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.

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.

Divergent lineage of a novel hantavirus in the banana pipistrelle (Neoromicia nanus) in Côte d'Ivoire.

Recently identified hantaviruses harbored by shrews and moles (order Soricomorpha) suggest that other mammals having shared ancestry may serve as reservoirs. To investigate this possibility, archival tissues from 213 insectivorous bats (order Chiroptera) were analyzed for hantavirus RNA by RT-PCR. Following numerous failed attempts, hantavirus RNA was detected in ethanol-fixed liver tissue from two banana pipistrelles (Neoromicia nanus), captured near Mouyassué village in Côte d'Ivoire, West Africa, in June 2011. Phylogenetic analysis of partial L-segment sequences using maximum-likelihood and Bayesian methods revealed that the newfound hantavirus, designated Mouyassué virus (MOUV), was highly divergent and basal to all other rodent- and soricomorph-borne hantaviruses, except for Nova virus in the European common mole (Talpa europaea). Full genome sequencing of MOUV and further surveys of other bat species for hantaviruses, now underway, will provide critical insights into the evolution and diversification of hantaviruses.

Molecular evolution of Azagny virus, a newfound hantavirus harbored by the West African pygmy shrew (Crocidura obscurior) in Côte d'Ivoire.

BACKGROUND: Tanganya virus (TGNV), the only shrew-associated hantavirus reported to date from sub-Saharan Africa, is harbored by the Therese's shrew (Crocidura theresae), and is phylogenetically distinct from Thottapalayam virus (TPMV) in the Asian house shrew (Suncus murinus) and Imjin virus (MJNV) in the Ussuri white-toothed shrew (Crocidura lasiura). The existence of myriad soricid-borne hantaviruses in Eurasia and North America would predict the presence of additional hantaviruses in sub-Saharan Africa, where multiple shrew lineages have evolved and diversified. METHODS: Lung tissues, collected in RNAlater®, from 39 Buettikofer's shrews (Crocidura buettikoferi), 5 Jouvenet's shrews (Crocidura jouvenetae), 9 West African pygmy shrews (Crocidura obscurior) and 21 African giant shrews (Crocidura olivieri) captured in Côte d'Ivoire during 2009, were systematically examined for hantavirus RNA by RT-PCR. RESULTS: A genetically distinct hantavirus, designated Azagny virus (AZGV), was detected in the West African pygmy shrew. Phylogenetic analysis of the S, M and L segments, using maximum-likelihood and Bayesian methods, under the GTR+I+Γ model of evolution, showed that AZGV shared a common ancestry with TGNV and was more closely related to hantaviruses harbored by soricine shrews than to TPMV and MJNV. That is, AZGV in the West African pygmy shrew, like TGNV in the Therese's shrew, did not form a monophyletic group with TPMV and MJNV, which were deeply divergent and basal to other rodent- and soricomorph-borne hantaviruses. Ancestral distributions of each hantavirus lineage, reconstructed using Mesquite 2.74, suggested that the common ancestor of all hantaviruses was most likely of Eurasian, not African, origin. CONCLUSIONS: Genome-wide analysis of many more hantaviruses from sub-Saharan Africa are required to better understand how the biogeographic origin and radiation of African shrews might have contributed to, or have resulted from, the evolution of hantaviruses.

Comparative phylogeography of African fruit bats (Chiroptera, Pteropodidae) provide new insights into the outbreak of Ebola virus disease in West Africa, 2014-2016.

Both Ebolavirus and Marburgvirus were detected in several fruit bat species of the family Pteropodidae, suggesting that this taxon plays a key role in the life cycle of filoviruses. After four decades of Zaire Ebolavirus (ZEBOV) outbreaks in Central Africa, the virus was detected for the first time in West Africa in 2014. To better understand the role of fruit bats as potential reservoirs and circulating hosts between Central and West Africa, we examine here the phylogeny and comparative phylogeography of Pteropodidae. Our phylogenetic results confirm the existence of four independent lineages of African fruit bats: the genera Eidolon and Rousettus, and the tribes Epomophorini and Scotonycterini, and indicate that the three species suspected to represent ZEBOV reservoir hosts (Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata) belong to an African clade that diversified rapidly around 8-7 Mya. To test for phylogeographic structure and for recent gene flow from Central to West Africa, we analysed the nucleotide variation of 675 cytochrome b gene (Cytb) sequences, representing eight fruit bat species collected in 48 geographic localities. Within Epomophorina, our mitochondrial data do not support the monophyly of two genera (Epomops and Epomophorus) and four species (Epomophorus gambianus, Epomops franqueti, Epomops buettikoferi, and Micropteropus pusillus). In Epomops, however, we found two geographic haplogroups corresponding to the Congo Basin and Upper Guinea forests, respectively. By contrast, we found no genetic differentiation between Central and West African populations for all species known to make seasonal movements, Eidolon helvum, E. gambianus, H. monstrosus, M. pusillus, Nanonycteris veldkampii, and Rousettus aegyptiacus. Our results suggest that only three fruit bat species were able to disperse directly ZEBOV from the Congo Basin to Upper Guinea: E. helvum, H. monstrosus, and R. aegyptiacus.

The comparative phylogeography of fruit bats of the tribe Scotonycterini (Chiroptera, Pteropodidae) reveals cryptic species diversity related to African Pleistocene forest refugia.

The hypothesis of Pleistocene forest refugia was tested using comparative phylogeography of Scotonycterini, a fruit bat tribe endemic to Africa containing four species: Scotonycteris zenkeri, Casinycteris argynnis, C. campomaanensis, and C. ophiodon. Patterns of genetic structure were assessed using 105 Scotonycterini (including material from three holotypes) collected at 37 localities, and DNA sequences from the mitochondrial cytochrome b gene (1140 nt) and 12 nuclear introns (9641 nt). Phylogenetic trees and molecular dating were inferred by Bayesian methods. Multilocus analyses were performed using supermatrix, SuperTRI, and *BEAST approaches. Mitochondrial analyses reveal strong phylogeographical structure in Scotonycteris, with four divergent haplogroups (4.9-8.7%), from Upper Guinea, Cameroon, western Equatorial Africa, and eastern Democratic Republic of the Congo (DRC). In C. argynnis, we identify two mtDNA haplogroups corresponding to western and eastern Equatorial Africa (1.4-2.1%). In C. ophiodon, the mtDNA haplotypes from Cameroon and Ivory Coast differ by only 1.3%. Nuclear analyses confirm the validity of the recently described C. campomaanensis and indicate that western and eastern populations of C. argynnis are not fully isolated. All mtDNA clusters detected in Scotonycteris are found to be monophyletic based on the nuclear dataset, except in eastern DRC. In the nuclear tree, the clade from western Equatorial Africa is closely related to individuals from eastern DRC, whereas in the mitochondrial tree it appears to be the sister-group of the Cameroon clade. Migrate-n analyses support gene flow from western Equatorial Africa to eastern DRC. Molecular dating indicates that Pleistocene forest refugia have played an important role in shaping the evolution of Scotonycterini, with two phases of allopatric speciation at approximately 2.7 and 1.6 Mya, resulting from isolation in three main forest areas corresponding to Upper Guinea, Cameroon, and Equatorial Africa. Two cryptic species and two subspecies are described herein in the genus Scotonycteris. Female philopatry and male biased dispersal are supported for the smallest taxa, i.e., the three species of Scotonycteris and C. argynnis. The Congo, Ntem, and Sanaga rivers are identified as biogeographic barriers to the dispersal of Scotonycteris during interglacial periods. A greater capacity for long-distance dispersal is inferred for the largest species, C. ophiodon.

Molecular phylogeny of hantaviruses harbored by insectivorous bats in Côte d'Ivoire and Vietnam.

The recent discovery of genetically distinct hantaviruses in multiple species of shrews and moles prompted a further exploration of their host diversification by analyzing frozen, ethanol-fixed and RNAlater®-preserved archival tissues and fecal samples from 533 bats (representing seven families, 28 genera and 53 species in the order Chiroptera), captured in Asia, Africa and the Americas in 1981-2012, using RT-PCR. Hantavirus RNA was detected in Pomona roundleaf bats (Hipposideros pomona) (family Hipposideridae), captured in Vietnam in 1997 and 1999, and in banana pipistrelles (Neoromicia nanus) (family Vespertilionidae), captured in Côte d'Ivoire in 2011. Phylogenetic analysis, based on the full-length S- and partial M- and L-segment sequences using maximum likelihood and Bayesian methods, demonstrated that the newfound hantaviruses formed highly divergent lineages, comprising other recently recognized bat-borne hantaviruses in Sierra Leone and China. The detection of bat-associated hantaviruses opens a new era in hantavirology and provides insights into their evolutionary origins.