Evolutionary Analysis And Identification Of Elephantid Species Using Mitochondrial Protein Coding Genes

Mitochondrial protein coding genes(MPCGs)sequences have been widely used to assess evolutionary adaptations,evolutionary relationships,and interspecific variations necessary for species identification.The effectiveness of these genes has been assessed in a wide range of taxa.However,in elephantids,which comprises of three living species of elephants(i.e.Asian elephants,African savanna and African forest elephants)and the extinct mammoths including woolly mammoths,there still remain several unresolved issues.Firstly,the existence of mitochondrial variants in woolly mammoths and their role in adaptive evolution essential for tolerance to cold and dry environments has not been analyzed in detail;secondly,no comprehensive comparison has been done between cytochrome b(Cyt-b)and COI genes in regards to their effectiveness in revealing interspecific variations necessary for distinguishing elephantids.Thirdly,there is an urgent need for a highly specific and sensitive method to robustly separate elephantid species(including their haplotypes)while simultaneously excluding other species and contamination from human DNA.These issues were addressed in this study through:comprehensive analysis of thirteen complete MPCGs sequences of elephantids to test for evidence of adaptive selection in woolly mammoths;comparative assessment of effectiveness of reduced sequence lengths of cyt-b and COI genes in distinguishing elephantids;and development of a PCR-based method to separate elephantids.Firstly,we analysed thirteen mitochondrial protein-coding genes sequences from seventy five broadly distributed woolly mammoths to test for signatures of positive selection.Biochemical and protein structure analysis.Biochemical and homology protein analyses revealed that:eleven amino acid sites in six MPCGs genes(ND1,ND4,ND5,ND6,ATP6 and Cyt-b)show strong evidence of positive selection(adaptive changes)with some mutations being predicted to have influenced protein-protein interaction and oxidative phosphorylation process in mitochondrial complex I.Mutations in ATP6 and Cyt-b residues were predicted to have influenced metabolic regulatory chain.These findings suggest metabolic adaptations in mammoth's mitogenome in relation to extremely cold environments.Secondly,a comparative analysis of ninety five Cyt-b and COI gene sequences from the woolly mammoth and the three extant elephant species revealed that Cyt-b and COI contain sufficient interspecific variations to separate elephantids,although Cyt-b provides more accurate identifications.However,sequence lengths affect the overall effectiveness of both genes with COI being ineffective at short fragment sizes(<200 bp).Cyt-b can still provide correct identification when using fragment sizes as short as 100 bp,demonstrating its effectiveness in revealing species resolution.Thirdly,we developed a PCR-sequencing method based on a short sequence of the Cyt-b gene(116bp)to specifically amplify elephantids DNA while simultaneously avoiding human DNA contamination,and while excluding non-elephantid species and ivory substitutes was designed.The effectiveness of this method was tested using fecal DNA samples from Asian elephants(n=61),African savanna elephant(n=20),white rhino(Ceratotherium simum)(n=6)and common hippopotamus(Hippopotamus amphibius)(n=4),and ivory DNA samples from the woolly mammoth(n=61),forest elephant(n=8)and savanna elephant(n=2).This method showed a high success rate of amplification,sequencing and correct elephantids species identification.The amplification and sequencing success rate reached 100%for African savanna elephant and African Forest elephant ivory DNA,and 96.7%for fossilised mammoth ivory DNA.The detection limit of the PCR system was as low as 10 copy numbers of the target elephantid DNA,demonstrating its suitability for analysis of degraded samples.These findings suggest:(1)Inferences made regarding the role of positively selected mutations in metabolic adaptations can be further tested to provide more insights into significance and relevancy of these variations on other systems;(2)Cyt-b is better suited to highlight interspecific relationships among elephantids,and should be given priority over COI in this regard;(3)elephantid identification using assays targeting short yet highly informative gene fragments(e.g.116 bp Cyt-b assay designed in this study)is suitable for testing DNA derived from degraded and fossilized samples;(4)Owing matrilineal inheritance of mtDNA,hybrid elephants cannot be distinguished using mtDNA genes alone.Nuclear genes should therefore be examined in conjunction with mtDNA for more accurate assignment.