Using D-Loop region to study the genetic structure and phylogenetic analysis of Iranian red deer

Introduction: The breeds and subpopulations of any species, which are the result of mutation processes, genetic drift, natural selection and gene-environment interactions, are very precious. They have been inherited down to the present generation, and their preservation is of great value and importance (Alijani, 2009). Genetic analysis of wild populations is essential for conserving biodiversity, increasing knowledge about the survival of these species, and finding the factors that threaten or contribute to the survival of these populations. The red deer is one of the biggest free-ranging mammals of central Europe and as it is not endangered in terms of population numbers, is the perfect model for studying the genetic population effects of a multitude of deliberate and unintentional anthropogenic influences on natural populations over a long period of time (Kiabi et al, 1998). Red deer gene pools are affected by habitat fragmentation, keeping of populations in enclosures, translocations, (re)introductions, and trophy hunting (Bruford et al, 2003). Various schedules of population regulation by hunting are applied throughout Europe. Many autochthonous stocks have been hybridized with the introduced animals, thus blurring the historical boundaries between formerly natural populations (Hiendleder et al, 1998). The objectives of the present studies were to gain insight into phylogeographic history; to characterize and quantify the genetic diversity within and among populations to implement conservation and management strategies, and to compare different molecular marker systems with regard to their respective resolution power (Cavalli –Sforza, 2003). For this purpose, a research program was designed to characterize populations of Iranian red deer (Cervus elaphus), using the mitochondrial control region (CR). In order to understand the origin, phylogeny, and phylogeography of the species C. elaphus, the DNA sequence variation of the mitochondrial D-loop gene of five populations of deer was examined from the entire distribution area of Cervinae with an emphasis on Iran. Several methods, including maximum parsimony, maximum likelihood, and nested clade analysis revealed that red deer originated from the area between Noshahr and other populations. The mitochondrial DNA (mtDNA) data do not support the traditional classification of red deer as only one species nor its division into numerous subspecies. The discrepancies between the geographical pattern of differentiation based on mtDNA D-loop and the existing specific and sub-specific taxonomy based on morphology are discussed. The present study was carried out to identify the existing gene pool, study the relationship and genetic variation of the Maral (Caspian red deer, C. elaphus) using the D-loop sequence data from the mitochondrial genome.
Material and methods: Blood, hair or tissue samples were taken from 78 Maral and their DNA was extracted. Sampling was carried out in coordination with Environmental and Wildlife Organization of the provinces of Ardebil, East Azarbaijan, Qazvin, Golestan and Mazandaran, as well as the Museum of Natural Resources and the reserves of the Gene Bank of Tehran. Blood samples were taken from those animals that were captured for population health check or treatments to use fumigant drugs and pneumatic gun in vacuum tubes contained EDTA. Hair samples were also taken directly from live animals and other tissue samples were provided from dead animals which newly dehorned. The D-loop region of the mitochondrial genome was amplified using specific primers with PCR and all PCR products were sequenced. A total of 130 sequences of D-Loopregion from deer species (52 sequences from NCBI and 78 sequences from Maral populations were studied) wereused for bioinformatics analysis between the species. The sequences were aligned and compared using BioEdit software to find nucleotide diversity. Using Mega software, a phylogenetic tree was drawn.  Haplotypes and genetic diversity parameters including nucleotide diversity, haplotype diversity, genetic distance, genetic differentiation, nucleotide frequency, etc., were performed using the DnaSp 5.10 software. DnaSP software was used to analyze the genetic data of the population, haplotypes and genetic diversity parameters, specific alleles, Rst, Fst values, and genetic distances of the 6-population hierarchy including Arasbaran, Ziaran, Semeskandeh, Fandoghlu, Ghorgh, Noshahr. Finally, NETWORK software applied to analyze the network to identify haplotypes.
Results and discussion: The results of Maral sequence analysis using DnaSP software led to the identification of mutations that resulted in the creation of 5 haplotypes for the D-loop region. The haplotype diversity, nucleotide diversity and mean nucleotide difference for the D-loop region were 0.218, 0.0007 and 0.491, respectively. The negative and significant Tajima D test result demonstrated an inbreeding among the populations. Results showed that the Noshahr population was in highest genetic distance from other populations. The genetic distance estimated for other populations were low. The phylogenetic tree using the D-Loop region showed that the Maral's populations is divided into two main branches. The main branch of the first consists of the population of Noshahr and its second main branch is divided into two branches, the first branch of which is Arasbaran, Ziaran, Semaskand, Gorgan, and Fandoghlo, and the second branch of the population is Noshahr. It revealed that there were a close relationship among Arasbaran, Ziaran, Semaskand, Gorgan and Fandoghlo populations, while Noshahr population showed a higher diversity. The genetic diversity indices showed that the 6 investigated populations of Maral have probably experienced a bottleneck (Yuasa et al, 2006). Results of analysis D-loop sequences of Maral and other deer species showed that red deer, fawn deer and yellow deer were in a branch, but each was in separate groups, and Shoka was a separate branch. In this study, all Iranian Maral in a separate group of European, Asian and American red deer placed in one branch (Zamani, 2014). Placement of Polish red deer in the Maral Cluster, in network analysis, was an interesting result (Lorenzini, 2015); however, no historical evidence was found to support this result.
Conclusion: In conclusion, the recent fluctuations in population size and interruption in the gene flow are due to the past geographical transfer of red deer from a population to other habitats. A high risk of inbreeding was observed in Maral populations. Therefore, for conserving the populations, it is necessary to consider a program for introducing new blood from other populations and increasing their genetic diversity.