The role of Molossidae and Vespertilionidae in shaping the diversity of alphacoronaviruses in the Americas
[Gimme Shelter (and some coronaviruses too!) ]
Four of the seven alphaCoV clades found in the Americas are maintained by Phyllostomidae, which was pointed out as a key bat family in the origins and diversification of alphaCoVs, based on high CST rates. The remaining three alphaCoV clades are maintained by members of the Vespertilionidae and Molossidae, suggesting a possible link between these two bat families.
In this study, we explored the epidemiological links between molossid and vespertilionid bats in the Americas, also contributing the first CoV data set from Argentina.
Phyllostomidae clades
Vespertilionidae-Molossidae clades
The American bat alphaCoVs clustered into seven clades (A to G). The 21 sequences from Argentina pertain to clades A and B and were isolated from Tadarida, Molossus, and Myotis.
Clade A includes a sequence isolated from Molossus (Argentina) related to a large group including Brazilian sequences from this genus, and a novel Tadarida lineage from Argentina. The ancestral state reconstruction revealed 3 spillovers from Molossus to Glossophaga and Eptesicus.
Clade B contains alphaCoV sequences from Myotis, Tadarida, and Molossus from Argentina. A Tadarida-to-Myotis spillover and a host shift from Tadarida to Molossus were identified.
The samples isolated from Molossus form a monophyletic group suggesting that this variant is also maintained by mastiff bats.
The ancestral state reconstruction suggests that the ancestral host for clade B is Myotis, which is also supported by the higher phylogenetic diversity.
Clades A and B show marked host and geographic structures. Spillovers took place in geographic regions that correspond to overlapping host ranges.
Although both viral clades are maintained by vespers and molossids, only one of these viral lineages circulate in most bat genera.
However, there is an interesting exception. We found that two nonrelated lineages (A and B) co-circulate in Tadarida and Molossus in the same region (Argentina and Brazil).
These bat genera share different variants and are capable of maintaining high levels of CoV diversity.
Myotis is probably the major evolutionary reservoir of alphaCoVs in the Americas. Two of the three vespertilionid/molossid clades are maintained totally (clade C) or partially (clade B) by Myotis, covering the complete geographic distribution of the studied region.
Its widespread occurrence, together with being the richest host in terms of viral phylogenetic diversity, and that it has several representative lineages across the phylogeny, including basally splitting lineages, pose Myotis as the most likely ancestor for clades B and C.
The role of Myotis as a reservoir and a key link in the evolution of CoVs appears to be a global trend. It has been involved in several significant host switches during alphaCoV evolution in China and harbors different alphaCoVs in America, Asia, Europe, and Oceania.
Taken together, these results confirm that the widespread genera Myotis, Molossus, and Tadarida are determinant hosts in the origin and dissemination, via cross-species transmission, of alphaCoVs in the Americas.
Cross-species transmission of bat coronaviruses in the Americas
[Welcome to the jungle (where there are more bat species and higher coronavirus transmission rates!)]
Coronaviruses (CoVs) have a strong zoonotic potential due to their high rates of evolvavility and their capacity for overcoming host-specific barriers. Bats harbor the largest number of CoV species among mammals, with the highest CoV diversity found in areas with the highest levels of bat species richness. Understanding their origin and patterns of cross-species transmission is crucial for pandemic preparedness. This study aimed to understand how Americas’ bat-CoVs diversify, circulate among, and transmit between bat families and genera, and ultimately to identify bat hosts and regions where the risk of CoV spillover is the highest.
alpha and betaCoV ancestral host reconstruction
alphaCoV and bat hosts co-phylogeny
betaCoV and bat hosts co-phylogeny
Substantial levels of CST were only found among alphaCoVs. In contrast, cospeciation prevailed along the evolution of betaCoVs. Brazil is the center of diversification for both alpha and betaCoVs, with the highest levels of bat species richness. The bat family Phyllostomidae has played a key role in the evolution of American bat-CoVs, supported by the highest values of host transition rates.
Global bat species distributions (upper panel), and distribution of bat-CoV hosts in the Americas (lower panel).
Host transition rates among American bat genera
Phylogenetic reconciliation between betaCoVs and bat hosts trees
Identification of a novel Rabies virus lineage circulating in mesocarnivores in South America
[Barking dogs never bite?]
The rabies virus (RABV) is characterized by a history dominated by host shifts within and among bats and carnivores. One of the main outcomes of long-term RABV maintenance in dogs was the establishment of variants in a wide variety of mesocarnivores.
We carried out a phylogenetic and phylogeographic analysis, contributing to a better understanding of the origins, diversification, and the role of different host species in the evolution and diffusion of a dog-related variant endemic of South America. A total of 237 complete Nucleoprotein gene sequences were studied, corresponding to wild and domestic species, performing selection analyses, ancestral states reconstructions, and recombination analyses.
This variant originated in Brazil and disseminated through Argentina and Paraguay, where a previously unknown lineage was found. A single host shift was identified in the phylogeny, from dog to the crab-eating fox (Cerdocyon thous) in the Northeast of Brazil. Although this process occurred in a background of purifying selection, there is evidence of adaptive evolution -or selection of sub-consensus sequences- in internal branches after the host shift. The interaction of domestic and wild cycles persisted after host switching, as revealed by spillovers and putative recombination events.
Phylogenetic position of the novel RABV lineage (Am3c)
Ancestral host recosntruction of the AgV2 variant.
Phylogeographic reconstruction
Selection analysis and recombination events
Use and limitations of cytochrome b for bat species identification
[One gene to know them all?]
Bats (order Chiroptera) include more than 1400 species representing 20% of total mammal diversity. Species identification is not trivial among such a diverse taxonomic group. Morphological traits, echolocation call patterns, and molecular phylogenetic approaches frequently produce conficting clusterings, since species limits are not obvious in morphologically cryptic species, or in recently deverged taxa (which fail to produce reciprocally monophyletic groups when studied on the basis of a limited number of genes). These phenomena are likely to complicate species identification at fieldwork, especially between closely related species, where external morphology-based approaches are the only immediate means for a priori species assignment.
To discuss the extent of the use of the mitochondrial cytochrome b gene for species identification, and current bat systematics, we used as a case study a set of samples collected in a Rabies virus eco-epidemiological survey involving five localities in the province of Santa Fe (Argentina).
We contrasted our results with current systematics of all studied genera based on molecular, morphological, integrative, or genomic approaches showing that Cytb is useful for species-level delimitation in non-conflicting genera (Eumops, Dasypterus, Molossops) and has infrageneric resolution in taxonomically challenging lineages (Eptesicus, Myotis, Molossus). This is an important outcome since, in most ecological and eco-epidemiological studies, the objective is to capture and release animals after taking small biopsies for their identification.
Molecular processes (low genetic differentiation, incomplete lineage sorting), biological events (introgression), limitations in morphological identification, and errors in current taxonomy, may act in isolation or combination, having more impact in taxonomically challenging groups. In these cases, the use of a limited number of loci and/or the application of morphological approaches would be insufficient for species determination. The continuously increasing use and decreasing costs of genomic approaches (eg. GBS) may be the step forward to enhance accurate species delimitation and identification.
Cytochrome b-based Bayesian phylogeny of bats at the genus/family level.
When sequence variation precludes virus diagnosis
[Fishing with the wrong bait]
It is widely known that RNA viruses have the highest evolutionary rates observed. The Rabies virus (RABV) has been historically detected through Direct Imnunofluorescence (FAT, Fluorescence Antibody Test) but recently TaqMan probe-based quantitative RT-PCR has been considered also a gold standard technique.
A key aspect that may impede efficient positive diagnosis in RABV (and other RNA viruses) is a direct consequence of rapid evolvavility: sequence variation at the sites of primers or probes annealing.
We tested two widely used TaqMan probe-based quantitative RT-PCR assays targeting the nucleoprotein gene (LysGT1 assay) and leader sequences (LN34 qRT-PCR assay) of RABV genomes, across all RABV variants circulating in Argentina.
Samples obtained from bats, dogs, cattle, and horses, that were previously tested for rabies by FAT and conventional RT-PCR, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The LysGT1 assay failed to detect three bat-related variants. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of three or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results, and in consequence, the LN34 assay would ensure more effective detection of RABV in Argentina.
Bayesian phylogeny of 87 sequences based on 191 bp involving the 3′ terminus of the RABV N gene and 51 downstream nucleotides. Bayesian posterior probability of main clades is shown. In the left panel, the subclade (V3, V3A) is expanded.
Alignment showing variability of Argentinian RABV genetic variants in the region comprising the amplicon used in the LysGT1 qRT-PCR assay. Primers JW12 and N165-146 and the probe LysGT1 were mapped in the resulting alignment. Dots depict conserved nucleotides. Mismatches of the primers and probe are indicated with black empty boxes.
Model organisms, non-model genes
Approximately one-third of all cellular proteins are imported into the lumen of the endoplasmic reticulum (ER) or integrated into its membranes and most of them are glycoproteins. The UDP- glucose: glycoprotein glucosyltransferase (UGGT) is a protein that operates as the gatekeeper for the ER quality control (QC) mechanism of glycoprotein folding. The QC allows cells to differentiate between native and non-native protein conformations, exporting properly folded proteins to their destination, and eliminating those which fail to fold adequately.
UGGT function is widely conserved across eukaryotes and only a few organisms lack UGGT activity. Two genes encode UGGT-like proteins in humans (HUGT1 and HUGT2), and in Caenorhabditis: Ce-uggt-a and Ce-uggt-b. On the contrary, genomes of other model species as Drosophila melanogaster, Arabidopsis thaliana, and also Trypanosoma cruzi carry a single uggt gene.
It has been demonstrated that Ce-UGGT-a and Ce-UGGT-b have different functions in C. elegans. Whereas Ce-UGGT-a displays canonical UGGT activity, Ce-UGGT-b proved to be completely inactive. In contrast, evidence supports the notion that both human, and all vertebrate UGGT1 and UGGT2 are active.
Through a Bayesian phylogenetic analysis based on 195 UGGT and UGGT-like protein sequences of an ample spectrum of eukaryotic species we demonstrated that uggt genes went through independent duplications in Caenorhabditis and vertebrates.
We further estimated rates of non-synonymous (dN) to synonymous (dS) susbtitution rates to assess if natural selection has affected the evolution of uggt paralogs. The results reflect a generalized background of purifying selection in all UGGTs, suggesting that both duplicates, in vertebrates as well as in Caenorhabditis, have been subjected to functional constraints. The catalytic domain is highly conserved as a product of a strong negative selective pressure and shows almost no evidence of positive selection. In contrast, the recognition domain has undergone a positive diversifying selection process in UGGT2/UGGT-b of both lineages.
We found conservation of the UGGT activity in the catalytic domain and a putative divergent function of the recognition domain for the UGGT2 protein in vertebrates, which would have gone through a specialization process. In Caenorhabditis, uggt-b evolved under different constraints compared to uggt-a which, by means of a putative neofunctionalization process, resulted in a non-redundant paralog. The non-canonical function of uggt-b in the worm lineage highlights the need to take precautions before generalizing gene functions in model organisms.
Phylogenetic tree showing independent origins of UGGT paralogues in vertebrates and Caenorhabditis.
Differential domain conservation in UGGTs.
Spatio-temporal dynamics of the torquatus species group of the subterranean rodents of the genus Ctenomys
[Rise and reign of an underground dynasty]
Subterranean rodents of the genus Ctenomys have experienced an explosive radiation and rapidly colonized the southern cone of South America. The torquatus group, one of the main groups of the genus, comprises several species and species complexes which inhabit the eastern part of the distribution of Ctenomys including southern Brazil, northern and central Uruguay and northeastern Argentina. This group has undergone a high chromosomal diversification with diploid numbers varying from 41 to 70.
Timetree showing main lineages within Ctenomys.
The torquatus group originated during the early Pleistocene between 1.25 and 2.32 million years from the present in a region that includes the northwest of Uruguay and the southeast of the Brazilian state of Río Grande do Sul. Most lineages have dispersed early towards their present distribution areas going through subsequent range expansions in the last 800,000-700,000 years. Ctenomys torquatus went through a rapid range expansion for the last 200,000 years, becoming the most widespread species of the group. The colonization of the Corrientes and Entre Ríos Argentinean provinces supposes at least two crossing events across the Uruguay River between 1.0 and 0.5 million years before the present, in the context of a cold and dry paleoenvironment. The resulting temporal and geographic frame enables the comprehension of the incidence of both, the amplitude of distribution areas and divergence times into the patterns of chromosomal diversification found in the group.
We investigated the origins of the torquatus group as well as its diversification patterns in relation to geography and cladogenesis, based on mitochondrial cytochrome b nucleotide sequences. We conducted a Bayesian multi-calibrated relaxed clock analysis to estimate the ages of the torquatus group and its main lineages. Using the estimated evolutionary rate we performed a continuous phylogeographic analysis, to reconstruct the geographic diffusion of the torquatus group in a temporal frame.
Species ranges of the torquatus group.
Spatio-temporal reconstruction of the torquatus group spread.
Reassessing the causal connection between satDNA dynamics and chromosomal evolution in Ctenomys
[The Y is why]
Fulltext link: https://doi.org/10.1163/18759866-bja10052
Methodology:
Males have 3x RPCS compared with females
The evolution of RPCS copy number followed different trajectories in males and females.
The shifts in RPCS copy number would not explain the most extensive karyotypic repatterning events (red branches).
It is crucial to consider the influence of sex crhomosomes in the study of satDNA evolution.
Integrative lineage delimitation in the most chromosomally variable group of Ctenomys
[Not so cryptic after all]
In past decades, specialists have advocated for distinct and partially incompatible species concepts, leading to different conclusions about species boundaries and numbers of species. These incompatibilities emerge from considering different biological properties upon which several alter- native concepts are based. The reason why different biological features lead to incompatible species concepts is that they evolve at different rates, and not even necessarily in the same order during the process of speciation. Before the acquisition of the first distinct biological property, there would be overall agreement in the existence of a single species. At the other extreme, where putative species become phenotipically distinguishable, reproductively incompatible, diagnosable, ecologically distinct, etc, there would be full agreement on the fact that there are two different species. Lineage delimitation becomes more confusing as divergence times are shorter, not only because there is little differentiation, but also because there is a greater discordance among different character sources.
Any property that provides evidence of lineage separation is relevant to the inference of species boundaries and the number of species, a highly corroborated hypothesis of lineage separation will require multiple lines of evidence.
The tuco-tucos of the Corrientes group inhabit a vast area under the influence of the Iberá wetland, the second largest in South America, a geological depression dominated by lakes, swamps, marshes, grasslands, and forests. Demes of tuco-tucos of the Corrientes group are patchy distributed in sandy soils, predominant in this area. However, proximity to the wetlands makes these habitats unstable as the system’s water-level fluctuates depending on rainfall and surface as well as underground rivers flow.
To date, 26 populations have been thoroughly studied in the Corrientes group, which showed high levels of chromosomal variability, with more than 17 different karyomorphs described, which suggests that chromosomal evolution is an ongoing and recurrent process in this group. We also studied the Corrientes group from a phylogenetic point of view using mitocondrial markers to get insight into chrosmosomal evolution in the group and its relationship with the closely related Ctenomys pearsoni complex.
The most likely scenario is that diploid number went through several reductions via centric fusions. However, we did not find interstitial telomeric sequences (ITS), suggesting that if fusions prevailed, these sequences degenerated or were lost.
Integrating karyotypes, phylogenetics and population genetics, we delimited a total of seven independently evolving lineages in the Corrientes group.
Tuco-tucos from the Corrientes group.
Distribution of independent lineages from the Ctenomys Corrientes group.
Karyomorphs of the Ctenomys Corrientes group
Overlap between species distributions and Protected Areas as a tool for conservation
[Engineer saved, ecosystem secured]
South American rodents of the genus Ctenomys, popularly known as tuco-tucos, inhabit the southern cone of the continent, from Perú and Brazil to Tierra del Fuego in Argentina. Tuco-tucos are the most speciose subterranean rodent genus and one of the most among mammals, counting more than 60 species. Enhanced sampling and taxonomic efforts are still needed (but being carried out) to assess the status of many nominal forms, to clarify the number of living species as well as to guide ulterior conservation and management programs in Protected Areas.
As we can only protect what we know, and especially where we know that entity occurs, we decided to analyze the extent of overlap of Ctenomys species distributions with Protected Areas with two main objectives: 1) to assess which species occur solely in non-protected areas and 2) which of them have, at least potential, overlap with such areas. We analyzed the current distribution of all extant Ctenomys species, their conservation status and the proportion of their distributions in Protected Areas, and we showed as a case study that this analysis may function as a tool not only for conserving species we know that indeed occur in a Protected Area, but also to seek for "expected species" in such areas.
Knowing for caring
[Knowing for caring]
South American subterranean rodents of the genus Ctenomys (Rodentia, Ctenomyidae, tuco-tuco) are one of the most diverse genera among mammals. Recently described species, new taxonomic revisions, and new distribution range delimitation made the revision of distribution areas and conservation status of these mammals mandatory.
In this study, we integrated potential for conservation in protected areas, and levels of habitat transformation to revise previous conservation status assessments and propose the first assessment for all Data Deficient or not evaluated species of tuco-tucos. Our results indicate that 53 (78%) of these species are threatened and that 47 (69%) have little or no overlap with protected areas, emphasizing the urgent need to conduct conservation efforts.
Here, 18 of 22 species previously classified as Data Deficient resulted in them being put in an at-risk category (VU, EN, CR). In addition, nine species that have not been previously evaluated were classified as threatened, with these two groups comprising more than 47% of the known species. These results posit that the Ctenomyidae are the rodent family with the greatest number of species at risk of extinction. Finally, a total of 33 (49%) species have been reported from three or fewer localities; all considered threatened through the approach implemented in this study. These geographically restricted taxa should be given more attention in conservation programs since the richness of this genus relies on the survival of such species.
See cool interactive maps and tables here: https://lamarck.unl.edu/Tuco-2/
