VNTR • AFLP • SNP • RAPD • RAPD appears most suitable because of its • Simple and Cost effectiveness • Versatility • Does not require prior sequence information • Does not need the radioactivity

12. Different categories of molecular markers Protein-based DNA-based (DNA source – mitochondria/nucleus) PCR-based Non-PCR-based Allozyme mtDNA RAPD VNTR RFLP AFLP SNP Inheritance pattern Prior sequence information required? Applications Mendelian codominant Yes Species identification, linkage mapping, population studies, Maternal inheritance No* Maternal lineage Mendelian codominant Yes Paternity analysis, linkage mapping, population studies Mendelian codominant Yes Linkage mapping, population studies, disease diagnosis Mendelian dominant No Species and hybrid identification, fingerprinting for population studies Mendelian dominant No Population studies, linkage mapping Mendelian codominant Yes Linkage mapping, population studies *Conserved PCR primer sequence information can be adopted from a related species. Schematic division of various categories of molecular markers and their essential features.

13. Status of different molecular markers for study of Fish population genetics • Globally about 100 fish species from 30 families were genetically analysed by RAPD method. • Indian Scenario • 48 fish species studied by RAPD markers • 37 species by Allozymemarker • 20 species byRFLP/ mtDNA/ VNTR Assay

14. Great diversity of Channaspecies • Snakehead fishes of Channidae family, endemic to freshwater, are distributed only in Asian and African countries. They are represented by 30 species of which 27 are confined to Asian countries (Amback et al., 2006). • In the Indian subcontinent alone, there are 12 different species of this group and 04 of them are ubiquitously distributed across the country. • These fishes are considered highly relished food fishes, fetch good market price and are regarded as consumer’s choice. • Scientifically, they provide an interesting target for phylogenetic analysis due to the existence of several species and availability of multi species of this genus in a particular area. • Despite great economic importance and rich species diversity of these fishes, most of the Channa species are showing declining trend and hence included in the comprehensive list of different categories of declining fresh water fishes prepared at CAMP workshop (1998).

15. Distribution of Channa species in India C. striatus India: Ubiquitous Other Places: Southern China and Thailand 1 2 C. punctatus India: Ubiquitous Other Places: Indian Sub-continent, China 3 C. marulius India: Ubiquitous Other Places: Bangladesh, China, Thailand and Cambodia, Sri Lanka and Pakistan C. gachua India: Ubiquitous Other Places: Indian Sub-continent 4

16. 5 C. amphibius India: North Bengal Other Places: Bhutan C. barca India: West Bengal,Brahmaputra river in Assam, Ganges- Other Places: Bhutan 6 7 C. orientalis India: India Other Places: Afghanistan, Iran, Pakistan, Nepal, Sri Lanka, Bangladesh, Burma C. aurantimaculata India: Brahmaputra River basin, northern Assam, Other Places: - 8

17. 9 C. bleheri India: The Brahmaputra River basin at northern Assam, Other Places: - 10 C. micropeltes India: The Brahmaputra River basin at northern Assam, Kerala Other Places: Malaysia, Thailand, Vietnam C. stewartii India: Eastern Himalaya Inhabits both running and standing waters Other Places: Nepal 11 12 C. Diplogramma India:Western Ghats, Kerala Other Places: -

18. C. punctatus C. marulius C. striatus C. gachua Four different species of genus Channa

19. Heteropneustes fossilis Clarias batrachus Clarias gariepinus Three catfish species

20. Focal Theme of the study • Out of the 7 selected species in the present study, 6 species were placed into different declining categorized according to IUCN criteria (International Union for Conservation of Nature) in Conservation Assessment and management Plan (CAMP, 1997) workshop. Hence, the focal theme of this study were: • 1: To identify species specific DNA profiles and bands, if any, from the RAPD fingerprints. • 2: To evaluate comparative densitometric analysis of RAPD band profile for additional assistance in species identification. • 3: To establish inter-specific phylogenetic relationship among the four species of the genus Channa and intra-specific phylogenetic analysis among all seven species using the neighbor-joining method. • Continued……….

21. continued………. ‘Focal Theme of the study’ • 4: To quantify gene diversity of the population of investigated fish species by RAPD fingerprinting. • 5: To assess the Genetic Similarity (SI) and Genetic Distance (GD) of the population of investigated fish species. • 6: To estimate polymorphic band contents (PIC) to assess its feasibility for use in population genetics. • 7: To identify sex-specific RAPD markers, if any.

22. Primers yielding clear and reproducible DNA fingerprinting • Total 22 primers were initially screened (Operon technologies) • OPA Kit (20 primers) • OPB Kit (02 primers)

23. Significance of generated RAPD band profiles • There are three main applications of generated RAPD band profiles. • Species identification at molecular level. • Phylogenetic relationship among the four species of Channa. • Assessment of genetic variations within the genome of investigated fish species.

24. CpCgh CmCs Kb 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 0.3 0.2 M 1 2 3 4 5 6 7 8 9 10 11 M 1 2 3 4 5 6 7 8 9 10 11 12 13 OPA12 RAPD Profiling of four species of Channa amplified by OPA 4 (Cp= C. punctatus, Cgh = C. gachua, Cm = C. marulius and Cs = C. striatus)

25. Kb 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 0.3 0.2 M 1 2 3 4 5 6 7 8 9 10 11 12 13 CpCgh CmCs RAPD Profiling of four species of Channa amplified by OPA19 (Cp= C. punctatus, Cgh = C. gachua, Cm = C. marulius and Cs = C. striatus)

26. H. fossilis C. batrachus C. gariepinus M 1 2 3 4 5 6 7 8 9 10 11 12 N M 1 2 3 4 5 6 7 8 9 10 11 N 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 0.3 0.2 RAPD bands profile of catfish species obtained by primer OPA1

27. H. fossilis C. gariepinus C. batrachus M 1 2 3 4 5 6 7 8 9 10 11 12 N M 1 2 3 4 5 6 7 8 9 10 11 12 N 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 0.3 0.2 RAPD bands profile of catfish species obtained by primer OPA19

28. Kb 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 0.3 0.2 M 1 2 3 4 5 6 7 8 9 10 11 12 13 CpCgh Cm Cs OPA 12 RAPD Profiling of four species of Channa amplified by OPA12 (Cp= C. punctatus, Cgh = C. gachua, Cm = C. marulius and Cs = C. striatus)

29. Cgh Cgh Cm Cp Cs M 1 2 3 4 5 6 7 8 9 10 11 12 N KbM 1 2 3 4 5 6 7 8 9 10 11 12 13 10.0 7.0 5.0 3.0 2.0 1.5 1.0 .7 .5 .3 .2 RAPD bands profile of Channagachua obtained by primer OPA12

30. Cm Cgh Cm Cp Cs KbM 1 2 3 4 5 6 7 8 9 10 11 12 13 M 1 2 3 4 5 6 7 8 9 10 11 12 N 10.0 7.0 5.0 3.0 2.0 1.5 1.0 .7 .5 .3 .2 RAPD bands profile of Channamarulius obtained by primer OPA12

31. Cs Cgh Cm Cp Cs M 1 2 3 4 5 6 7 8 9 10 11 12 N KbM 1 2 3 4 5 6 7 8 9 10 11 12 13 10.0 7.0 5.0 3.0 2.0 1.5 1.0 .7 .5 .3 .2 RAPD bands profile of Channa striatus obtained by primer OPA12

32. Cp Cp Cgh Cm Cs M 1 2 3 4 5 6 7 8 9 10 11 12 N M 1 2 3 4 5 6 7 8 9 10 11 12 N Kb 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 0.3 0.2 RAPD bands profile of Channa punctatus obtained by primer OPA7

33. Kb 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 0.3 0.2 M 1 2 3 4 5 6 7 8 9 10 11 12 13 CpCgh CmCs RAPD Profiling of four species of Channa amplified by OPA 20 (Cp= C. punctatus, Cgh = C. gachua, Cm = C. marulius and Cs = C. striatus)

34. H. fossilis C. gariepinus C. batrachus M 1 2 3 4 5 6 7 8 9 10 11 12 N M 1 2 3 4 5 6 7 8 9 10 11 12 N 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 0.3 0.2 RAPD bands profile of catfish species obtained by primer OPA19

35. C. gariepinus C. batrachus M 1 2 3 4 5 6 7 8 9 10 11 12 N 10.0 7.0 5.0 3.0 2.0 1.5 1.0 0.7 0.5 OPB12 0.3 0.2 RAPD bands profile of catfish species obtained by primer OPB12

36. Profile height Profile height 1 160 160 4 2 120 120 2 80 80 3 3 1 40 40 4 0 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Cm Cs Peaks of RAPD band profiles of C. marulius & C. striatus showing relative quantitative PCR products with OPA12

37. C. marulius 0.495 C. gachua 0.505 C. punctatus 0.179 0.102 C. striatus 0.321 Cluster Analysis showing genetic relatedness of four different Channa species with OPA4

38. C. marulius 0.550 C. gachua 0.450 C. punctatus 0.236 0.164 C. striatus 0.336 Cluster Analysis showing genetic relatedness of four different Channa species with OPA7

39. C. marulius 0.490 C. gachua 0.510 C. punctatus 0.223 0.066 C. striatus 0.243 Cluster Analysis showing genetic relatedness of four different Channa species with OPA19

40. C. gachua C. punctatus C. striatus C. marulius Polymorphic band content of Channa species obtained with different primers

41. Genetic analyses values of four Channa species. C. punctatus C. gachua C. marulius C. striatus GS = Genetic similarity; GD = Genetic distance; I = Shannon’s information index; H = Gene diversity; PC% = Polymorphic band content

42. C. batrachus C. gariepinus H. fossilis Polymorphic band content of catfish species obtained with different primers

43. Genetic analyses values of three catfish species. C. batrachus C. gariepinus H. fossilis GS = Genetic similarity; GD = Genetic distance; I = Shannon’s information index; H = Gene diversity; PC% = Polymorphic band content

44. CONCLUSIONS The present study has clearly established the molecular basis of the identification of all seven investigated fish species. Among all the primers OPA12 & OPA 7 is nearly regarded most suitable primers for Channa species, primer OPA8 for all catfish species and OPA11 and OPB12 for two Clarias species. C. gariepinusshows the highest polymorphism and hence regarded as very hardy whereasC. batrachusshows great degree of vulnerability. Among the Channid group. C. punctatusappears hardy and C. maruliusshows highest degree of susceptibility. The other two species i.e., C. gachuaandC. striatusfall in between the two. 5. Based on molecular phylogeny C. punctatus and C. striatus are in one cluster hence close to each other compared to C. marulius and C. gachuawhich shows relatedness between themselves hence put in another cluster. 6. This study has generated highly significant baseline data on the economically important food fishes of India and can be utilized in the effective management of successful propagation of these species by fishery biologists.

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