Congruence between co-occurrence and trait-based networks is scale-dependent: a case study with flea parasites of small mammalian hosts.

We applied a novel framework based on network theory and a concept of modularity that estimates congruence between trait-based ( = functional) co-occurrence networks, thus allowing the inference of co-occurrence patterns and the determination of the predominant mechanism of community assembly. The aim was to investigate the relationships between species co-occurrence and trait similarity in flea communities at various scales (compound communities: across regions within a biogeographic realm or across sampling sites within a geographic region; component communities: across sampling sites within a geographic region; and infracommunities: within a sampling site). We found that compound communities within biogeographic realms were assembled via environmental or host-associated filtering. In contrast, functional and spatial/host-associated co-occurrence networks, at the scale of regional compound communities, mostly indicated either stochastic processes or the lack of dominance of any deterministic process. Analyses of congruence between functional and either spatial (for component communities) or host-associated (for infracommunities) co-occurrence networks demonstrated that assembly rules in these communities varied among host species. In component communities, stochastic processes prevailed, whereas environmental filtering was indicated in 4 and limiting similarity/competition in 9 of 31 communities. Limiting similarity/competition processes dominated in infracommunities, followed by stochastic mechanisms. We conclude that assembly processes in parasite communities are scale-dependent, with different mechanisms acting at different scales.

Local genetic adaptation to habitat in wild chimpanzees.

How populations adapt to their environment is a fundamental question in biology. Yet we know surprisingly little about this process, especially for endangered species such as non-human great apes. Chimpanzees, our closest living relatives, are particularly interesting because they inhabit diverse habitats, from rainforest to woodland-savannah. Whether genetic adaptation facilitates such habitat diversity remains unknown, despite having wide implications for evolutionary biology and conservation. Using 828 newly generated exomes from wild chimpanzees, we find evidence of fine-scale genetic adaptation to habitat. Notably, adaptation to malaria in forest chimpanzees is mediated by the same genes underlying adaptation to malaria in humans. This work demonstrates the power of non-invasive samples to reveal genetic adaptations in endangered populations and highlights the importance of adaptive genetic diversity for chimpanzees.

Human interactions with bats and bat coronaviruses in rural Côte d'Ivoire.

Bats are presumed reservoirs of diverse α- and ß- coronaviruses (CoVs) and understanding the diversity of bat-CoVs and the role bats play in CoV transmission is highly relevant in the context of the current COVID pandemic. We sampled bats in Côte d'Ivoire (2016-2018) living at ecotones between anthropogenic and wild habitats in the Marahoué National Park, a recently encroached protected area, to detect and characterize the CoVs circulating in bats and humans. A total of 314 bats were captured, mostly during the rainy season (78%), and CoV RNA was detected in three of the bats (0.96%). A CoV RNA sequence similar to Chaerephon bat coronavirus/Kenya/KY22/2006 (BtKY22) was found in a Chaerephon cf. pumilus and a Mops sp. fecal swab, while a CoV RNA sequence similar to the two almost identical Kenya bat coronaviruses BtKY55 and BtKY56 (BtKY55/56) was detected in an Epomops buettikoferi oral swab. Phylogenetic analyses indicated differences in the degree of evolutionary host-virus co-speciation for BtKY22 and BtKY55/56. To assess potential for human exposure to these viruses, we conducted human syndromic and community-based surveillance in clinics and high-risk communities. We collected data on participant characteristics, livelihoods, animal contact, and high-risk behaviors that may be associated with exposure to zoonotic diseases. We then collected biological samples for viral testing from 401 people. PCR testing of these biological samples revealed no evidence of CoV infection among the enrolled individuals. We identified higher levels of exposure to bats in people working in crop production and in hunting, trapping and fishing. Finally, we used the 'Spillover' risk-ranking tool to assess the potential for viral spillover and concluded that, while there is no evidence to suggest imminent risk of spillover for these CoVs, their host range and other traits suggest caution and vigilance are warranted in people with high exposure risk.

Identification of the onchocerciasis vector in the Kakoi-Koda focus of the Democratic Republic of Congo.

BACKGROUND: The objective of this study was to characterise the vector in a small hyper-endemic focus of onchocerciasis (the Kakoi-Koda focus) which has recently been discovered on the western slopes of the rift valley above Lake Albert. METHODOLOGY/PRINCIPAL FINDINGS: Aquatic stages of blackflies were collected by hand from streams and rivers, and anthropophilic adult females were collected by human landing catches. Using a combination of morphotaxonomy and DNA barcoding, the blackflies collected biting humans within the focus were identified as Simulium dentulosum and Simulium vorax, which were also found breeding in local streams and rivers. Simulium damnosum s.l., Simulium neavei and Simulium albivirgulatum were not found (except for a single site in 2009 where crabs were carrying S. neavei). Anthropophilic specimens from the focus were screened for Onchocerca DNA using discriminant qualitative real-time triplex PCR. One specimen of S. vorax was positive for Onchocerca volvulus in the body, and out of 155 S. dentulosum, 30% and 11% were infected and infective (respectively). CONCLUSIONS/SIGNIFICANCE: Simulium dentulosum currently appears to be the main vector of human onchocerciasis within the Kakoi-Koda focus, and S. vorax may be a secondary vector. It remains possible that S. neavei was the main (or only) vector in the past having now become rare as a result of the removal of tree-cover and land-use changes. Simulium vorax has previously been shown to support the development of O. volvulus in the laboratory, but this is the first time that S. dentulosum has been implicated as a probable vector of onchocerciasis, and this raises the possibility that other blackfly species which are not generally considered to be anthropophilic vectors might become vectors under suitable conditions. Because S. dentulosum is not a vector in endemic areas surrounding the Kakoi-Koda focus, it is probable that the Kakoi-Koda focus is significantly isolated.

Enhanced surveillance of monkeypox in Bas-Uélé, Democratic Republic of Congo: the limitations of symptom-based case definitions.

BACKGROUND: Following an outbreak of cases of vesicular-pustular rash with fever, evocative of human monkeypox, in Bas-Uélé province, Democratic Republic of Congo, surveillance was strengthened. METHODS: Households with at least one active generalized vesicular-pustular rash case were visited, and contact and clinical history information were collected from all household members. Whenever possible, skin lesions were screened by polymerase chain reaction for the monkeypox virus, followed by the varicella-zoster virus, when negative for the former. RESULTS: Polymerase chain reaction results were obtained for 77 suspected cases, distributed in 138 households, of which 27.3% were positive for monkeypox, 58.4% positive for chickenpox, and 14.3% negative for both. Confirmed monkeypox cases presented more often with monomorphic skin lesions on the palms of the hands and on the soles of the feet. Integrating these three features into the case definition raised the specificity to 85% but would miss 50% of true monkeypox cases. A predictive model fit on patient demographics and symptoms had 97% specificity and 80% sensitivity but only 80% and 33% in predicting out-of-sample cases. CONCLUSION: Few discriminating features were identified and the performance of clinical case definitions was suboptimal. Rapid field diagnostics are needed to optimize worldwide early detection and surveillance of monkeypox.

Correction: Dusabimana et al. Surveillance for Onchocerciasis-Associated Epilepsy and Ov16 IgG4 Testing of Children 6-10 Years Old Should Be Used to Identify Areas Where Onchocerciasis Elimination Programs Need Strengthening. Pathogens 2022, 11, 281.

There was an error in the original publication [...].

Population dynamics and genetic connectivity in recent chimpanzee history.

Knowledge on the population history of endangered species is critical for conservation, but whole-genome data on chimpanzees (Pan troglodytes) is geographically sparse. Here, we produced the first non-invasive geolocalized catalog of genomic diversity by capturing chromosome 21 from 828 non-invasive samples collected at 48 sampling sites across Africa. The four recognized subspecies show clear genetic differentiation correlating with known barriers, while previously undescribed genetic exchange suggests that these have been permeable on a local scale. We obtained a detailed reconstruction of population stratification and fine-scale patterns of isolation, migration, and connectivity, including a comprehensive picture of admixture with bonobos (Pan paniscus). Unlike humans, chimpanzees did not experience extended episodes of long-distance migrations, which might have limited cultural transmission. Finally, based on local rare variation, we implement a fine-grained geolocalization approach demonstrating improved precision in determining the origin of confiscated chimpanzees.

Surveillance for Onchocerciasis-Associated Epilepsy and OV16 IgG4 Testing of Children 6-10 Years Old Should Be Used to Identify Areas Where Onchocerciasis Elimination Programs Need Strengthening.

To eliminate onchocerciasis-associated morbidity, it is important to identify areas where there is still high ongoing Onchocerca volvulus transmission. Between 2015 and 2021, door-to-door surveys were conducted in onchocerciasis-endemic villages in Cameroon, the Democratic Republic of Congo (DRC), Nigeria, South Sudan, and Tanzania to determine epilepsy prevalence and incidence, type of epilepsy and ivermectin therapeutic coverage. Moreover, children aged between six and 10 years were tested for anti-Onchocerca antibodies using the Ov16 IgG4 rapid diagnostic test (RDT). A mixed-effect binary logistic regression analysis was used to assess significantly associated variables of Ov16 antibody seroprevalence. A high prevalence and incidence of epilepsy was found to be associated with a high Ov16 antibody seroprevalence among 6-10-year-old children, except in the Logo health zone, DRC. The low Ov16 antibody seroprevalence among young children in the Logo health zone, despite a high prevalence of epilepsy, may be explained by a recent decrease in O. volvulus transmission because of a decline in the Simulium vector population as a result of deforestation. In the Central African Republic, a new focus of O. volvulus transmission was detected based on the high Ov16 IgG4 seropositivity among children and the detecting of nodding syndrome cases, a phenotypic form of onchocerciasis-associated epilepsy (OAE). In conclusion, Ov16 IgG4 RDT testing of 6-10-year-old children is a cheap and rapid method to determine the level of ongoing O. volvulus transmission and to assess, together with surveillance for OAE, the performance of onchocerciasis elimination programs.

Orbivirus RNA in a Banana Serotine (Afronycteris nanus) Bat in the Republic of the Congo.

Orbiviruses are arthropod borne viruses of vertebrates, with some of them being important pathogens of veterinary, conservation and economic importance, while others are occasionally associated with human disease. Some apparently bat specific orbiviruses have been detected, but little is known about their distribution and diversity. We thus sampled and screened 52 bats living in the Congo Basin, and detected RNA indicative of a novel orbivirus in a single banana serotine (Afronycteris nanus) by PCR. The detected RNA clusters with epizootic haemorrhagic disease virus, bluetongue virus, and others. The findings highlight the need for more studies into arbovirus presence and diversity in bat species.

Quantitative estimates of glacial refugia for chimpanzees (Pan troglodytes) since the Last Interglacial (120,000 BP).

Paleoclimate reconstructions have enhanced our understanding of how past climates have shaped present-day biodiversity. We hypothesize that the geographic extent of Pleistocene forest refugia and suitable habitat fluctuated significantly in time during the late Quaternary for chimpanzees (Pan troglodytes). Using bioclimatic variables representing monthly temperature and precipitation estimates, past human population density data, and an extensive database of georeferenced presence points, we built a model of changing habitat suitability for chimpanzees at fine spatio-temporal scales dating back to the Last Interglacial (120,000 BP). Our models cover a spatial resolution of 0.0467° (approximately 5.19 km2 grid cells) and a temporal resolution of between 1000 and 4000 years. Using our model, we mapped habitat stability over time using three approaches, comparing our modeled stability estimates to existing knowledge of Afrotropical refugia, as well as contemporary patterns of major keystone tropical food resources used by chimpanzees, figs (Moraceae), and palms (Arecacae). Results show habitat stability congruent with known glacial refugia across Africa, suggesting their extents may have been underestimated for chimpanzees, with potentially up to approximately 60,000 km2 of previously unrecognized glacial refugia. The refugia we highlight coincide with higher species richness for figs and palms. Our results provide spatio-temporally explicit insights into the role of refugia across the chimpanzee range, forming the empirical foundation for developing and testing hypotheses about behavioral, ecological, and genetic diversity with additional data. This methodology can be applied to other species and geographic areas when sufficient data are available.

No Evidence Known Viruses Play a Role in the Pathogenesis of Onchocerciasis-Associated Epilepsy. An Explorative Metagenomic Case-Control Study.

Despite the increasing epidemiological evidence that the Onchocerca volvulus parasite is strongly associated with epilepsy in children, hence the name onchocerciasis-associated epilepsy (OAE), the pathophysiological mechanism of OAE remains to be elucidated. In June 2014, children with unprovoked convulsive epilepsy and healthy controls were enrolled in a case control study in Titule, Bas-Uélé Province in the Democratic Republic of the Congo (DRC) to identify risk factors for epilepsy. Using a subset of samples collected from individuals enrolled in this study (16 persons with OAE and 9 controls) plasma, buffy coat, and cerebrospinal fluid (CSF) were subjected to random-primed next-generation sequencing. The resulting sequences were analyzed using sensitive computational methods to identify viral DNA and RNA sequences. Anneloviridae, Flaviviridae, Hepadnaviridae (Hepatitis B virus), Herpesviridae, Papillomaviridae, Polyomaviridae (Human polyomavirus), and Virgaviridae were identified in cases and in controls. Not unexpectedly, a variety of bacteriophages were also detected in all cases and controls. However, none of the identified viral sequences were found enriched in OAE cases, which was our criteria for agents that might play a role in the etiology or pathogenesis of OAE.

Molecular detection and genomic characterization of diverse hepaciviruses in African rodents.

Hepatitis C virus (HCV; genus Hepacivirus) represents a major public health problem, infecting about three per cent of the human population. Because no animal reservoir carrying closely related hepaciviruses has been identified, the zoonotic origins of HCV still remain unresolved. Motivated by recent findings of divergent hepaciviruses in rodents and a plausible African origin of HCV genotypes, we have screened a large collection of small mammals samples from seven sub-Saharan African countries. Out of 4,303 samples screened, eighty were found positive for the presence of hepaciviruses in twenty-nine different host species. We, here, report fifty-six novel genomes that considerably increase the diversity of three divergent rodent hepacivirus lineages. Furthermore, we provide strong evidence for hepacivirus co-infections in rodents, which were exclusively found in four sampled species of brush-furred mice. We also detect evidence of recombination within specific host lineages. Our study expands the available hepacivirus genomic data and contributes insights into the relatively deep evolutionary history of these pathogens in rodents. Overall, our results emphasize the importance of rodents as a potential hepacivirus reservoir and as models for investigating HCV infection dynamics.

Citizen science and online data: Opportunities and challenges for snake ecology and action against snakebite.

The secretive behavior and life history of snakes makes studying their biology, distribution, and the epidemiology of venomous snakebite challenging. One of the most useful, most versatile, and easiest to collect types of biological data are photographs, particularly those that are connected with geographic location and date-time metadata. Photos verify occurrence records, provide data on phenotypes and ecology, and are often used to illustrate new species descriptions, field guides and identification keys, as well as in training humans and computer vision algorithms to identify snakes. We scoured eleven online and two offline sources of snake photos in an attempt to collect as many photos of as many snake species as possible, and attempt to explain some of the inter-species variation in photograph quantity among global regions and taxonomic groups, and with regard to medical importance, human population density, and range size. We collected a total of 725,565 photos-between 1 and 48,696 photos of 3098 of the world's 3879 snake species (79.9%), leaving 781 "most wanted" species with no photos (20.1% of all currently-described species as of the December 2020 release of The Reptile Database). We provide a list of most wanted species sortable by family, continent, authority, and medical importance, and encourage snake photographers worldwide to submit photos and associated metadata, particularly of "missing" species, to the most permanent and useful online archives: The Reptile Database, iNaturalist, and HerpMapper.

Coronavirus surveillance in wildlife from two Congo basin countries detects RNA of multiple species circulating in bats and rodents.

Coronaviruses play an important role as pathogens of humans and animals, and the emergence of epidemics like SARS, MERS and COVID-19 is closely linked to zoonotic transmission events primarily from wild animals. Bats have been found to be an important source of coronaviruses with some of them having the potential to infect humans, with other animals serving as intermediate or alternate hosts or reservoirs. Host diversity may be an important contributor to viral diversity and thus the potential for zoonotic events. To date, limited research has been done in Africa on this topic, in particular in the Congo Basin despite frequent contact between humans and wildlife in this region. We sampled and, using consensus coronavirus PCR-primers, tested 3,561 wild animals for coronavirus RNA. The focus was on bats (38%), rodents (38%), and primates (23%) that posed an elevated risk for contact with people, and we found coronavirus RNA in 121 animals, of which all but two were bats. Depending on the taxonomic family, bats were significantly more likely to be coronavirus RNA-positive when sampled either in the wet (Pteropodidae and Rhinolophidae) or dry season (Hipposideridae, Miniopteridae, Molossidae, and Vespertilionidae). The detected RNA sequences correspond to 15 alpha- and 6 betacoronaviruses, with some of them being very similar (>95% nucleotide identities) to known coronaviruses and others being more unique and potentially representing novel viruses. In seven of the bats, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E or NL63 (>80% nucleotide identities). The findings highlight the potential for coronavirus spillover, especially in regions with a high diversity of bats and close human contact, and reinforces the need for ongoing surveillance.

Using MALDI-TOF mass spectrometry to identify ticks collected on domestic and wild animals from the Democratic Republic of the Congo.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) has recently emerged as an alternative to morphological and molecular tools to identify tick species. In this study, we set out to evaluate and confirm the ability of MALDI-TOF MS to identify different species of ticks collected in the Democratic Republic of the Congo and preserved in 70% ethanol. A total of 575 ticks, of which 530 were collected from domestic pigs and 45 from wild animals, were subjected to MALDI-TOF MS analysis to evaluate the intraspecies reproducibility and interspecies specificity of MS profiles obtained from the different species. Morphologically, the ticks belonged to seven different species, namely Rhipicephalus complanatus, Rhipicephalus congolensis, Haemaphysalis muhsamae, Ixodes cumulatimpunctatus, Amblyomma exornatum, Amblyomma compressum and an unidentified Rhipicephalus sp. A total of 535/575 (93%) of the spectra obtained were of good enough quality to be used for our analyses. Our home-made MALDI-TOF MS arthropod database was upgraded with spectra obtained from between one and five randomly selected specimens per species. For these reference specimens, molecular identification of the ticks was also made using 16S, 12S rDNA genes and the Cox1 mtDNA gene sequencing. The remaining good quality spectra were then queried against the upgraded MALDI-TOF MS database, showing that 100% were in agreement with the morphological identification, with logarithmic score values (LSVs) between 1.813 and 2.51. The consistency between our morphological, molecular and MALDI-TOF MS identification confirms the capability and precision of MALDI-TOF MS for tick identification.

Socializing One Health: an innovative strategy to investigate social and behavioral risks of emerging viral threats.

In an effort to strengthen global capacity to prevent, detect, and control infectious diseases in animals and people, the United States Agency for International Development's (USAID) Emerging Pandemic Threats (EPT) PREDICT project funded development of regional, national, and local One Health capacities for early disease detection, rapid response, disease control, and risk reduction. From the outset, the EPT approach was inclusive of social science research methods designed to understand the contexts and behaviors of communities living and working at human-animal-environment interfaces considered high-risk for virus emergence. Using qualitative and quantitative approaches, PREDICT behavioral research aimed to identify and assess a range of socio-cultural behaviors that could be influential in zoonotic disease emergence, amplification, and transmission. This broad approach to behavioral risk characterization enabled us to identify and characterize human activities that could be linked to the transmission dynamics of new and emerging viruses. This paper provides a discussion of implementation of a social science approach within a zoonotic surveillance framework. We conducted in-depth ethnographic interviews and focus groups to better understand the individual- and community-level knowledge, attitudes, and practices that potentially put participants at risk for zoonotic disease transmission from the animals they live and work with, across 6 interface domains. When we asked highly-exposed individuals (ie. bushmeat hunters, wildlife or guano farmers) about the risk they perceived in their occupational activities, most did not perceive it to be risky, whether because it was normalized by years (or generations) of doing such an activity, or due to lack of information about potential risks. Integrating the social sciences allows investigations of the specific human activities that are hypothesized to drive disease emergence, amplification, and transmission, in order to better substantiate behavioral disease drivers, along with the social dimensions of infection and transmission dynamics. Understanding these dynamics is critical to achieving health security--the protection from threats to health-- which requires investments in both collective and individual health security. Involving behavioral sciences into zoonotic disease surveillance allowed us to push toward fuller community integration and engagement and toward dialogue and implementation of recommendations for disease prevention and improved health security.

Sequences of Previously Unknown Rhabdoviruses Detected in Bat Samples from the Republic of the Congo.

The family Rhabdoviridae contains diverse viruses, including vector-borne and nonvector-borne viruses, some that are human pathogens, including rabies virus and also nonpathogenic viruses. Bats, which are a known reservoir of viruses with zoonotic potential including coronaviruses, also carry multiple rhabdoviruses such as but not limited to lyssaviruses. We collected samples from 193 insectivorous and frugivorous bats in the Republic of the Congo and tested them for rhabdovirus RNA. Four samples were found positive for viral RNA representing sequences of four different, not previously described rhabdoviruses. Although phylogenetic and taxonomic placement of the novel sequences is uncertain, similarities with previously detected rhabdovirus sequences in bats suggest that these could represent vertebrate viruses. Considering the pathogenic risks some rhabdoviruses pose for humans, these results highlight the need for more research and surveillance regarding rhabdoviruses and bats.

A Descriptive-Multivariate Analysis of Community Knowledge, Confidence, and Trust in COVID-19 Clinical Trials among Healthcare Workers in Uganda.

Background-misinformation and mistrust often undermines community vaccine uptake, yet information in rural communities, especially of developing countries, is scarce. This study aimed to identify major challenges associated with coronavirus disease 2019 (COVID-19) vaccine clinical trials among healthcare workers and staff in Uganda. Methods-a rapid exploratory survey was conducted over 5 weeks among 260 respondents (66% male) from healthcare centers across the country using an online questionnaire. Twenty-seven questions assessed knowledge, confidence, and trust scores on COVID-19 vaccine clinical trials from participants in 46 districts in Uganda. Results-we found low levels of knowledge (i.e., confusing COVID-19 with Ebola) with males being more informed than females (OR = 1.5, 95% CI: 0.7-3.0), and mistrust associated with policy decisions to promote herbal treatments in Uganda and the rushed international clinical trials, highlighting challenges for the upcoming Oxford-AstraZeneca vaccinations. Knowledge, confidence and trust scores were higher among the least educated (certificate vs. bachelor degree holders). We also found a high level of skepticism and possible community resistance to DNA recombinant vaccines, such as the Oxford-AstraZeneca vaccine. Preference for herbal treatments (38/260; 14.6%, 95% CI: 10.7-19.3) currently being promoted by the Ugandan government raises major policy concerns. High fear and mistrust for COVID-19 vaccine clinical trials was more common among wealthier participants and more affluent regions of the country. Conclusion-our study found that knowledge, confidence, and trust in COVID-19 vaccines was low among healthcare workers in Uganda, especially those with higher wealth and educational status. There is a need to increase transparency and inclusive participation to address these issues before new trials of COVID-19 vaccines are initiated.

Recent genetic connectivity and clinal variation in chimpanzees.

Much like humans, chimpanzees occupy diverse habitats and exhibit extensive behavioural variability. However, chimpanzees are recognized as a discontinuous species, with four subspecies separated by historical geographic barriers. Nevertheless, their range-wide degree of genetic connectivity remains poorly resolved, mainly due to sampling limitations. By analyzing a geographically comprehensive sample set amplified at microsatellite markers that inform recent population history, we found that isolation by distance explains most of the range-wide genetic structure of chimpanzees. Furthermore, we did not identify spatial discontinuities corresponding with the recognized subspecies, suggesting that some of the subspecies-delineating geographic barriers were recently permeable to gene flow. Substantial range-wide genetic connectivity is consistent with the hypothesis that behavioural flexibility is a salient driver of chimpanzee responses to changing environmental conditions. Finally, our observation of strong local differentiation associated with recent anthropogenic pressures portends future loss of critical genetic diversity if habitat fragmentation and population isolation continue unabated.

The evolutionary history of manatees told by their mitogenomes.

The manatee family encompasses three extant congeneric species: Trichechus senegalensis (African manatee), T. inunguis (Amazonian manatee), and T. manatus (West Indian manatee). The fossil record for manatees is scant, and few phylogenetic studies have focused on their evolutionary history. We use full mitogenomes of all extant manatee species to infer the divergence dates and biogeographical histories of these species and the effect of natural selection on their mitogenomes. The complete mitochondrial genomes of T. inunguis (16,851 bp), T. senegalensis (16,882 bp), and T. manatus (16,882 bp), comprise 13 protein-coding genes, 2 ribosomal RNA genes (rRNA - 12S and 16S), and 22 transfer RNA genes (tRNA), and (D-loop/CR). Our analyses show that the first split within Trichechus occurred during the Late Miocene (posterior mean 6.56 Ma and 95% HPD 3.81-10.66 Ma), followed by a diversification event in the Plio-Pleistocene (posterior mean 1.34 Ma, 95% HPD 0.1-4.23) in the clade composed by T. inunguis and T. manatus; T. senegalensis is the sister group of this clade with higher support values (pp > 0.90). The branch-site test identified positive selection on T. inunguis in the 181st position of the ND4 amino acid gene (LRT = 6.06, p = 0.0069, BEB posterior probability = 0.96). The ND4 gene encodes one subunit of the NADH dehydrogenase complex, part of the oxidative phosphorylation machinery. In conclusion, our results provide novel insight into the evolutionary history of the Trichechidae during the Late Miocene, which was influenced by geological events, such as Amazon Basin formation.