4. Results. 34 P a g e

Transcription

1 4. Results 4.1 Morphological classification In this study a total of 630 specimens were collected and were identified to 91 species, 67 genera, 39 families and 12 orders based on morphological taxonomy as mentioned in table Order Family Individuals Studied Osteoglossiformes Notopteridae 2 Elopiformes Megalopidae 2 Anguilliformes Anguillidae 7 Clupeiformes Clupeidae 1 Engraulidae 2 Pristigasteridae 1 Gonorynchiformes Chanidae 1 Cypriniformes Cobitidae 5 Cyprinidae 85 Siluriformes Ariidae 4 Bagridae 14 Clariidae 4 Heteropneustidae 4 Pangasiidae 2 Schilbeidae 2 Siluridae 11 Sisoridae 1 Beloniformes Belonidae 2 Synbranchiformes Mastacembelidae 8 Perciformes Ambassidae 1 Anabantidae 2 Carangidae 3 Channidae 5 Cichlidae 5 Gerreidae 2 Gobiidae 5 Haemulidae 1 Lutjaninae 2 Mugilidae 2 Mullidae 2 Nandidae 1 Sciaenidae 4 Sillaginidae 2 Sphyraenidae 1 Teraponidae 1 Pleuronectiformes Cynoglossidae 1 Paralichthyidae 1 Scorpaeniformes Platiycephalidae 1 Table 4.1.1: Family wise number of Individuals Studied. 34 P a g e

2 4.2 DNA processing results Identified fishes were then digitally photographed for digital records (for illustrative images see appendix-i). A complete database was then uploaded to Barcode of Life Data System. The tissue subsamples were then used for DNA isolation. The quality of Isolated DNA was checked on agarose gel as shown in Figure Figure 4.2.1: Quality check for Genomic DNA (1% agarose gel) All DNA samples were then screened for amplification of COI gene with the primer pair FF2d (forward) and FR1d (reverse) (Ivanova et al., 2007) as shown in Figure Figure PCR amplified product The samples were then sequences and the sequences obtained were then compared in SeqScape software v2.7 with the standard reference file for possible NUMTs, stop codon. Every single base of each of the sequence aligned was inspected in the software carefully the subsequent contig assemblies generated using them had no stop codons. 4.3 COI gene based studies A total of 201 specimens were studies for their cytochrome oxidase 1 gene based species identification. The details of species employed for molecular studies are depicted in table 2. COI amplicons were recovered from all 201 individuals and there was no stop codon or indels observed which might be the signal for amplification of NUMT. Average sequence length of >500 bp is obtained. The sequences were deposited to BOLD and NCBI with all supportive data. 35 P a g e

3 Table Details of fishes studies for their molecular taxonomy using DNA barcoding Code Order Family Name of fish BESH08 Cypriniformes Cyprinidae Amblypharyngodon mola GF679 Cypriniformes Cyprinidae Amblypharyngodon mola PSSH02 Cypriniformes Cyprinidae Amblypharyngodon mola BESH02 Cypriniformes Cyprinidae Amblypharyngodon mola GF675 Cypriniformes Cyprinidae Amblypharyngodon mola PRUM01 Cypriniformes Cyprinidae Amblypharyngodon mola COYN01 Perciformes Anabantidae Anabas testudineus ABRJ01 Perciformes Anabantidae Anabas testudineus AGIS01 Anguilliformes Anguillidae Anguilla bengalensis bengalensis AGMJ02 Anguilliformes Anguillidae Anguilla bengalensis bengalensis AGIS02 Anguilliformes Anguillidae Anguilla bengalensis bengalensis AGIS03 Anguilliformes Anguillidae Anguilla bengalensis bengalensis AGRJ01 Anguilliformes Anguillidae Anguilla bengalensis bengalensis AGMJ01 Anguilliformes Anguillidae Anguilla bengalensis bengalensis AGPW02 Anguilliformes Anguillidae Anguilla bengalensis bengalensis ANPA04 Cypriniformes Cyprinidae Hypophthalmichthys nobilis 33V Cypriniformes Cyprinidae Hypophthalmichthys nobilis HMPA01 Cypriniformes Cyprinidae Hypophthalmichthys nobilis HHRJ01 Siluriformes Ariidae Arius arius GF783 Siluriformes Ariidae Arius arius RSYN03 Siluriformes Ariidae Arius arius RSYN01 Siluriformes Ariidae Arius arius MSNW01 Siluriformes Bagridae Batasio tengra 13V Perciformes Carangidae Caranx hippos CCND01 Cypriniformes Cyprinidae Catla catla CCSH07 Cypriniformes Cyprinidae Catla catla CCGA14 Cypriniformes Cyprinidae Catla catla CCND11 Cypriniformes Cyprinidae Catla catla CNGA05 Perciformes Ambassidae Chanda nama MUND01 Perciformes Channidae Channa marulia MUND02 Perciformes Channidae Channa marulia CSSH02 Perciformes Channidae Channa punctate COPW02 Perciformes Channidae Channa punctate Code Order Family Name of fish COPA10 Perciformes Channidae Channa punctate CZYN01 Gonorynchiformes Chanidae Chanos chanos LZYN01 Mugiliformes Mugilidae Chelon parsia BEGA02 Siluriformes Clariidae Clarias gariepinus CLGA01 Siluriformes Clariidae Clarias gariepinus GF722 Siluriformes Schilbeidae Clupisoma prateri 4V Clupeiformes Engraulidae Coilia ramacarti 7V Pleuronectiformes Cynoglossidae Cynoglossus elongetus CYBD01 Cypriniformes Cyprinidae Cyprinus carpio CYPA13 Cypriniformes Cyprinidae Cyprinus carpio CYRJ01 Cypriniformes Cyprinidae Cyprinus carpio DVKW02 Cypriniformes Cyprinidae Danio devario DVKW01 Cypriniformes Cyprinidae Danio devario CEGA03 Cypriniformes Cyprinidae Danio devario CEGA02 Cypriniformes Cyprinidae Danio devario 31V Perciformes Sciaenidae Dendrophysa russelii GF789 Perciformes Sciaenidae Dendrophysa russelii CAKA04 Cypriniformes Cyprinidae Devario cf. devario NDKA01 Cypriniformes Cyprinidae Devario cf. devario ESND02 Perciformes Cichlidae Etroplus suratensis ESND03 Perciformes Cichlidae Etroplus suratensis 42V Siluriformes Sichilbeidae Eutropiicthys goongawarii PAND01 Cypriniformes Cyprinidae Garra mullya CSKA02 Cypriniformes Cyprinidae Garra mullya GGGA01 Cypriniformes Cyprinidae Garra mullya GMND02 Cypriniformes Cyprinidae Garra mullya GF767 Perciformes Gerreidae Gerres sinurus GF765 Perciformes Gerreidae Gerres sinurus GGNW01 Perciformes Gobidae Glossogobius giuris giuris GGND02 Perciformes Gobidae Glossogobius giuris giuris GGRJ01 Perciformes Gobidae Glossogobius giuris giuris GGPW03 Perciformes Gobidae Glossogobius giuris giuris GHPW02 Siluriformes Sisoridae Glyptothorax lonah 36 P a g e

4 Code Order Family Name of fish HFPA01 Siluriformes Heteropneustidae Heteropneustes fossilis HFNW01 Siluriformes Heteropneustidae Heteropneustes fossilis HFPA03 Siluriformes Heteropneustidae Heteropneustes fossilis POND06 Cypriniformes Cyprinidae Labeo bata LBHN01 Cypriniformes Cyprinidae Labeo boga LBYN02 Cypriniformes Cyprinidae Labeo calbasu LCND03 Cypriniformes Cyprinidae Labeo calbasu LCND02 Cypriniformes Cyprinidae Labeo calbasu LBYN01 Cypriniformes Cyprinidae Labeo calbasu LCBS03 Cypriniformes Cyprinidae Labeo calbasu LBKA01 Cypriniformes Cyprinidae Labeo calbasu LBYN03 Cypriniformes Cyprinidae Labeo calbasu LRND03 Cypriniformes Cyprinidae Labeo rohita LRPO04 Cypriniformes Cyprinidae Labeo rohita LRPO01 Cypriniformes Cyprinidae Labeo rohita GF685 Cypriniformes Cyprinidae Labeo rohita GF686 Cypriniformes Cyprinidae Labeo rohita LRSH11 Cypriniformes Cyprinidae Labeo rohita NBGA04 Cypriniformes Cobitidae Lepidocephalichthys guntea NBGA06 Cypriniformes Cobitidae Lepidocephalichthys guntea OSKA01 Cypriniformes Cobitidae Lepidocephalichthys guntea NBKA02 Cypriniformes Cobitidae Lepidocephalichthys guntea GF771 Perciformes Lutjaninae Lutjanus johnii GF769 Perciformes Lutjaninae Lutjanus johnii GF720 Synbranchiformes Mastacembelidae Macrognathus aculeate GF721 Synbranchiformes Mastacembelidae Macrognathus aculeate MQRJ01 Synbranchiformes Mastacembelidae Macrognathus aculeate MCYN06 Perciformes Sciaenidae Macrospinosa cuja MCYN07 Perciformes Sciaenidae Macrospinosa cuja MPPA06 Synbranchiformes Mastacembelidae Macrognathus pancalus MABS02 Synbranchiformes Mastacembelidae Macrognathus pancalus MPPA05 Synbranchiformes Mastacembelidae Macrognathus pancalus MAUM01 Synbranchiformes Mastacembelidae Macrognathus pancalus MPBS01 Synbranchiformes Mastacembelidae Macrognathus pancalus GF723 Elopiformes Megalopidae Megalops cordyla 6V Elopiformes Megalopidae Megalops cundigs Code Order Family Name of fish 10V Elopiformes Megalopidae Megalops cyprinoides MNND02 Siluriformes Bagridae Mystus bleekeri MNSH02 Siluriformes Bagridae Mystus bleekeri MTGD01 Siluriformes Bagridae Mystus vittatus NDRJ01 Perciformes Nandidae Nandus nandus CHPA01 Osteoglossiformes Notopteridae Notopteus notopterus CLPA05 Osteoglossiformes Notopteridae Notopteus notopterus OBND03 Siluriformes Siluridae Ompok bimaculatus OBND01 Siluriformes Siluridae Ompok bimaculatus OBND07 Siluriformes Siluridae Ompok bimaculatus WAPU01 Siluriformes Siluridae Ompok bimaculatus OPKW01 Siluriformes Siluridae Ompok bimaculatus WAPU02 Siluriformes Siluridae Ompok bimaculatus OKPA01 Siluriformes Siluridae Ompok bimaculatus OPKA03 Siluriformes Siluridae Ompok pabda GF715 Siluriformes Siluridae Ompok pabda OMND02 Perciformes Cichlidae Oreochromis mossambicus OMGD02 Perciformes Cichlidae Oreochromis mossambicus OMGD01 Perciformes Cichlidae Oreochromis niloticus WAPA01 Siluriformes Pangasiidae Pangasius hypophthalmus PPYN02 Siluriformes Pangasiidae Pangasius pangasius GF775 Clupeiformes Pristigasteridae Pellona ditchela GF797 Scorpaeniformes Platiycephalidae Platycephalus cf. indicus GF751 Perciformes Haemulidae Pomadasys kaakan 1V Perciformes Gobiidae Pseudapocryptes lanceolatus GF793 Pleuronectiformes Paralichthyidae Pseudorhombus arsius PSGA01 Cypriniformes Cyprinidae Crossocheilus latius GF656 Cypriniformes Cyprinidae Puntius conchonius GF729 Cypriniformes Cyprinidae Puntius conchonius GF665 Cypriniformes Cyprinidae Puntius conchonius PSHN03 Cypriniformes Cyprinidae Puntius fraceri PUGD01 Cypriniformes Cyprinidae Puntius sarana PDND01 Cypriniformes Cyprinidae Puntius sarana PSPA01 Cypriniformes Cyprinidae Puntius sarana 44V Cypriniformes Cyprinidae Puntius sarana PSPA01 Cypriniformes Cyprinidae Puntius sarana 37 P a g e

5 Code Order Family Name of fish PSSH01 Cypriniformes Cyprinidae Puntius sophore PSHN01 Cypriniformes Cyprinidae Puntius sophore PSNW01 Cypriniformes Cyprinidae Puntius sophore BSGD01 Cypriniformes Cyprinidae Puntius sophore RSKG01 Cypriniformes Cyprinidae Puntius conchonius GF668 Cypriniformes Cyprinidae Puntius ticto RDGA03 Cypriniformes Cyprinidae Rasbora daniconius RDGA01 Cypriniformes Cyprinidae Rasbora daniconius RLKW01 Cypriniformes Cyprinidae Rasbora labiosa PMUM01 Cypriniformes Cyprinidae Rasbora rasbora RRND01 Siluriformes Bagridae Rita pavimentatus SAPW03 Siluriformes Bagridae Rita pavimentatus RRND02 Siluriformes Bagridae Rita pavimentatus MAGD01 Siluriformes Bagridae Rita pavimentatus RRND03 Siluriformes Bagridae Rita pavimentatus SNPA14 Cypriniformes Cyprinidae Salmophasia boopis SNND02 Cypriniformes Cyprinidae Salmophasia novacula SNND01 Cypriniformes Cyprinidae Salmophasia novacula SNND12 Cypriniformes Cyprinidae Salmophasia novacula SNGA01 Cypriniformes Cyprinidae Salmophasia novacula SHRJ04 Cypriniformes Cyprinidae Salmostoma bacaila SHRN05 Cypriniformes Cyprinidae Salmostoma bacaila SCYN09 Mugiliformes Mugilidae Sicamugil cascasia GF779 Perciformes Sillaginidae Sillago sihama GF781 Perciformes Sillaginidae Sillago vincenti ASND01 Siluriformes Bagridae Sperata aor MYRJ02 Siluriformes Bagridae Sperata seenghala SYYN01 Perciformes Sphyraenidae Sphyraena acutipinnis STYN02 Beloniformes Belonidae Strongylura strongylura GF741 Perciformes Teraponidae Terapon jarbua 37V Clupeiformes Engraulidae Thyrssa gautmensis TSPA01 Cypriniformes Cyprinidae Thynnichthys sandkhol PGND01 Cypriniformes Cyprinidae Thynnichthys sandkhol CYNW01 Cypriniformes Cyprinidae Thynnichthys sandkhol TSPA03 Cypriniformes Cyprinidae Thynnichthys sandkhol APHN01 Cypriniformes Cyprinidae Thynnichthys sandkhol PEGA03 Cypriniformes Cyprinidae Tor tor Code Order Family Name of fish BBGA02 Cypriniformes Cyprinidae Tor tor 45V Cypriniformes Cyprinidae Tor tor SXPW01 Cypriniformes Cyprinidae Tor tor GF755 Perciformes Mullidae Upeneus sulphureus WAPA04 Siluriformes Siluridae Wallago attu WAPA03 Siluriformes Siluridae Wallago attu GF683 Beloniformes Belonidae Xenentodon cancila 38 P a g e

6 All together 201 COI sequences having good and reliable quality were having length of >500bp were included for analysis. The sequences were deposited to BOLD having unique BOLD ID and NCBI accession numbers as shown in Table GC content Only three (1.9 %) sequences out of 201 samples analysed were failed to yield DNA barcode. The overall GC content found to be 44.64% which is higher at 1 st codon position (55.97%) and lowest at codon position 3 rd (35.04%) (Table 4.3.2), (Figure 4.3.1) Table 4.3.2: GC Percentage of the COI sequences under study (201 sequences) Min Mean Max SE G % C % A % T % GC % GC % Codon Pos GC % Codon Pos GC % Codon Pos We have observed slightly higher GC content in some selected carnivores taxa versus herbivorous taxa (44.49% versus 44.34%) suggest the possibility of higher transition rate in herbivores mostly Cyprinids (table and figure 3). A. Piscivorus fishes (total sequences 47) Min Mean Max SE G % C % A % T % GC % GC % Codon Pos GC % Codon Pos GC % Codon Pos P a g e

7 B. Cyprinids (obligate herbivores) (Number of Sequences : 84) Min Mean Max SE G % C % A % T % GC % GC % Codon Pos GC % Codon Pos GC % Codon Pos Table 4.3.3: Comparative GC percentage A B Figure 4.3.1: Percept GC content comparison in Carnivores (A) and Herbivores fishes (B) Transition/transversion bias The nucleotide composition, transition sites and transversion sites were mentioned in table 5. The transition of G to A and C to T is 15.85% whereas T to C and G to C to T is 4.57 % with average GC content of 44.77% From\To A T C G A - T C G Table: 4.3.4: MCL transition/ transversion bias. Some species as mentioned in table were difficult in their taxonomic identifications due to morphological similarities where DNA barcode data proved to be very useful in their identifications as depicted in table P a g e

8 Table 4.3.5: NCBI accession numbers Code Name of organism BOLD id NCBI accession BESH08 Amblypharyngodon GDK JX GF679 Amblypharyngodon mola GDK JX PSSH02 Amblypharyngodon mola GDK JX BESH02 Amblypharyngodon mola GDK JX GF675 Amblypharyngodon mola GDK JX PRUM01 Amblypharyngodon mola GDK JX COYN01 Anabas testudineus GDK JX ABRJ01 Anabas testudineus GDK JX AGIS01 Anguilla bengalensis bengalensis GDK JX AGMJ02 Anguilla bengalensis bengalensis GDK JX AGIS02 Anguilla bengalensis bengalensis GDK JX AGIS03 Anguilla bengalensis bengalensis GDK JX AGRJ01 Anguilla bengalensis bengalensis GDK JX AGMJ01 Anguilla bengalensis bengalensis GDK JX AGPW02 Anguilla bengalensis bengalensis GDK JX ANPA04 Hypophthalmichthys nobilis GDK JX V Hypophthalmichthys nobilis GDK JX HMPA01 Hypophthalmichthys nobilis GDK JX HHRJ01 Arius arius GDK JX GF783 Arius arius GDK JX RSYN03 Arius arius GDK JX RSYN01 Arius arius GDK JX MSNW01 Batasio tengra GDK JX V Caranx hippos GDK JX CCND01 Catla catla GDK JX CCSH07 Catla catla GDK JX CCGA14 Catla catla GDK JX CCND11 Catla catla GDK JX CNGA05 Chanda nama GDK JX MUND01 Channa marulia GDK JX MUND02 Channa marulia GDK JX CSSH02 Channa punctate GDK JX COPW02 Channa punctate GDK JX Code Name of organism BOLD id NCBI accession COPA10 Channa punctate GDK JX CZYN01 Chanos chanos GDK JX SQRJ01 Cirrhina reba GDK JX NBGA05 Lepidocephalichthys guntea GDK CTGA03 Cirrhinus cirrhosis GDK JX BEGA03 Clarias batrachus GDK JX BEGA01 Clarias batrachus GDK JX CLGA01 Clarias gariepinus GDK JX BEGA02 Clarias gariepinus GDK JX GF722 Clupisoma prateri GDK JX V Coilia ramacarti GDK JX V Cynoglossus elongetus GDK JX CYBD01 Cyprinus carpio GDK JX CYPA13 Cyprinus carpio GDK JX CYRJ01 Cyprinus carpio GDK JX DVKW02 Danio devario GDK JX DVKW01 Danio devario GDK JX CEGA03 Danio devario GDK JX CEGA02 Danio devario GDK JX V Dendrophysa russelii GDK JX GF789 Dendrophysa russelii GDK JX CAKA04 Devario cf. devario GDK JX NDKA01 Devario cf. devario GDK JX ESND02 Etroplus suratensis GDK JX ESND03 Etroplus suratensis GDK JX V Eutropiicthys goongawarii GDK JX PAND01 Garra mullya GDK JX CSKA02 Garra mullya GDK JX GGGA01 Garra mullya GDK JX GMND02 Garra mullya GDK JX GF767 Gerres sinurus GDK JX GF765 Gerres sinurus GDK JX GGNW01 Glossogobius giuris giuris GDK JX P a g e

9 Code Name of organism BOLD id NCBI accession GGRJ01 Glossogobius giuris giuris GDK JX GGPW03 Glossogobius giuris giuris GDK JX GHPW02 Glyptothorax lonah GDK JX HFPA01 Heteropneustes fossilis GDK JX HFNW01 Heteropneustes fossilis GDK JX HFPA03 Heteropneustes fossilis GDK JX HFPA02 Heteropneustes fossilis GDK JX V Hilsa ilisa GDK JX POND04 Labeo bata GDK JX POND06 Labeo bata GDK JX LBHN01 Labeo boga GDK JX LBYN02 Labeo calbasu GDK JX LCND03 Labeo calbasu GDK JX LCND02 Labeo calbasu GDK JX LBYN01 Labeo calbasu GDK JX LCBS03 Labeo calbasu GDK JX LBKA01 Labeo calbasu GDK JX LBYN03 Labeo calbasu GDK JX LRND03 Labeo rohita GDK JX LRPO04 Labeo rohita GDK JX LRPO01 Labeo rohita GDK JX GF685 Labeo rohita GDK JX GF686 Labeo rohita GDK JX LRSH11 Labeo rohita GDK JX NBGA04 Lepidocephalichthys guntea GDK JX NBGA06 Lepidocephalichthys guntea GDK JX OSKA01 Lepidocephalichthys guntea GDK JX NBKA02 Lepidocephalichthys guntea GDK JX GF771 Lutjanus johnii GDK JX GF769 Lutjanus johnii GDK JX GF720 Macrognathus aculeate GDK JX GF721 Macrognathus aculeate GDK JX MQRJ01 Macrognathus aculeate GDK JX MCYN06 Macrospinosa cuja GDK JX Code Name of organism BOLD id NCBI accession MPPA06 Macrognathus pancalus GDK JX MABS02 Macrognathus pancalus GDK JX MPPA05 Macrognathus pancalus GDK JX MAUM01 Macrognathus pancalus GDK JX MPBS01 Macrognathus pancalus GDK JX GF723 Megalops cordyla GDK JX V Megalops cundigs GDK JX V Megalops cyprinoides GDK JX MNND02 Mystus bleekeri GDK JX MNSH02 Mystus bleekeri GDK JX PGPW02 Mystus bleekeri GDK JX SAPA07 Mystus cavasius GDK JX MTGD02 Mystus vittatus GDK JX MTGD01 Mystus vittatus GDK JX NDRJ01 Nandus nandus GDK JX CHPA01 Notopteus notopterus GDK JX CLPA05 Notopteus notopterus GDK JX OBND03 Ompok bimaculatus GDK JX OBND01 Ompok bimaculatus GDK JX OBND07 Ompok bimaculatus GDK JX WAPU01 Ompok bimaculatus GDK JX OPKW01 Ompok bimaculatus GDK JX WAPU02 Ompok bimaculatus GDK JX OKPA01 Ompok bimaculatus GDK JX OPKA03 Ompok pabda GDK JX GF715 Ompok pabda GDK JX OMND02 Oreochromis mossambicus GDK JX OMGD02 Oreochromis mossambicus GDK JX OMGD01 Oreochromis niloticus GDK JX WAPA01 Pangasius hypophthalmus GDK JX PPYN02 Pangasius pangasius GDK JX GF775 Pellona ditchela GDK JX GF797 Platycephalus cf. indicus GDK JX GF751 Pomadasys kaakan GDK JX P a g e

10 Code Name of organism BOLD id NCBI accession GF793 Pseudorhombus arsius GDK JX PSGA01 Crossocheilus latius GDK JX GF656 Puntius conchonius GDK JX GF729 Puntius conchonius GDK JX GF665 Puntius conchonius GDK JX PSHN03 Puntius fraceri GDK JX PUGD01 Puntius sarana GDK JX PDND01 Puntius sarana GDK JX PSPA01 Puntius sarana GDK JX V Puntius sarana GDK JX PSPA01 Puntius sarana GDK JX PSSH01 Puntius sophore GDK JX PSHN01 Puntius sophore GDK JX PSNW01 Puntius sophore GDK JX PTGA03 Puntius sophore GDK JX PSHN02 Puntius sophore GDK JX PSNW02 Puntius sophore GDK JX BSGD01 Puntius sophore GDK JX RSKG01 Puntius conchonius GDK JX GF668 Puntius ticto GDK JX RDGA03 Rasbora daniconius GDK JX RDGA01 Rasbora daniconius GDK JX RLKW01 Rasbora labiosa GDK JX PMUM01 Rasbora rasbora GDK JX RRND01 Rita pavimentatus GDK JX SAPW03 Rita pavimentatus GDK JX RRND02 Rita pavimentatus GDK JX MAGD01 Rita pavimentatus GDK JX RRND03 Rita pavimentatus GDK JX SNPA14 Salmophasia boopis GDK JX SNND02 Salmophasia novacula GDK JX SNND01 Salmophasia novacula GDK JX SNND12 Salmophasia novacula GDK JX SNGA01 Salmophasia novacula GDK JX Code Name of organism BOLD id NCBI accession SHRN05 Salmostoma bacaila GDK JX SCYN09 Sicamugil cascasia GDK JX GF779 Sillago sihama GDK JX GF781 Sillago vincenti GDK JX ASND01 Sperata aor GDK JX MYRJ02 Sperata seenghala GDK JX SYYN01 Sphyraena acutipinnis GDK JX STYN02 Strongylura strongylura GDK JX GF741 Terapon jarbua GDK JX V Thyrssa gautmensis GDK JX TSPA01 Thynnichthys sandkhol GDK JX PGND01 Thynnichthys sandkhol GDK JX CYNW01 Thynnichthys sandkhol GDK JX TSPA03 Thynnichthys sandkhol GDK JX APHN01 Thynnichthys sandkhol GDK JX PEGA03 Tor tor GDK JX BBGA02 Tor tor GDK JX V Tor tor GDK JX SXPW01 Tor tor GDK JX BBGA01 Tor tor GDK JX TBYN02 Trachinotus baillonii GDK JX GF761 Upeneus cf. sulphureus GDK JX GF755 Upeneus sulphureus GDK JX WAPA04 Wallago attu GDK JX WAPA03 Wallago attu GDK JX GF683 Xenentodon cancila GDK JX P a g e

11 Table 4.3.6: Fishes with morphologies ambiguities Code of Name of fish Image of fish Closely resembling with Image of morphologically fish resembling fish OMND01 Oreochromis mossambica Oreochromis niloticus CHPA01 Notopterus notopterus Notopterus chitala MNSH02 Mystus bleekeri Mystus cavasius OPKA03 Ompok pabda Ompok bimacualtus STYN02 Strongylura strongylura Xenentodon cancila 45v Tor tor Puntius vittatus 44 P a g e

12 TSPA01 Thyninchthys sandkhol Labeo boga CLGA01 Clarias gariepinus Clarias batrachus MUND01 Channa punctate CHanna marulia SORJ01 Cirrhina reba Sicamugil cascasia PSHN03 Puntius fraceri Puntius sophore PSGA01 Crosschelius latius Psilorhynchus sucatio 45 P a g e

13 4.4 Deep divergence between the species The phylogenetic tree generated based on K2P/NJ model is shown at figure 5 (same tree can be generated using BOLD). Average intraspecific variation was unrelated to the number of individuals analysed. COI based species identification study separated all 91 species, with 13.74% mean distance among individuals of congeneric species (table 4.4.1). This result is summarized in K2P NJ tree (figure 4.6.1) to provide an overview of sequence divergences between all species of Godavari river. For species within higher taxonomic ranks like families, orders, and classes, pairwise genetic divergences are in increasing order like 19.31, , and % respectively (table and figure 4.4.1). The rate of increase declines in the higher taxonomic categories due to substitutional saturation. Table : Genetic distance at within taxonomic ranks n Taxa Comparis ons Min Dist (%) Mean Dist(%) Max Dist(%) SE Dist(%) Within Species Within Genus Within Family Within Order Within Class A B C D Figure 4.4.1: Genetic divergence among Godavari fishes (A- Mean Intra-Specific; B- Distance to Nearest Neighbor; C: NN distance within species, Individuals studied per species) 46 P a g e

14 The analysis of the nearest neighbour distance (NN), which is the genetic distance between a species and its closest congeneric relative, revealed that while only 2.18% of the nearest neighbour was lower than 1% in all cases (Figure 4.4.1A). Congeneric nearest neighbour averaged % (Figure 4.4.1B) which is 16-fold higher than the maximum intraspecific distance of 0.85% and 5-fold higher than maximum intraspecific distance Minimal divergence but phylogeographic variation within species The K2P NJ tree shows that distinction between Puntius species is well known for morphological complexities, but seven species in this study are separated very well unambiguously. Similarly, some Cyprinids like Crossochelius were having ambiguities reported in many studies. DNA barcode can distinguish them very clearly. Some ambiguities reported in this study are in distinction between some species of Sisoridae and Bagaridae, Anabantidae and Carangidae, Mugilidae and Teropontidae and may suggest shared haplotypes, because genetic distance between these families is only 0.65, 0.22, 0.20 respectively. Such cases of shared haplotypes or low inter-specific variation may be due to introgressive hybridization, incomplete lineage sorting, taxonomic over-splitting, recent radiation or possibly misidentification of the original specimens (Won et al. 2005; Hubert et al. 2008; Steinke et al. 2009b). This may be due to wrong identification or may be distance between the sampling regions which may have imposed pressure for speciation. Either of the possibilities need in depth studies. Some taxa, however showed very high genetic divergence than others like, the average within genus divergence of the Salmostoma was 24.17%, which is considerably larger than 13.74% of the genus Ompok which is itself larger than the within Rasbora divergence of 1.13% (Figure 4.6.1). The divergence within species is evident in species: Amblypharyngodon mola, Anguilla bengalensis bengalensis, Glossogobious giuris giuris, Labeo rohita, Labeo calbasu and Lepidocephalichthys guntea. In these cases specimens were sampled 47 P a g e

15 from more than one sampling regions. This suggests that some regions in the river are isolated for fish migrations. Family Genetic Distance Species (n) < >2.5 Cyprinidae Ambassidae Anguillidae Ariidae Bagridae Channidae Cichlidae Clariidae Cobitidae Gerreidae Gobiidae Heterbpneustidae Lutjanidae Mastacemballidae Notopteridae Sciaenidae Silluridae Table 4.5.1: Summary of Godavari River fish diversity and distribution of the genetic distance to the nearest neighbour; COI sequences of 91 species analysed with K2P model. 48 P a g e

16 The deep divergence is recorded in case of Anguilla bengalensis bengalensis where within species deep divergence values were highest (4.18) as shown in table & figure This may be correlated to ecosystem dependent adaptive radiation. Table 4.5.2: Distance Matrix of individuals of Anguilla bengalensis bengalensis. Species ID AGIS03 AGIS01 AGIS02 AGMJ02 AGPW02 AGMJ01 AGRJ01 AGIS03 AGIS AGIS AGMJ AGPW AGMJ AGRJ Figure Conspecific divergence between Anguilla bengalensis bengalensis individuals 4.6DNA Barcode diagnostic information Based on barcode sequence analysis some diagnostic characters can be pointed out at various taxonomic levels like grouping by species (table 4.6.1) and grouping by families (table 4.6.2), etc. This grouping pattern can be observed at NJ tree also. 49 P a g e

17 These characters can be used in analysing unknown taxon for consensus verification. Table 4.6.1: Grouping by Species : DNA barcode dignostic characters (BOLD Sys v3) Group Name (# sequences) Diagnostic Characters Diagnostic or Partial Partial Characters Partial or Uninformative Invalid Characters Characters Characters Amblypharyngodon mola (6) Anguilla bengalensis bengalensis (7) Arius arius (4) Catla catla (4) Channa punctata (3) Cyprinus carpio (3) Danio devario (4) Garra mullya (4) Glossogobius giuris giuris (4) Heteropneustes fossilis (4) Hypophthalmichthys nobilis (3) Labeo calbasu (6) Labeo rohita (6) Lepidocephalichthys guntea (5) Macrognathus aculeata (3) Macrognathus pancalus (5) Mystus bleekeri (3) Ompok bimaculatus (7) Puntius conchonius (4) Puntius sarana (5) Puntius sophore (7) Rita pavimentatus (5) Salmophasia novacula (4) Thynnichthys sandkhol (5) Tor tor (4) Table 4.6.2: Grouping by family : DNA barcode dignostic characters (BOLD Sys v3) Group Name (sequences) Diagnostic Characters Diagnostic or Partial Characters Partial Characters Partial or Uninformativ e Characters Invalid Characters Cyprinidae (84) Anguillidae (7) Ariidae (4) Bagridae (14) Carangidae (3) Channidae (5) Clariidae (4) Sciaenidae (4) Cichlidae (5) Gobiidae (5) Heteropneustidae (4) Cobitidae (5) Mastacembelidae (8) Siluridae (11) P a g e

18 Clarias batrachus(n=2) Clarias gariepinus(n=2) Heteropneustes fossilis(n=4) Mystus vittatus(n=2) Batasio tengra(n=1) Mystus bleekeri(n=3) Clupisoma prateri(n=1) Eutropiicthys goongawarii(n=1) Pangasius hypophthalmus(n=1) Pangasius pangasius(n=1) Mystus cavasius(n=1) Sperata aor(n=1) Sperata seenghala(n=1) Arius arius(n=4) Glyptothorax lonah(n=1) Rita pavimentatus(n=5) Oreochromis mossambicus(n=2) Oreochromis niloticus(n=1) Wallago attu(n=2) Ompok pabda(n=2) Ompok bimaculatus(n=7) Dendrophysa russelii(n=2) Macrospinosa cuja(n=2) Lutjanus johnii(n=2) Anabas testudineus(n=2) Trachinotus baillonii(n=1) Pomadasys kaakan(n=1) Chelon parsia(n=1) Terapon jarbua(n=1) Platycephalus cf. indicus(n=1) Sicamugil cascasia(n=1) Channa punctata(n=3) Channa marulia(n=2) Macrognathus aculeata (n=3) Macrognathus pancalus(n=5) Strongylura strongylura(n=1) Xenentodon cancila(n=1) Sphyraena acutipinnis(n=1) Nandus nandus(n=1) Pseudorhombus arsius(n=1) Chanda nama(n=1) Caranx hippos(n=1) Megalaspis cordyla(n=1) Glossogobius giuris giuris(n=4) Notopterus notopterus(n=2) Cynoglossus elongetus(n=1) Pseudapocryptes lanceolatus(n=1) Etroplus suratensis(n=2) Coilia ramacarti(n=1) Thyrssa gautmensis Pellona ditchela (n=1) Megalops cundigs(n=1) Megalops cyprinoides(n=1) Anguilla bengalensis bengalensis(n=7) Sillago sihama(n=1) Sillago vincenti(n=1) Upeneus cf. sulphureus(n=1) Upeneus sulphureus(n=1) Hilsa ilisa(n=1) Gerres sinurus(n=2) Chanos chanos(n=1) Amblypharyngodon mola(n=6) Rasbora daniconius (n=2) Rasbora labiosa(n=1) Rasbora rasbora (n=1) Devario cf. devario(n=2) Danio devario(n=4) Lepidocephalichthys guntea(n=5) Salmophasia novacula(n=4) Hypophthalmichthys nobilis(n=3) Salmostoma bacaila(n=2) Puntius sophore(n=7) Puntius fraceri(n=1) Salmophasia boopis(n=1) Puntius sarana(n=5) Tor tor(n=5) Garra mullya(n=4) Crossocheilus latius Puntius ticto(n=1) Puntius conchonius(n=4) Cirrhina reba(n=3) Labeo bata(n=2) Cyprinus carpio carpio(n=3) Cirrhinus cirrhosus(n=1) Thynnichthys sandkhol(n=5) Labeo boga(n=1) Labeo rohita(n=6) Catla catla(n=4) Labeo fimbriatus(n=1) Labeo calbasu(n=6) 0.02 Figure : NJ distance tree with 91 fishes from river Godavari 51 P a g e

19 4.7 Barcode of life data search and analysis Bold data generated under the project can be seen in following manner for species comparisons as shown in Figure 7. ( Figure 4.7.1: Image of the project Submitted to BOLD Systems. On the barcode of life data system the records regarding all the specimens can be seen as illustrated in Appendix I. On BOLD systems it will be possible to see the DNA sequences, trace files, images of specimen, geographic informations, taxonomic details as well we can have comparision of species using phylogenic tree so as to place the organism to its correct taxonomic position. 52 P a g e