MATERIALS AND METHODS

Samples Collection
Totally 197 samples (2-3 g dorsal fin tissues) of adult stellate sturgeon were collected from four regions including 52 samples from region 1(Astara-Anzali), 43 samples, from region 2 (Kiyashahr-Ramsar), 50 samples from region 3 (Nowshahr-Babolsar), 52 samples from region 4 (Miyankale-Gomishan) in 2006 by the International Sturgeon Research Institute from South Caspian Sea in Iran (Fig. 1).

DNA Extraction
Genomic DNA was extracted from fin tissue following the method described by Hillis and Moritz (1990) with some modifications (Pourkazemi, 1996). Approximately 100 mg of fin tissue from the dorsal fin was cut into small pieces. These pieces were then placed in 1 mL STE buffer (10 mM Tris/HCl, pH 8.0; 50 mM EDTA, 200 mM NaCl and 0.5% SDS) and digested with 0.5 mg mL^-1 proteinase K at 55°C overnight. The resulting solution was centrifuged and the DNA in supernatant that was precipitated with ethanol and dissolved in TE buffer. DNA was purified using the phenol-chloroform extraction.

Fig. 1:	Map shows the sampling sites of stellate sturgeon where 1, 2, 3 and 4 represent each region and shows sturgeon fishing station

Table 1:	Loci, repeat motif, primers sequence, gene bank number and primer sources used at present study in four stellate sturgeon collections

The quality and concentration of DNA were assessed by agarose gel electrophoresis and spectrophotometry (model CECIL CE2040) stored at -20°C until use.

PCR Profiles and Primer Sequences
Nuclear DNA was amplified using 15 microsatellites primers designed for Acipenser and Scaphirhynchus (May et al., 1997; McQuown et al., 2000, Table 1). Polymerases Chain Reaction (PCR) condition for each primer set were optimized for stellate sturgeon. Experimental condition tested included varying the annealing temperature (49-61.2°C), MgCl₂ concentration (1-2.5 mM), dinucleotide triphosphate concentration (200 μM), 1 U Taq DNA polymerase and primer concentration (0.5-1 pμ) and approximately 100 ng of template DNA. All reactions were performed in 20 μL.

PCR products were separated on 6% polyacrylamid gels (29:1 acrylamid:bis-acrylamid; 1X TBE buffer) and followed by silver-staining. Gels were run at 170 V for 2 h and 30 min. Alleles were sized using BioCapt software and each gel contained an allelic ladder (50 bp) to assist in consistent scoring of alleles.

Data Analysis
Allelic frequencies, observed and expected heterozygosities, genetic distance (Nei, 1978), genetic identity (Nei, 1978) were computed in GeanAlex 6.0 software (Peakall and Smouse, 2005). This package was also used to calculate F_ST and R_ST, Nm, Hardy-Weinberg (HW) tests of equilibrium, AMOVA (Analysis of Molecular Variance). The dendrograms of genetic distance (Nei, 1972, 1978) UPGMA computed in TFPGA (version 1.3).

RESULTS

Amplification and Banding Patterns
Out of 15 sets of microsatellite primers, four sets (LS-69, LS-57, LS-62 and Spl-168) have not shown any flanking sites on stellate sturgeon genome. Eleven sets of primers were successfully amplified where one sets (Spl-113; Table 2) produce 2 loci and one set (LS-39) showed monomorphic pattern. Therefore totally 11 loci were investigated at present study. All microsatellite primers where able to produce DNA bands displayed a characteristic disomic banding pattern, where heterozygous individuals showed two bands of equal intensity on the gels and some times with one band darker than the others. These banding patterns may be a result of the presence of a homologous locus that is detected under certain PCR conditions.

Genetic Variation Within Sampling Sites (Region)
The average number of alleles found at each locus was 13 and ranged from 8 (Spl-105) to 18 (Spl-113b) alleles (Table 3). The total number of alleles found in each population ranged from 122 (region two) to 154 (region four).

Out of 202 observed alleles, 128 occurred at frequencies of <0.05 in all samples. Spl-170 showed the highest allele number (30 allele) of which 24 alleles at frequencies of <0.05 and LS-34 showed the maximum variability ranging in frequency.

Table 2:	PCR condition and reaction, locus and allele size (bp) on stellate sturgeon

Table 3:	Absolute numbers of alleles observed within 4 sampling sites (region) using 12 sets of microsatellite primers
N = Mean number of alleles observed per locus. *Loci = Loci in accordance with H-W equilibrium (p≤0.01)

Table 4:	Observed (Ho) and expected (He) heterozygosities at 11 loci in four sampling regions

Fig. 2:	UPGMA dendrogram based on the genetic distance computed by Nei’s (1972) between A. stellatus population, according to microsatellite DNA analysis

Average heterozygosity over the loci was 0.650 and ranged from 0.624 region one to 0.662 in Region four (Table 4).

Deviation from Hardy-Weinberg equilibrium were in all cases (p≤0.01) expect Spl-170 in region one and LS-68 in region two and Spl-173 in region three (Table 3).

Genetic Variation Among Sampling Regions
Genetic distance calculated between each pair of collections ranged from 0.337 (between Regions one and four) to 0.606 (between Regions two and four, Table 5). In general, similarities in more geographically close areas were higher than more distance one (Fig. 1, Table 5). Results of analysis of molecular variance of genetic variation, among regions was 0.0127, among population/regions was 0.035 and individual within groups was 0.047 (p≤0.01).

Table 5:	Pairwise population of genetic distance (below diagonal) and genetic identity (above diagonal) (Nei, 1972) detected at 11 loci in stellate sturgeon samples

Table 6:	Pairwise estimates of genetic differentiation detected at 11 loci in stellate sturgeon samples, using unbiased RST (below diagonal) and FST values (above diagonal)
Probabilities of RST or FST determined by AMOVA tests, p≤0.01

The highest range of genetic difference were observed between regions 2 and 4 (F_ST = 0.063, R_ST = 0.287, p≤0.01) and the lowest between regions 3 and 4 (F_ST = 0.033, R_ST = 0.301, p≤0.01). However, F_ST and R_ST estimates often differ in a pronounced manner (Balloux and Lugon-Moulin, 2002).Values of pairwise R_ST among samples were consistently much higher (as much as an order of magnitude) than equivalent F_ST values (Table 6).

Molecular Phylogeny
The dendrogram based on genetic distance Nei (1972, 1978) revealed three major cluster. One cluster consists of Region two alone, a second consists of Region three, while the third consists of Region one and four together (Fig. 2).

DISCUSSION

Out of 15 pairs of studied primers, 4 pairs was not amplified in the PCR reaction, which may be because of the lack of flanking sites in these primers due to high genetic distance of primers source species and the stellate sturgeon. One primer pair was monomorphic and the rest of them were polymorph. One pairs of polymorphic primers amplificated two loci. Few samples showed few additional bands in some loci, which may related to non-exclusive primers of this species. The long-term persistence of an endangered fish species can be investigated by allelic diversity, gene diversity, effective population size and population structure (Yue et al., 2004). At present study, comparison of the Ho values of A. stellatus (0.650, Table 4) it was slight lower than anadromus species (Ho = 0.68, Dewoody and Avise, 2000) and the mean number of alleles in this study (13.6) was higher than anadromus species (11.3) for microsatellites. The high genetic variability of the stellate sturgeon from region two, may reflect different population colonization origins. Region two populations not only presented the least similar genetically to other regions, but also showed the lowest number of alleles (122 alleles) due to bottleneck population such as over fishing and deterioration in the breeding environment resulting in less recruitment, dams, pollution. Many alleles with low frequency (<0.05) confirm that. The number of alleles for a given locus is more sensitive to demographic fluctuations (Nei et al., 1975; Maruyama and Fuerst, 1985) and is therefore more appropriate for testing whether a bottleneck has occurred in a population (Luikart and Cornuet, 1998). Therefore it needs immediate attention for its genetic conservation and overall variability (Table 3). AMOVA test revealed the populations of each region differ gather presenting a significant difference (p≤0.01) among the samples of the various regions in the Iranian coast line South Caspian These results stand in stark contrast to previous studies used mtDNA. Low levels of variation were detected and little differentiation among sampling site was detected over a comparable geographic range (Pourkazemi, 1996; Shabani, 2005). This may result from the more rapid rate of mutational change at microsatellite loci compared with that at even the most rapidly evolving area of the mitochondrial genome (Wirgin et al., 2002). At present study deviation from the Hardy-Weinberg equilibrium observed in most loci. Maybe, it is due to presence of null alleles. Similar results have been reported the null alleles in inheritance of microsatellite loci in lake and white sturgeons (Rodzen et al., 2004; Zhao et al., 2005; McQuown et al., 2003; Pyatskowit et al., 2001; Welsh et al., 2003) and it also may be related to not using species specific primers and the most important reason sampling from mixtures of migrating population and sampling methodology. To detect such a population structure, samples must be collected from spawning sites. Samples from non-spawning adults may reflect mixtures of migrating population (McQuown et al., 2003). At present study F_ST in all sampling site was low but significant (p<0.001). So in each river in the South Caspian sea have different population. The lowest of F_ST on AMOVA was between region 4 and 3 with the high gene flow. But the highest of genetic distance and lower genetic identity was between region 2 and 4 (Table 5). Regarding the fishery returning, it is possible that the caught samples are a combination of the various generation and different birth places which have formed a gathering to feed.

Phylogenetic tree of stellate sturgeon using 12 microsatellites loci in four regions of south of Caspian Sea showed three clarified clusters included cluster of region two, three, one and four. As it shown in Fig. 2, the samples of the region two is in a separate cluster, which is possibly the samples that have caught before entering spawning sturgeon into the Sefidrude river and suggested the existence of a separate population in Sefidrud. The obtained results of the phylogeny show that 3 populations of stellate sturgeon exist on South of Caspian Sea originated from Sefidrud, one of population is in Sefidrud and other populations are derived from it. However, it should be mentioned that usually phylogeny sanders has not recommended distinguishing different population for a species. Stellate sturgeon is an anadromus species which immigrates to river for spawning and returns to the sea for feeding and growth, but the comprehensive study has not been performed up yet. In comparison with other molecular methods performed in the stellate sturgeon species, microsatellites studies showed the higher genetic variation and heterozygosity than mtDNA. Although this fish has the various allelic number in each of the under-studied regions in the Iranian cost line south Caspian Sea varies greatly. As microsatellites method has higher capacity to show the genetic variation, using above primers compared to other methods and with regard to the severe reduction of these fishes reserves before performing the artificial reproduction, identify the caught sample, then take the required actions for artificial reproduction and transfer among the various workshops. The present study showed that the genetic variation in the stellate sturgeon compared to the other sturgeons is in acceptable level, but the effect of over fishing have caused the reduction stocks in this species. Over fishing and ecologic changes in the Caspian Sea have altered the population structure of this species effectively. With population differentiation in region two especially Sefidrud drainage, we suggest special consideration on conservation policy and restocking program of this species in Sefidrud area.

ACKNOWLEDGMENTS

We are grateful to all support provided by Iranian Fishery Research Organization (Project Cod 82-0710441000-53), Islamic Azad University, Science and Research Branch, Tehran. This research was performed in the Molecular Genetic Lab of the Dr. Dadman International Sturgeon Research Institute-Rasht. The honorable persons in charge and those who are involved in the institute and the export of the genetic lab. We would like to extended our appreciation to, Mr. Nuruzfashkhami head of genetic department, special thanks to Mr. Qasemi, Mr. Chakmeduz, for their great operation.