Seasonal Patterns of Mpox Index Cases, Africa, 1970-2021.

Across 133 confirmed mpox zoonotic index cases reported during 1970-2021 in Africa, cases occurred year-round near the equator, where climate is consistent. However, in tropical regions of the northern hemisphere under a dry/wet season cycle, cases occurred seasonally. Our findings further support the seasonality of mpox zoonotic transmission risk.

Phylogeography of horseshoe bat sarbecoviruses in Vietnam and neighbouring countries. Implications for the origins of SARS-CoV and SARS-CoV-2.

Previous studies on horseshoe bats (Rhinolophus spp.) have described many coronaviruses related to SARS-CoV (SARSCoVr) in China and only a few coronaviruses related to SARS-CoV-2 (SARSCoV2r) in Yunnan (southern China), Cambodia, Laos and Thailand. Here, we report the results of several field missions carried out in 2017, 2021 and 2022 across Vietnam during which 1218 horseshoe bats were sampled from 19 locations. Sarbecoviruses were detected in 11% of faecal RNA extracts, with much more positives among Rhinolophus thomasi (46%). We assembled 38 Sarbecovirus genomes, including 32 SARSCoVr, four SARSCoV2r, and two recombinants of SARSCoVr and SARSCoV2r (RecSar), one showing a Spike protein very similar to SARS-CoV-2. We detected a bat co-infected with four coronaviruses, including two sarbecoviruses. Our analyses revealed that Sarbecovirus genomes evolve in Vietnam under strong geographical and host constraints. First, we found evidence for a deep separation between viruses from northern Vietnam and those from central and southern Vietnam. Second, we detected only SARSCoVr in Rhinolophus thomasi, both SARSCoVr and SARSCoV2r in Rhinolophus affinis, and only RecSar in Rhinolophus pusillus captured close to the border with China. Third, the bias in favour of Uracil in synonymous third codon positions of SARSCoVr extracted from R. thomasi showed a negative correlation with latitudes. Our results also provided support for an emergence of SARS-CoV in horseshoe bats from northern Yunnan and emergence of SARS-CoV-2 in horseshoe bats from northern Indochina subtropical forests (southern Yunnan, northern Laos and north-western Vietnam).

Phylogeny of sea spiders (Arthropoda: Pycnogonida) inferred from mitochondrial genome and 18S ribosomal RNA gene sequences.

The phylogeny of sea spiders has been debated for more than a century. Despite several molecular studies in the last twenty years, interfamilial relationships remain uncertain. In the present study, relationships within Pycnogonida are examined in the light of a new dataset composed of 160 mitochondrial genomes (including 152 new sequences) and 130 18S rRNA gene sequences (including 120 new sequences), from 141 sea spider morphospecies representing 26 genera and 9 families. Node congruence between mitochondrial and nuclear markers was analysed to identify the most reliable relationships. We also reanalysed a multilocus dataset previously published and showed that the high percentages of missing data make phylogenetic conclusions difficult and uncertain. Our results support the monophyly of most families currently accepted, except Callipallenidae and Nymphonidae, the monophyly of the superfamilies Ammotheoidea (Ammotheidae + Pallenopsidae), Nymphonoidea (Nymphonidae + Callipallenidae), Phoxichilidioidea (Phoxichilidiidae + Endeidae) and Colossendeoidea (Colossendeidae + Pycnogonidae + Rhynchothoracidae), and the sister-group relationship between Ammotheoidea and Phoxichilidioidea. We discuss the morphological evolution of sea spiders, identifying homoplastic characters and possible synapomorphies. We also discuss the palaeontological and phylogenetic arguments supporting either a radiation of sea spiders prior to Jurassic or a progressive diversification from Ordovician or Cambrian.

National Monkeypox Surveillance, Central African Republic, 2001-2021.

We analyzed monkeypox disease surveillance in Central African Republic (CAR) during 2001-2021. Surveillance data show 95 suspected outbreaks, 40 of which were confirmed as monkeypox, comprising 99 confirmed and 61 suspected monkeypox cases. After 2018, CAR's annual rate of confirmed outbreaks increased, and 65% of outbreaks occurred in 2 forested regions bordering the Democratic Republic of the Congo. The median patient age for confirmed cases was 15.5 years. The overall case-fatality ratio was 7.5% (12/160) for confirmed and suspected cases, 9.6% (8/83) for children <16 years of age. Decreasing cross-protective immunity from smallpox vaccination and recent ecologic alterations likely contribute to increased monkeypox outbreaks in Central Africa. High fatality rates associated with monkeypox virus clade I also are a local and international concern. Ongoing investigations of zoonotic sources and environmental changes that increase human exposure could inform practices to prevent monkeypox expansion into local communities and beyond endemic areas.

The genome of the lowland anoa (Bubalus depressicornis) illuminates the origin of river and swamp buffalo.

Two types of domestic water buffalo are currently recognized: the river buffalo from the Indian subcontinent and Mediterranean countries and the swamp buffalo from China and Southeast Asia. To test the hypothesis of two separate species of water buffalo, we sequenced the genome of the lowland anoa, Bubalus depressicornis, which is a dwarf wild buffalo endemic to Sulawesi, and two genomes of swamp buffalo, and made comparisons with 12 additional genomes. Three genomic data sets were constructed to infer phylogenetic relationships: the mitochondrial genome (15,468 bp; maternal transmission), two concatenated Y-chromosomal genes, AMELY and DDX3Y (20,036 bp; paternal transmission), and a selection of 30 nuclear genes representing all cattle chromosomes (364,887 bp; biparental transmission). The comparisons between our 30 nuclear gene sequences obtained by read mapping and those directly extracted from Bos taurus and Bubalus bubalis genome assemblies show that the mapping approach revealed higher levels of heterozygosity at both nucleotide sites and indels (insertions and deletions) (0.09-0.15%), as well as several sequence errors (0.07%). Our phylogenetic and molecular dating analyses provide strong evidence that the lowland anoa, river buffalo, and swamp buffalo are three distinct taxa which separated rapidly from each other during the Pleistocene epoch. We therefore conclude that two species of domestic water buffalo should be distinguished: Bubalus bubalis for the river buffalo and Bubalus kerabau for the swamp buffalo. The new classification can have deep implications for understanding the evolution and selection of domesticated forms and for the conservation and management of wild buffalo populations in South and Southeast Asia.

Highly rearranged mitochondrial genome in Nycteria parasites (Haemosporidia) from bats.

Haemosporidia parasites have mostly and abundantly been described using mitochondrial genes, and in particular cytochrome b (cytb). Failure to amplify the mitochondrial cytb gene of Nycteria parasites isolated from Nycteridae bats has been recently reported. Bats are hosts to a diverse and profuse array of Haemosporidia parasites that remain largely unstudied. There is a need to obtain more molecular data from chiropteran parasites. Such data would help to better understand the evolutionary history of Haemosporidia, which notably include the Plasmodium parasites, malaria's agents. We use next-generation sequencing to obtain the complete mitochondrial genome of Nycteria parasites from African Nycteris grandis (Nycteridae) and Rhinolophus alcyone (Rhinolophidae) and Asian Megaderma spasma (Megadermatidae). We report four complete mitochondrial genomes, including two rearranged mitochondrial genomes within Haemosporidia. Our results open outlooks into potentially undiscovered Haemosporidian diversity.

Multi-locus phylogeny of the tribe Tragelaphini (Mammalia, Bovidae) and species delimitation in bushbuck: Evidence for chromosomal speciation mediated by interspecific hybridization.

The bushbuck is the most widespread bovid species in Africa. Previous mitochondrial studies have revealed a polyphyletic pattern suggesting the possible existence of two distinct species. To assess this issue, we have sequenced 16 nuclear genes and one mitochondrial fragment (cytochrome b gene + control region) for most species of the tribe Tragelaphini, including seven bushbuck individuals belonging to the two divergent mtDNA haplogroups, Scriptus and Sylvaticus. Our phylogenetic analyses show that the Scriptus lineage is a sister-group of Sylvaticus in the nuclear tree, whereas it is related to Tragelaphus angasii in the mitochondrial tree. This mito-nuclear discordance indicates that the mitochondrial genome of Scriptus was acquired by introgression after one or several past events of hybridization between bushbuck and an extinct species closely related to T. angasii. The division into two bushbuck species is supported by the analyses of nuclear markers and by the karyotype here described for T. scriptus (2n = 57 M/58F), which is strikingly distinct from the one previously found for T. sylvaticus (2n = 33 M/34F). Molecular dating estimates suggest that the two species separated during the Early Pleistocene after an event of interspecific hybridization, which may have mediated massive chromosomal rearrangements in the common ancestor of T. scriptus.

Phylogeny, biogeography and character evolution in the tribe Desmodieae (Fabaceae: Papilionoideae), with special emphasis on the New Caledonian endemic genera.

The nearly cosmopolitan tribe Desmodieae (Fabaceae) includes many important genera for medicine and forage. However, the phylogenetic relationships among the infratribal groups circumscribed using morphological traits are still poorly known. In this study, we used chloroplast (rbcL, psbA-trnH) and nuclear (ITS-1) DNA sequences to investigate the molecular phylogeny and historical biogeography of Desmodieae, and infer ancestral states for several vegetative and reproductive traits. Three groups, corresponding to the Desmodium, Lespedeza, and Phyllodium groups sensu Ohashi were retrieved in the phylogenetic analyses. Conflicts in the topologies inferred from the chloroplast and nuclear datasets were detected. For instance, the Lespedeza clade was sister to the groups Phyllodium+Desmodium based on chloroplast DNA, but nested within the Desmodium group based on ITS-1. Moreover, the New Caledonian endemic genera Arthroclianthus and Nephrodesmus were not monophyletic but together formed a clade, which also included Hanslia and Ohwia based on chloroplast DNA. The hypothetical common ancestor of Desmodieae was dated to the Middle Oligocene (ca. 28.3Ma) and was likely an Asian shrub or tree producing indehiscent loments. Several colonization events towards Oceania, America, and Africa occurred (all less than ca. 17.5Ma), most probably through long distance dispersal. The fruits of Desmodieae repeatedly evolved from indehiscence to dehiscence. We also showed that indehiscent loments allow for more variability in the number of seeds per fruit than indehiscent legumes. Modularity seems here to allow variability in the number of ovules produced in a single ovary.

Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants.

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation and likely contribute to the remarkable diversity of placental structures. Independent capture events have been identified in primates, rodents, lagomorphs, and carnivores, where they are involved in the formation of a syncytium layer at the fetomaternal interface via trophoblast cell-cell fusion. We searched for similar genes within the suborder Ruminantia where the placenta lacks an extended syncytium layer but displays a heterologous cell-fusion process unique among eutherian mammals. An in silico search for intact envelope genes within the Bos taurus genome identified 18 candidates belonging to five endogenous retrovirus families, with one gene displaying both placenta-specific expression, as assessed by quantitative RT-PCR analyses of a large panel of tissues, and conservation in the Ovis aries genome. Both the bovine and ovine orthologs displayed fusogenic activity by conferring infectivity on retroviral pseudotypes and triggering cell-cell fusion. In situ hybridization of placenta sections revealed specific expression in the trophoblast binucleate cells, consistent with a role in the formation--by heterologous cell fusion with uterine cells--of the trinucleate cells of the cow and the syncytial plaques of the ewe. Finally, we show that this gene, which we named "Syncytin-Rum1," is conserved among 16 representatives of higher ruminants, with evidence for purifying selection and conservation of its fusogenic properties, over 30 millions years of evolution. These data argue for syncytins being a major driving force in the emergence and diversity of the placenta.

Bat Rhinacoviruses Related to Swine Acute Diarrhoea Syndrome Coronavirus Evolve under Strong Host and Geographic Constraints in China and Vietnam.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV; Coronaviridae, Rhinacovirus) was detected in 2017 in Guangdong Province (China), where it caused high mortality rates in piglets. According to previous studies, SADS-CoV evolved from horseshoe bat reservoirs. Here, we report the first five Rhinacovirus genomes sequenced in horseshoe bats from Vietnam and their comparisons with data published in China. Our phylogenetic analyses provided evidence for four groups: rhinacoviruses from Rhinolphus pusillus bats, including one from Vietnam; bat rhinacoviruses from Hainan; bat rhinacoviruses from Yunnan showing a divergent synonymous nucleotide composition; and SADS-CoV and related bat viruses, including four rhinacoviruses from Vietnam sampled in Rhinolophus affinis and Rhinolophus thomasi. Our phylogeographic analyses showed that bat rhinacoviruses from Dien Bien (Vietnam) share more affinities with those from Yunnan (China) and that the ancestor of SADS-CoVs arose in Rhinolophus affinis circulating in Guangdong. We detected sequencing errors and artificial chimeric genomes in published data. The two SADS-CoV genomes previously identified as recombinant could also be problematic. The reliable data currently available, therefore, suggests that all SADS-CoV strains originate from a single bat source and that the virus has been spreading in pig farms in several provinces of China for at least seven years since the first outbreak in August 2016.

Combining multiple autosomal introns for studying shallow phylogeny and taxonomy of Laurasiatherian mammals: Application to the tribe Bovini (Cetartiodactyla, Bovidae).

Mitochondrial sequences are widely used for species identification and for studying phylogenetic relationships among closely related species or populations of the same species. However, many studies of mammals have shown that the maternal history of the mitochondrial genome can be discordant with the true evolutionary history of the taxa. In such cases, the analyses of multiple nuclear genes can be more powerful for deciphering interspecific relationships. Here, we designed primers for amplifying 13 new exon-primed intron-crossing (EPIC) autosomal loci for studying shallow phylogeny and taxonomy of Laurasiatherian mammals. Three criteria were used for the selection of the markers: gene orthology, a PCR product length between 600 and 1200 nucleotides, and different chromosomal locations in the bovine genome. Positive PCRs were obtained from different species representing the orders Carnivora, Cetartiodactyla, Chiroptera, Perissodactyla and Pholidota. The newly developed markers were analyzed in a phylogenetic study of the tribe Bovini (the group containing domestic and wild cattle, bison, yak, African buffalo, Asian buffalo, and saola) based on 17 taxa and 18 nuclear genes, representing a total alignment of 13,095 nucleotides. The phylogenetic results were compared to those obtained from analyses of the complete mitochondrial genome and Y chromosomal genes. Our analyses support a basal divergence of the saola (Pseudoryx) and a sister-group relationship between yak and bison. These results contrast with recent molecular studies but are in better agreement with morphology. The comparison of pairwise nucleotide distances shows that our nuDNA dataset provides a good signal for identifying taxonomic levels, such as species, genera, subtribes, tribes and subfamilies, whereas the mtDNA genome fails because of mtDNA introgression and higher levels of homoplasy. Accordingly, we conclude that the genus Bison should be regarded as a synonym of Bos, with the European bison relegated to a subspecies rank within Bos bison. We compared our molecular dating estimates to the fossil record in order to propose a biogeographic scenario for the evolution of Bovini during the Neogene.

Molecular systematics and phylogeography of the tribe Myonycterini (Mammalia, Pteropodidae) inferred from mitochondrial and nuclear markers.

The tribe Myonycterini comprises five fruit bat species of the family Pteropodidae, which are endemic to tropical Africa. Previous studies have produced conflicting results about their interspecific relationships. Here, we performed a comparative phylogeographic analysis based on 148 complete cytochrome b gene sequences from the three species distributed in West Africa and Central Africa (Myonycteris torquata, Lissonycteris angolensis and Megaloglossus woermanni). In addition, we investigated phylogenetic relationships within the tribe Myonycterini, using a matrix including 29 terminal taxa and 7235 nucleotide characters, corresponding to an alignment of two mitochondrial genes and seven nuclear introns. Our phylogenetic analyses confirmed that the genus Megaloglossus belongs to the tribe Myonycterini. Further, the genus Rousettus is paraphyletic, with R. lanosus, sometimes placed in the genus Stenonycteris, being the sister-group of the tribes Myonycterini and Epomophorini. Our phylogeographic results showed that populations of Myonycteris torquata and Megaloglossus woermanni from the Upper Guinea Forest are highly divergent from those of the Congo Basin Forest. Based on our molecular data, we recommended several taxonomic changes. First, Stenonycteris should be recognized as a separate genus from Rousettus and composed of S. lanosus. This genus should be elevated to a new tribe, Stenonycterini, within the subfamily Epomophorinae. This result shows that the evolution of lingual echolocation was more complicated than previously accepted. Second, the genus Lissonycteris is synonymised with Myonycteris. Third, the populations from West Africa formerly included in Myonycteris torquata and Megaloglossus woermanni are now placed in two distinct species, respectively, Myonycteris leptodon and Megaloglossus azagnyi sp. nov. Our molecular dating estimates show that the three phases of taxonomic diversification detected within the tribe Myonycterini can be related to three distinct decreases in tree cover vegetation, at 6.5-6, 2.7-2.5, and 1.8-1.6Ma. Our results suggest that the high nucleotide distance between Ebolavirus Côte d'Ivoire and Ebolavirus Zaire can be correlated with the Plio/Pleistocene divergence between their putative reservoir host species, i.e., Myonycteris leptodon and Myonycteris torquata, respectively.

Retracing Phylogenetic, Host and Geographic Origins of Coronaviruses with Coloured Genomic Bootstrap Barcodes: SARS-CoV and SARS-CoV-2 as Case Studies.

Phylogenetic trees of coronaviruses are difficult to interpret because they undergo frequent genomic recombination. Here, we propose a new method, coloured genomic bootstrap (CGB) barcodes, to highlight the polyphyletic origins of human sarbecoviruses and understand their host and geographic origins. The results indicate that SARS-CoV and SARS-CoV-2 contain genomic regions of mixed ancestry originating from horseshoe bat (Rhinolophus) viruses. First, different regions of SARS-CoV share exclusive ancestry with five Rhinolophus viruses from Southwest China (RfYNLF/31C: 17.9%; RpF46: 3.3%; RspSC2018: 2.0%; Rpe3: 1.3%; RaLYRa11: 1.0%) and 97% of its genome can be related to bat viruses from Yunnan (China), supporting its emergence in the Rhinolophus species of this province. Second, different regions of SARS-CoV-2 share exclusive ancestry with eight Rhinolophus viruses from Yunnan (RpYN06: 5.8%; RaTG13: 4.8%; RmYN02: 3.8%), Laos (RpBANAL103: 3.3%; RmarBANAL236: 1.7%; RmBANAL52: 1.0%; RmBANAL247: 0.7%), and Cambodia (RshSTT200: 2.3%), and 98% of its genome can be related to bat viruses from northern Laos and Yunnan, supporting its emergence in the Rhinolophus species of this region. Although CGB barcodes are very useful in retracing the origins of human sarbecoviruses, further investigations are needed to better take into account the diversity of coronaviruses in bats from Cambodia, Laos, Myanmar, Thailand and Vietnam.

Identifying the Most Probable Mammal Reservoir Hosts for Monkeypox Virus Based on Ecological Niche Comparisons.

Previous human cases or epidemics have suggested that Monkeypox virus (MPXV) can be transmitted through contact with animals of African rainforests. Although MPXV has been identified in many mammal species, most are likely secondary hosts, and the reservoir host has yet to be discovered. In this study, we provide the full list of African mammal genera (and species) in which MPXV was previously detected, and predict the geographic distributions of all species of these genera based on museum specimens and an ecological niche modelling (ENM) method. Then, we reconstruct the ecological niche of MPXV using georeferenced data on animal MPXV sequences and human index cases, and conduct overlap analyses with the ecological niches inferred for 99 mammal species, in order to identify the most probable animal reservoir. Our results show that the MPXV niche covers three African rainforests: the Congo Basin, and Upper and Lower Guinean forests. The four mammal species showing the best niche overlap with MPXV are all arboreal rodents, including three squirrels: Funisciurus anerythrus, Funisciurus pyrropus, Heliosciurus rufobrachium, and Graphiurus lorraineus. We conclude that the most probable MPXV reservoir is F. anerythrus based on two niche overlap metrics, the areas of higher probabilities of occurrence, and available data on MPXV detection.

Detection and genetic characterization of circoviruses in more than 80 bat species from eight countries on four continents.

Several bat-associated circoviruses and circular rep-encoding single-stranded DNA (CRESS DNA) viruses have been described, but the exact diversity and host species of these viruses are often unknown. Our goal was to describe the diversity of bat-associated circoviruses and cirliviruses, thus, 424 bat samples from more than 80 species were collected on four continents. The samples were screened for circoviruses using PCR and the resulting amino acid sequences were subjected to phylogenetic analysis. The majority of bat strains were classified in the genus Circovirus and some strains in the genus Cyclovirus and the clades CRESS1 and CRESS3. Some strains, however, could only be classified at the taxonomic level of the order and were not classified in any of the accepted or proposed clades. In the family Circoviridae, 71 new species have been predicted. This screening of bat samples revealed a great diversity of circoviruses and cirliviruses. These studies underline the importance of the discovery and description of new cirliviruses and the need to establish new species and families in the order Cirlivirales.

Nucleotide composition of CO1 sequences in Chelicerata (Arthropoda): detecting new mitogenomic rearrangements.

Here we study the evolution of nucleotide composition in third codon-positions of CO1 sequences of Chelicerata, using a phylogenetic framework, based on 180 taxa and three markers (CO1, 18S, and 28S rRNA; 5,218 nt). The analyses of nucleotide composition were also extended to all CO1 sequences of Chelicerata found in GenBank (1,701 taxa). The results show that most species of Chelicerata have a positive strand bias in CO1, i.e., in favor of C nucleotides, including all Amblypygi, Palpigradi, Ricinulei, Solifugae, Uropygi, and Xiphosura. However, several taxa show a negative strand bias, i.e., in favor of G nucleotides: all Scorpiones, Opisthothelae spiders and several taxa within Acari, Opiliones, Pseudoscorpiones, and Pycnogonida. Several reversals of strand-specific bias can be attributed to either a rearrangement of the control region or an inversion of a fragment containing the CO1 gene. Key taxa for which sequencing of complete mitochondrial genomes will be necessary to determine the origin and nature of mtDNA rearrangements involved in the reversals are identified. Acari, Opiliones, Pseudoscorpiones, and Pycnogonida were found to show a strong variability in nucleotide composition. In addition, both mitochondrial and nuclear genomes have been affected by higher substitution rates in Acari and Pseudoscorpiones. The results therefore indicate that these two orders are more liable to fix mutations of all types, including base substitutions, indels, and genomic rearrangements.

Molecular phylogenetics unveils the ancient evolutionary origins of the enigmatic fairy armadillos.

Fairy armadillos or pichiciegos (Xenarthra, Dasypodidae) are among the most elusive mammals. Due to their subterranean and nocturnal lifestyle, their basic biology and evolutionary history remain virtually unknown. Two distinct species with allopatric distributions are recognized: Chlamyphorus truncatus is restricted to central Argentina, while Calyptophractus retusus occurs in the Gran Chaco of Argentina, Paraguay, and Bolivia. To test their monophyly and resolve their phylogenetic affinities within armadillos, we obtained sequence data from modern and museum specimens for two mitochondrial genes (12S RNA [MT-RNR1] and NADH dehydrogenase 1 [MT-ND1]) and two nuclear exons (breast cancer 1 early onset exon 11 [BRCA1] and von Willebrand factor exon 28 [VWF]). Phylogenetic analyses provided a reference phylogeny and timescale for living xenarthran genera. Our results reveal monophyletic pichiciegos as members of a major armadillo subfamily (Chlamyphorinae). Their strictly fossorial lifestyle probably evolved as a response to the Oligocene aridification that occurred in South America after their divergence from Tolypeutinae around 32 million years ago (Mya). The ancient divergence date (∼17Mya) for separation between the two species supports their taxonomic classification into distinct genera. The synchronicity with Middle Miocene marine incursions along the Paraná river basin suggests a vicariant origin for pichiciegos by the disruption of their ancestral range. Their phylogenetic distinctiveness and rarity in the wild argue in favor of high conservation priority.

Variation in synonymous nucleotide composition among genomes of sarbecoviruses and consequences for the origin of COVID-19.

The subgenus Sarbecovirus includes two human viruses, SARS-CoV and SARS-CoV-2, respectively responsible for the SARS epidemic and COVID-19 pandemic, as well as many bat viruses and two pangolin viruses. Here, the synonymous nucleotide composition (SNC) of Sarbecovirus genomes was analysed by examining third codon-positions, dinucleotides, and degenerate codons. The results show evidence for the eight following groups: (i) SARS-CoV related coronaviruses (SCoVrC including many bat viruses from China), (ii) SARS-CoV-2 related coronaviruses (SCoV2rC; including five bat viruses from Cambodia, Thailand and Yunnan), (iii) pangolin sarbecoviruses, (iv) three bat sarbecoviruses showing evidence of recombination between SCoVrC and SCoV2rC genomes, (v) two highly divergent bat sarbecoviruses from Yunnan, (vi) the bat sarbecovirus from Japan, (vii) the bat sarbecovirus from Bulgaria, and (viii) the bat sarbecovirus from Kenya. All these groups can be diagnosed by specific nucleotide compositional features except the one concerned by recombination between SCoVrC and SCoV2rC. In particular, SCoV2rC genomes have less cytosines and more uracils at third codon-positions than other sarbecoviruses, whereas the genomes of pangolin sarbecoviruses show more adenines at third codon-positions. I suggest that taxonomic differences in the imbalanced nucleotide pools available in host cells during viral replication can explain the eight groups of SNC here detected among Sarbecovirus genomes. A related effect due to hibernating bats and their latitudinal distribution is also discussed. I conclude that the two independent host switches from Rhinolophus bats to pangolins resulted in convergent mutational constraints and that SARS-CoV-2 emerged directly from a horseshoe bat sarbecovirus.

Genomic Bootstrap Barcodes and Their Application to Study the Evolution of Sarbecoviruses.

Recombination creates mosaic genomes containing regions with mixed ancestry, and the accumulation of such events over time can complicate greatly many aspects of evolutionary inference. Here, we developed a sliding window bootstrap (SWB) method to generate genomic bootstrap (GB) barcodes to highlight the regions supporting phylogenetic relationships. The method was applied to an alignment of 56 sarbecoviruses, including SARS-CoV and SARS-CoV-2, responsible for the SARS epidemic and COVID-19 pandemic, respectively. The SWB analyses were also used to construct a consensus tree showing the most reliable relationships and better interpret hidden phylogenetic signals. Our results revealed that most relationships were supported by just a few genomic regions and confirmed that three divergent lineages could be found in bats from Yunnan: SCoVrC, which groups SARS-CoV related coronaviruses from China; SCoV2rC, which includes SARS-CoV-2 related coronaviruses from Southeast Asia and Yunnan; and YunSar, which contains a few highly divergent viruses recently described in Yunnan. The GB barcodes showed evidence for ancient recombination between SCoV2rC and YunSar genomes, as well as more recent recombination events between SCoVrC and SCoV2rC genomes. The recombination and phylogeographic patterns suggest a strong host-dependent selection of the viral RNA-dependent RNA polymerase. In addition, SARS-CoV-2 appears as a mosaic genome composed of regions sharing recent ancestry with three bat SCoV2rCs from Yunnan (RmYN02, RpYN06, and RaTG13) or related to more ancient ancestors in bats from Yunnan and Southeast Asia. Finally, our results suggest that viral circular RNAs may be key molecules for the mechanism of recombination.