Study of Genetic Similarities and Phylogenetic Analysis of Iranian Dromedary and Bactrian Camels with Seven Major Species of the Camelidae Family Based on Mitochondrial Genome

Introduction: Mitochondrial DNA (mtDNA), also referred to as the mitogenome, has emerged as a pivotal tool in evolutionary biology and phylogenetics due to its maternal inheritance, high mutation rate, and lack of recombination. These characteristics make it an ideal molecular marker for tracing lineage divergences and understanding genetic relationships among species. This study leverages complete mitochondrial genome sequences, protein-coding genes (PCG), and single nucleotide polymorphism (SNP) data to elucidate the genetic similarities and phylogenetic relationships between Iranian dromedary (Camelus dromedarius) and Bactrian (Camelus bactrianus) camels and seven major species of the Camelidae family, including wild and domesticated members (including Vicugna vicugna, Lama guanicoe, Vicugna pacos, Lama glama, Camelus ferus, Camelus dromedarius and Camelus bactrianus). The Iranian dromedary and Bactrian camels play a vital role in desert ecosystems and local livelihoods, yet their populations face challenges due to habitat loss and declining traditional pastoralism The primary objectives of this research were to: 1) compare the complete mitochondrial genomes of Iranian camels with those of other Camelidae species to identify evolutionary divergences and conserved genetic regions, 2) analyze nucleotide and amino acid sequences of 13 protein-coding mitochondrial genes per each mitogenomes to assess functional conservation and divergence, 3) utilize SNP-based genomic data generated based on complete mitogenomes to construct a Genomic Relationship Matrix (GRM) and validate phylogenetic inferences, and 4) resolve the taxonomic classification of Camelidae species and clarify the evolutionary position of Iranian camels within the family.
Materials and Methods: Complete mitochondrial genomes of seven Camelidae species (Vicugna vicugna, Lama guanicoe, Vicugna pacos, Lama glama, Camelus ferus, Camelus dromedarius and Camelus bactrianus) and 10 Iranian camel haplotypes (four Camelus dromedary i.e., Haplotypes 1, 2, 3, and 4, and six Camelus Bactrian i.e., Haplotypes 1, 2, 3, 4, 5, and 6) were retrieved from NCBI. In following nucleotide and amino acid sequences of 13 PCGs (ATP6, ATP8, COX1, COX2, COX3, ND1, ND2, ND3, ND4, ND5, ND6, ND4L and CYTB) were extracted for comparative analysis. Also, a total of 3100 SNPs were derived from whole mitogenome alignments, filtered for quality, and used for population genomic analyses. Maximum Likelihood (ML) method were applied to whole mitogenome sequences and PCG alignments to reconstruct evolutionary relationships. Nucleotide diversity were calculated for PCGs and mitogenome regions. GRM was generated to quantify pairwise genetic distances. Principal Component Analysis (PCA), Heatmap clustering and Level Plot of common nucleotides were performed on the SNP dataset to visualize genetic structuring. A heatmap is a data visualization method based on color coding that is widely used to uncover hidden patterns in genomic data. A Level Plot is a graphical tool for displaying nucleotide similarity or differences between various samples based on SNP data, and is depicted as a color-coded or spectral matrix
Results and discussion: Phylogenetic trees revealed two primary clades: Clade/Claster A: Comprised Vicugna and Lama species (Vicugna vicugna, Lama guanicoe, Vicugna pacos and Lama glama), forming a distinct group. Clade/Claster B: Included Camelus ferus, Camelus dromedarius and Camelus bactrianus with Iranian dromedaries and bactrians forming subclusters closely related to Camelus ferus. Sequence identity between Iranian camels and other species ranged from 82.4% to 100%, with the highest similarity observed among C. dromedarius haplotypes. Analysis of nucleotide and amino acid sequences of all protein-coding genes (PCGs) showed a clustering pattern similar to that of the complete mitochondrial genomes. However, ATP8 exhibited the highest variability, while COX1 was the most conserved, reflecting differential evolutionary pressures. Heatmap, PCA, and Level Plot analyses of SNP-based nucleotide similarities and GRM data confirmed the results from the complete mtDNA analysis (the separation of Vicugna/Lama and Camelus camelids, with Iranian camels grouping distinctly within the latter), demonstrating that SNPs are an effective tool for phylogenetic and evolutionary studies. SNP-based analyses proved highly congruent with traditional mtDNA methods, validating their utility for high-resolution phylogenetic studies.
Conclusion: This study provides a comprehensive genomic framework for understanding the evolutionary history of Camelidae, demonstrating the power of mitochondrial genomics and SNP-based approaches in phylogenetics as comparative genomic analysis of mtDNA can robustly classify the major species of the Camelidae family and clarify their evolutionary relationships. The results of the present study not only refine the classification of Iranian camels but also offer a foundation for future research on camelid adaptation, domestication, and conservation.