Phylogenomic and molecular markers based studies on clarifying the evolutionary relationships among Peptoniphilus species. Identification of several Genus-Level clades of Peptoniphilus species and transfer of some Peptoniphilus species to the genus Aedoeadaptatus.

To clarify the evolutionary relationships among Peptoniphilus species, whose members show association with increased risk for prostate cancer, detailed phylogenomic and comparative analyses were conducted on their genome sequences. In phylogenetic trees based on core genome proteins and 16S rRNA gene sequences, Peptoniphilus species formed eight distinct clades, with Aedoeadaptatus and Anaerosphaera species branching between them. The observed clades designated as Peptoniphilus sensu stricto (encompassing its type species), Harei, Lacrimalis, Duerdenii, Mikwangii, Stercorisuis, Catoniae and Aedoeadaptatus, show genus level divergence based on 16S rRNA similarity and average amino acid identity (AAI). The Genome Taxonomy Database also assigns most of these clades to distinct taxa. Several Peptoniphilus species (viz. P. coxii, P. ivorii, P. nemausensis and some non-validly published species) grouped reliably with the type species of Aedoeadaptatus (A. acetigenes) and are affiliated to this genus based on 16S rRNA similarity, AAI, and multiple uniquely shared molecular signatures. Hence, we are proposing the transfer of these species into the emended genus Aedoeadaptatus. Our analyses on protein sequences from Peptoniphilus genomes have also identified 54 novel molecular markers consisting of conserved signature indels (CSIs), which are specific for different Peptoniphilus species clades and provide reliable means for their demarcation in molecular terms. Lastly, we also show that based on the shared presence of these CSIs in the genomes of uncharacterized Peptoniphilus spp. (cultured and uncultured), their affiliations to the specific Peptoniphilus clades can be accurately predicted. These results should prove useful in understanding the potential involvement of Peptoniphilus-related species in diseases.

Robust demarcation of the family Peptostreptococcaceae and its main genera based on phylogenomic studies and taxon-specific molecular markers.

The family Peptostreptococcaceae, which contains 15 genera including Clostridioides, presently lacks proper circumscription. Using 52 available genomes for Peptostreptococcaceae species, we report comprehensive phylogenomic and comparative analyses to reliably discern their evolutionary relationships. In phylogenetic trees based on core genome proteins and 16S rRNA gene sequences, the examined species formed a strongly supported clade designated as Peptostreptococcaceae sensu stricto. This clade encompassed the genera Peptostreptococcus (type genus), Asaccharospora, Clostridioides, Intestinibacter, Paeniclostridium, Paraclostridium, Peptacetobacter, Romboutsia and Terrisporobacter, and two misclassified species (viz. Eubacterium tenue and 'Clostridium dakarense'). The distinctness of this clade is strongly supported by eight identified conserved signature indels (CSIs), which are specific for the species from this clade. Based on the robust evidence provided by presented studies, we are proposing the emendment of family Peptostreptococcaceae to only the genera within the Peptostreptococcaceae sensu stricto clade. We also report 67 other novel CSIs, which reliably demarcate different Peptostreptococcaceae species clades and clarify the classification of some misclassified species. Based on the consistent evidence obtained from different presented studies, we are making the following proposals to clarify the classification of Peptostreptococcaceae species: (i) transfer of Eubacterium tenue, Paeniclostridium ghonii and Paeniclostridium sordellii as comb. nov. into the genus Paraclostridium; (ii) transfer of Clostridioides mangenotii as a comb. nov. into Metaclostridioides gen. nov.; (iii) classification of 'Clostridium dakarense' as a novel species Faecalimicrobium dakarense gen. nov., sp. nov. (type strain FF1T; genome and 16S rRNA accession numbers GCA_000499525.1 and KC517358, respectively); (iv) transfer of two misclassified species, Clostridium paradoxum and Clostridium thermoalcaliphilum, into Alkalithermobacter gen. nov.; and (v) proposals for two novel families, Peptoclostridiaceae fam. nov. and Tepidibacteraceae fam. nov., to accommodate remaining unclassified Peptostreptococcaceae genera. The described CSIs specific for different families and genera provide novel and reliable means for the identification, diagnostics and biochemical studies on these bacteria.

Update on the genus Robertmurraya: a bacterial genus honoring Dr. Robert G.E. Murray (with some personal reminiscences).

The genus Robertmurraya was created by my group in 2020 to recognize the contributions of Dr. Robert G.E. Murray to the field of prokaryotic taxonomy. This manuscript updates the information regarding this genus. In addition to the seven Robertmurraya species with validly published names, the work presented here shows that two species with effectively published names, "Bacillus yapensis" and "Bacillus dakarensis", and an uncharacterized Bacillus sp. Y1 are also affiliated with this genus. Based on these results, reclassification of "Bacillus yapensis" as a novel species Robertmurraya yapensis sp. nov. is proposed. It is also suggested that "Bacillus dakarensis", for which strains are not available from culture collections, should also be recognized as "Robertmurraya dakarensis". This article also reflects on the serendipitous way I came to know Dr. Murray and his extensive interactions with me and strong support for our work for more than 10 years. Dr. Murray also introduced me and our work to his friend and contemporary Dr. Peter Sneath, who like him also contributed extensively to the field of prokaryotic taxonomy. This introduction led to a fruitful collaboration with Dr. Sneath leading to a joint publication describing the use of the Character Compatibility approach to molecular sequence data.

Phylogenomic and molecular markers based studies on Staphylococcaceae and Gemella species. Proposals for an emended family Staphylococcaceae and three new families (Abyssicoccaceae fam. nov., Salinicoccaceae fam. nov. and Gemellaceae fam. nov.) harboring four new genera, Lacicoccus gen. nov., Macrococcoides gen. nov., Gemelliphila gen. nov., and Phocicoccus gen. nov.

The family Staphylococcacae and genus Gemella contain several organisms of clinical or biotechnological importance. We report here comprehensive phylogenomic and comparative analyses on 112 available genomes from species in these taxa to clarify their evolutionary relationships and classification. In a phylogenomic tree based on 678 core proteins, Gemella species were separated from Staphylococcacae by a long branch indicating that they constitute a distinct family (Gemellaceae fam. nov.). In this tree, Staphylococcacae species formed two main clades, one encompassing the genera Aliicoccus, Jeotgalicoccus, Nosocomiicoccus and Salinicoccus (Family "Salinicoccaceae"), while the other clade consisted of the genera Macrococcus, Mammaliicoccus and Staphylococcus (Family Staphylococcaceae emend.). In this tree, species from the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus each formed two distinct clades. Two species clades for these genera are also observed in 16S rRNA gene trees and supported by average amino acid identity analysis. We also report here detailed analyses on protein sequences from Staphylococcaceae and Gemella genomes to identify conserved signature indels (CSIs) which are specific for different genus and family-level clades. These analyses have identified 120 novel CSIs robustly demarcating different proposed families and genera. The identified CSIs provide independent evidence that the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus consist of two distinct clades, which can be reliably distinguished based on multiple exclusively shared CSIs. We are proposing transfers of the species from the novel clades of the above four genera into the genera Gemelliphila gen. nov., Phocicoccus gen. nov., Macrococcoides gen. nov. and Lacicoccus gen. nov., respectively. The identified CSIs also provide strong evidence for division of Staphylococcaceae into an emended family Staphylococcaceae and two new families, Abyssicoccaceae fam. nov. and Salinicoccaceae fam. nov. All of these families can be reliably demarcated based on several exclusively shared CSIs.

AppIndels.com server: a web-based tool for the identification of known taxon-specific conserved signature indels in genome sequences. Validation of its usefulness by predicting the taxonomic affiliation of >700 unclassified strains of Bacillus species.

Taxon-specific conserved signature indels (CSIs) in genes/proteins provide reliable molecular markers (synapomorphies) for unambiguous demarcation of taxa of different ranks in molecular terms and for genetic, biochemical and diagnostic studies. Because of their predictive abilities, the shared presence of known taxon-specific CSIs in genome sequences has proven useful for taxonomic purposes. However, the lack of a convenient method for identifying the presence of known CSIs in genome sequences has limited their utility for taxonomic and other studies. We describe here a web-based tool/server (AppIndels.com) that identifies the presence of known and validated CSIs in genome sequences and uses this information for predicting taxonomic affiliation. The utility of this server was tested by using a database of 585 validated CSIs, which included 350 CSIs specific for ≈45 Bacillales genera, with the remaining CSIs being specific for members of the orders Neisseriales, Legionellales and Chlorobiales, family Borreliaceae, and some Pseudomonadaceae species/genera. Using this server, genome sequences were analysed for 721 Bacillus strains of unknown taxonomic affiliation. Results obtained showed that 651 of these genomes contained significant numbers of CSIs specific for the following Bacillales genera/families: Alkalicoccus, 'Alkalihalobacillaceae', Alteribacter, Bacillus Cereus clade, Bacillus Subtilis clade, Caldalkalibacillus, Caldibacillus, Cytobacillus, Ferdinandcohnia, Gottfriedia, Heyndrickxia, Lederbergia, Litchfieldia, Margalitia, Mesobacillus, Metabacillus, Neobacillus, Niallia, Peribacillus, Priestia, Pseudalkalibacillus, Robertmurraya, Rossellomorea, Schinkia, Siminovitchia, Sporosarcina, Sutcliffiella, Weizmannia and Caryophanaceae. Validity of the taxon assignment made by the server was examined by reconstructing phylogenomic trees. In these trees, all Bacillus strains for which taxonomic predictions were made correctly branched with the indicated taxa. The unassigned strains likely correspond to taxa for which CSIs are lacking in our database. Results presented here show that the AppIndels server provides a useful new tool for predicting taxonomic affiliation based on shared presence of the taxon-specific CSIs. Some caveats in using this server are discussed.

Phylogenomics studies and molecular markers reliably demarcate genus Pseudomonas sensu stricto and twelve other Pseudomonadaceae species clades representing novel and emended genera.

Genus Pseudomonas is a large assemblage of diverse microorganisms, not sharing a common evolutionary history. To clarify their evolutionary relationships and classification, we have conducted comprehensive phylogenomic and comparative analyses on 388 Pseudomonadaceae genomes. In phylogenomic trees, Pseudomonas species formed 12 main clusters, apart from the "Aeruginosa clade" containing its type species, P. aeruginosa. In parallel, our detailed analyses on protein sequences from Pseudomonadaceae genomes have identified 98 novel conserved signature indels (CSIs), which are uniquely shared by the species from different observed clades/groups. Six CSIs, which are exclusively shared by species from the "Aeruginosa clade," provide reliable demarcation of this clade corresponding to the genus Pseudomonas sensu stricto in molecular terms. The remaining 92 identified CSIs are specific for nine other Pseudomonas species clades and the genera Azomonas and Azotobacter which branch in between them. The identified CSIs provide strong independent evidence of the genetic cohesiveness of these species clades and offer reliable means for their demarcation/circumscription. Based on the robust phylogenetic and molecular evidence presented here supporting the distinctness of the observed Pseudomonas species clades, we are proposing the transfer of species from the following clades into the indicated novel genera: Alcaligenes clade - Aquipseudomonas gen. nov.; Fluvialis clade - Caenipseudomonas gen. nov.; Linyingensis clade - Geopseudomonas gen. nov.; Oleovorans clade - Ectopseudomonas gen. nov.; Resinovorans clade - Metapseudomonas gen. nov.; Straminea clade - Phytopseudomonas gen. nov.; and Thermotolerans clade - Zestomonas gen. nov. In addition, descriptions of the genera Azomonas, Azotobacter, Chryseomonas, Serpens, and Stutzerimonas are emended to include information for the CSIs specific for them. The results presented here should aid in the development of a more reliable classification scheme for Pseudomonas species.

Phylogenomic Analyses and Molecular Signatures Elucidating the Evolutionary Relationships amongst the Chlorobia and Ignavibacteria Species: Robust Demarcation of Two Family-Level Clades within the Order Chlorobiales and Proposal for the Family Chloroherpetonaceae fam. nov.

Evolutionary relationships amongst Chlorobia and Ignavibacteria species/strains were examined using phylogenomic and comparative analyses of genome sequences. In a phylogenomic tree based on 282 conserved proteins, the named Chlorobia species formed a monophyletic clade containing two distinct subclades. One clade, encompassing the genera Chlorobaculum, Chlorobium, Pelodictyon, and Prosthecochloris, corresponds to the family Chlorobiaceae, whereas another clade, harboring Chloroherpeton thalassium, Candidatus Thermochlorobacter aerophilum, Candidatus Thermochlorobacteriaceae bacterium GBChlB, and Chlorobium sp. 445, is now proposed as a new family (Chloroherpetonaceae fam. nov). In parallel, our comparative genomic analyses have identified 47 conserved signature indels (CSIs) in diverse proteins that are exclusively present in members of the class Chlorobia or its two families, providing reliable means for identification. Two known Ignavibacteria species in our phylogenomic tree are found to group within a larger clade containing several Candidatus species and uncultured Chlorobi strains. A CSI in the SecY protein is uniquely shared by the species/strains from this "larger Ignavibacteria clade". Two additional CSIs, which are commonly shared by Chlorobia species and the "larger Ignavibacteria clade", support a specific relationship between these two groups. The newly identified molecular markers provide novel tools for genetic and biochemical studies and identification of these organisms.

Conserved Molecular Signatures in the Spike, Nucleocapsid, and Polymerase Proteins Specific for the Genus Betacoronavirus and Its Different Subgenera.

The genus Betacoronavirus, consisting of four main subgenera (Embecovirus, Merbecovirus, Nobecovirus, and Sarbecovirus), encompasses all clinically significant coronaviruses (CoVs), including SARS, MERS, and the SARS-CoV-2 virus responsible for current COVID-19 pandemic. Very few molecular characteristics are known that are specific for the genus Betacoronavirus or its different subgenera. In this study, our analyses of the sequences of four essential proteins of CoVs, viz., spike, nucleocapsid, envelope, and RNA-dependent RNA polymerase (RdRp), identified ten novel molecular signatures consisting of conserved signature indels (CSIs) in these proteins which are specific for the genus Betacoronavirus or its subgenera. Of these CSIs, two 14-aa-conserved deletions found within the heptad repeat motifs 1 and 2 of the spike protein are specific for all betacoronaviruses, except for their shared presence in the highly infectious avian coronavirus. Six additional CSIs present in the nucleocapsid protein and one CSI in the RdRp protein are distinctive characteristics of either the Merbecovirus, Nobecovirus, or Sarbecovirus subgenera. In addition, a 4-aa insert is present in the spike protein, which is uniquely shared by all viruses from the subgenera Merbecovirus, Nobecovirus, and Sarbecovirus, but absent in Embecovirus and all other genera of CoVs. This molecular signature provides evidence that viruses from the three subgenera sharing this CSI are more closely related to each other, and they evolved after the divergence of embecoviruses and other CoVs. As all CSIs specific for different groups of CoVs are flanked by conserved regions, their sequences provide novel means for identifying the above groups of CoVs and for developing novel diagnostic tests. Furthermore, our analyses of the structures of the spike and nucleocapsid proteins show that all identified CSIs are localized in the surface-exposed loops of these protein. It is postulated that these surface loops, through their interactions with other cellular proteins/ligands, play important roles in the biology/pathology of these viruses.

Conserved Signatures in Protein Sequences Reliably Demarcate Different Clades of Rodents/Glires Species and Consolidate Their Evolutionary Relationships.

The grandorder Glires, consisting of the orders Rodentia and Lagomorpha, encompasses a significant portion of the extant mammalian species including Rat, Mouse, Squirrel, Guinea pig and Beaver. Glires species play an important role in the ecosystem and provide valuable animal models for genetic studies and animal testing. Thus, it is important to reliably determine their evolutionary relationships and identify molecular characteristics that are specific for different species groups within the Glires. In this work, we have constructed a phylogenetic tree for >30 genome sequenced Glires species based on concatenated sequences of 25 conserved proteins. In this tree, members of different orders, suborders, and families within Glires formed strongly supported clades, and their interrelationships were also generally reliably resolved. In parallel, we conducted comparative analyses on more than 1500 protein sequences from Glires species to identify highly conserved molecular markers. These markers were comprised of conserved signature indels (CSIs) in proteins, which are specific for different Rodentia/Glires clades. Of the 41 novel CSIs identified in this work, some are specific for the entire Glires, Rodentia, or Lagomorpha clades, whereas many others reliably demarcate different family/suborder level clades of Rodentia (viz. Myomorpha, Castorimorpha, Sciuromorpha, Hystricomorpha, and Muroidea). Additionally, some of the CSIs also provide information regarding the interrelationships among Rodentia subgroups. Our analysis has also identified one CSI that is commonly shared by the Glires and Scandentia species (tree shrew), however, its evolutionary significance is unclear. Several of the identifed rodents-specific CSIs are present in conserved disease-related proteins. Thus, they provide novel molecular markers for genetic and biochemical studies on the functions of these proteins.

Phylogenomics and molecular signatures support division of the order Neisseriales into emended families Neisseriaceae and Chromobacteriaceae and three new families Aquaspirillaceae fam. nov., Chitinibacteraceae fam. nov., and Leeiaceae fam. nov.

The order Neisseriales contains 37 genera harboring 122 species with validly published names, which are placed into two families, Neisseriaceae and Chromobacteriaceae. Genome sequences are now available for 35 of the 37 Neisseriales genera for reliably determining their evolutionary relationships and taxonomy. We report here comprehensive phylogenomic and comparative analyses on protein sequences from 110 Neisseriales genomes plus 3 Chitinimonas genomes using multiple approaches. In a phylogenomic tree based on 596 core proteins, Neisseriales species formed 5 strongly supported clades. In addition to the clades for Neisseriaceae and Chromobacteriaceae families, three novel species clades designated as the "Chitinibacteraceae", "Aquaspirillaceae", and "Leeiaceae" were observed. The genus Chitinimonas grouped reliably with members of the "Chitinibacteraceae" clade. The major clades within the order Neisseriales can also be distinguished based on average amino acid identity analysis. In parallel, our comparative genomic studies have identified 30 conserved signature indels (CSIs) that are specific for members of the order Neisseriales or its five main clades. One of these CSIs is uniquely shared by all Neisseriales, whereas 8, 4, 9, 3 and 5 CSIs are distinctive characteristics of the Neisseriaceae, Chromobacteriaceae, "Chitinibacteraceae", "Aquaspirillaceae" and "Leeiaceae" clades, respectively. Based on the strong phylogenetic and molecular evidence presented here, we are proposing that the three newly identified clades should be recognized as novel families (Chitinibacteraceae fam. nov., Aquaspirillaceae fam. nov. and Leeiaceae fam. nov.) within the order Neisseriales. In addition, we are also emending descriptions of the families Neisseriaceae and Chromobacteriaceae regarding their constituent genera and other distinguishing characteristics.

Phylogenomic and comparative genomic analyses of species of the family Pseudomonadaceae: Proposals for the genera Halopseudomonas gen. nov. and Atopomonas gen. nov., merger of the genus Oblitimonas with the genus Thiopseudomonas, and transfer of some misclassified species of the genus Pseudomonas into other genera.

The evolutionary relationships among species of the family Pseudomonadaceae were examined based on 255 available genomes representing >85 % of the species from this family. In a phylogenetic tree based on concatenated sequences of 118 core proteins, most species of the genus Pseudomonas grouped within one large cluster which also included members of the genera Azotobacter and Azomonas. Within this large cluster 18-30 clades/subclades of species of the genus Pseudomonas consisting of between 1 and 36 species, were observed. However, a number of species of the genus Pseudomonas branched outside of this main cluster and were interspersed among other genera of the family Pseudomonadaceae. This included a strongly supported clade (Pertucinogena clade) consisting of 19 mainly halotolerant species. The distinctness of this clade from all other members of the family Pseudomonadaceae is strongly supported by 24 conserved signature indels (CSIs) in diverse proteins that are exclusively found in all members of this clade. Nine uncharacterized members of the genus Pseudomonas also shared these CSIs and they branched within the Pertucinogena clade, indicating their affiliation to this clade. On the basis of the strong evidence supporting the distinctness of the Pertucinogena clade, we are proposing transfer of species from this clade into a novel genus Halopseudomonas gen. nov. Pseudomonas caeni also branches outside of the main cluster and groups reliably with Oblitimonas alkaliphila and Thiopseudomonas denitrificans. Six identified CSIs are uniquely shared by these three species and we are proposing their integration into the emended genus Thiopseudomonas, which has priority over the name Oblitimonas. We are also proposing transfer of the deep-branching Pseudomonas hussainii, for which 22 exclusive CSIs have been identified, into the genus Atopomonas gen. nov. Lastly, we present strong evidence that the species Pseudomonas cissicola and Pseudomonas geniculata are misclassified into the genus Pseudomonas and that they are specifically related to the genera Xanthomonas and Stenotrophomonas, respectively. In addition, we are also reclassifying 'Pseudomonas acidophila' as Paraburkholderia acidicola sp. nov. (Type strain: G-6302=ATCC 31363=BCRC 13035).

The Family Borreliaceae (Spirochaetales), a Diverse Group in Two Genera of Tick-Borne Spirochetes of Mammals, Birds, and Reptiles.

Spirochetes of the family Borreliaceae are, with one exception, tick-borne pathogens of a variety of vertebrates. The family at present comprises two genera: Borrelia (Swellengrebel), which includes the agents of relapsing fever, avian spirochetosis, and bovine borreliosis, and Borreliella (Gupta et al.), which includes the agents of Lyme disease and was formerly known as 'Borrelia burgdorferi sensulato complex'. The two genera are distinguished not only by their disease associations but also biological features in the tick vector, including tissue location in unfed ticks and transovarial transmission. Borrelia species transmitted by argasid (soft) ticks tend to have more exclusive relationships with their tick vectors than do other Borrelia species and all Borreliella species that have ixodid (hard) ticks as vectors. The division of genera is supported by phylogenomic evidence from whole genomes and by several specific molecular markers. These distinguishing phylogenetic criteria also applied to three new species or isolates of Borrelia that were discovered in ixodid ticks of reptiles, a monotreme, and birds. Although the deep branching of the family from other spirochetes has been a challenge for inferences about evolution of the family, the discovery of related microorganisms in the gut microbiota of other arachnids suggests an ancestral origin for the family as symbionts of ticks and other arachnids.

A robust phylogenetic framework for members of the order Legionellales and its main genera (Legionella, Aquicella, Coxiella and Rickettsiella) based on phylogenomic analyses and identification of molecular markers demarcating different clades.

The order Legionellales contains several clinically important microorganisms. Although members of this order are well-studied for their pathogenesis, there is a paucity of reliable characteristics distinguishing members of this order and its constituent genera. Genome sequences are now available for 73 Legionellales species encompassing ≈90% of known members from different genera. With the aim of understanding evolutionary relationships and identifying reliable molecular characteristics that are specific for this order and its constituent genera, detailed phylogenetic and comparative analyses were conducted on the protein sequences from these genomes. A phylogenomic tree was constructed based on 393 single copy proteins that are commonly shared by the members of this order to delineate the evolutionary relationships among its members. In parallel, comparative analyses were performed on protein sequences from Legionellales genomes to identify novel molecular markers consisting of conserved signature indels (CSIs) that are specific for different clades and genera. In the phylogenomic tree and in an amino acid identity matrix based on core proteins, members of the genera Aquicella, Coxiella, Legionella and Rickettsiella formed distinct clades confirming their monophyly. In these studies, Diplorickettsia massiliensis exhibited a close relationship to members of the genus Rickettsiella. The results of our comparative genomic analyses have identified 59 highly specific molecular markers consisting of CSIs in diverse proteins that are uniquely shared by different members of this order. Four of these CSIs are specific for all Legionellales species, except the two deeper-branching "Candidatus Berkiella" species, providing means for identifying members of this order in molecular terms. Twenty four, 7 and 6 CSIs are uniquely shared by members of the genera Legionella, Coxiella and Aquicella, respectively, identifying these groups in molecular terms. The descriptions of these three genera are emended to include information for their novel molecular characteristics. We also describe 12 CSIs that are uniquely shared by D. massiliensis and different members of the genus Rickettsiella. Based on these results, we are proposing an integration of the genus Diplorickettsia with Rickettsiella. Three other CSIs suggest that members of the genera Coxiella and Rickettsiella shared a common ancestor exclusive of other Legionellales. The described molecular markers, due to their exclusivity for the indicated taxa/genera, provide important means for the identification of these clinically important microorganisms and for discovering novel properties unique to them.

Conserved molecular signatures in the spike protein provide evidence indicating the origin of SARS-CoV-2 and a Pangolin-CoV (MP789) by recombination(s) between specific lineages of Sarbecoviruses.

Both SARS-CoV-2 and SARS coronaviruses (CoVs) are members of the subgenus Sarbecovirus. To understand the origin of SARS-CoV-2, sequences for the spike and nucleocapsid proteins from sarbecoviruses were analyzed to identify molecular markers consisting of conserved inserts or deletions (termed CSIs) that are specific for either a particular clade of Sarbecovirus or are commonly shared by two or more clades of these viruses. Three novel CSIs in the N-terminal domain (NTD) of the spike protein S1-subunit (S1-NTD) are uniquely shared by SARS-CoV-2, Bat-CoV-RaTG13 and most pangolin CoVs (SARS-CoV-2r clade). Three other sarbecoviruses viz. bat-CoVZXC21, -CoVZC45 and -PrC31 (forming CoVZC/PrC31 clade), and a pangolin-CoV_MP789 also contain related CSIs in the same positions. In contrast to the S1-NTD, both SARS and SARS-CoV-2r viruses contain two large CSIs in the S1-C-terminal domain (S1-CTD) that are absent in the CoVZC/PrC31 clade. One of these CSIs, consisting of a 12 aa insert, is also present in the RShSTT clade (Cambodia-CoV strains). Sequence similarity studies show that the S1-NTD of SARS-CoV-2r viruses is most similar to the CoVZC/PrC31 clade, whereas their S1-CTD exhibits highest similarity to the RShSTT- (and the SARS-related) CoVs. Results from the shared presence of CSIs and sequence similarity studies on different CoV lineages support the inference that the SARS-CoV-2r cluster of viruses has originated by a genetic recombination between the S1-NTD of the CoVZC/PrC31 clade of CoVs and the S1-CTD of RShSTT/SARS viruses, respectively. We also present compelling evidence, based on the shared presence of CSIs and sequence similarity studies, that the pangolin-CoV_MP789, whose receptor-binding domain is most similar to the SARS-CoV-2 virus, has resulted from another independent recombination event involving the S1-NTD of the CoVZC/PrC31 CoVs and the S1-CTD of an unidentified SARS-CoV-2r related virus. The SARS-CoV-2 virus involved in this latter recombination event is postulated to be most similar to the SARS-CoV-2. Several other CSIs reported here are specific for other clusters of sarbecoviruses including a clade consisting of bat-SARS-CoVs (BM48-31/BGR/2008 and SARS_BtKY72). Structural mapping studies show that the identified CSIs form distinct loops/patches on the surface of the spike protein. It is hypothesized that these novel loops/patches on the spike protein, through their interactions with other host components, should play important roles in the biology/pathology of SARS-CoV-2 virus. Lastly, the CSIs specific for different clades of sarbecoviruses including SARS-CoV-2r clade provide novel means for the identification of these viruses and other potential applications.