View of Morphometric and Genetic Variation in Three Populations of Indian Salmon (Polydactylus plebeius)

Morphometric and Genetic Variation in Three Populations of Indian Salmon ( Polydactylus plebeius )

Ramakrishnan THIRUMARAISELVI, Muthusamy THANGARAJ*, Vellaichamy RAMANADEVI

Annamalai University, Faculty of Marine Sciences, Centre of Advanced study in Marine Biology, Parangipettai, TamilNadu, 608 502, India; [email protected] (*corresponding author)

Abstract

Morphometric character analyses and RAPD was used to discriminate and ratify the status of three populations of Indian salmon, Polydactylus plebeius along the coromandel coast of India. Morphometric analyses showed a clear pattern of differentiation between the stocks and revealed the discreteness of two groups, southern stock (Pazhayar) and northern stock (Cuddalore). The univariate analysis of variance showed significant differences between means of the samples for most morphometric descriptors. A total of 1077 scorable bands were produced using all ten arbitrary primers in three populations. An un-weighted pair-group method with arithmetic mean (UPGMA) dendrogram was constructed based on genetic values to show the genetic relationship among the three populations.

The genetic diversity (H) of P. plebeius in Cuddalore was more (0.0733 ± 0.0648) than Pazhayar (0.0609 ± 0.0416) and Vellar (0.0613

± 0.0344) populations. All the three populations had significantly (p<0.001) higher interpopulation genetic distance value than the intrapopulation value. Further molecular studies, comprising more markers and populations are still required to precisely evaluate the genetic structure of threadfin fishes throughout the Indian coast.

Keywords: genetic variation, Indian salmon, morphometric, Polydactylus, population structure

Introduction

Threadfin fish belongs to the family Polynemidae, which forms part of the order Perciformes. They typically inhabit marine coastal waters, estuaries and freshwater riv- er mouths. This family has a wide distribution in the tropi- cal parts of Atlantic, Indian and Pacific Oceans (Kagwade, 1970). The potential annual yield of polynemids in India is estimated around 9,000 tones (Srinath and Balan, 2003).

Present catch of polynemids have gone down mainly be- cause of the introduction of shrimp trawlers which has destroyed most of the young-ones of the larger varieties of polynemids grow to more than a meter (Prasad et al., 2005).

Morphometric analysis has been applied to many stock differentiation and life-history problems in many fish spe- cies (Bronte et al., 1999). If shape differences in different populations of the same species can be used to discrimi- nate morphotypes, they may also be useful in examining the stock structure within a morphotype (Joseph and Jayasankar, 2001). Detection of differences within a mor- photype may indicate geographically separated stocks, whose shapes may be predicted on local environmental conditions or have genetic bases (Joseph and Jayasankar, 2001). They discriminate two Nemipterus populations through morphometric and meristic characters in India.

Turan et al. (2006) reported the genetic and morphologi- cal variation of Pomatomus saltatrix throughout the Black Seas, Marmara, Aegean and eastern Mediterranean Seas.

Erguden et al. (2009) underwent morphometric and mer- istic analyses of chub mackerel Scomber japonicus to dis- criminate stocks throughout the Black, Marmara, Aegean, and northeastern Mediterranean Seas.

Genetic markers are generally oversensitive to a low level of gene flow, relatively low level of exchange between stocks, which are quite negligible from a management per- spective, and may be sufficient to ensure genetic homogene- ity (Carvalho and Hauser, 1994; Ward and Grewe, 1994).

RAPDs have gained considerable attention particularly in population genetics (Lu and Rank, 1996), species and sub- species identification (Bardakci and Skibinski, 1994), phy- logenetics, linkage group identification, chromosome and genome mapping, analysis of interspecific gene flow and hybrid speciation, and analysis of mixed genome samples (Hadrys et al., 1992), breeding analysis and as a potential marker for single-locus genetic fingerprints (Brown and Epifanio, 2003).

In finfishes, size and morphometric variations among populations continue to play an important role in stock identification, despite the advent of biochemical and mo- lecular genetics technique which help in identification of genetic differences between groups (Swain and Foote, 1999). Considering the importance of morphometric, ge- netic variation and as there are no attempts to study in P.

plebeius, in spite of its significant contribution in Indian marine fish landing the present study was undertaking.

The objective of this study was to evaluate patterns of mor- phological and genetic variation in the Indian salmon, P.

Received 07 May 2013; accepted 16 June 2013

ing a UV spectrophotometer. The DNA was diluted in TE buffer to a final concentration of 100 ng/μL. Ten com- mercially available decamer random primers (An1-An10) from Chromous Biotech Pvt Ltd (Bangalore, India) were used for this study. The amplification reaction was car- ried out in a 25 μL reaction volume containing 10 mM Tris-HCl, 50 mM KCl (pH 8.5), 2.5 mM MgCl₂, 0.001%

gelatin, 100 μM of dNTP mix, 0.2 μM of each primer, 1 U of Taq DNA polymerase (Bangalore Genei, India) and 25 ng of template DNA. The PCR was performed in a thermocycle (TechGene, UK) for 40 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 35°C for 30 seconds, and extension at 72°C for 60 seconds. The final extension was carried out at the same temperature for 5 minutes. The resulting products were electrophoretically analyzed through 1.5% agarose gels, stained with ethidi- um bromide, and visualized using a UV transilluminator.

Subsequently the gel was photographed using a gel docu- mentation system (Lark, India).

Data analysis

Genetic similarity/distance between the three stripped threadfin populations was estimated using Popgene Soft- ware (Version 1.31) (Yeh et al., 1999). Nei and Li’s (1979) genetic similarity (GS) among the three populations was computed and converted by Popgene into genetic dis- tance (GD) according to Hillis and Mortiz’s (1990) for- mula, GD = 1-GS. The GS reflects the proportion of the bands shared between individuals and values range from (1) when present to (0) when absent. Phylogenetic rela- tionship was estimated based on genetic distance values generated from RAPD data among the three populations.

A neighbor-joining dendrogram also generated based on Nei’s procedure (Nei, 1978) using Popgene.

plebeius in three estuarine regions, by analyzing morpho- metric characters and banding pattern using ten arbitrary primers.

Materials and methods Sample collection

The striped threadfin, P. plebeius were collected from three stations such as, (S1) Cuddalore estuary (Lat. 11°42’

N; Long. 74°46’ E), (S2) Vellar estuary (Lat. 11°29’ N;

Long. 79°46’ E) and (S3) Pazhayar estuary (Lat. 11°21’

N; Long. 79°49’ E) given in Fig. 1. Totally fifty individuals from each station were sampled for morphometric charac- ter measurements and tissue samples DNA isolation.

Morphometrics

Forty nine morphometric characters were measured as per earlier report (Motomura et al., 2001) and used for further analysis. The morphometric data were analysed us- ing sheared PCA as per Bookstein et al. (1985). This tech- nique, which quantifies shape differences independent of size, as previously been used to distinguish fish species (Browers and Stauffer, 1993). Morphometric measure- ments were log transferred to preserve allometrics, stan- dardize variance and produce a scale invariant covariance matrix before analysis. To ensure comprehensive analyses of the data for more powerful discrimination between populations, sheared principal components of represented morphometric measurements were scattered against the first principal components in SPSS (V.14.0).

DNA isolation and PCR

The DNA was isolated by standard Proteinase-K/Phe- nol-Chloroform-ethanol method (Sambrook et al., 1989) and the concentration of isolated DNA was estimated us-

Fig. 1. Map showing the sample collection sites in Coromandel coast of India

Differences in intrapopulation and interpopulation genetic distance coefficients among the three populations were tested by one way analysis of variance (ANOVA).

Intrapopulation and interpopulation genetic distance val- ues were compared by paired t-test. The statistical analyses were performed by the software SYSTAT (version 7.0).

Results

Morphometrics

Morphometric values obtained from the three popula- tions of P. plebeius are shown in the Tab. 1. Many of the characters in all the three populations are not significantly deviate. Vellar and Pazhayar populations are showing more morphological similar characters value when comparing to Cuddalore population. Except total length, fork length, maximum body height, overall caudal fin length, height and length of longest pectoral fin filament, the Cuddalore population also mingled with other two populations. The principal components analysis score plot also exhibited a similar trend of clustering of Cuddalore and Pazhayar population separately. But, the Vellar population individu- als are shared with Pazhayar and Cuddalore populations (Fig. 2).

Genetic diversity

By using the ten random primers (An1-An10) in three populations, totally 1077 scorable bands were observed. In Cuddalore population, total numbers of bands were 330 and in Pazhayar, Vellar it was 417 and 330 respectively.

Nei’s (1978) unbiased genetic distances and genetic simi- larity between three populations of P. plebeius are given in Tab. 2. The genetic distance between Pazhayar and Cudda- lore was more (0.0034) than Vellar and Pazhayar (0.0033).

The genetic identity between Cuddalore and Pazhayar was 0.9966, genetic identity between Pazhayar and Vellar was 0.9967 based on the RAPD data. The overall observed and expected polymorphic loci in three populations are given in Tab. 3. The genetic diversity (H) of P. plebeius in Cuddalore population was more (0.0733 ± 0.0648) than Pazhayar (0.0609 ± 0.0416) and Vellar (0.0613 ± 0.0344) populations. The intrapopulation genetic distance values for the three populations were tested by one way ANOVA and found to be significantly slight different (p<0.0001) (Tab. 4). But the interpopulation genetic distance values estimated for the three populations also tested by one way ANOVA and found to be significantly much different (p<0.001) (Tab. 4).

Fig. 2. Principal component score plot of morphometric data in three populations

Tab. 1. Morphometric characters observed in three populations of P. plebeius

No. Morphometric characters (mm) Vellar Pazhayar Cuddalore

1 TL 130 ± 15.20^a 156 ± 28.1^a 187 ± 11.63^a

2 FL 108.7 ± 10.26^a 125.8±30.32^b 152.3± 18.87^a

3 STL 98.14 ±11.59^a 114.4±21.64^b 133.5± 8.12^a

4 1^st P-DL 34.71±3.61^a,c 44.4±10.42^a 44.1±2.58^b,c

5 2^nd P-DL 61.51± 3.9^a 71.34±14.38^b 79.3±4.92^a

6 P-PTL 26.17±4.05^a,c 32.25±5.78^a 37.52±2.06^b,c

7 P-PVL 38.37±3.33^a,c 43.40±4.30^a 48.80±6.51^b,c

8 P-AL 63.4±5.80^a 72.96±12.94^b 85.66±4.94^a,b

9 HL 29.37±3.13^a,c 36.06±6.59^a 41.57±2.89^b,c

10 HH 17.97±2.8^a,c 28.06±7.20^a 28.14±1.79^b,c

11 HW 5.25±0.56^a,c 6.93±1.56^a 7.66±0.85^b,c

12 LMC 5.71±0.71^a,c 7.71±2.38^a 9.61±0.74^b,c

13 LMO 9.22±1.13^a,c 11.34±2.75^a 14±1.64^b,c

14 UJL 12.62±1.69^a,c 14.15±2.12^a 14.95±0.49^b,c

15 MH 17.4±2.25^a 15.31±1.83^a,b 19.33±2.26^b

16 EH 12.59±1.17^a 14.56±2.15^a 18.61±1.56^a

17 OD 7.03±0.93^a 8.46±1.64^a 10.5±0.70^a

18 DEO 3.64±0.52^a,c 4.98±0.98^a 5.57±0.53^b,c

19 EA 21.64±2.83^a 24.78±5.42^b 32.61±2.37^a,b

20 MBH 30.02±2.36^a 35.21±6.72^a 43.04±3.52^a

21 MBW 5.45±0.70^a 6.53±1.39^b 5.80±0.60^c

22 B 1st-DFL 13.85±1.71^a 16.37±3.56^b 19.19±1.99^a

23 B 2^nd-DFL 19.11±2.25^a,c 22.59±4.25^a 25.14±1.82^b,c

24 B-AFL 19.82±1.82^a 21.40±3.67^b 24.04±1.80^a

25 B-PTFL 5.25±0.56^a 5.56±0.91^b 6.95±0.66^a,b

26 B-PVFL 4.71±0.51^a 4.56±0.71^b 5.19±0.40^c

27 1^st-DFH 19.4±2.58^a 23.31±4.50^a 28.61±2.29^a

28 2^nd-DFH 18.74±2.52^a 23.40±4.17^a 28.23±2.02^a

29 AFH 17.4±2.08^a 20.68±3.88^a 25.33±1.90^a

30 PTFH 19.34±2.38^a 22.78±3.77^a 27.66±2.55^a

31 PVFH 14.82±1.38^a 16.12±2.87^b 20.85±1.52^a,b

32 L.L 1^st-DFR 19.4±2.58^a 23.31±4.50^a 28.61±2.29^a

33 L.L 2^nd-DFR 18.77±2.53^a 23.40±4.17^a 28.23±2.02^a

34 L.L-AFR 17.4±2.08^a 20.68±3.88^a 25.33±1.90^a

35 L.L-PTFR 19.37±2.38^a 22.78±3.77^a 27.66±2.55^a

36 L.L-PVFR 14.54±2.41^a 16.12±2.87^b 20.85±1.52^a,b

37 O 1^st-DFR-AFL 41.82±4.05^a 50.65±9.14^a 62.04±4.99^a

38 O 2^nd-DFR-AFL 30.57±2.44^a 35.84±6.57^a 43.28±3.22^a

39 O PVFR-AFL 27.34±3.10^a 31.75±4.57^a 38.28±4.45^a

40 2^nd DF SPINE 7.37±1.37^a,c 9.65±2.29^a 10.66±1.06^b,c

41 L.L AF SPINE 6.68±1.49^a 7.93±1.58^b 9.85±0.96^a,b

42 CPL 11.05±1.02^a 12.40±2.56^b 12.57±1.36^a

43 CPH 13.31±1.40^a,c 16.90±3.55^a 19.28±1.67^b,c

44 CPW 4.65±0.68^a 5.5±1.10^b 5.66±0.65^a

45 UCFL 36.28±3.34^a 40.75±7.96^b 50.23±3.61^a,b

46 LCFL 33.31±3.48^a 38.78±6.59^a 46.04±3.26^a

47 CFH 45.65±4.56^a,c 54.56±10.81^a 57.42±3.13^b,c

48 L.L PT F.FILAMENT 36.05±2.36^a 40.21±6.99^b 49.76±2.48^a,b

49 BMH 15.37 ±1.43^a 19.03 ±3.76^a 23.66 ±2.45^a

The common superscripts sharing the row is not significantly different (p<0.001)

(TL: Total length; FL: Fork length; STL: Standard length;1st. P-DL: 1st Pre-dorsal length; 2nd. P-DL: 2ndPre-dorsal length; P-PTL: Pre-pectoral length; P-PVL: Pre- pelvic length; P-AL: Pre-anal length; HL: Head length; HH: Head height; HW: Head width; LMC: Length of snout with mouth closed; LMO: Length of snout with mouth opened; UJL: Upper jaw length; MH: Mouth height; EH: Eye height; OD: Orbit diameter; DEO: Dermal eye opening; EA: Eye area; MBH: Maximum body height; MBW: Maximum body width; B. 1st-DFL: Base of 1st dorsal fin length; B. 2nd-DFL: Base of 2nd dorsal fin length; B-AFL: Base of anal fin length; B-PTFL: Base of pectoral fin length; B-PVFL: Base of pelvic fin length; 1st-DFH: 1st Dorsal fin height; 2nd-DFH: 2nd Dorsal fin height; AFH: Anal fin height; PTFH: Pectoral fin height; PVFH: Pelvic fin height; L. L. 1st-DFR: Length of longest 1st dorsal fin ray; L. L. 2nd-DFR: Length of longest 2nd dorsal fin ray; L. L-AFR: Length of longest anal fin ray; L. L-PTFR: Length of longest pectoral fin ray; L. L-PVFR: Length of longest pelvic fin ray; O. 1st DFL-AFL: Origin of 1st dorsal fin length to anal fin length; O.

2nd DFL-AFL: Origin of 2nd dorsal fin length to anal fin length; O. PVFL-AFL: Origin of pelvic fin length to anal fin length; 2nd DF SPINE: 2nd dorsal fin spine length;

L. L. AF SPINE: Length of longest anal fin spine; CPL: Caudal peduncle length; CPH: Caudal peduncle height; CPW: Caudal peduncle width; UCFL: Upper caudal fin length; LCFL: Lower caudal fin length; CFH: Caudal fin height; L. L. PT. F. FILAMENT: Length of longest pectoral fin filament; BMH: Body mid line height)

Discussion

As per the present study, Vellar and Pazhayar popu- lations are close enough when compared to Cuddalore population and exhibiting low phenotypic differentia- tion in PCA scatter plot analysis. The obtained p-values denote morphometric results are insignificant to support

the established differentiation between these three popu- lations that often leads to taxonomic uncertainity. Some of the Pazhayar individuals clustered with Vellar population and some individuals clustered in a separate place in the plot. Only some individuals in Vellar population placed in between the Cuddalore and Pazhayar populations and remaining all the individuals dropped in a separate cluster in the plot.

The close distribution of these samples may be ac- counted for recent separation due to ecological altera- tions. Discrimination of the three populations as a single entity could be confirmed statistically by the insignificant difference observed from the morphometric data. Turan et al. (2006) studied the morphological variation of Po- matomus saltatrix based on morphometric and meristic analyses of samples collected throughout the Black Sea, Marmara, Aegean and eastern Mediterranean Seas and the results indicated existence of three morphologically differentiated groups. Erguden et al. (2009) underwent morphometric and meristic analyses of chub mackerel, Scomber japonicus throughout the Black, Marmara, Aege- Based on Nei’s genetic distance value, an UPGMA den-

drogram was constructed and given in (Fig. 3). The cluster values indicated distinct relationship between the three populations of P. plebeius. In this dendrogram Pazhayar and Vellar populations are closely related than Cuddalore population.

Tab. 3. Overall observed number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity (H), Shannon’s information index (I), Number of polymorphic loci (Np) and Percentage of polymorphic loci (Pp) in three populations of P. plebeius

Populations Na Ne H I Np Pp (%)

Cuddalore 1.3347±0.4721 1.0447±0.0832 0.0733±0.0648 0.1173±0.0737 332 33.47

Pazhayar 1.4183±0.4935 1.0483±0.0777 0.0609±0.0416 0.1124±0.0839 415 41.83

Vellar 1.3327±0.4714 1.0407±0.0804 0.0613±0.0344 0.1107±0.0684 330 33.27

Overall 0.0401±0.0007 0.0379±0.0006 0.0540±0.0317 8.7672 1077 92.10

Tab. 4. Summary of results of one way ANOVA to test for differences in intrapopulation and interpopulation genetic distance value calculated based on RAPD markers among the three populations

Source of Variation Sum of Squares df Mean squares F P

Within Cuddalore population 1.0958 9 0.1217 12.2173 0.0001

Error 0.0985 58 0.0896

Total 1.1943 67 0.2113

Within Pazhayar population 1.4883 9 1.6537 10.2890 0.0001

Error 0.0894 62 0.8903

Total 1.5777 71 2.5440

Within Vellar population 1.5423 9 1.7137 10.2362 0.0001

Error 0.9225 55 0.6742

Total 2.4648 64 2.3879

Between three populations 2.9215 15 2.5632 34.4703 0.001

Error 1.1264 561 0.1633

Total 4.0479 576 2.7265

Fig. 3. Neighbour joining tree (1000 replications) generated from RAPD data of the three populations

Tab. 2. Nei’s genetic identity (above diagonal) and genetic distance (below diagonal) of three populations of P. plebeius

Populations Cuddalore Pazhayar Vellar

Cuddalore **** 0.9966 0.9966

Pazhayar 0.0034 **** 0.9967

Vellar 0.0034 0.0033 ****

alosa ilisha and Lates calcarifer (Shifat et al., 2003; Rah- man et al., 2009; Chandra et al., 2010; Mehrotra et al., 2010; Rajasekar et al., 2012).

Conclusions

Statistical inference on morphometric data in this study revealed that it is insignificant to consider all three fish populations are widely distinct. RAPD gene diversity indices and genetic identity and distance values go accord- ingly same as mentioned with morphometric data. This was again corroborated with PCA and scatter plot analysis as well as dendrograms structured using UPGMA meth- od. From the inferences claimed with previous studies in other fishes, it is feasible to consider all three populations as a single genetic structure. This presumption could be authenticated henceforth with other molecular markers.

Further molecular studies, comprising more markers with other populations are still required to precisely evaluate the genetic structure of threadfin fishes along the Indian coast.

Acknowledgements

The authors are grateful to the University Authorities, The Director, CAS in Marine Biology, Annamalai Univer- sity for the encouragement and facilities provided.

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