| چکیده انگلیسی مقاله |
The Namak chub (Squalius namak) is an endemic species of the inland waters of Iran. However, no study has been conducted on the structure and genetic diversity of its populations. To address this, 103 Namak chub (S. namak) were sampled from four stations in the Jajrood River, and five microsatellite loci (Sluc4, Sluc5, Sluc7, Sluc13, and SarN2) were used. GenAlEx and CERVUS genetic software were used to analyze the data. The number of alleles ranged from 2 to 37 (average 12.43), and expected heterozygosity ranged from 0 to 0.95 (average 0.74), both of which were higher than the averages typically reported for freshwater fish. The average FST index for genetic differentiation was estimated to be 0.19, and the mean gene flow (Nm) was 1.30. Analysis of molecular variance revealed high genetic diversity within populations and low diversity among them. All loci exhibited a pattern of deviation from Hardy–Weinberg equilibrium (HWE), likely due to the use of non-specific primers, the presence of null alleles, and possibly small sample sizes. According to the findings of the present study, the genetic diversity observed in the populations of the Namak chub at these four sites was high, and considering the relatively low level of gene flow and the high FST, there appears to be significant differentiation among the populations of this species in the studied areas. These results can support efforts in biodiversity conservation and management planning. Keywords: Population genetics, Genetic differentiation, Microsatellite, Chub, Molecular marker. Introduction The genetic diversity of a species determines its adaptive capacity and evolutionary potential. Small populations of species with restricted distributions often have low genetic diversity within populations but high genetic differentiation between populations due to genetic drift and limited gene flow (Zhai et al., 2019). Genetic diversity is now a concern in many conservation programs, as low genetic diversity is associated with increased inbreeding, accumulation of deleterious mutations, and reduced adaptive potential (McCusker & Bentzen, 2010). Microsatellite loci are standard genetic markers used for population genetic analysis (Coates et al., 2009). In freshwater ecosystems, fish biodiversity is a good indicator of ecosystem health (Zhou et al., 2024). Freshwater fish species constitute 40% of the world's total fish diversity and are one of the most endangered animal groups (Costa et al., 2021). In terms of diversity and endemism, Iran has a high richness of freshwater fish. According to the latest studies, a total of 300 fish species belonging to 38 families and 110 genera live in the inland waters of Iran (Sayyadzadeh & Esmaeili, 2024). The genus Squalius is one of the important genera in the family Leuciscidae, with five species of this genus identified in the inland waters of Iran (Sayyadzadeh & Esmaeili, 2024), one of which is the Namak chub (Squalius namak Khaefi et al., 2016). This endemic species was recently described and as a result, few studies have been conducted on it (Mouludi-Saleh & Keivany, 2018). Materials & Methods The study area was the Jajroud River, one of the important aquatic ecosystems in the northeast of Tehran (Naderi et al., 2020). Using a Samus 725 electrofishing device, 103 S. namak were collected. Fish were anesthetized using a clove oil solution. Then, caudal fin clips were obtained from the samples and transferred into the labeled tubes containing 96% ethanol. DNA was extracted from the fin tissue using the phenol-chloroform method (Sambrook & Russel, 2001). PCR reaction was used to amplify five pairs of primers for this work. Since no specific primer was designed for this species, the primers from related species were used. For better separation of PCR products, electrophoresis on 8% polyacrylamide gel was used. Data obtained from AlphaEaseC software were first transferred to Excel. Then genetic indices such as number of alleles per locus (Na), number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), degree of differentiation (FST) and gene flow (Nm) were calculated using GenAlEx 6.5 software (Peakall & Smouse, 2006). Also, to determine the genetic diversity within and between populations based on FST and RST, the analysis of molecular variance (AMOVA) in the GenAlEx software package was used. Finally, principal component analysis (PCA) was performed to examine the genetic relationship of the four populations and the Hardy-Weinberg equilibrium test for the four loci used with Bonferroni correction in CERVUS 3.0.7 software. Research Findings The total number of alleles at the loci level ranged from 2 to 37, with the Sluc5 locus showing the highest number of alleles (37) and the Sluc4 locus showing the lowest number (2). The average number of alleles per locus was 10.25 in the Khojir 1 population, 26.25 in the Khojir 2 population, 11.75 in the Jajrood 1 population, and 1.5 in the Jajrood 2 population. The average observed heterozygosity in the Khojir 1 population was 0.65, in the Khojir 2 population was 0.68, in the Jajrood 1 population was 0.51, and in the Jajrood 2 population was 0.75. Expected heterozygosity values (He) ranged from 0.50 to 0.96 (average across all four populations: 0.74). At the population level, the highest and lowest average values were 0.94 and 0.37, observed in the Khojir 2 and Jajrood 2 populations, respectively. At the locus level, the degree of differentiation (FST) and gene flow (Nm) were calculated, and the average gene flow among the populations was 1.30, with the highest value observed at the Sluc5 locus (1.93) and the lowest at the SarN2 locus (0.49). The average genetic differentiation was 0.19, with the highest differentiation observed at the SarN2 locus (0.33). AMOVA based on RST indicated that 93% of the observed genetic variation was related to differences among individuals within populations, and 7% was due to differences among the four studied populations. The chi-square test with Bonferroni correction, used to examine Hardy–Weinberg equilibrium, showed that all loci deviated from equilibrium. Discussion of Results & Conclusion In this study, the average heterozygosity observed in the four populations was 0.64, which is higher than the values observed in freshwater fish (0.46) (Hedayati et al., 2017). If heterozygosity values are higher than the average, the level of genetic diversity is considered high; if they are lower, genetic diversity in that population is reduced (Farasati et al., 2020). The average total number of alleles for the four populations was 12.43, which was higher than the value calculated for freshwater fish (7.5) (Hedayati et al., 2017). This value is strongly affected by the number of samples (Gorjipoor & Nazari, 2014). In the present study, the average gene flow rate was 1.30, and the reason for the high genetic diversity observed in this species can be attributed to the existence of gene flow between populations. When Nm < 1, gene flow is the dominant factor causing differentiation, and when Nm > 1, genetic drift is the main factor causing genetic differentiation (Karami Nasab et al., 2014). However, high gene flow values do not necessarily guarantee genetic diversity, because a low number of parents and populations, and consequently interbreeding among small populations, can reduce diversity (Farasati et al., 2020). However, the number obtained in this study does not represent all real populations. Another point is that the FST rate does not reach unity in most cases because the effect of polymorphism (caused by mutations) effectively reduces the FST rate (Silavi et al., 2024). Based on the available analyses, it appears that the S. namak species exhibits favorable genetic diversity in the studied areas. Given the ecological importance of this endemic species in rivers, preserving its genetic diversity is deemed necessary and important. |