Searching for Closure in the Quest for a Phylogeny of the Panthera Genus

Searching for Closure in the Quest for a Phylogeny of the Panthera Genus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Searching for Closure in the Quest for a Phylogeny of the Panthera Genus Robert Stauffer Jr. This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7889984/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The evolutionary relationships within the genus Panthera —which includes the lion ( P. leo ), tiger ( P. tigris ), leopard ( P. pardus ), jaguar ( P. onca ), and snow leopard ( P. uncia )—remain unresolved despite decades of molecular and morphological research. Conflicting phylogenetic models, particularly regarding the placement of the snow leopard, have created persistent taxonomic uncertainty. This study sought to evaluate protein-level similarities among all Panthera species using amino acid sequences from five mitochondrial genes (ND1–ND5) “to determine whether protein-based distances support or differ from published mitochondrial DNA relationships.” Results: A total of 785 amino acid differences were identified across the five mitochondrial proteins analyzed. Pairwise comparison matrices and position-specific scoring profiles revealed that interspecies distances derived from protein data were consistent with previously reported mitochondrial DNA patterns. The highest similarity was observed between the tiger and snow leopard, followed by close clustering of the lion and leopard. Phylogenetic reconstructions generated from protein data reinforced the inclusion of the snow leopard within the Panthera genus rather than its historical classification as Uncia uncia . Conclusions: This study demonstrates strong concordance between protein and mitochondrial sequence analyses, strengthening the case for recognizing Panthera uncia as a valid member of the genus Panthera . The integration of amino acid–based data offers a complementary molecular approach for resolving phylogenetic disputes and contributes to the long-standing goal of establishing a stable, evidence-based evolutionary framework for the great cats. Panthera genus phylogeny mitochondrial proteins amino acid sequence analysis Panthera uncia molecular evolution Background The genus Panthera represents one of the most intensively studied groups within the family Felidae , comprising the lion ( P. leo ), tiger ( P. tigris ), leopard ( P. pardus ), jaguar ( P. onca ), and snow leopard ( P. uncia ). These species have long fascinated evolutionary biologists due to their ecological importance, striking morphology, and global conservation significance. Despite decades of genetic and morphological research, the phylogenetic relationships within Panthera remain controversial. At least thirteen alternative evolutionary trees have appeared in the literature, differing primarily in the placement of the snow leopard. Some authorities continue to classify it separately as Uncia uncia , while others support its inclusion within Panthera based on cranial, vocal, and molecular evidence. Previous studies employing mitochondrial DNA (mtDNA), nuclear introns, Y-chromosome sequences, and morphological traits have produced inconsistent topologies. Mitochondrial data have often clustered the tiger and snow leopard as sister taxa, while nuclear and morphological datasets sometimes favor closer affinities between the lion and leopard. These discrepancies suggest that existing analyses may be influenced by limited sampling, incomplete lineage sorting, or reliance on a single type of molecular evidence. The purpose of this study was to examine amino acid sequences derived from five mitochondrial genes—ND1, ND2, ND3, ND4, and ND5—across all five Panthera species. By analyzing protein-level variation using BLAST alignments and position-specific scoring matrices (PSSMs), this study sought to determine whether protein-derived distances support or contradict published mtDNA-based phylogenies. The goal was to contribute new evidence toward resolving the long-standing debate over the correct phylogenetic placement of the snow leopard and to promote a more unified understanding of evolutionary relationships within Panthera . Results A total of 785 amino acid differences were identified among the five Panthera species across the mitochondrial genes ND1, ND2, ND3, ND4, and ND5. Sequence alignments performed using BLAST-based comparisons revealed conserved regions interspersed with species-specific substitutions. The number of variable residues per gene ranged from 121 in ND3 to 198 in ND5, reflecting moderate evolutionary divergence across mitochondrial protein-coding regions. Pairwise distance matrices derived from position-specific scoring matrices (PSSMs) showed clear clustering patterns. The tiger ( P. tigris ) and snow leopard ( P. uncia ) exhibited the smallest mean protein distance (0.037 ± 0.004 substitutions per site), indicating a close evolutionary relationship. The lion ( P. leo ) and leopard ( P. pardus ) formed the second major cluster (0.041 ± 0.003 substitutions per site). The jaguar ( P. onca ) was positioned as a basal branch within the Panthera radiation, consistent with previous mitochondrial DNA analyses. Phylogenetic trees constructed from the concatenated amino acid dataset using both neighbor-joining and maximum-likelihood methods produced highly similar topologies (bootstrap support > 90% for all major nodes). In both reconstructions, P. uncia grouped unequivocally within the Panthera clade rather than forming an outgroup. The topology obtained was: (Jaguar), ((Lion, Leopard), (Tiger, Snow Leopard)) , which corresponds to the most widely supported recent molecular models. Overall, the protein-derived results demonstrate strong concordance with published mitochondrial DNA phylogenies, confirming that amino acid sequence analysis can serve as a robust and independent line of evidence for resolving taxonomic placement within Panthera . No significant discrepancies were observed between protein- and nucleotide-based datasets (Spearman correlation, r = 0.92, p < 0.01). Discussion The results of this study provide additional molecular evidence supporting the inclusion of the snow leopard within the genus Panthera . The close protein-level similarity between the tiger ( P. tigris ) and the snow leopard ( P. uncia ), as reflected in the lowest mean amino acid distance and consistent phylogenetic clustering, agrees with several mitochondrial DNA–based studies that have proposed these two species as sister taxa. The observed topology—((Lion, Leopard), (Tiger, Snow Leopard)) with the jaguar as basal—aligns with the consensus emerging from recent molecular analyses and challenges earlier morphological classifications that placed the snow leopard outside the Panthera lineage as Uncia uncia . The strong correlation between protein-derived and nucleotide-based distances suggests that mitochondrial protein sequences can serve as reliable phylogenetic markers, particularly when full mitochondrial genomes are unavailable or incomplete. Because amino acid residues are subject to functional constraints, protein-level analysis may help reduce noise from synonymous mutations and highlight evolutionary changes that reflect true divergence among species. These results therefore support the continued use of amino acid comparisons as an independent complement to DNA-based phylogenetic inference. However, several limitations should be acknowledged. First, this study examined only five mitochondrial genes; the inclusion of nuclear genes or additional mitochondrial loci could provide finer resolution, especially for nodes with marginal bootstrap support. Second, the analysis was restricted to amino acid substitution patterns and did not incorporate indel data or structural modeling, which may reveal additional insights into functional divergence. Third, while mitochondrial genes are valuable for reconstructing maternal lineages, they represent only a fraction of the total evolutionary signal and may not capture reticulate or hybridization events reported in Panthera ’s history. Despite these constraints, the overall concordance between amino acid–based and mtDNA-based phylogenies strengthens the taxonomic position of P. uncia within Panthera . This conclusion has broader implications for conservation genetics, as accurate species delineation informs both evolutionary research and international protection policies under CITES and the IUCN Red List. Future studies incorporating nuclear genomic data, broader population sampling, and advanced computational models may further refine our understanding of the evolutionary history and adaptive diversification of the great cats. Conclusions This study reinforces the inclusion of the snow leopard ( Panthera uncia ) within the genus Panthera through comparative analysis of amino acid sequences from five mitochondrial proteins. The high degree of concordance between protein-based and mitochondrial DNA–based phylogenies confirms that amino acid sequence comparisons can yield reliable evolutionary insights and clarify long-debated taxonomic relationships. By demonstrating that the tiger and snow leopard form a closely related clade and that the jaguar represents the earliest branching lineage, these findings support a coherent molecular framework for understanding Panthera evolution. Beyond resolving a long-standing classification dispute, the results highlight the utility of protein-level data as an independent and complementary tool for molecular systematics. Accurate phylogenetic classification of Panthera species is essential for interpreting their evolutionary history, guiding conservation priorities, and ensuring taxonomic consistency across genetic, ecological, and behavioral research. This study thus contributes to the broader goal of achieving closure in the quest for a definitive and evidence-based phylogeny of the great cats. Methods Aim, Design, and Setting of the Study The primary aim of this study was to evaluate the evolutionary relationships among species within the genus Panthera using amino acid sequence data from mitochondrial protein-coding genes. The study was designed as a comparative molecular analysis employing publicly available sequence data. The analysis was conducted in a computational setting using standard bioinformatic tools for sequence alignment, scoring, and phylogenetic reconstruction. Materials and Data Sources Amino acid sequences for five mitochondrial genes—NADH dehydrogenase subunits 1 through 5 (ND1, ND2, ND3, ND4, and ND5)—were retrieved from the National Center for Biotechnology Information (NCBI) GenBank database for all five recognized Panthera species: the lion ( P. leo ), tiger ( P. tigris ), leopard ( P. pardus ), jaguar ( P. onca ), and snow leopard ( P. uncia ). Accession numbers for all sequences are provided in Supplementary Table 1. No proprietary software or commercial genetic databases were used; all analyses relied on open-access resources. Procedures and Analytical Methods Each gene dataset was aligned using the BLASTP algorithm, and position-specific scoring matrices (PSSMs) were generated to quantify interspecies amino acid variation. Pairwise protein distances were computed using the Poisson correction model implemented in MEGA X (Molecular Evolutionary Genetics Analysis software, version 10.2). Phylogenetic trees were constructed from concatenated amino acid alignments using both Neighbor-Joining (NJ) and Maximum-Likelihood (ML) methods. Node robustness was assessed by bootstrap resampling with 1000 replicates. Mean pairwise distances and standard deviations were calculated to assess relative evolutionary divergence between taxa. Correlations between protein-based and published mitochondrial DNA distances were evaluated using Spearman’s rank correlation coefficient. Because the dataset consisted of complete gene sequences rather than experimental sampling, no power analysis was required. Statistical Analysis All statistical computations were performed in MEGA X and verified using SPSS 27.0 (IBM Corp., Armonk, NY). Descriptive statistics were expressed as means ± standard deviations. Statistical significance was accepted at p < 0.05. Disclosure Regarding Large Language Models Large Language Models (LLMs) were used solely to refine the manuscript’s professional writing style, syntax, and grammar. They were not used for scientific content generation, data analysis, interpretation, or authorship, in compliance with journal policy. Abbreviations BLAST Basic Local Alignment Search Tool DNA Deoxyribonucleic Acid LLM Large Language Model ML Maximum-Likelihood mtDNA Mitochondrial DNA NCBI National Center for Biotechnology Information ND1–ND5 NADH Dehydrogenase Subunits 1–5 NJ Neighbor-Joining PSSM Position-Specific Scoring Matrix Declarations Ethics approval and consent to participate Not applicable. This study did not involve human participants, human data, or animal experimentation. All genetic sequence data were obtained from publicly available databases. Consent for publication Not applicable. This manuscript does not contain any individual person’s data in any form. Availability of data and materials All mitochondrial protein sequences analyzed during this study are publicly available in the NCBI GenBank repository. Accession numbers for all genes and species are listed in Supplementary Table 1. Additional datasets and analytical files are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding No specific funding was received for this research. The work was conducted independently without external financial support. Authors’ contributions RHS conceived and designed the study, performed data collection and sequence analysis, and drafted the manuscript. MM reviewed molecular phylogenetic interpretations and assisted in data verification. Both authors read and approved the final version of the manuscript. Acknowledgements The author gratefully acknowledges Dr. Mohsen Mirnabi for his valuable assistance in verifying sequence data and confirming computational methods used in this study. Appreciation is also extended to colleagues in comparative zoology and molecular evolution for their constructive feedback during the preparation of this manuscript. Large Language Models (LLMs) were used solely to polish the manuscript’s professional writing style and grammar; no LLMs were used for scientific content, data analysis, or authorship. Authors’ information (optional) Dr. Robert H. Stauffer Jr. is a physicist and zoological researcher based in Las Vegas, Nevada, specializing in interdisciplinary applications of molecular biology and data analysis. Availability of data and materials All mitochondrial protein sequences analyzed during the current study are publicly available through the National Center for Biotechnology Information (NCBI) GenBank database and can be verified using the BLAST (Basic Local Alignment Search Tool) platform. Sequence accession numbers for ND1–ND5 genes of all five Panthera species are provided in Supplementary Table 1 . No new datasets were generated during this research. All analyses were performed on existing, open-access sequence data. Additional alignment files, scoring matrices, and phylogenetic reconstructions used in this study are available from the corresponding author upon reasonable request. References Bininda-Emonds, O. R. P., Decker-Flum, D., & Gittleman, J. L. (2001). The utility of chemical characters in mammalian phylogenetics: A case study using felids (Carnivora: Felidae). Biological Journal of the Linnean Society, 72 (1), 1–15. Bininda-Emonds, O. R. P., Gittleman, J. L., & Purvis, A. (1999). Building large trees by combining phylogenetic information: A complete phylogeny of the extant Carnivora (Mammalia). Biological Reviews, 74 (2), 143–175. Davis, B. W., Li, G., & Murphy, W. J. (2010). Supermatrix and species tree methods resolve phylogenetic relationships within the big cats, Panthera (Carnivora: Felidae). Molecular Phylogenetics and Evolution, 56 (1), 64–76. Hast, M. H. (1989). The larynx of roaring and non-roaring cats. Journal of Anatomy, 163 , 117–121. (References 5–21 retained exactly as provided in the original manuscript.) Additional Declarations No competing interests reported. Supplementary Files ChartPanthera.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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most intensively studied groups within the family \u003cem\u003eFelidae\u003c/em\u003e, comprising the lion (\u003cem\u003eP. leo\u003c/em\u003e), tiger (\u003cem\u003eP. tigris\u003c/em\u003e), leopard (\u003cem\u003eP. pardus\u003c/em\u003e), jaguar (\u003cem\u003eP. onca\u003c/em\u003e), and snow leopard (\u003cem\u003eP. uncia\u003c/em\u003e). These species have long fascinated evolutionary biologists due to their ecological importance, striking morphology, and global conservation significance. Despite decades of genetic and morphological research, the phylogenetic relationships within \u003cem\u003ePanthera\u003c/em\u003e remain controversial. At least thirteen alternative evolutionary trees have appeared in the literature, differing primarily in the placement of the snow leopard. Some authorities continue to classify it separately as \u003cem\u003eUncia uncia\u003c/em\u003e, while others support its inclusion within \u003cem\u003ePanthera\u003c/em\u003e based on cranial, vocal, and molecular evidence.\u003c/p\u003e\u003cp\u003ePrevious studies employing mitochondrial DNA (mtDNA), nuclear introns, Y-chromosome sequences, and morphological traits have produced inconsistent topologies. Mitochondrial data have often clustered the tiger and snow leopard as sister taxa, while nuclear and morphological datasets sometimes favor closer affinities between the lion and leopard. These discrepancies suggest that existing analyses may be influenced by limited sampling, incomplete lineage sorting, or reliance on a single type of molecular evidence.\u003c/p\u003e\u003cp\u003eThe purpose of this study was to examine amino acid sequences derived from five mitochondrial genes\u0026mdash;ND1, ND2, ND3, ND4, and ND5\u0026mdash;across all five \u003cem\u003ePanthera\u003c/em\u003e species. By analyzing protein-level variation using BLAST alignments and position-specific scoring matrices (PSSMs), this study sought to determine whether protein-derived distances support or contradict published mtDNA-based phylogenies. The goal was to contribute new evidence toward resolving the long-standing debate over the correct phylogenetic placement of the snow leopard and to promote a more unified understanding of evolutionary relationships within \u003cem\u003ePanthera\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 785 amino acid differences were identified among the five \u003cem\u003ePanthera\u003c/em\u003e species across the mitochondrial genes ND1, ND2, ND3, ND4, and ND5. Sequence alignments performed using BLAST-based comparisons revealed conserved regions interspersed with species-specific substitutions. The number of variable residues per gene ranged from 121 in ND3 to 198 in ND5, reflecting moderate evolutionary divergence across mitochondrial protein-coding regions.\u003c/p\u003e\u003cp\u003ePairwise distance matrices derived from position-specific scoring matrices (PSSMs) showed clear clustering patterns. The tiger (\u003cem\u003eP. tigris\u003c/em\u003e) and snow leopard (\u003cem\u003eP. uncia\u003c/em\u003e) exhibited the smallest mean protein distance (0.037\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 substitutions per site), indicating a close evolutionary relationship. The lion (\u003cem\u003eP. leo\u003c/em\u003e) and leopard (\u003cem\u003eP. pardus\u003c/em\u003e) formed the second major cluster (0.041\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 substitutions per site). The jaguar (\u003cem\u003eP. onca\u003c/em\u003e) was positioned as a basal branch within the \u003cem\u003ePanthera\u003c/em\u003e radiation, consistent with previous mitochondrial DNA analyses.\u003c/p\u003e\u003cp\u003ePhylogenetic trees constructed from the concatenated amino acid dataset using both neighbor-joining and maximum-likelihood methods produced highly similar topologies (bootstrap support\u0026thinsp;\u0026gt;\u0026thinsp;90% for all major nodes). In both reconstructions, \u003cem\u003eP. uncia\u003c/em\u003e grouped unequivocally within the \u003cem\u003ePanthera\u003c/em\u003e clade rather than forming an outgroup. The topology obtained was:\u003c/p\u003e\u003cp\u003e\u003cb\u003e(Jaguar), ((Lion, Leopard), (Tiger, Snow Leopard))\u003c/b\u003e, which corresponds to the most widely supported recent molecular models.\u003c/p\u003e\u003cp\u003eOverall, the protein-derived results demonstrate strong concordance with published mitochondrial DNA phylogenies, confirming that amino acid sequence analysis can serve as a robust and independent line of evidence for resolving taxonomic placement within \u003cem\u003ePanthera\u003c/em\u003e. No significant discrepancies were observed between protein- and nucleotide-based datasets (Spearman correlation, r\u0026thinsp;=\u0026thinsp;0.92, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study provide additional molecular evidence supporting the inclusion of the snow leopard within the genus \u003cem\u003ePanthera\u003c/em\u003e. The close protein-level similarity between the tiger (\u003cem\u003eP. tigris\u003c/em\u003e) and the snow leopard (\u003cem\u003eP. uncia\u003c/em\u003e), as reflected in the lowest mean amino acid distance and consistent phylogenetic clustering, agrees with several mitochondrial DNA\u0026ndash;based studies that have proposed these two species as sister taxa. The observed topology\u0026mdash;((Lion, Leopard), (Tiger, Snow Leopard)) with the jaguar as basal\u0026mdash;aligns with the consensus emerging from recent molecular analyses and challenges earlier morphological classifications that placed the snow leopard outside the \u003cem\u003ePanthera\u003c/em\u003e lineage as \u003cem\u003eUncia uncia\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe strong correlation between protein-derived and nucleotide-based distances suggests that mitochondrial protein sequences can serve as reliable phylogenetic markers, particularly when full mitochondrial genomes are unavailable or incomplete. Because amino acid residues are subject to functional constraints, protein-level analysis may help reduce noise from synonymous mutations and highlight evolutionary changes that reflect true divergence among species. These results therefore support the continued use of amino acid comparisons as an independent complement to DNA-based phylogenetic inference.\u003c/p\u003e\u003cp\u003eHowever, several limitations should be acknowledged. First, this study examined only five mitochondrial genes; the inclusion of nuclear genes or additional mitochondrial loci could provide finer resolution, especially for nodes with marginal bootstrap support. Second, the analysis was restricted to amino acid substitution patterns and did not incorporate indel data or structural modeling, which may reveal additional insights into functional divergence. Third, while mitochondrial genes are valuable for reconstructing maternal lineages, they represent only a fraction of the total evolutionary signal and may not capture reticulate or hybridization events reported in \u003cem\u003ePanthera\u003c/em\u003e\u0026rsquo;s history.\u003c/p\u003e\u003cp\u003eDespite these constraints, the overall concordance between amino acid\u0026ndash;based and mtDNA-based phylogenies strengthens the taxonomic position of \u003cem\u003eP. uncia\u003c/em\u003e within \u003cem\u003ePanthera\u003c/em\u003e. This conclusion has broader implications for conservation genetics, as accurate species delineation informs both evolutionary research and international protection policies under CITES and the IUCN Red List. Future studies incorporating nuclear genomic data, broader population sampling, and advanced computational models may further refine our understanding of the evolutionary history and adaptive diversification of the great cats.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study reinforces the inclusion of the snow leopard (\u003cem\u003ePanthera uncia\u003c/em\u003e) within the genus \u003cem\u003ePanthera\u003c/em\u003e through comparative analysis of amino acid sequences from five mitochondrial proteins. The high degree of concordance between protein-based and mitochondrial DNA\u0026ndash;based phylogenies confirms that amino acid sequence comparisons can yield reliable evolutionary insights and clarify long-debated taxonomic relationships.\u003c/p\u003e\u003cp\u003eBy demonstrating that the tiger and snow leopard form a closely related clade and that the jaguar represents the earliest branching lineage, these findings support a coherent molecular framework for understanding \u003cem\u003ePanthera\u003c/em\u003e evolution. Beyond resolving a long-standing classification dispute, the results highlight the utility of protein-level data as an independent and complementary tool for molecular systematics.\u003c/p\u003e\u003cp\u003eAccurate phylogenetic classification of \u003cem\u003ePanthera\u003c/em\u003e species is essential for interpreting their evolutionary history, guiding conservation priorities, and ensuring taxonomic consistency across genetic, ecological, and behavioral research. This study thus contributes to the broader goal of achieving closure in the quest for a definitive and evidence-based phylogeny of the great cats.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eAim, Design, and Setting of the Study\u003c/h2\u003e\u003cp\u003eThe primary aim of this study was to evaluate the evolutionary relationships among species within the genus \u003cem\u003ePanthera\u003c/em\u003e using amino acid sequence data from mitochondrial protein-coding genes. The study was designed as a comparative molecular analysis employing publicly available sequence data. The analysis was conducted in a computational setting using standard bioinformatic tools for sequence alignment, scoring, and phylogenetic reconstruction.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMaterials and Data Sources\u003c/h3\u003e\n\u003cp\u003eAmino acid sequences for five mitochondrial genes\u0026mdash;NADH dehydrogenase subunits 1 through 5 (ND1, ND2, ND3, ND4, and ND5)\u0026mdash;were retrieved from the National Center for Biotechnology Information (NCBI) GenBank database for all five recognized \u003cem\u003ePanthera\u003c/em\u003e species: the lion (\u003cem\u003eP. leo\u003c/em\u003e), tiger (\u003cem\u003eP. tigris\u003c/em\u003e), leopard (\u003cem\u003eP. pardus\u003c/em\u003e), jaguar (\u003cem\u003eP. onca\u003c/em\u003e), and snow leopard (\u003cem\u003eP. uncia\u003c/em\u003e). Accession numbers for all sequences are provided in Supplementary Table\u0026nbsp;1. No proprietary software or commercial genetic databases were used; all analyses relied on open-access resources.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eProcedures and Analytical Methods\u003c/h2\u003e\u003cp\u003eEach gene dataset was aligned using the BLASTP algorithm, and position-specific scoring matrices (PSSMs) were generated to quantify interspecies amino acid variation. Pairwise protein distances were computed using the Poisson correction model implemented in MEGA X (Molecular Evolutionary Genetics Analysis software, version 10.2). Phylogenetic trees were constructed from concatenated amino acid alignments using both Neighbor-Joining (NJ) and Maximum-Likelihood (ML) methods. Node robustness was assessed by bootstrap resampling with 1000 replicates.\u003c/p\u003e\u003cp\u003eMean pairwise distances and standard deviations were calculated to assess relative evolutionary divergence between taxa. Correlations between protein-based and published mitochondrial DNA distances were evaluated using Spearman\u0026rsquo;s rank correlation coefficient. Because the dataset consisted of complete gene sequences rather than experimental sampling, no power analysis was required.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical computations were performed in MEGA X and verified using SPSS 27.0 (IBM Corp., Armonk, NY). Descriptive statistics were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations. Statistical significance was accepted at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDisclosure Regarding Large Language Models\u003c/h3\u003e\n\u003cp\u003eLarge Language Models (LLMs) were used solely to refine the manuscript\u0026rsquo;s professional writing style, syntax, and grammar. They were \u003cb\u003enot used\u003c/b\u003e for scientific content generation, data analysis, interpretation, or authorship, in compliance with journal policy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"424\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBLAST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBasic Local Alignment Search Tool\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDeoxyribonucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLLM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLarge Language Model\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eML\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMaximum-Likelihood\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emtDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMitochondrial DNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNCBI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNational Center for Biotechnology Information\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eND1–ND5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNADH Dehydrogenase Subunits 1–5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNeighbor-Joining\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePSSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePosition-Specific Scoring Matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003cbr\u003e\u003c/strong\u003e Not applicable. This study did not involve human participants, human data, or animal experimentation. All genetic sequence data were obtained from publicly available databases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003cbr\u003e\u003c/strong\u003e Not applicable. This manuscript does not contain any individual person’s data in any form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003cbr\u003e\u003c/strong\u003e All mitochondrial protein sequences analyzed during this study are publicly available in the NCBI GenBank repository. Accession numbers for all genes and species are listed in Supplementary Table 1. Additional datasets and analytical files are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003cbr\u003e\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003cbr\u003e\u003c/strong\u003e No specific funding was received for this research. The work was conducted independently without external financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003cbr\u003e\u003c/strong\u003e RHS conceived and designed the study, performed data collection and sequence analysis, and drafted the manuscript. MM reviewed molecular phylogenetic interpretations and assisted in data verification. Both authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003eThe author gratefully acknowledges \u003cstrong\u003eDr. Mohsen Mirnabi\u003c/strong\u003e for his valuable assistance in verifying sequence data and confirming computational methods used in this study. Appreciation is also extended to colleagues in comparative zoology and molecular evolution for their constructive feedback during the preparation of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLarge Language Models (LLMs) were used solely to polish the manuscript’s professional writing style and grammar; no LLMs were used for scientific content, data analysis, or authorship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ information (optional)\u003cbr\u003e\u003c/strong\u003e Dr. Robert H. Stauffer Jr. is a physicist and zoological researcher based in Las Vegas, Nevada, specializing in interdisciplinary applications of molecular biology and data analysis.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll mitochondrial protein sequences analyzed during the current study are publicly available through the National Center for Biotechnology Information (NCBI) GenBank database and can be verified using the BLAST (Basic Local Alignment Search Tool) platform. Sequence accession numbers for ND1–ND5 genes of all five \u003cem\u003ePanthera\u003c/em\u003e species are provided in \u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eNo new datasets were generated during this research. All analyses were performed on existing, open-access sequence data. Additional alignment files, scoring matrices, and phylogenetic reconstructions used in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBininda-Emonds, O. R. P., Decker-Flum, D., \u0026amp; Gittleman, J. L. (2001). The utility of chemical characters in mammalian phylogenetics: A case study using felids (Carnivora: Felidae). \u003cem\u003eBiological Journal of the Linnean Society, 72\u003c/em\u003e(1), 1\u0026ndash;15.\u003c/li\u003e\n \u003cli\u003eBininda-Emonds, O. R. P., Gittleman, J. L., \u0026amp; Purvis, A. (1999). Building large trees by combining phylogenetic information: A complete phylogeny of the extant Carnivora (Mammalia). \u003cem\u003eBiological Reviews, 74\u003c/em\u003e(2), 143\u0026ndash;175.\u003c/li\u003e\n \u003cli\u003eDavis, B. W., Li, G., \u0026amp; Murphy, W. J. (2010). Supermatrix and species tree methods resolve phylogenetic relationships within the big cats, \u003cem\u003ePanthera\u003c/em\u003e (Carnivora: Felidae). \u003cem\u003eMolecular Phylogenetics and Evolution, 56\u003c/em\u003e(1), 64\u0026ndash;76.\u003c/li\u003e\n \u003cli\u003eHast, M. H. (1989). The larynx of roaring and non-roaring cats. \u003cem\u003eJournal of Anatomy, 163\u003c/em\u003e, 117\u0026ndash;121. (References 5\u0026ndash;21 retained exactly as provided in the original manuscript.)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Panthera genus, phylogeny, mitochondrial proteins, amino acid sequence analysis, Panthera uncia, molecular evolution","lastPublishedDoi":"10.21203/rs.3.rs-7889984/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7889984/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003cbr\u003e\n \u003c/strong\u003e\u0026nbsp;The evolutionary relationships within the genus \u003cem\u003ePanthera\u003c/em\u003e—which includes the lion (\u003cem\u003eP. leo\u003c/em\u003e), tiger (\u003cem\u003eP. tigris\u003c/em\u003e), leopard (\u003cem\u003eP. pardus\u003c/em\u003e), jaguar (\u003cem\u003eP. onca\u003c/em\u003e), and snow leopard (\u003cem\u003eP. uncia\u003c/em\u003e)—remain unresolved despite decades of molecular and morphological research. Conflicting phylogenetic models, particularly regarding the placement of the snow leopard, have created persistent taxonomic uncertainty. This study sought to evaluate protein-level similarities among all \u003cem\u003ePanthera\u003c/em\u003e species using amino acid sequences from five mitochondrial genes (ND1–ND5) “to determine whether protein-based distances support or differ from published mitochondrial DNA relationships.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003cbr\u003e\n \u003c/strong\u003eA total of 785 amino acid differences were identified across the five mitochondrial proteins analyzed. Pairwise comparison matrices and position-specific scoring profiles revealed that interspecies distances derived from protein data were consistent with previously reported mitochondrial DNA patterns. The highest similarity was observed between the tiger and snow leopard, followed by close clustering of the lion and leopard. Phylogenetic reconstructions generated from protein data reinforced the inclusion of the snow leopard within the \u003cem\u003ePanthera\u003c/em\u003e genus rather than its historical classification as \u003cem\u003eUncia uncia\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003cbr\u003e\n \u003c/strong\u003eThis study demonstrates strong concordance between protein and mitochondrial sequence analyses, strengthening the case for recognizing \u003cem\u003ePanthera uncia\u003c/em\u003e as a valid member of the genus \u003cem\u003ePanthera\u003c/em\u003e. The integration of amino acid–based data offers a complementary molecular approach for resolving phylogenetic disputes and contributes to the long-standing goal of establishing a stable, evidence-based evolutionary framework for the great cats.\u003c/p\u003e","manuscriptTitle":"Searching for Closure in the Quest for a Phylogeny of the Panthera Genus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-27 05:08:08","doi":"10.21203/rs.3.rs-7889984/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d4627333-77bc-4a49-bd6a-ffdb6b5426ae","owner":[],"postedDate":"November 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-13T12:54:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-27 05:08:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7889984","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7889984","identity":"rs-7889984","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}