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

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.


Introduction
Threadfin fish belongs to the family Polynemidae, which forms part of the order Perciformes.They typically inhabit marine coastal waters, estuaries and freshwater river mouths.This family has a wide distribution in the tropical 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 because 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 species (Bronte et al., 1999).If shape differences in different populations of the same species can be used to discriminate morphotypes, they may also be useful in examining the stock structure within a morphotype ( Joseph and Jayasankar, 2001).Detection of differences within a morphotype 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 morphological variation of Pomatomus saltatrix throughout the Black Seas, Marmara, Aegean and eastern Mediterranean Seas.Erguden et al. (2009) underwent morphometric and meristic analyses of chub mackerel Scomber japonicus to discriminate 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 perspective, and may be sufficient to ensure genetic homogeneity (Carvalho and Hauser, 1994;Ward and Grewe, 1994).RAPDs have gained considerable attention particularly in population genetics (Lu and Rank, 1996), species and subspecies identification (Bardakci and Skibinski, 1994), phylogenetics, 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 molecular genetics technique which help in identification of genetic differences between groups (Swain and Foote, 1999).Considering the importance of morphometric, genetic 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 morphological and genetic variation in the Indian salmon, P.
ing a UV spectrophotometer.The DNA was diluted in TE buffer to a final concentration of 100 ng/μL.Ten commercially available decamer random primers (An1-An10) from Chromous Biotech Pvt Ltd (Bangalore, India) were used for this study.The amplification reaction was carried out in a 25 μL reaction volume containing 10 mM Tris-HCl, 50 mM KCl (pH 8.5), 2.5 mM MgCl 2 , 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 ethidium bromide, and visualized using a UV transilluminator.Subsequently the gel was photographed using a gel documentation system (Lark, India).

Data analysis
Genetic similarity/distance between the three stripped threadfin populations was estimated using Popgene Software (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 distance (GD) according to Hillis and Mortiz's (1990) formula, 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 relationship 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 morphometric characters and banding pattern using ten arbitrary primers.

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 using sheared PCA as per Bookstein et al. (1985).This technique, which quantifies shape differences independent of size, as previously been used to distinguish fish species (Browers and Stauffer, 1993).Morphometric measurements were log transferred to preserve allometrics, standardize 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/Phenol-Chloroform-ethanol method (Sambrook et al., 1989) and the concentration of isolated DNA was estimated us- 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 values were compared by paired t-test.The statistical analyses were performed by the software SYSTAT (version 7.0).

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 similarity between three populations of P. plebeius are given in Tab. 2. The genetic distance between Pazhayar and Cuddalore 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).

Discussion
As per the present study, Vellar and Pazhayar populations are close enough when compared to Cuddalore population and exhibiting low phenotypic differentiation in PCA scatter plot analysis.The obtained p-values denote morphometric results are insignificant to support the established differentiation between these three populations 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 accounted for recent separation due to ecological alterations.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 Pomatomus 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 dendrogram 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.

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 accordingly same as mentioned with morphometric data.This was again corroborated with PCA and scatter plot analysis as well as dendrograms structured using UPGMA method.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.
an, and northeastern Mediterranean Seas.They observed a clear pattern of morphometric and meristic differentiation between the stocks.The present study also showed a clear pattern of differentiation between the stocks and revealed two groups, the southern estuary stock (Pazhayar) and the northern estuary stock (Cuddalore).
The RAPD profile showed 92.10% polymorphic loci among the three estuarine populations in this study.As compared to previous studies, 68% of polymorphic loci were reported in three populations of catfish, Clarias batrachus in three water bodies of Bhopal in India (Mehrotra et al., 2010).Rahman et al. (2009) studied genetic variations of wild and hatchery populations of Catla catla revealed by RAPD markers and found overall 54.55% polymorphism.Shifat et al. (2003) have also reported RAPD Jaccards dissimilarity coefficient in 34 individuals of Tenualosa ilisha and observed 20.41% polymorphism.Rajasekar et al. (2012) investigated that the RAPD profile showed 93.12% polymorphic loci among the three populations of Lates calcarifer.The present study inferred from these RAPD data that showed relatively high level (92.10%) of genetic polymorphism and is possibly due to the small sampling size.
While considering the Nei's gene diversity (H), it was high (0.0733 ± 0.0648) in Cuddalore population compared to the Vellar (0.0613 ± 0.0344) and Pazhayar (0.0609 ± 0.0416) populations, which are more or less similar.This result also coincides with previous study revealed by RAPD marker, where Nemipterus nemurus (0.0652 ± 0.0520) showed similar gene diversity than N. cyanomus (0.0354 ± 0.0208) and P. caeruleus (0.0283 ± 0.0197) (Parveen et al., 2011).Their results illustrate that genetic structure of damselfish population differ upon at the genus level not at the species level.
Nei's genetic identities between the three populations were high and it is evident that those three distinct populations were recently isolated or they are inhabited with weak geographical barriers.Similar genetic identity and genetic distance values were observed in the genetic diversity analysis of two Gangetic riverine populations of Eutropiichthys vacha using RAPD (Chandra et al., 2010).They confirmed eventhough these two riverine populations vary spatially but originate from the same drainage system.In theory, the intrapopulation genetic distance values are expected to be lower than the interpopulation genetic distance.In this study also all the three populations had significantly (p<0.001) higher interpopulation genetic distance value than the intrapopulation value.
The clustering pattern obtained by UPGMA method both the Pazhayar and Vellar populations were fell in one clade and Cuddalore population displayed in another clade.This clustering pattern is directly propotional to the geographical distance.The same type clustering pattern regarding the geographic distance was obtained with the RAPD profiling analysis study on wild fish populations of Catla catla, Eutropiichthys vacha, Clarias batrachus, Tenu-

Fig. 1 .
Fig. 1.Map showing the sample collection sites in Coromandel coast of India Morphometric values obtained from the three populations 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 individuals are shared with Pazhayar and Cuddalore populations (Fig. 2).

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