Phylogenetic supertree reveals detailed evolution of SARS-CoV-2

Phylogenetic supertree reveals detailed evolution of SARS-CoV-2 | 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 Phylogenetic supertree reveals detailed evolution of SARS-CoV-2 Tingting Li, Dongxia Liu, Yadi Yang, Jiali Guo, Yujie Feng, Xinmo Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-33194/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Dec, 2020 Read the published version in Scientific Reports → Version 1 posted You are reading this latest preprint version Abstract Corona Virus Disease 2019 (COVID-19) caused by the emerged coronavirus SARS-CoV-2 is spreading globally. The origin of SARS-Cov-19 and its evolutionary relationship is still ambiguous. Several reports attempted to figure out this critical issue by genome-based phylogenetic analysis, with limited progress. Here we applied phylogenetic supertree analysis to study the origin and evolution of SARS-CoV-2. Phylogenetic supertree analysis firmly disputes the accuracy of bat coronavirus RaTG13 be the last common ancestor of SARS- CoV-2s reported in other phylogenetic tree analysis based on viral genome sequences, although RaTG13 shows 96.5% similarity with SARS-CoV-2 in the genome. Therefore, viewing RaTG13 as the last common ancestor of SARS-CoV-2 would seriously mislead phylogenetic inference of SARS-CoV-2. Importantly, the discovery of evolution and mutation in SARS-CoV-2s was achieved by phylogenetic supertree analysis. Taken together, the phylogenetic supertree showed extraordinary priority on the SARS-CoV-2 evolution inference relative to the normal phylogenetic tree based on full-length genomic sequences. Evolutionary Genetics SARS-CoV-2 COVID-19 coronavirus supertree phylogenetics Figures Figure 1 Figure 2 Introduction Severe acute respiratory syndrome coronavirus–2 (SARS-CoV–2), a novel coronavirus emerged in December 2019, causes an ongoing outbreak of Corona Virus Disease 2019 (COVID–19). COVID–19 has caused a global pandemic, and as of 15 April 2020, 198,1239 cases of infections have been confirmed in more than 110 countries, with 12,6681 deaths 1. Currently, three coronaviruses known for causing severe human disease, are SARS-CoV–2, SARS-CoV, and MERS-CoV, with the last two viruses caused epidemics in the past. Although three viruses are identified as beta-coronavirus within the subgenus of the sarbecoronavirus achieved by the full-length genomic sequence analysis, SARS-CoV–2 is divergent from SARS-CoV and MERS-CoV, and it belongs to a distinct lineage 2. More and more genomes of SARS-CoV–2 isolates were sequenced all around the world, which creates the opportunity for precise analysis of phylogeny and evolution of SARS-CoV–2 2–6. However, the detailed evolution of SARS-CoV–2 is still under the veil, ascribed to the reality that SARS-CoV–2 isolates displayed barely detected phylogenetic distance among each other in the phylogenetic tree with further related coronavirus as outgroup that was constructed based on a single gene in the whole genome or one gene composed of the full-length genomic sequence. In particular, there are several limitations to the methods for phylogenetic tree construction mentioned before. Phylogenetic methods using single genes to determine the relationships between viruses face the problem of gene selection, with different trees yielded from different genes, which are not always consistent but often conflict. Considering the high diversity in sizes of genes consisting of coronavirus genome, the phylogenetic analysis with full-length SARS-CoV–2 genomic sequence hardly provide reliable evolutionary information, based on the probability that it will drown out plenty of phylogenetic information retained in some proteins coded by the smaller size of genes, which are vital for viral infection and virulence. For example, the gene of ORF1ab (21,290 bp) in SARS-CoV–2 genome comprises about 75% of the whole genome sequence, while the genes of key functional and structural proteins, including S (3,822 bp), E (228 bp), M (668 bp), and N (1,260 bp), take up less than 22%. Furthermore, the above two approaches for phylogenetic analysis require orthologous genes in the species to allow a meaningful comparison, which would limit the employment of species with large phylogenetic distance as outgroup. For example, at least five proteins of SARS-CoV–2 fail to get corresponding orthologous proteins in beta coronavirus MERS-CoV, and the ORF8 of SARS-CoV–2 has no orthologous proteins in sarbecoronavirus SARS-CoV. Therefore, such phylogenies can seriously mislead evolutionary events in between. Supertree method, whose embryonic theory was described in the 1980s 7,8, can merge a set of consistent or inconsistent phylogenetic trees based on different genes in the genome, into one comprehensive phylogenetic tree reasonably 9. The phylogenetic network based on supertree method revealed non-vertical evolution scenario during the evolutionary history of haloarchaea, which was improbable for phylogenetic approaches relied on a single gene or the full-length genome sequences 10. Given its superiority, the supertree method was employed here for phylogenetic analysis of SARS-CoV–2 virus, by combining the phylogenetic information from ten genes of the coronavirus. Our study aims to figure out the origin and evolution of SARS-CoV–2 through phylogenetic supertree analysis. Material And Methods Dataset construction The full-length genomic sequences and protein-coding sequences (CDSs) of 102 SARS-CoV- 2, 5 SARS-CoV, 2 MERS-CoV, and 11 bat coronaviruses were downloaded from NCBI Severe acute respiratory syndrome coronavirus 2 data hub (https ://www . ncbi.nlm.nih.gov/labs/virus/vssi/#/ ) and GenBank ( http://www.ncbi.nlm.nih.gov/genbank/ ) (Table S1). Among genomic sequences of SARS- CoV and bat coronaviruses, those showing high similarity with genomic sequences of SARS- CoV–2 were chosen. The integrity of sequences was checked, and the fragmented sequences were reconstructed. Finally, the datasets were constructed by labeling the sequences with the region of sampling and collection date. Construction of phylogenetic tree with the whole genomic sequences The full-length genomic sequences of 120 coronaviruses were aligned using the L-INS-i method of MAFFT v7.310 11. Aligned sequences were converted into phylip file format by Clustal W 12. Maximum likelihood (ML) trees based on full-length genomic sequences were constructed and estimated by PhyML program version 3.0 13 with 100 bootstraps resampling. The phylogenetic trees were visualized by FigTree v1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/) . Construction of phylogenetic supertrees Ten groups of CDSs for orthologous proteins in selected coronaviruses were organized using the OrthoMCL program 14, and the repeated sequences were removed from the orthologous groups. The CDSs of 120 coronaviruses were extracted and assigned to their corresponding orthologous protein groups by custom-made scripts. They were then aligned by MAFFT 11 with the L-INS-i method, followed with formation into phylip file by Clustal W 12. ML phylogenies were employed to build the phylogenetic trees based on each CDSs, with 100 bootstrap replications using PhyML13. The matrix representation with parsimony approach (MRP) method 15,16 was applied to construct the supertree of coronaviruses. We adapted custom-made scripts to build the Baum-Ragan matrix by searching the well-supported (above 55% bootstrap support) bipartitions in each source ML phylogenetic tree. For further analysis, two clades of the bipartitions were coded by A and T respectively and were transformed to pseudo-sequences (Baum-Ragan matrix) as reported in previous study 10. The A/T substitutions were treated equally in the following analysis, without systematic bias imported. The pseudo-sequences of the coronaviruses were used to re-construct the phylogenetic supertree using PhyML 13. Mutation analysis of the SARS-CoV–2 clades in the supertree Amino acid sequences of the viral genes were aligned by MAFFT 11 and displayed in MEGA X 17. Mutation sites on sequences of SARS-CoV–2 positioned in subclades in the phylogenetic supertree were identified manually. Results And Discussion Comparison of phylogenetic supertree and ML tree To accurately determine the evolutionary relationships among SARS-CoV–2, approaches of phylogenetic supertree and ML tree were employed for phylogenetic analysis of 102 SARS- CoV–2 isolated all over the world together with 5 SARS-CoV, 2 MERS-CoV, and 11 bat coronaviruses as outgroups. In the phylogenetic supertree (Figure 1), SARS-CoV and MERS- CoV were placed on one major branch, while SARS-CoV–2 belonged to another major branch. The divergent location of SARS-CoV–2 relative to SARS-CoV and MERS-CoV on the phylogenetic supertree was consistent with the results from the phylogenetic ML tree in this study (Figure S1) and with previous reports about the phylogeny of SARS-CoV–2 constructed with the whole genome 3,4,6. However, some discrepancies present between the phylogenetic supertree and the ML tree. Distinctive phylogenetic distances observed on clades of SARS-CoV and SARS- CoV–2 in phylogenetic supertree, explicitly presented evolutionary relationships among coronaviruses. By contrast, coronaviruses clustered tightly on clades of SARS-CoV and SARS-CoV–2 in phylogenetic ML tree (Figure S1), with barely discerned branch length (less than 0.001). Furthermore, the phylogenetic supertree successfully identified coronavirus AY572035 sampled from civet the closest ancestor of the SARS-CoVs (Figure 1) with a distinct branch length, which was highly consistent with the previous study 18. It is worth noting that some bat coronavirus sampled from the same animal host or/and same sampling location, displayed closer genetic distance in phylogenetic supertree, which is rational and logical from the perspective of evolutionary progress. However, bat coronavirus had no definitive evolution relationship in the phylogenetic ML tree. Therefore, the phylogenetic ML tree was less suitable for phylogenetic inference, at least for coronavirus listed above. The major factor that determines phylogenetic ML tree topology appears to be the orf1ab gene that is about 75% of the genome. It is readily explained by the similar evolution relationship obtained in the phylogenetic ML tree relative to the source phylogenetic ML tree based on the sequence of ORF1ab (Figure S1, Figure 2A). Taken together, the phylogenetic supertree displayed significant superiority for deciphering evolutionary relationships among coronavirus. Clues to the origin of the SARS-CoV–2 As the phylogenetic supertree and ML tree exhibited, RaTG13 (MN996532), bat-SL- CoVZC45 (MG772933), bat-SL-CoVZXC21 (MG772934) and SARS-CoV–2s formed one major clade (Figure 1, Figure S1). In particular, RaTG13 isolated from bat Rhinolophus affinis ( Yunnan, China), is the closest relative of SARS-CoV–2s located on different branches, which substantiates the previously reported phylogeny of SARS-CoV–2s constructed with the whole genome. The phylogenetic distance of SARS-CoV–2s and RaTG13 was distinct exhibited in the phylogenetic supertree (Figure 1); by contrast, it was barely observed in the phylogenetic tree constructed in this study (Figure S1) or previous report 19. To interpret the disparate proximity between SARS-CoV–2s and RaTG13 in phylogenetic supertree relative to ML tree, we examined and evaluated the 10 source ML trees (Figure 2), based on which the phylogenetic supertree was built. Consistent with the results of supertree and ML tree, RaTG13 (MN996532) is identified as adjacent coronavirus to SARS-CoV–2s in source ML trees based on phylogenetic analysis of five CDSs, including ORF1ab, spike protein, N protein, ORF6 and ORF7a (Figure 2A, 2B, 2D, 2G, 2H). By contrast, bat coronavirus MG772933 and MG772934, both of which are isolated from bat Rhinolophus sinicus ( Zhejiang, China) 20, were the nearest relatives of SARS-CoV–2s in source ML trees based on M protein, ORF3a, and ORF8 (Figure 2C, 2F, 2I). In addition, phylogenetic analysis of E protein sequence showed that SARS-CoV–2s, MN996532, MG772933, and MG772934 are pinpointed on the same branch (Figure 2E). The above distinct phylogenetic analysis results showed beyond a reasonable doubt that there are highly non-uniform rates of evolution on sequences of varied proteins in SARS-CoV–2s, with no clear consensus phylogeny within coronavirus could be determined, which makes single gene based phylogenetic analysis a relatively weak tool to study viral phylogeny. The conflict phylogeny reflected by 10 source ML trees suggests a possibility of another bat coronavirus in divergent species be the adjacent ancestor of SARS-CoV–2, and/or SARS-CoV–2s already made advanced evolution in its animal host. What is clear is that the actual validity of RaTG13 be the direct ancestor of SARS-CoV–2 is seriously questioned, although they share 96.5% identical genome sequence. Therefore, it is misleading in phylogenetic inference to taking RaTG13 as the direct ancestor of SARS-CoV–2. Mutants and evolution of SARS-CoV–2 Within phylogenetic supertree, nine sub-branches were resolved in SARS-CoV–2 clades, labeled from clade A until clade I in Figure 1, which were absent in phylogenetic ML tree based on full-length genomic sequence analysis (Figure S1). The sub-branches displayed an evolutionary scenario of the SARS-CoV–2s in human hosts from December 2019 to March 2020 all around the world, at least based on 102 SARS-CoV–2 isolates in this study. By interrogating ten CDSs of SARS-CoV–2s, diverse mutations are disseminated within five viral proteins, which are ORF1ab, N protein, spike protein, ORF3a, and ORF8 (Table 1). Within most mutation sites described in this study, the original amino acid was substituted by another one possessing altered chemical properties, except L1599F in ORF1ab (clade A), V62L in ORF8 (clade H), and I1606V in ORF1ab (clade D1). Most strikingly, SARS-CoV–2s from the USA displayed common mutation in clades of A, C, D, F, H, and I, covering a large number of countries listed in this study, including Spain, Finland, Sweden, Italy, Brazil, Australia, and South Korea. In particular, detection of the identical mutation in ORF3a protein (G251V) in clade I indicated the spread of the G251V mutant happened at least in January 2020 or earlier, in Sweden, Italy, Brazil, Australia, and the USA. The gene of ORF1ab, taking up 75% of the whole genome size of coronavirus, produces a series of non-structural proteins (nsp), which assemble to facilitate viral replication and transcription. Mutations in ORF1ab present in the majority of clades, including clades A, B, C, D1 in D, and E, which are involved in SARS-CoV–2s from Spain, USA, China, but no identical mutation site was detected. Among them was a mutation from proline to leucine (P4715L) in ORF1ab, which was located on Nsp12 that is considered a primary target for nucleotide analog antiviral inhibitors such as remdesivir, thus the mutation would possibly make anti-coronavirus treatment less effective 21,22. The viral spike protein, responsible for virus entry into the host cell, exhibited two mutated sites distributed in clade A (D614G) and F (H49Y), respectively. The mutation site D614G in spike protein is located between the receptor-binding domain (451–509) and the polybasic cleavage site (682–685) 23, which possibly can regulate binding capability of the virus with human host ACE2 receptor or capability of viral infection. Further studies and clinical observations are needed to find out whether mutation sites on various proteins could change the viral ability to infect and its pathogenicity. Conclusion The phylogenetic supertree is a powerful approach applied in the phylogenetic analysis of coronavirus. The distinct phylogenetic distance on SARS-CoV–2 clade was only can be detected by phylogenetic supertree. Rely on this approach, this study rationally questioned the reliability of RaTG13 be the last common ancestor of SARS-CoV–2s, and revealed various common mutations in SARS-CoV–2s. Timely monitoring the variation and evolution of SARS-CoV–2s would be favorable to treatment and control of COVID19 and prevent its future outbreak. Declarations Acknowledgments This study was supported by the Chinese National Natural Science Foundation (81902099). Author contributions JF and TL conceived the study, JF, TL, DL, YY, JG, YF analyzed the data, TL and JF wrote the paper, DL, YY, XZ, SC collected the data. Conflicts of Interest : The authors declare no conflict of interest. References Dong, E., Du, H. & Gardner, L. An interactive web-based dashboard to track COVID- 19 in real time. Lancet Infect. Dis. , doi:10.1016/S1473-3099(20)30120-1 (2020). Zhu, N. et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. Engl. J. Med. 382 , 727-733, doi:10.1056/NEJMoa2001017 (2020). Wu, F. et al. A new coronavirus associated with human respiratory disease in Nature 579 , 265-269, doi:10.1038/s41586-020-2008-3 (2020). Paraskevis, D. et al. Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event. Genet. 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Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 30 , 269-271, doi:10.1038/s41422- 020-0282-0 (2020). Grein, J. et al. Compassionate Use of Remdesivir for Patients with Severe Covid-19. Engl. J. Med. , doi:10.1056/NEJMoa2007016 (2020). Andersen, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C. & Garry, R. F. The proximal origin of SARS-CoV-2. Med. , doi:10.1038/s41591-020-0820-9 (2020). Table Due to technical limitations, Table 1 is provided in the Supplementary Files section. Supplementary Files Supplementarymaterial.pdf Table1.docx Cite Share Download PDF Status: Published Journal Publication published 22 Dec, 2020 Read the published version in Scientific Reports → 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-33194","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638354,"identity":"6a19dc83-d173-43ae-babc-a42e1fdd03ea","order_by":1,"name":"Tingting Li","email":"","orcid":"","institution":"Institute of Immunology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Li","suffix":""},{"id":638360,"identity":"4f855ced-6cad-40b1-ac1b-23ca7cb7745b","order_by":2,"name":"Dongxia Liu","email":"","orcid":"","institution":"Institute of Immunology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dongxia","middleName":"","lastName":"Liu","suffix":""},{"id":638365,"identity":"18a5aa31-7766-43ec-8c41-732c1bc52b49","order_by":3,"name":"Yadi Yang","email":"","orcid":"","institution":"Institute of Immunology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yadi","middleName":"","lastName":"Yang","suffix":""},{"id":638366,"identity":"44049001-5c12-4d2f-a174-215e48963716","order_by":4,"name":"Jiali Guo","email":"","orcid":"","institution":"The Second Clinical Medical School, Lanzhou University, Lanzhou, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Guo","suffix":""},{"id":638367,"identity":"01aefc2e-1fd0-4aef-9ffd-8a5f43ca305e","order_by":5,"name":"Yujie Feng","email":"","orcid":"","institution":"The Second Clinical Medical School, Lanzhou University, Lanzhou, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Feng","suffix":""},{"id":638368,"identity":"6244e2d0-f585-4eb6-b946-3bfb9b678ca7","order_by":6,"name":"Xinmo Zhang","email":"","orcid":"","institution":"The Second Clinical Medical School, Lanzhou University, Lanzhou, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinmo","middleName":"","lastName":"Zhang","suffix":""},{"id":638369,"identity":"8d113327-14b7-4264-9a72-9c12d0ec8897","order_by":7,"name":"Shilong Cheng","email":"","orcid":"","institution":"The First Clinical Medical School, Lanzhou University, Lanzhou, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shilong","middleName":"","lastName":"Cheng","suffix":""},{"id":638370,"identity":"9cce7fd2-b0e3-427c-837d-e58062d6ab8c","order_by":8,"name":"Jie Feng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACxuYDQLLCAsyRIE5LWwKQPCNBghYGtgSQPlK0MLcxH3v4dZ6EvcEB5oO3eRjs8ohwGFu6sew2CWaDA2zJ1jwMycWEtczvMZOW3CbBZnCAx0yah+FAYgNhW/i/SUvOkeAxOABkEKmFh03yY4OEBNAWNmK1sJlJMxyTMJA8zGZsOccgmbAWwzbmZ5I/amzs+Y43P7zxpsKOCC1AFcw8IBYziDAgpB4I5EGO+0GEwlEwCkbBKBjBAACfWTJL5s9oWAAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Pathology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, China","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2020-06-03 11:13:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-33194/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-33194/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-020-79484-8","type":"published","date":"2020-12-22T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1254249,"identity":"b6c452dd-3500-4aa0-b0bb-dc8aabf9a275","added_by":"auto","created_at":"2020-06-04 16:54:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":303278,"visible":true,"origin":"","legend":"MRP supertree for the SARS-CoV-2. The host and sampling locations of animal coronaviruses are shown in the corresponding brackets. The label of SARS-CoV-2 is coded by the abbreviation of sampling location, sampling time, and Genbank accession. MERS- CoV clade, SARS-CoV clade, and nine clades of SARS-CoV-2 were highlighted with labels. The numbers along the branches mark the bootstrap values percentage out of 1000 bootstrap resamplings.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-33194/v1/fig1.png"},{"id":1254250,"identity":"9a02c69d-6158-4a26-8d26-744966e646dd","added_by":"auto","created_at":"2020-06-04 16:54:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145498,"visible":true,"origin":"","legend":"Source phylogenetic ML trees (A: ORF1ab, B: Spike protein, C: M protein, D: N protein, E: E protein, F: ORF3a, G: ORF6, H: ORF7a, I: ORF8) for phylogenetic supertree construction. SARS-CoV-2 clade was in bold. Bat viruses of MG996532 (red), MG772933 (blue), and MG772934 (blue) were in colors. Clades of SARS-CoV and MERS-CoV were highlighted with green and purple, respectively.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-33194/v1/fig2.png"},{"id":13537095,"identity":"3978a279-651c-46e1-a186-f432c2c14da3","added_by":"auto","created_at":"2021-09-17 01:34:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":708902,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-33194/v1/05792df1-7fb3-4108-83ca-3b12404fc98f.pdf"},{"id":1254252,"identity":"0db29dbd-8182-437b-8446-d05c045fd52d","added_by":"auto","created_at":"2020-06-04 16:54:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":988987,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-33194/v1/Supplementarymaterial.pdf"},{"id":1254253,"identity":"c50b417d-48f1-426c-b4d4-bfc6daa2857c","added_by":"auto","created_at":"2020-06-04 16:54:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33396,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-33194/v1/Table1.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePhylogenetic supertree reveals detailed evolution of SARS-CoV-2\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSevere acute respiratory syndrome coronavirus–2 (SARS-CoV–2), a novel coronavirus emerged in December 2019, causes an ongoing outbreak of Corona Virus Disease 2019 (COVID–19). COVID–19 has caused a global pandemic, and as of 15 April 2020, 198,1239 cases of infections have been confirmed in more than 110 countries, with 12,6681 deaths 1. Currently, three coronaviruses known for causing severe human disease, are SARS-CoV–2, SARS-CoV, and MERS-CoV, with the last two viruses caused epidemics in the past.\u003c/p\u003e\n\u003cp\u003eAlthough three viruses are identified as beta-coronavirus within the subgenus of the sarbecoronavirus achieved by the full-length genomic sequence analysis, SARS-CoV–2 is divergent from SARS-CoV and MERS-CoV, and it belongs to a distinct lineage 2. More and more genomes of SARS-CoV–2 isolates were sequenced all around the world, which creates the opportunity for precise analysis of phylogeny and evolution of SARS-CoV–2 2–6.\u003c/p\u003e\n\u003cp\u003eHowever, the detailed evolution of SARS-CoV–2 is still under the veil, ascribed to the reality that SARS-CoV–2 isolates displayed barely detected phylogenetic distance among each other in the phylogenetic tree with further related coronavirus as outgroup that was constructed based on a single gene in the whole genome or one gene composed of the full-length genomic sequence. In particular, there are several limitations to the methods for phylogenetic tree construction mentioned before. Phylogenetic methods using single genes to determine the relationships between viruses face the problem of gene selection, with different trees yielded from different genes, which are not always consistent but often conflict. Considering the high diversity in sizes of genes consisting of coronavirus genome, the phylogenetic analysis with full-length SARS-CoV–2 genomic sequence hardly provide reliable evolutionary information, based on the probability that it will drown out plenty of phylogenetic information retained in some proteins coded by the smaller size of genes, which are vital for viral infection and virulence. For example, the gene of ORF1ab (21,290 bp) in SARS-CoV–2 genome comprises about 75% of the whole genome sequence, while the genes of key functional and structural proteins, including S (3,822 bp), E (228 bp), M (668 bp), and N (1,260 bp), take up less than 22%. Furthermore, the above two approaches for phylogenetic analysis require orthologous genes in the species to allow a meaningful comparison, which would limit the employment of species with large phylogenetic distance as outgroup. For example, at least five proteins of SARS-CoV–2 fail to get corresponding orthologous proteins in beta coronavirus MERS-CoV, and the ORF8 of SARS-CoV–2 has no orthologous proteins in sarbecoronavirus SARS-CoV. Therefore, such phylogenies can seriously mislead evolutionary events in between.\u003c/p\u003e\n\n\u003cp\u003eSupertree method, whose embryonic theory was described in the 1980s 7,8, can merge a set of consistent or inconsistent phylogenetic trees based on different genes in the genome, into one comprehensive phylogenetic tree reasonably 9. The phylogenetic network based on supertree method revealed non-vertical evolution scenario during the evolutionary history of haloarchaea, which was improbable for phylogenetic approaches relied on a single gene or the full-length genome sequences 10. Given its superiority, the supertree method was employed here for phylogenetic analysis of SARS-CoV–2 virus, by combining the phylogenetic information from ten genes of the coronavirus. Our study aims to figure out the origin and evolution of SARS-CoV–2 through phylogenetic supertree analysis.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003ch2\u003eDataset construction\u003c/h2\u003e\n\u003cp\u003eThe full-length genomic sequences and protein-coding sequences (CDSs) of 102 SARS-CoV- 2, 5 SARS-CoV, 2 MERS-CoV, and 11 bat coronaviruses were downloaded from NCBI Severe acute respiratory syndrome coronavirus 2 data hub (https\u003ca href=\"http://www.ncbi.nlm.nih.gov/labs/virus/vssi/%23/)\"\u003e://www\u003c/a\u003e.\u003ca href=\"http://www.ncbi.nlm.nih.gov/labs/virus/vssi/%23/)\"\u003e\u003ca href=\"http://ncbi.nlm.nih.gov/labs/virus/vssi/#/\"\u003encbi.nlm.nih.gov/labs/virus/vssi/#/\u003c/a\u003e) \u003c/a\u003eand GenBank (\u003ca href=\"http://www.ncbi.nlm.nih.gov/genbank/)\"\u003e\u003ca href=\"http://www.ncbi.nlm.nih.gov/genbank/\"\u003ehttp://www.ncbi.nlm.nih.gov/genbank/\u003c/a\u003e) \u003c/a\u003e(Table S1). Among genomic sequences of SARS- CoV and bat coronaviruses, those showing high similarity with genomic sequences of SARS-\u003c/p\u003e\n\n\u003cp\u003eCoV–2 were chosen. The integrity of sequences was checked, and the fragmented sequences were reconstructed. Finally, the datasets were constructed by labeling the sequences with the region of sampling and collection date.\u003c/p\u003e\n\n\u003ch2\u003eConstruction of phylogenetic tree with the whole genomic sequences\u003c/h2\u003e\n\u003cp\u003eThe full-length genomic sequences of 120 coronaviruses were aligned using the L-INS-i method of MAFFT v7.310 11. Aligned sequences were converted into phylip file format by Clustal W 12. Maximum likelihood (ML) trees based on full-length genomic sequences were constructed and estimated by PhyML program version 3.0 13 with 100 bootstraps resampling. The phylogenetic trees were visualized by FigTree v1.4.4 (\u003ca href=\"http://tree.bio.ed.ac.uk/software/figtree/)\"\u003e\u003ca href=\"http://tree.bio.ed.ac.uk/software/figtree/)\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/)\u003c/a\u003e.\u003c/a\u003e\u003c/p\u003e\n\n\u003ch2\u003eConstruction of phylogenetic supertrees\u003c/h2\u003e\n\u003cp\u003eTen groups of CDSs for orthologous proteins in selected coronaviruses were organized using the OrthoMCL program 14, and the repeated sequences were removed from the orthologous groups. The CDSs of 120 coronaviruses were extracted and assigned to their corresponding orthologous protein groups by custom-made scripts. They were then aligned by MAFFT 11 with the L-INS-i method, followed with formation into phylip file by Clustal W 12. ML phylogenies were employed to build the phylogenetic trees based on each CDSs, with 100 bootstrap replications using PhyML13. The matrix representation with parsimony approach (MRP) method 15,16 was applied to construct the supertree of coronaviruses. We adapted custom-made scripts to build the Baum-Ragan matrix by searching the well-supported (above 55% bootstrap support) bipartitions in each source ML phylogenetic tree. For further analysis, two clades of the bipartitions were coded by A and T respectively and were transformed to pseudo-sequences (Baum-Ragan matrix) as reported in previous study 10. The A/T substitutions were treated equally in the following analysis, without systematic bias imported. The pseudo-sequences of the coronaviruses were used to re-construct the phylogenetic supertree using PhyML 13.\u003c/p\u003e\n\n\u003ch2\u003eMutation analysis of the SARS-CoV–2 clades in the supertree\u003c/h2\u003e\n\u003cp\u003eAmino acid sequences of the viral genes were aligned by MAFFT 11 and displayed in MEGA X 17. Mutation sites on sequences of SARS-CoV–2 positioned in subclades in the phylogenetic supertree were identified manually.\u003c/p\u003e\n"},{"header":"Results And Discussion","content":"\u003ch2\u003eComparison of phylogenetic supertree and ML tree\u003c/h2\u003e\n\u003cp\u003eTo accurately determine the evolutionary relationships among SARS-CoV–2, approaches of phylogenetic supertree and ML tree were employed for phylogenetic analysis of 102 SARS- CoV–2 isolated all over the world together with 5 SARS-CoV, 2 MERS-CoV, and 11 bat coronaviruses as outgroups. In the phylogenetic supertree (Figure 1), SARS-CoV and MERS- CoV were placed on one major branch, while SARS-CoV–2 belonged to another major branch. The divergent location of SARS-CoV–2 relative to SARS-CoV and MERS-CoV on the phylogenetic supertree was consistent with the results from the phylogenetic ML tree in this study (Figure S1) and with previous reports about the phylogeny of SARS-CoV–2 constructed with the whole genome 3,4,6. However, some discrepancies present between the phylogenetic supertree and the ML tree.\u003c/p\u003e\n\n\u003cp\u003eDistinctive phylogenetic distances observed on clades of SARS-CoV and SARS- CoV–2 in phylogenetic supertree, explicitly presented evolutionary relationships among coronaviruses. By contrast, coronaviruses clustered tightly on clades of SARS-CoV and SARS-CoV–2 in phylogenetic ML tree (Figure S1), with barely discerned branch length (less than 0.001). Furthermore, the phylogenetic supertree successfully identified coronavirus AY572035 sampled from civet the closest ancestor of the SARS-CoVs (Figure 1) with a distinct branch length, which was highly consistent with the previous study 18. It is worth noting that some bat coronavirus sampled from the same animal host or/and same sampling location, displayed closer genetic distance in phylogenetic supertree, which is rational and logical from the perspective of evolutionary progress. However, bat coronavirus had no definitive evolution relationship in the phylogenetic ML tree. Therefore, the phylogenetic ML tree was less suitable for phylogenetic inference, at least for coronavirus listed above. The major factor that determines phylogenetic ML tree topology appears to be the orf1ab gene that is about 75% of the genome. It is readily explained by the similar evolution relationship obtained in the phylogenetic ML tree relative to the source phylogenetic ML tree based on the sequence of ORF1ab (Figure S1, Figure 2A). Taken together, the phylogenetic supertree displayed significant superiority for deciphering evolutionary relationships among coronavirus.\u003c/p\u003e\n\u003ch2\u003eClues to the origin of the SARS-CoV–2\u003c/h2\u003e\n\u003cp\u003eAs the phylogenetic supertree and ML tree exhibited, RaTG13 (MN996532), bat-SL- CoVZC45 (MG772933), bat-SL-CoVZXC21 (MG772934) and SARS-CoV–2s formed one major clade (Figure 1, Figure S1). In particular, RaTG13 isolated from bat \u003cem\u003eRhinolophus affinis (\u003c/em\u003eYunnan, China), is the closest relative of SARS-CoV–2s located on different branches, which substantiates the previously reported phylogeny of SARS-CoV–2s constructed with the whole genome. The phylogenetic distance of SARS-CoV–2s and RaTG13 was distinct exhibited in the phylogenetic supertree (Figure 1); by contrast, it was barely observed in the phylogenetic tree constructed in this study (Figure S1) or previous report 19.\u003c/p\u003e\n\n\u003cp\u003eTo interpret the disparate proximity between SARS-CoV–2s and RaTG13 in phylogenetic supertree relative to ML tree, we examined and evaluated the 10 source ML trees (Figure 2), based on which the phylogenetic supertree was built. Consistent with the results of supertree and ML tree, RaTG13 (MN996532) is identified as adjacent coronavirus to SARS-CoV–2s in source ML trees based on phylogenetic analysis of five CDSs, including ORF1ab, spike protein, N protein, ORF6 and ORF7a (Figure 2A, 2B, 2D, 2G, 2H). By contrast, bat coronavirus MG772933 and MG772934, both of which are isolated from bat \u003cem\u003eRhinolophus sinicus (\u003c/em\u003eZhejiang, China) 20, were the nearest relatives of SARS-CoV–2s in source ML trees based on M protein, ORF3a, and ORF8 (Figure 2C, 2F, 2I). In addition, phylogenetic analysis of E protein sequence showed that SARS-CoV–2s, MN996532, MG772933, and MG772934 are pinpointed on the same branch (Figure 2E). The above distinct phylogenetic analysis results showed beyond a reasonable doubt that there are highly non-uniform rates of evolution on sequences of varied proteins in SARS-CoV–2s, with no clear consensus phylogeny within coronavirus could be determined, which makes single gene based phylogenetic analysis a relatively weak tool to study viral phylogeny. The conflict phylogeny reflected by 10 source ML trees suggests a possibility of another bat coronavirus in divergent species be the adjacent ancestor of SARS-CoV–2, and/or SARS-CoV–2s already made advanced evolution in its animal host. What is clear is that the actual validity of RaTG13 be the direct ancestor of SARS-CoV–2 is seriously questioned, although they share 96.5% identical genome sequence. Therefore, it is misleading in phylogenetic inference to taking RaTG13 as the direct ancestor of SARS-CoV–2.\u003c/p\u003e\n\n\u003ch2\u003eMutants and evolution of SARS-CoV–2\u003c/h2\u003e\n\u003cp\u003eWithin phylogenetic supertree, nine sub-branches were resolved in SARS-CoV–2 clades, labeled from clade A until clade I in Figure 1, which were absent in phylogenetic ML tree based on full-length genomic sequence analysis (Figure S1). The sub-branches displayed an evolutionary scenario of the SARS-CoV–2s in human hosts from December 2019 to March 2020 all around the world, at least based on 102 SARS-CoV–2 isolates in this study. By interrogating ten CDSs of SARS-CoV–2s, diverse mutations are disseminated within five viral proteins, which are ORF1ab, N protein, spike protein, ORF3a, and ORF8 (Table 1).\u003c/p\u003e\n\u003cp\u003eWithin most mutation sites described in this study, the original amino acid was substituted by another one possessing altered chemical properties, except L1599F in ORF1ab (clade A), V62L in ORF8 (clade H), and I1606V in ORF1ab (clade D1). Most strikingly, SARS-CoV–2s from the USA displayed common mutation in clades of A, C, D, F, H, and I, covering a large number of countries listed in this study, including Spain, Finland, Sweden, Italy, Brazil, Australia, and South Korea. In particular, detection of the identical mutation in ORF3a protein (G251V) in clade I indicated the spread of the G251V mutant happened at least in January 2020 or earlier, in Sweden, Italy, Brazil, Australia, and the USA.\u003c/p\u003e\n\u003cp\u003eThe gene of ORF1ab, taking up 75% of the whole genome size of coronavirus, produces a series of non-structural proteins (nsp), which assemble to facilitate viral replication and transcription. Mutations in ORF1ab present in the majority of clades, including clades A, B, C, D1 in D, and E, which are involved in SARS-CoV–2s from Spain, USA, China, but no identical mutation site was detected. Among them was a mutation from proline to leucine (P4715L) in ORF1ab, which was located on Nsp12 that is considered a primary target for nucleotide analog antiviral inhibitors such as remdesivir, thus the mutation would possibly make anti-coronavirus treatment less effective 21,22.\u003c/p\u003e\n\n\u003cp\u003eThe viral spike protein, responsible for virus entry into the host cell, exhibited two mutated sites distributed in clade A (D614G) and F (H49Y), respectively. The mutation site D614G in spike protein is located between the receptor-binding domain (451–509) and the polybasic cleavage site (682–685) 23, which possibly can regulate binding capability of the virus with human host ACE2 receptor or capability of viral infection. Further studies and clinical observations are needed to find out whether mutation sites on various proteins could change the viral ability to infect and its pathogenicity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe phylogenetic supertree is a powerful approach applied in the phylogenetic analysis of coronavirus. The distinct phylogenetic distance on SARS-CoV–2 clade was only can be detected by phylogenetic supertree. Rely on this approach, this study rationally questioned the reliability of RaTG13 be the last common ancestor of SARS-CoV–2s, and revealed various common mutations in SARS-CoV–2s. Timely monitoring the variation and evolution of SARS-CoV–2s would be favorable to treatment and control of COVID19 and prevent its future outbreak.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Chinese National Natural Science Foundation (81902099).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJF and TL conceived the study, JF, TL, DL, YY, JG, YF analyzed the data, TL and JF wrote the paper, DL, YY, XZ, SC collected the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDong, E., Du, H. \u0026amp; Gardner, L. An interactive web-based dashboard to track COVID- 19 in real time. \u003cem\u003eLancet Infect. 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J. Med.\u003c/em\u003e, doi:10.1056/NEJMoa2007016 (2020).\u003c/li\u003e\n\u003cli\u003eAndersen, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C. \u0026amp; Garry, R. F. The proximal origin of SARS-CoV-2. \u003cem\u003e Med.\u003c/em\u003e, doi:10.1038/s41591-020-0820-9 (2020).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eDue to technical limitations, Table 1 is provided in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"SARS-CoV-2, COVID-19, coronavirus, supertree, phylogenetics","lastPublishedDoi":"10.21203/rs.3.rs-33194/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-33194/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCorona Virus Disease 2019 (COVID-19) caused by the emerged coronavirus SARS-CoV-2 is spreading globally. The origin of SARS-Cov-19 and its evolutionary relationship is still ambiguous. Several reports attempted to figure out this critical issue by genome-based phylogenetic analysis, with limited progress. Here we applied phylogenetic supertree analysis to study the origin and evolution of SARS-CoV-2. Phylogenetic supertree analysis firmly disputes the accuracy of bat coronavirus RaTG13 be the last common ancestor of SARS- CoV-2s reported in other phylogenetic tree analysis based on viral genome sequences, although RaTG13 shows 96.5% similarity with SARS-CoV-2 in the genome. Therefore, viewing RaTG13 as the last common ancestor of SARS-CoV-2 would seriously mislead phylogenetic inference of SARS-CoV-2. Importantly, the discovery of evolution and mutation in SARS-CoV-2s was achieved by phylogenetic supertree analysis. Taken together, the phylogenetic supertree showed extraordinary priority on the SARS-CoV-2 evolution inference relative to the normal phylogenetic tree based on full-length genomic sequences.\u003c/p\u003e","manuscriptTitle":"Phylogenetic supertree reveals detailed evolution of SARS-CoV-2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-06-04 16:54:11","doi":"10.21203/rs.3.rs-33194/v1","editorialEvents":[],"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":"be27d223-6372-4de0-ac79-69750a5970dc","owner":[],"postedDate":"June 4th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":112408,"name":"Evolutionary Genetics"}],"tags":[],"updatedAt":"2021-01-18T13:37:59+00:00","versionOfRecord":{"articleIdentity":"rs-33194","link":"https://doi.org/10.1038/s41598-020-79484-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2020-12-22 12:00:00","publishedOnDateReadable":"December 22nd, 2020"},"versionCreatedAt":"2020-06-04 16:54:11","video":"","vorDoi":"10.1038/s41598-020-79484-8","vorDoiUrl":"https://doi.org/10.1038/s41598-020-79484-8","workflowStages":[]},"version":"v1","identity":"rs-33194","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-33194","identity":"rs-33194","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}