Teresa Kearney

The taxonomy of the small, sub-Saharan, insectivorous bat, Afronycteris helios (Heller, 1912), has been unresolved for decades. The name A. cf. helios was introduced in the literature to recognise bats found in east and southern Africa that were like A. helios but had glands on the uropatagium. Cranio-dental morphology, bacular morphology, and molecular genetics (albeit the latter two being without representation of A. helios), provided evidence to formally describe “A. cf. helios”, which is currently known from Kenya, Mozambique, and South Africa. Bayesian analyses based on cytochrome oxidase b, cytochrome c oxidase subunit 1, and 12S rRNA confirm that it belongs to the genus Afronycteris with A. nanus (Peter, 1852) and A. helios. These analyses also revealed genetic, bacular, and cranio-dental morphological differences within A. nanus, which are described here. Pending a more thorough geographic analysis, including all existing synonyms, A. cf. nanus has been introduced to refer to bats that were smaller than A. nanus. Although co-occurring in north-eastern parts of South Africa, A. cf. nanus has a more westerly distribution extending to west Africa, relative to the more easterly distribution of A. nanus. Afronycteris sp. nov. showed at least a 6.9%, 3.4% and 2.9% nucleotide difference to its nearest relative based on cytochrome oxidase b, cytochrome c oxidase subunit 1, and 12S rRNA respectively. It is cranio-dentally smaller than A. helios, slightly smaller than A. nanus, and larger than A. cf. nanus, with a distinct baculum, and a unique pair of glands on the uropatagium.

Hosting 1460 plant and 126 vertebrate endemic species, the Great Escarpment (hereafter, Escarpment) forms a semi‐circular “amphitheater” of mountains girdling southern Africa from arid west to temperate east. Since arid and temperate biota are usually studied separately, earlier studies overlooked the biogeographical importance of the Escarpment as a whole. Bats disperse more widely than other mammalian taxa, with related species and intraspecific lineages occupying both arid and temperate highlands of the Escarpment, providing an excellent model to address this knowledge gap. We investigated patterns of speciation and micro‐endemism from modeled past, present, and future distributions in six clades of southern African bats from three families (Rhinolophidae, Cistugidae, and Vespertilionidae) having different crown ages (Pleistocene to Miocene) and biome affiliations (temperate to arid). We estimated mtDNA relaxed clock dates of key divergence events across the six clades in relation both to biogeographical features and patterns of phenotypic variation in crania, bacula and echolocation calls. In horseshoe bats (Rhinolophidae), both the western and eastern “arms” of the Escarpment have facilitated dispersals from the Afrotropics into southern Africa. Pleistocene and pre‐Pleistocene “species pumps” and temperate refugia explained observed patterns of speciation, intraspecific divergence and, in two cases, mtDNA introgression. The Maloti‐Drakensberg is a center of micro‐endemism for bats, housing three newly described or undescribed species. Vicariance across biogeographic barriers gave rise to 29 micro‐endemic species and intraspecific lineages whose distributions were congruent with those identified in other phytogeographic and zoogeographic studies. Although Köppen–Geiger climate models predict a widespread replacement of current temperate ecosystems in southern Africa by tropical or arid ecosystems by 2070–2100, future climate Maxent models for 13 bat species (all but one of those analyzed above) showed minimal range changes in temperate species from the eastern Escarpment by 2070, possibly due to the buffering effect of mountains to climate change.

This study assessed and related quantitative age determination methods based on cranial bone fusion and dental development to linear morphometrics in Rousettus aegyptiacus. Five growth development stages were identified based on cranial suture fusion and degree of second molar tooth eruption. Expressing these growth development stages in measurement size showed a linear growth pattern, with little overlap between smaller (stages 1, 2, and 3) and larger (stages 4 and 5) individuals. Total skull length (TSL), mastoid breadth (MB) and forearm length (FAL) had the highest influence on variation along the first and second principal components, accounting for 93% of variation. Advanced size was confirmed to relate to aging owing to development of cranial suture fusions and dental development. The smallest and largest individuals were significantly (P < 0.05) separated by measurements of TSL, MB and FAL. Meanwhile, some intermediate sized individuals overlapped despite being in different stages of cranial suture development. Species specific reliability in morphological approaches to age determination can be achieved by establishing a baseline reference, which may be directly related to the quantitative cementum growth assessment method.

Mammarenaviruses are hosted by several rodent species, a small number of which have been known to be zoonotic. Host surveillance among small mammals has identified a large diversity of previously undescribed mammarenaviruses. Intensified biosurveillance is warranted to better understand the diversity of these agents. Longitudinal host surveillance involving non-volant small mammals at a site in the Limpopo province, South Africa, was conducted. The study reports on the screening results of 563 samples for the presence of mammarenavirus RNA. PCR-positive samples were subjected to sequencing using Miseq amplicon sequencing. Sequences with close similarity to Mariental and Lunk viruses were identified from two rodent species, Micaelamys namaquensis and Mus minutoides. This represents the first description of these viruses from South Africa. The genomic sequences reported here partially satisfied the requirements put forward by the International Committee on the Taxonomy of Viruses’ criteria for species delineation, suggesting that these may be new strains of existing species. The known distribution of these mammarenaviruses is thus expanded further south in Africa.

The reservoir host of Mokola virus (MOKV), a rabies-related lyssavirus species endemic to Africa, remains unknown. Only sporadic cases of MOKV have been reported since its first discovery in the late 1960s, which subsequently gave rise to various reservoir host hypotheses. One particular hypothesis focusing on non-volant small mammals (e.g. shrews, sengis and rodents) is buttressed by previous MOKV isolations from shrews (Crocidura sp.) and a single rodent (Lophuromys sikapusi). Although these cases were only once-off detections, it provided evidence of the first known lyssavirus species has an association with non-volant small mammals. To investigate further, retrospective surveillance was conducted in 575 small mammals collected from South Africa. Nucleic acid surveillance using a pan-lyssavirus quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay of 329 brain samples did not detect any lyssavirus ribonucleic acid (RNA). Serological surveillance using a micro-neutralisation test of 246 serum samples identified 36 serum samples that were positive for the presence of MOKV neutralising antibodies (VNAs). These serum samples were all collected from Gerbilliscus leucogaster (Bushveld gerbils) rodents from Meletse in Limpopo province (South Africa). Mokola virus infections in Limpopo province have never been reported before, and the high MOKV seropositivity of 87.80% in these gerbils may indicate a potential rodent reservoir.

Migration and hibernation are survival strategies that require physiological preparation using fattening. Bats employ both strategies in times of resource shortages. However, because males and females vary seasonally in their reproductive physiological needs, they may employ different fattening patterns. Whilst fattening, migration and hibernation are common in temperate bats, little is known about subtropical migratory insectivores. This study investigated seasonal variation in body mass of the regionally migrating Natal long-fingered bat Miniopterus natalensis to determine if males and females show fattening in preparation for migration/hibernation. Seasonal change best explained the variation observed in overall body mass, whilst sex and forearm length explained the variation to a lesser extent. Body mass between males and females differed significantly by reproductive category among the four seasons. Forearm length was a significant predictor of the body mass of males. Scrotal males had a higher body mass in summer compared to autumn. This pattern of mass gain was not observed in non-scrotal males. The summer body mass of nonpregnant and post-lactating females was not significantly higher than the autumn body mass of nonpregnant females, which did not support the hypothesis that females would exhibit fattening during summer before migration. Results suggest that males and females employ different mass-gain strategies related to reproductive investment rather than fattening preparation for migration or hibernation.

Most vertebrates host a wide variety of haematophagous parasites, which may play an important role in the transmission of vector-borne microorganisms to hosts. Surveillance is usually performed by collecting blood and/or tissue samples from vertebrate hosts. There are multiple methods to obtain samples, which can be stored for decades if properly kept. However, blood sampling is considered an invasive method and may possibly be harmful to the sampled individual. In this study, we investigated the use of ectoparasites as a tool to acquire molecular information about the presence and diversity of infectious microorganism in host populations. We tested the presence of three distinct vector-borne microorganisms in both bat blood and bat flies: Bartonella bacteria, malaria-like Polychromophilus sp. (Apicomplexa), and Trypanosoma sp. (Kinetoplastea). We detected the presence of these microorganisms both in bats and in their bat flies, with the exception of Trypanosoma sp. in South African bat flies. Additionally, we found Bartonella sp. in bat flies from one population in Spain, suggesting its presence in the host population even if not detected in bats. Bartonella and Polychromophilus infection showed the highest prevalence in both bat and bat fly populations. Single, co- and triple infections were also frequently present in both. We highlight the use of haematophagous ectoparasites to study the presence of infectious microorganism in host blood and its use as an alternative, less invasive sampling method.

Seasonal reproduction is a period of extreme physiological and behavioural changes, yet we know little about how it may affect host microbial communities (i.e. microbiota) and pathogen transmission. Here, we investigated shifts of the bacterial microbiota in saliva, urine and faeces during the seasonal reproduction of bats in South Africa, and test for an interaction in shedding patterns of both bacterial ( Leptospira ) and viral (adeno- and herpesviruses) agents. Based on a comparative approach in two cave-dwelling bat species and high-throughput sequencing of the 16S rRNA gene, we demonstrated a clear signature in microbiota changes over the reproduction season, consistent across the multiple body habitats investigated, and associated with the sex, age and reproductive condition of bats. We observed in parallel highly dynamic shedding patterns for both bacteria and viruses, but did not find a significant association between viral shedding and bacterial microbiota composition. Indeed, only Leptospira shedding was associated with alterations in both the diversity and composition of the urinary microbiota. These results illustrate how seasonal reproduction in bats substantially affects microbiota composition and infection dynamics, and have broad implications for the understanding of disease ecology in important reservoir hosts, such as bats.

Species within the Neoromicia bat genus are abundant and widely distributed in Africa. It is common for these insectivorous bats to roost in anthropogenic structures in urban regions. Additionally, Neoromicia capensis have previously been identified as potential hosts for Middle East respiratory syndrome (MERS)-related coronaviruses. This study aimed to ascertain the gastrointestinal virome of these bats, as viruses excreted in fecal material or which may be replicating in rectal or intestinal tissues have the greatest opportunities of coming into contact with other hosts. Samples were collected in five regions of South Africa over eight years. Initial virome composition was determined by viral metagenomic sequencing by pooling samples and enriching for viral particles. Libraries were sequenced on the Illumina MiSeq and NextSeq500 platforms, producing a combined 37 million reads. Bioinformatics analysis of the high throughput sequencing data detected the full genome of a novel species of the Circoviridae family, and also identified sequence data from the Adenoviridae, Coronaviridae, Herpesviridae, Parvoviridae, Papillomaviridae, Phenuiviridae, and Picornaviridae families. Metagenomic sequencing data was insufficient to determine the viral diversity of certain families due to the fragmented coverage of genomes and lack of suitable sequencing depth, as some viruses were detected from the analysis of reads-data only. Follow up conventional PCR assays targeting conserved gene regions for the Adenoviridae, Coronaviridae, and Herpesviridae families were used to confirm metagenomic data and generate additional sequences to determine genetic diversity. The complete coding genome of a MERS-related coronavirus was recovered with additional amplicon sequencing on the MiSeq platform. The new genome shared 97.2% overall nucleotide identity to a previous Neoromicia-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and herpesvirus sequences that were widespread throughout Neoromicia populations in South Africa. Furthermore, similar adenovirus sequences were detected within these populations throughout several years. With the exception of the coronaviruses, the study represents the first report of sequence data from several viral families within a Southern African insectivorous bat genus; highlighting the need for continued investigations in this regard.