Seahorse

GENETIC DIVERGENCE STUDIES IN SEAHORSE

BASED ON MITOCHONDRIAL DNA & MICROSATELLITE

SEQUENCING

Karan Veer Singh

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OBJECTIVES: 1. Morphological & molecular identification of Seahorse fish species from India using traditional and molecular approaches.(16s rDNA, Cyt b and Co I gene segments). 2. Development of microsatellite markers in Seahorse species. a. Identification of microsatellite loci in H. kuda, H. trimaculatus, through cross species amplification. b. Sequencing of identified microsatellite loci to confirm repeats.4 3. Population genetics of seahorse- Genotyping of individuals from natural populations with i. identified microsatellite markers and ii. mitochondrial DNA markers.

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Clear species identification will make it easier:-

• To develop marking systems to distinguish aquacultured seahorses from wild-caught specimens.• To modify fishing practices appropriately, • Design protective marine reserves, & To assess captive breeding potential for seahorses. • Genetic studies provide valuable information for baseline management decision. • Identifying remnant population and determining the extent to which native genetic pools are being changed by hybridization. • The population structure of threatened or endangered species.

Out Comes & Out Put :

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Present Status

Government of India has banned the collection (fishing) of seahorses since July 2001,Under Schedule – I of the Wildlife Protection Act 1972.

Seahorse characteristics of low fecundity, limited mobility, structured mating patterns and site fidelity, make them particularly vulnerable to heavy fishing pressure.

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S.no Scientific Name Complex of Species

Place

1 H. borboniensis Kochi / Palk *

2 H. fuscus -- Palk

3 H. histrix 4 Expected

4 H. kuda 10 Kochi / Palk

5 H. trimaculatus 2 Kochi / Palk

6 H. spinosissimus Palk

7 H. kelloggi Not well described Expected

8 H. mohnikei New 2007

Present Status

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Phylum ChordataSubphylum PiscesSuperclasss GnathostomataClass OsteichthyesSubclass TeleosteiOrder Syngnathiformes (syn:Together, with + gnathos: Jaw)

Super Family SyngnathoideaFamily Syngnathidae & SolenostomideaSub Family Hippocampus & Syngnathinae (pipefishes)Genus Hippocampus (hippo:Horse + campus:Sea animal)

Taxonomy

includes pipefishes, pipehorses and seadragons

Families Pegasidae Tube Mouths / Sea mouthsAulostomidae Trumpet FishesFistularidae Cornet FishesCentriscidae Shrimp Fishes

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RESEARCH METHODOLOGY: 

1.Sample acquisition and identification•Indian Ocean along the eastern and western side. 

2.DNA extraction, PCR amplification, and sequencing•Sample preparation using fin clippings and or tail region for DNA extraction.•(Taggart et al., (1992) and Cenis et al., (1993) with minor modifications)•Seahorse Species specific primers development. •PCR amplification based on standard protocols for selected Microsatellite & Mt DNA.•Sequencing of the sample on DNA sequencer.

3.Molecular Data Analysis using Genetic software•Sequence editing /Processing/ Submission•Molecular data analysis (EditSeq, MegaBACE, CLUSTALW,BIOEDIT, MEGA 4, Arlequin)•Phylogenetic analysis (PAUP,MOLPHY,MEGA,AMOVA)

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Sample Collection:

•Thondi, Mullimunai and Pamban Palk Bay.•Mandapam, Tuticorin (Vellipatti, Thirespuram & Tiruchendur) Gulf of Mannar (east coast of India). •Vizhinjam near Trivandrum, Shakthikulangara and Cochin along Kerala Coast. •Karwar, Kumta in Karnataka; & Panjim and Goa estuary.

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Sites of occurrences

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S.no Primer Name Sequence 5’ – 3’ References

1 L2510 F:CGCCTGTTTATCAAAAACAT

16 s Palumbi et al.,19912 H3058 R:CCGGTCTGAACTCAGATCACGT

3 COI F1 F:TCAACCAACCACAAAGACATTGGCAC

COI Ward R.D et al., 20054 COI R1 R:TAGACTTCTGGGTGGCCAAAGAATCA

5 Shf 2 F:TTGCAACCGCATTTTCTTCAG

Cyt b Lourie S.A. et al., 2005 6 Shr 2 R:CGGAAGGTGAGTCCTCGTTG

7 1027 R R:ACAGGTATTCCCCCAATTC

Mitochondrial DNA PCR primers used for Hippocampus spp.

GENETIC POPULATION STRUCTURE OF TWO CLOSELY RELATED SPECIES Hippocampus kuda and Hippocampus trimaculatus USING MtDNA SEQUENCES

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Genetic variability of Mitochondrial Markers (COI, 16S rRNA & Cyt b) in H.kuda and H. trimaculatus

COI 16S rRNA Cyt b

H. kuda H. trimaculatus H. kuda H. trimaculatus H. kuda H. trimaculatus

No. of Nucleotides

655 655 592 586 704 677

Variable Sites 1.71 1.62 0.76 0.68 2.13 2.05

Ti/Tv Ratio 2.47 2.27 1.92 1.80 3.46 3.30

Haplotypes Diversity

0.53 0.48 0.61 0.59 0.78 0.70

Nucleotide Diversity

1.29 1.13 0.59 0.52 0.95 0.87

Mean Genetic Divergence

0.57 0.60 0.47 0.41 0.70 0.68

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Hippocampus kuda, 16S rRNA

Haplotypes and Nucleotide Diversities

Haplotypes

351

415

591

592

PopulationsNumber of Sequences

Numbers of Haplotypes and Number

Haplotype diversity (h)

Nucleotide diversity (π)

H1 T C T C Palk Bay 6 2 (H1,H2) 0.59 0.003

H2 C . . . Gulf of Mannar 7 2 (H1,H2) 0.41 0.004

H3 . . C G

Kerala Coast 12 2 (H3,H4) 0.38 0.004H4 . T C G

H5 . . . G Goa 5 1 (H5) 1.00 0.000

Overall 30 5 0.595 0.003

Variable nucleotide positions of 16S rRNA haplotypes, nucleotide and haplotype diversities in different populations of Hippocampus kuda.

592 bp

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Variable nucleotide positions of COI haplotypes, nucleotide and haplotype diversity in Hippocampus kuda

655 bp

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Variable nucleotide positions of Cyt b haplotypes, nucleotide and haplotype diversity in Hippocampus kuda

Hippocampus kuda, Cyt b

Haplotypes

Haplotypes and Nucleotide Diversities

41

163

284

352

366

378

385

390

410

426

587

698

PopulationsNumber

of Sequences

Haplotypes and number

Haplotype diversity

(h)

Nucleotide diversity

(π)

H1 A C T C C T A T C C G G

Palk Bay

8 6 (H1,H2,H3,H4

,H5,H6)

0.51 0.006

H2 - - - - T - - - - T - -

H3 - - - - T C - - - - - A

H4 - - - - T - - C - - - AGulf of Mannar 6

6 (H1,H2,H3,H4, H5,H6)

0.59 0.005H5 - - - - T - - - - - - A

H6 - - - - T - - - - - - -

H7 G - C T T - G - T - A - Kerala coast 8 1(H7) 1.00 0.00

H8 G T - - T - G - T - A - Goa 8 1(H8) 1.00 0.00

Overall 30 8 0.78 0.003

704 bp

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Evolutionary relationship of H kuda population based on 16S rRNA, COI & Cyt b sequence data (Neighbor-Joining method) H5

H4 H3 H2

H1 Fistularia petimba

6565

46

0.02

H1 H2

H3 H4

H5 H6

H7 H8 Fistularia petimba

84

62

99

974651

0.05

H1 H2 H3 H4 H5 H6 H7 H8 Syngnathoides biaculeatus

49

95

0.000.020.040.060.080.100.12

3 ClustersHigh Boot strapMP similar TREE

Absence of common haplotype indicates the distinct genetic structure among four population

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Variable nucleotide positions of 16S rRNA haplotypes, nucleotide and haplotype diversity in Hippocampus trimaculatus

586 bp

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Variable nucleotide positions of COI haplotypes, nucleotide and haplotype diversity in Hippocampus trimaculatus

655 bp

Common Haplotype H5

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Variable nucleotide positions of Cyt b haplotypes, nucleotide and haplotype diversity in Hippocampus trimaculatus

677 bp

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Evolutionary relationship of Hippocampus trimaculatus population

16s

COI Cyt b

NJ & MP Tree2 ClustersHigh Boot Strap

Significant pair-wise comparison of ФST & AMOVA values indicates distinct genetic structure among east & west coast population

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Development of single locus microsatellite primer through cross species sequences amplification

HIGH RESULATION ANALYSIS OF POPULATION STRUCTURE USING MICROSATELLITE MARKERS

• PCR Amplification• PAGE & Visualisation• Calculation of Molecular weight• Image Master ID Elite, Marker (pBR322 DNA / MspI digest)• Final Selection• Sequencing PCR• Automated Genotyping• Analysis of Microsatellites Data

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Characteristics of 12 polymorphic microsatellite loci in H. kuda and H. trimaculatus

3 Species- 15 Loci80% Polymorphic lociNumber of repeats varied

Clear discreet band …Not as Ladder

5-6 Brown trout3-5 Northern Pike16-32 Red Seabream

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Observed number (na) of alleles for each population and overall populations

LocusPalk Bay

Gulf of Mannar

Kerala Coast

Goa Overall Populations

Han03 5 5 4 4 6

Han05 5 5 4 4 6

Han06 3 3 4 4 5

Han15 4 4 3 3 4

Hca08 5 5 5 6 6

Hca10 3 3 4 4 5

Hca11 3 3 6 4 6

Hca25 5 5 4 4 5

Hca27 4 4 4 4 4

Hca28 4 4 4 4 6

Hca34 4 4 4 4 5

Hca38 6 6 6 6 6

Total 51 51 52 51 64

Mean 4.2500 4.2500 4.3333 4.2500 5.334

S.D. 1.531 1.3093 1.2118 1.3025 0.9815

Range 3 – 6Average 4

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Total 23 alleles

Genetic Tags

Stock specific MarkersPartitioning of Breeding PopulationLimitation in Migration

No Gene Flow

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Observed and expected heterozygosity for each and overall population

LocusPopulations (n=40 each)

Palk Bay Gulf of Mannar Kerala Coast Goa

Han03H obs. H exp. H obs. H exp. H obs. H exp. H obs. H exp.

0.3571 0.3976 0.5310 0.5811 0.5501 0.5308 0.4502 0.4307

Han05 0.5943 0.5652 0.6927 0.6479 0.6571 0.6404 0.3344 0.2680

Han06 0.3336 0.3261 0.1731 0.1667 0.2571 0.2743 0.2663 0.2789

Han15 0.7098 0.6894 0.7857 0.7539 0.8037 0.7571 0.7571 0.7037

Hca08 0.5857 0.5291 0.4180 0.4168 0.4286 0.4221 0.3826 0.3322

Hca10 0.7429 0.6871 0.7387 0.7236 0.5014 0.5644 0.6017 0.6643

Hca11 0.6143 0.5949 0.1931 0.1867 0.6079 0.6068 0.6857 0.6539

Hca25 0.6271 0.6200 0.5857 0.5682 0.3714 0.3636 0.5271 0.5219

Hca27 0.6143 0.6949 0.4802 0.5674 0.6071 0.6698 0.6177 0.6000

Hca28 0.6143 0.5949 0.5802 0.5674 0.6171 0.6098 0.8718 0.8041

Hca34 0.6333 0.5987 0.4802 0.4674 0.6654 0.6598 0.3244 0.3077

Hca38 0.6283 0.6049 0.5802 0.5774 0.5555 0.5498 0.2148 0.2014

Mean Overall Loci

0.5148 0.5067 0.6130 0.5996 0.5239 0.5019 0.3965 0.3871

No significant association indicative of Linkage Disequilibrium between any pair of Loci for any population

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Populations Palk Bay Gulf of Mannar Kerala Coast Goa

Palk Bay ---- 0.9998 0.8259 0.7885

Gulf of Mannar 0.0023 ---- 0.9003 0.8201

Kerala Coast 0.1764 0.1085 ---- 0.9137

Goa 0.2165 0.1813 0.0863 ----

Genetic identity (above diagonal) & Genetic distance (below diagonal) using microsatellite markers in Hippocampus kuda

Nei’s Genetic IdentityLong Geographical Distances between population

AMOVA indicated significant genetic differentiation (FST 0.6511; P<0.0001)Among population 65%Within Population 34.89%

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PopulationsPalk Bay

Gulf of Mannar

Kerala Coast Goa

Palk Bay ---- 0.00230 NS 0.08881*** 0.10638***

Gulf of Mannar 0.00121NS ----- 0.08202*** 0.08990***

Kerala Coast 0.09915*** 0.08126*** ---- 0.06105***

Goa 0.11913*** 0.09740*** 0.06892*** ----

Pair-wise Fisher’s FST (θ) (above diagonal) and RST (below diagonal) between populations of Hippocampus kuda using microsatellite markers.

*** Significant after Bonferroni adjustment (P<0.0001)

Pair wise RST values between population differ significantly except GofM & Palk Bay

Pair wise & overall RST values were similar to FST

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Locus Palk Bay Gulf of Mannar Kerala Coast Overall Populations

Han03 6 (0.5333) 6 (0.4375) 6 (0.0123) 7

Han05 5 5 5 6

Han06 3 3 4 4

Han15 4 4 3 4

Hca08 4 4 5 5

Hca10 3 3 4 5

Hca11 3 3 3 4

Hca25 5 5 5 5

Hca27 4 4 4 4

Hca28 5 5 5 5

Hca34 4 4 4 (0.4895) 4

Hca38 5 (0.0104) 5 (0.0095) 5 5

Total 51 51 53 58

Mean 4.2500 4.2500 4.4167 4.834

S.D. 1.1612 1.3564 1.2304 1.2493

Observed number (na) of alleles for each population and overall populations

Allele frequency

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No Mixing of Gene Pool

* Stock- Specific markers * Genetic TAGs for selection programs

Null allele frequency was not significant (P<.05) at all 3 tested Loci

Total 11 Private Alleles

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Observed and expected heterozygosity for each and overall population

LocusPopulations (n=40 each)

Palk Bay Gulf of Mannar Kerala Coast

Han03H obs. H exp. H obs. H exp. H obs. H exp.

0.4352 0.4077 0.6524 0.6310 0.2378 0.2304

Han05 0.2439 0.2365 0.3100 0.2479 0.3826 0.3322

Han06 0.6309 0.6264 0.2735 0.2664 0.3984 0.4164

Han15 0.6378 0.6233 0.4861 0.4564 0.8069 0.7754

Hca08 0.5271 0.5219 0.4288 0.4100 0.4286 0.4221

Hca10 0.3298 0.3068 0.3391 0.3022 0.6079 0.6068

Hca11 0.6001 0.5978 0.4932 0.4865 0.6178 0.6644

Hca25 0.7234 0.7001 0.5857 0.5682 0.4432 0.4363

Hca27 0.6478 0.6233 0.5344 0.5199 0.5857 0.5491

Hca28 0.6667 0.7045 0.3398 0.3447 0.4358 0.4046

Hca34 0.6161 0.5911 0.5814 0.5696 0.4455 0.4641

Hca38 0.4081 0.4059 0.3826 0.3322 0.5718 0.5141

Mean Overall Loci

0.4264 0.4067 0.5363 0.5451 0.4087 0.4032

No significant association indicative of Linkage Disequilibrium between any pair of Loci for any population

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Populations Palk Bay Gulf of Mannar Kerala Coast

Palk Bay ---- 0.9972 0.8781

Gulf of Mannar 0.0019 ---- 0.8905

Kerala Coast 0.1302 0.1111 ----

Genetic identity (above diagonal) and Genetic distance (below diagonal) using microsatellite markers in Hippocampus trimaculatus

AMOVA indicated significant genetic differentiation (FST 0.6353; P<0.0001)Among population 63.53%Within Population 36.47%

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Populations Palk Bay Gulf of Mannar Kerala Coast

Palk Bay ---- 0.00266 NS 0.09997***

Gulf of Mannar 0.00201NS ----- 0.09005***

Kerala Coast 0.10445*** 0.098734*** ----

Pair-wise Fisher’s FST (θ) (above diagonal) and RST (below diagonal) between populations of Hippocampus trimaculatus using microsatellite markers.

*** Significant after Bonferroni adjustment (P<0.0001)

Pair wise & overall RST values were similar to FST

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Summary & conclusion Microsatellite genotyping and mitochondrial DNA sequence information have provided a clear evidence for the٭

existence of strong population differentiation.

.Haplotype Diversity very high as compared to the Nucleotide diversity in both species٭

.Distinct population of East & West coast / Reduced or No Gene flow٭

Along the west coast, two distinct populations of H. kuda indicate the absence of gene flow between Kerala and٭

Goa localities.

.Lack of Sub-structuring of H.trimaculatus between Kerala & TN. Range expansion from India To Java٭

High Genetic diversity in South East India (PB & GOM) may be the result of long-term stable environment٭

condition.

Coastal cold-water upwelling events characteristics of western India (Genetic Bottleneck) can be reason for the٭

presence of comparatively few Haplotypes.

However, significant number of common/shared haplotypes and the non-significant FST value between two east٭

coast populations of both the species suggests that some degree of gene flow exists between populations within

these areas.

All the classes of markers revealed high level of genetic variation in populations of this species as evinced from٭

values of heterozygosity, Fst and genetic distance. The study points out the need for stock-specific, propagation

assisted rehabilitation programme for this species.

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NBFGR X-Agricultural Science Congress 2011

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NBFGR News July – December 2008Molecular Genetic divergence studies in Indian seahorse

NBFGR Annual Report 2007 - 2008Genetic Diversity analysis in Indian seahorse, P 35-36

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Thanks

The Improbable seahorses,National Geography 1994