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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 4.1.1.

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.

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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 4.2.1.

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 4.2.2.

Figure 4.2.2. 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.

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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

Table 4.3.1. Details of fishes studies for their molecular taxonomy using DNA barcoding

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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

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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

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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

4.3.5.

4.3.1 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 1st

codon position (55.97%) and lowest at codon position 3rd (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 % 14.81 17.5 22.05 0.081 C % 22.61 27.14 33.16 0.126 A % 21.35 25.67 29.21 0.114 T % 25.4 29.34 34.3 0.11 GC % 39.18 44.64 52.14 0.159 GC % Codon Pos 1 50.57 55.97 59.91 0.128 GC % Codon Pos 2 39.97 42.92 44.92 0.054 GC % Codon Pos 3 23.16 35.04 54.36 0.417

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 4.3.3 and figure 3).

A. Piscivorus fishes (total sequences 47)

Min Mean Max SE G % 15.65 17.4 19.02 0.137 C % 24.48 27.09 30.41 0.252 A % 23.16 25.8 28.36 0.225 T % 26.53 29.59 32.56 0.207

GC % 40.15 44.49 48.93 0.351 GC % Codon Pos 1 52.43 55.93 59.45 0.285 GC % Codon Pos 2 41.47 42.78 44 0.099 GC % Codon Pos 3 23.16 34.63 47 1.007

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B. Cyprinids (obligate herbivores) (Number of Sequences : 84)

Min Mean Max SE G % 15.19 17.54 20.54 0.122 C % 24.55 26.8 29.77 0.131 A % 23.26 26.64 29.21 0.131 T % 25.4 28.91 32.73 0.146 GC % 40.5 44.34 49.08 0.198 GC % Codon Pos 1 51.48 56.37 59.36 0.162 GC % Codon Pos 2 41.03 43.21 45.61 0.078 GC % Codon Pos 3 25.15 33.3 46.51 0.489

Table 4.3.3: Comparative GC percentage

Figure 4.3.1: Percept GC content comparison in Carnivores (A) and Herbivores

fishes (B)

4.3.2 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 4.5735 -

C 4.5735 15.8530 - G 15.8530 4.5735 4.5735 -

Table: 4.3.4: MCL transition/ transversion bias.

Some species as mentioned in table 4.3.6 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 4.3.6.

A B

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Code Name of organism BOLD id NCBI

accession BESH08 Amblypharyngodon GDK025-11 JX260817 GF679 Amblypharyngodon mola GDK209-12 JX260818 PSSH02 Amblypharyngodon mola GDK044-11 JX260819 BESH02 Amblypharyngodon mola GDK024-11 JX260820 GF675 Amblypharyngodon mola GDK208-12 JX260821 PRUM01 Amblypharyngodon mola GDK090-11 JX260822 COYN01 Anabas testudineus GDK052-11 JX260823 ABRJ01 Anabas testudineus GDK169-12 JX260824 AGIS01 Anguilla bengalensis bengalensis GDK155-11 JX260825 AGMJ02 Anguilla bengalensis bengalensis GDK165-11 JX260826 AGIS02 Anguilla bengalensis bengalensis GDK162-11 JX260827 AGIS03 Anguilla bengalensis bengalensis GDK001-11 JX260828 AGRJ01 Anguilla bengalensis bengalensis GDK079-11 JX260829 AGMJ01 Anguilla bengalensis bengalensis GDK267-12 JX887590 AGPW02 Anguilla bengalensis bengalensis GDK156-11 JX887591 ANPA04 Hypophthalmichthys nobilis GDK082-11 JX260830 33V Hypophthalmichthys nobilis GDK195-12 JX260831 HMPA01 Hypophthalmichthys nobilis GDK059-11 JX260884 HHRJ01 Arius arius GDK174-12 JX260832 GF783 Arius arius GDK247-12 JX260833 RSYN03 Arius arius GDK003-11 JX260834 RSYN01 Arius arius GDK002-11 JX260835 MSNW01 Batasio tengra GDK172-12 JX260836 13V Caranx hippos GDK186-12 JX260837 CCND01 Catla catla GDK078-11 JX260838 CCSH07 Catla catla GDK264-12 JX887592 CCGA14 Catla catla GDK269-12 JX887593 CCND11 Catla catla GDK262-12 JX887594 CNGA05 Chanda nama GDK004-11 JX260839 MUND01 Channa marulia GDK068-11 JX260840 MUND02 Channa marulia GDK069-11 JX260841 CSSH02 Channa punctate GDK095-11 JX260842 COPW02 Channa punctate GDK005-11 JX260843

Code Name of organism BOLD id NCBI

accession COPA10 Channa punctate GDK051-11 JX260844 CZYN01 Chanos chanos GDK175-12 JX260845 SQRJ01 Cirrhina reba GDK046-11 JX260851 NBGA05 Lepidocephalichthys guntea GDK261-12 -- CTGA03 Cirrhinus cirrhosis GDK006-11 JX260850 BEGA03 Clarias batrachus GDK145-11 JX024330 BEGA01 Clarias batrachus GDK146-11 JX024335 CLGA01 Clarias gariepinus GDK034-11 JX260853 BEGA02 Clarias gariepinus GDK138-11 JX024327 GF722 Clupisoma prateri GDK227-12 JX260854 4V Coilia ramacarti GDK193-12 JX260855 7V Cynoglossus elongetus GDK196-12 JX260856 CYBD01 Cyprinus carpio GDK054-11 JX260857 CYPA13 Cyprinus carpio GDK055-11 JX260858 CYRJ01 Cyprinus carpio GDK009-11 JX887595 DVKW02 Danio devario GDK218-12 JX260860 DVKW01 Danio devario GDK217-12 JX260861 CEGA03 Danio devario GDK011-11 JX260862 CEGA02 Danio devario GDK010-11 JX260863 31V Dendrophysa russelii GDK185-12 JX260864 GF789 Dendrophysa russelii GDK248-12 JX260865 CAKA04 Devario cf. devario GDK035-11 JX260859 NDKA01 Devario cf. devario GDK164-11 JX260866 ESND02 Etroplus suratensis GDK223-12 JX260867 ESND03 Etroplus suratensis GDK224-12 JX260868 42V Eutropiicthys goongawarii GDK240-12 JX260869 PAND01 Garra mullya GDK198-12 JX260870 CSKA02 Garra mullya GDK256-12 JX887596 GGGA01 Garra mullya GDK257-12 JX887597 GMND02 Garra mullya GDK219-12 JX260871 GF767 Gerres sinurus GDK234-12 JX260872 GF765 Gerres sinurus GDK233-12 JX260873 GGNW01 Glossogobius giuris giuris GDK178-12 JX260874

Table 4.3.5: NCBI accession numbers

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Code Name of organism BOLD id NCBI accession

GGRJ01 Glossogobius giuris giuris GDK056-11 JX260876 GGPW03 Glossogobius giuris giuris GDK087-11 JX260877 GHPW02 Glyptothorax lonah GDK029-11 JX260878 HFPA01 Heteropneustes fossilis GDK097-11 JX260879 HFNW01 Heteropneustes fossilis GDK206-12 JX260880 HFPA03 Heteropneustes fossilis GDK058-11 JX260881 HFPA02 Heteropneustes fossilis GDK057-11 JX260882 36V Hilsa ilisa GDK188-12 JX260883 POND04 Labeo bata GDK042-11 JX260887 POND06 Labeo bata GDK043-11 JX260888 LBHN01 Labeo boga GDK191-12 JX260889 LBYN02 Labeo calbasu GDK036-11 JX260890 LCND03 Labeo calbasu GDK016-11 JX260891 LCND02 Labeo calbasu GDK037-11 JX260892 LBYN01 Labeo calbasu GDK015-11 JX260893 LCBS03 Labeo calbasu GDK060-11 JX260894 LBKA01 Labeo calbasu GDK038-11 JX260895 LBYN03 Labeo calbasu GDK161-11 JX260896 LRND03 Labeo rohita GDK063-11 JX260897 LRPO04 Labeo rohita GDK062-11 JX260898 LRPO01 Labeo rohita GDK061-11 JX260899 GF685 Labeo rohita GDK204-12 JX260900 GF686 Labeo rohita GDK205-12 JX260901 LRSH11 Labeo rohita GDK263-12 JX887598 NBGA04 Lepidocephalichthys guntea GDK084-11 JX260902 NBGA06 Lepidocephalichthys guntea GDK271-12 JX887599 OSKA01 Lepidocephalichthys guntea GDK258-12 JX887600 NBKA02 Lepidocephalichthys guntea GDK268-12 JX887601 GF771 Lutjanus johnii GDK236-12 JX260903 GF769 Lutjanus johnii GDK235-12 JX260904 GF720 Macrognathus aculeate GDK225-12 JX260905 GF721 Macrognathus aculeate GDK226-12 JX260906 MQRJ01 Macrognathus aculeate GDK184-12 JX260907 MCYN06 Macrospinosa cuja GDK012-11 JX260885

Code Name of organism BOLD id NCBI accession

MPPA06 Macrognathus pancalus GDK067-11 JX260912 MABS02 Macrognathus pancalus GDK065-11 JX260909 MPPA05 Macrognathus pancalus GDK066-11 JX260910 MAUM01 Macrognathus pancalus GDK017-11 JX260911 MPBS01 Macrognathus pancalus GDK064-11 JX887602 GF723 Megalops cordyla GDK228-12 JX260913 6V Megalops cundigs GDK192-12 JX260915 10V Megalops cyprinoides GDK190-12 JX260914 MNND02 Mystus bleekeri GDK039-11 JX260916 MNSH02 Mystus bleekeri GDK085-11 JX260917 PGPW02 Mystus bleekeri GDK128-11 JX260918 SAPA07 Mystus cavasius GDK074-11 JX260919 MTGD02 Mystus vittatus GDK018-11 JX260920 MTGD01 Mystus vittatus GDK019-11 JX260921 NDRJ01 Nandus nandus GDK170-12 JX260922 CHPA01 Notopteus notopterus GDK033-11 JX260847 CLPA05 Notopteus notopterus GDK077-11 JX260848 OBND03 Ompok bimaculatus GDK072-11 JX260923 OBND01 Ompok bimaculatus GDK071-11 JX260924 OBND07 Ompok bimaculatus GDK081-11 JX260925 WAPU01 Ompok bimaculatus GDK049-11 JX260926 OPKW01 Ompok bimaculatus GDK221-12 JX260927 WAPU02 Ompok bimaculatus GDK050-11 JX260928 OKPA01 Ompok bimaculatus GDK254-12 JX887604 OPKA03 Ompok pabda GDK040-11 JX260929 GF715 Ompok pabda GDK251-12 JX260930 OMND02 Oreochromis mossambicus GDK073-11 JX260931 OMGD02 Oreochromis mossambicus GDK270-12 JX887605 OMGD01 Oreochromis niloticus GDK099-11 JX260932 WAPA01 Pangasius hypophthalmus GDK080-11 JX260933 PPYN02 Pangasius pangasius GDK020-11 JX260934 GF775 Pellona ditchela GDK237-12 JX260935 GF797 Platycephalus cf. indicus GDK244-12 JX260936 GF751 Pomadasys kaakan GDK230-12 JX260937

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Code Name of organism BOLD id NCBI accession

GF793 Pseudorhombus arsius GDK243-12 JX260939 PSGA01 Crossocheilus latius GDK008-11 JX260940 GF656 Puntius conchonius GDK252-12 JX260946 GF729 Puntius conchonius GDK249-12 JX260948 GF665 Puntius conchonius GDK250-12 JX260958 PSHN03 Puntius fraceri GDK086-11 JX260949 PUGD01 Puntius sarana GDK176-12 JX260943 PDND01 Puntius sarana GDK021-11 JX260950 PSPA01 Puntius sarana GDK102-11 JX260951 44V Puntius sarana GDK189-12 JX260952 PSPA01 Puntius sarana GDK103-11 JX260956 PSSH01 Puntius sophore GDK104-11 JX260942 PSHN01 Puntius sophore GDK083-11 JX260944 PSNW01 Puntius sophore GDK180-12 JX260945 PTGA03 Puntius sophore GDK022-11 JX260953 PSHN02 Puntius sophore GDK088-11 JX260954 PSNW02 Puntius sophore GDK181-12 JX260955 BSGD01 Puntius sophore GDK023-11 JX260957 RSKG01 Puntius conchonius GDK207-12 JX260947 GF668 Puntius ticto GDK253-12 JX260959 RDGA03 Rasbora daniconius GDK027-11 JX260960 RDGA01 Rasbora daniconius GDK026-11 JX260961 RLKW01 Rasbora labiosa GDK212-12 JX887606 PMUM01 Rasbora rasbora GDK101-11 JX260962 RRND01 Rita pavimentatus GDK105-11 JX260963 SAPW03 Rita pavimentatus GDK130-11 JX260964 RRND02 Rita pavimentatus GDK028-11 JX260965 MAGD01 Rita pavimentatus GDK089-11 JX260966 RRND03 Rita pavimentatus GDK177-12 JX260967 SNPA14 Salmophasia boopis GDK272-12 JX887607 SNND02 Salmophasia novacula GDK242-12 JX260970 SNND01 Salmophasia novacula GDK241-12 JX260971 SNND12 Salmophasia novacula GDK260-12 JX887608 SNGA01 Salmophasia novacula GDK259-12 JX887609

Code Name of organism BOLD id NCBI accession

SHRN05 Salmostoma bacaila GDK168-11 JX260969 SCYN09 Sicamugil cascasia GDK126-11 JX260972 GF779 Sillago sihama GDK238-12 JX260974 GF781 Sillago vincenti GDK245-12 JX260973 ASND01 Sperata aor GDK220-12 JX260975 MYRJ02 Sperata seenghala GDK070-11 JX260976 SYYN01 Sphyraena acutipinnis GDK173-12 JX260977 STYN02 Strongylura strongylura GDK048-11 JX260978 GF741 Terapon jarbua GDK229-12 JX260979 37V Thyrssa gautmensis GDK187-12 JX260980 TSPA01 Thynnichthys sandkhol GDK091-11 JX260981 PGND01 Thynnichthys sandkhol GDK041-11 JX260982 CYNW01 Thynnichthys sandkhol GDK183-12 JX260983 TSPA03 Thynnichthys sandkhol GDK107-11 JX260984 APHN01 Thynnichthys sandkhol GDK014-11 JX260985 PEGA03 Tor tor GDK031-11 JX260986 BBGA02 Tor tor GDK032-11 JX260987 45V Tor tor GDK197-12 JX260988 SXPW01 Tor tor GDK045-11 JX260989 BBGA01 Tor tor GDK266-12 JX887610 TBYN02 Trachinotus baillonii GDK030-11 JX260990 GF761 Upeneus cf. sulphureus GDK232-12 JX260992 GF755 Upeneus sulphureus GDK231-12 JX260991 WAPA04 Wallago attu GDK076-11 JX260993 WAPA03 Wallago attu GDK075-11 JX260994 GF683 Xenentodon cancila GDK203-12 JX260995

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Table 4.3.6: Fishes with morphologies ambiguities

Code of fish

Name of fish Image of fish Closely resembling with Image of morphologically 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

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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

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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, 22.592, and

25.52 % respectively (table 4.4.1 and figure 4.4.1). The rate of increase declines in

the higher taxonomic categories due to substitutional saturation.

Table 4.4.1 : Genetic distance at within taxonomic ranks

n Taxa Comparisons

Min Dist (%)

Mean Dist(%)

Max Dist(%)

SE Dist(%)

Within Species 152 44 243 0 0.854 4.186 0.054 Within Genus 172 40 271 1.13 13.74 24.178 0.268 Within Family 186 26 3159 2.722 19.314 36.167 0.089 Within Order 197 10 1900 8.06 22.592 35.383 0.086 Within Class 199 1 14124 18.451 25.526 44.267 0.025

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)

A B

C D

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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 13.87 % (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 24.17.

4.5 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

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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 17 - 71 36 6

Ambassidae - - 1 - -

Anguillidae 2 - 1 9 9

Ariidae - - 2 4 -

Bagridae 4 - 6 4 -

Channidae 1 - 2 1 -

Cichlidae - - 1 1 -

Clariidae 1 - - 1 -

Cobitidae 1 - 3 6 -

Gerreidae - - 1 - -

Gobiidae - - 3 3 -

Heterbpneustidae 3 - 3 - -

Lutjanidae - - 1 - -

Mastacemballidae 3 - 6 4 -

Notopteridae - - 1 - -

Sciaenidae - - 1 1 -

Silluridae 2 - 14 7 -

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.

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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 4.5.2 &

figure 4.5.1. 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 AGIS01 0.00 AGIS02 3.28 1.32 -- AGMJ02 0.80 0.00 2.76 -- AGPW02 1.21 1.71 4.18 3.83 -- AGMJ01 1.61 1.03 3.96 33.76 2.20 -- AGRJ01 1.39 0.26 4.01 3.15 1.89 2.52 --

Figure 4.5.1. 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.

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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

Characters

Partial Characters

Partial or Uninformative

Characters

Invalid Characters

Amblypharyngodon mola (6) 3 0 2 0 0 Anguilla bengalensis bengalensis (7)

2 0 0 0 0

Arius arius (4) 4 0 1 0 0 Catla catla (4) 2 0 1 0 0 Channa punctata (3) 5 0 2 0 0 Cyprinus carpio (3) 1 0 1 0 0 Danio devario (4) 28 0 3 0 0 Garra mullya (4) 1 0 3 0 0 Glossogobius giuris giuris (4) 9 2 3 0 0 Heteropneustes fossilis (4) 5 0 1 0 0 Hypophthalmichthys nobilis (3) 2 1 15 1 0 Labeo calbasu (6) 0 0 0 0 0 Labeo rohita (6) 0 0 0 0 0 Lepidocephalichthys guntea (5)

2 0 1 0 0

Macrognathus aculeata (3) 2 1 0 0 0 Macrognathus pancalus (5) 5 0 0 0 0 Mystus bleekeri (3) 2 0 2 0 0 Ompok bimaculatus (7) 4 0 0 0 0 Puntius conchonius (4) 5 0 1 0 0 Puntius sarana (5) 3 0 0 0 0 Puntius sophore (7) 0 0 1 0 0 Rita pavimentatus (5) 3 0 0 0 0 Salmophasia novacula (4) 4 0 3 0 0 Thynnichthys sandkhol (5) 4 1 0 0 0 Tor tor (4) 2 0 1 0 0

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 Uninformative Characters

Invalid Characters

Cyprinidae (84) 0 0 0 0 0 Anguillidae (7) 0 0 17 0 0 Ariidae (4) 0 0 26 0 0 Bagridae (14) 0 0 0 0 0 Carangidae (3) 2 0 2 0 0 Channidae (5) 0 0 12 0 0 Clariidae (4) 0 1 9 0 0 Sciaenidae (4) 0 0 15 0 0 Cichlidae (5) 0 0 2 0 0 Gobiidae (5) 0 0 21 0 0 Heteropneustidae (4) 1 0 19 0 0 Cobitidae (5) 1 0 27 0 0 Mastacembelidae (8) 0 2 4 0 0 Siluridae (11) 0 0 2 0 0

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Figure 4.6.1.: NJ distance tree with 91 fishes from river Godavari

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

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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.

(http://www.barcodinglife.com/index.php/MAS_Management_RecordList)

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.