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Poxviruses are double stranded (ds) DNA viruses with large brick-shaped virions (~200x300nm) that can be seen by light microscopy. The Chordopoxvirus (ChPV) subfamily demonstrates a vast genetic diversity in poxvirus virulence and evolution, and infects a wide range of vertebrate hosts including human/primates, rodents, birds, squirrels, and many economically important ruminants. There are at least 14 distinct ChPV genera, whose members have genomes that range between 127-360 kbp, and can be either GC-rich (33-38% A+T base composition) or AT-rich (up to 76% A+T). My work in the assembly and annotation of novel poxviruses serves to enrich the poxvirus sequence repository and further virulence characterization, comparative analysis, and phylogenetic studies. Using a variety of programs, as well as tools developed by the Virus Bioinformatics Research Centre, a protocol is created, refined, and applied to the assembly and annotation of novel poxviruses: Pteropox virus (PTPV) from a south Australian megabat Pteropus scapulatus, Eptesipox virus (EPTV) from a north American microbat Eptesicus fuscus, sea otter poxvirus (SOPV) from the north American Enhydra lutris, and two Kangaroopox viruses western and eastern Kangaroopox viruses (WKPV, EKPV) from the Australian Macropus fuliginosus and Macropus giganteus. This is the first time poxviruses from these vertebrate hosts are assembled in full, and the result supports the establishment of 4 new ChPV genera. The two bat-isolated poxviruses, PTPV and EPTV, likely did not co-speciate with their hosts despite infection of related host species. Instead, EPTV forms a sister clade with the Clade II virus, and together forms a sister group with the orthopoxviruses. On the other hand, PTPV and SOPV are each other’s closest extant relatives despite the distant geographical location from which they were isolated; together they share a novel homolog of TRAIL (Tumor necrosis factor-Related Apoptosis-Inducing Ligand) never before seen in poxviruses. SOPV additionally encodes distinct interleukin (IL)-18 binding protein and tumor necrosis factor (TNF) receptor-like protein that could have novel immune-evasion roles. The KPVs present the first case of a putative viral cullin-like protein, which might be involved in regulating the host ubiquitination pathway. Altogether, these novel proteins can potentially serve as new virokines and viroceptors in the form of viromimicry pathogenesis; they demonstrate the capacity and diversity with which poxviruses modulate host immune responses in their favour, and should be studied further. / Graduate / 2019-04-11

O morcego Desmodus rotundus é uma das três espécies de morcegos hematófagos existentes. Possui ampla distribuição, ocorrendo do sul do México até Argentina e Chile. Além de seu hábito alimentar incomum, esta espécie possui particular interesse por ser transmissor da raiva bovina. Apesar dos métodos de controle da população, estudos estimaram em até 33 milhões de dólares ao ano os prejuízos causados por esta espécie a pecuária no Brasil. Ao mesmo tempo, segundo dados oficiais, cerca de 200.000 indivíduos da espécie podem ter sido mortos no Estado de São Paulo no ano de 2000 através dos métodos de controle populacional. Além deste controle não surtir o efeito desejado (o número de casos de raiva não diminuiu no período), não se conhece qual o efeito desta matança nas populações naturais do morcego. Apesar de sua ampla distribuição e reconhecida variação morfológica, nenhum estudo foi realizado para procurar entender como a variabilidade genética desta espécie está distribuída geograficamente. Este estudo se propôs a estudar a filogeografia do morcego vampiro comum analisando um marcador mitocondrial, dois marcadores nucleares e morfometria de crânio. O marcador mitocondrial identificou cinco clados monofiléticos sem haplótipos compartilhados nem zonas de contato, cada um representando uma região geográfica diferente. São eles: Mata Atlântica sul (MAS), Mata Atlântica norte (MAN), Amazônia e Cerrado (AMC), América Central (AC) e Pantanal (PAN), sendo que os clados da Mata Atlântica formam um clado monofilético a Leste, se contrapondo aos demais clados a Oeste. Os índices de divergência entre estes clados são comparáveis a distâncias descritas para espécies congenéricas. Os tempos de divergência estimados entre os clados através de métodos coalescentes e não-coalescentes apontam para uma divergência pleistocênica, além de testes de neutralidade apoiarem a idéia de fragmentação por refúgios. O padrão biogeográfico descrito para D. rotundus possui um paralelo em uma série de outros organismos. Os marcadores nucleares por sua vez mostraram baixa variabilidade, e extenso compartilhamento de haplótipos entre as localidades pertencentes a distintos clados mitocondriais, num padrão que contrasta com os resultados descritos anteriormente. Simulações coalescentes foram realizadas com os parâmetros calculados para o gene nuclear RAG2 e mostraram compatibilidade entre os dados observados e vicariância pleistocênica para um marcador nuclear com o Ne calculados para D. rotundus. Os dados de morfometria de crânio mostraram que existe pouca diferenciação ao longo de toda a distribuição da espécie. Dados de Fst, funções discriminantes e variáveis canônicas mostram uma grande afinidade entre indivíduos dos clados AC e AMC, que juntos formam a distribuição de uma antiga subespécie atribuída a este táxon, Desmodus rotundus murinus. As análises de distância de Mahalanobis também são concordantes com os resultados obtido para o marcador mitondrial. Por fim, uma análise realizada com o software treescan mostra existir uma correlação estatisticamente significativa entre a árvore de DNA mitocondrial e os dados multivariados de crânio. Assim, por fim propõe-se que se reconheçam duas linhagens hoje atribuídas a D. rotundus como espécies distintas: uma a Leste (Mata Atlântica) e uma a Oeste. Uma amostragem mais cuidadosa do interior do Brasil e do restante da América do Sul deve determinar corretamente a área de ocorrência de cada espécie. / The bat Desmodus rotundus is one of the three extant vampire bat species. It has a broad distribution, occurring from southern México until Argentina and Chile. Besides its unique feeding habit, this species is of particular interest for being the main vector of cattle rabies. Even with population control methods, studies have estimated in 33 million dollars per year the damage caused by this bat to cattle farming in Brazil. At the same time 200.000 specimens might have been killed in São Paulo state in the year 2000 using the population control methods. Besides the fact that this control did not diminish the number of rabies cases, the impact of this killing in the bats\' natural populations is unknown. Although this species has a broad distribution and recognized morphological variation, no effort was made thus far to understand how this species\' genetic variability is distributed geographically. This work is aimed at studying the common vapire bats\' phylogeographic pattern using a mitochondrial marker, two nuclear markers and skull morphometrics. The mitochondrial marker identified five monophiletic clades without shared haplotypes or contact zones. Each clade represents a distinct geographic region: South Atlantic Forest (SAF), North Atlantic Forest (NAF), Amazon and Cerrado (AMC), Central America (CA) and Pantanal (PAN). The Atlantic Forest clades form an Eastern monophiletic clade opposing the other clade that lies westwards. The nucleotide divergence between these clades is similar to the one described to congeneric species. The divergence times estimated by coalescent and non-coalescent methods point to a Pleistocene vicariant event. The neutrality tests also point to refugia allopatric fragmentation. The biogegraphic pattern described for D. rotundus has a parallel in many other organisms. The nuclear markers showed low variability and sharing of haplotypes among all localities, contrasting with the previous results. Coalescent simulations were carried with populational parameters estimated for the nuclear gene RAG2 and showed compatibility between the observed data and Pleistocene vicariance effect on a neutral nuclear marker. Skull morphometrics showed low differentiation throughout the bats\' distribution. Data on Fst, discriminant functions and canonic variables shows affinity between CA and AMC clades. These two clades together form the distribution of a subspecies previously described to this taxon, Desmodus rotundus murinus. The Mahalanobis distance analyses are also congruent with the results obtained withn the nuclear marker. The analysis done with the software treescan shows a statistic significant correlation between the mtDNA tree and the skull multivariate data. On the basis of the results presented, it is proposed that two lineages currently atributed to D. rotundus are to be recognized as different species: one to the east (Atlantic Forest) and one to the west. A detailed sampling of the Brazilian and South American country will determine the exact range of each species.

Studies involving geographic variation in the phenotypes of bats help scientists to explain why these mammals are the most species rich mammalian order second only to rodents, with well more than 1 300 species occurring worldwide. Such species richness or high diversity is the manifestation of the generation of biodiversity through the splitting of lineages within bat species. A lineage of bat species can diversify into several lineages which then differentiate from each other in allopatry. Thus, the spatial separation of a lineage into several lineages could be attributed to geographical, ecological and environmental factors across the distributional range of the species. Similarly, vicariant events may also play a role in separating lineages within species. The Damara horseshoe bat species, Rhinolophus damarensis, is widely distributed but restricted to the western half of southern Africa, where it occurs across several major biomes. Formerly regarded as the subspecies, R. darlingi damarensis, it was elevated to full species status on the basis of genetic and phenotypic differences between it and R. darlingi darlingi. Rhinolophus damarensis is itself made up of two ecologically separated genetic lineages. A total of 106 individuals of R. damarensis were sampled from seven localities across its distributional range, with a view to determining and documenting the extent of geographic variation in body size, echolocation parameters, wing parameters, cranial shape and postcranial morphology of individuals from populations of R. damarensis across the distributional range of the species. Firstly, an investigation into geographic variation in resting echolocation frequency (RF) of the horseshoe bat species, R. damarensis was carried out in the western half of southern Africa (Chapter 2). Three hypotheses were tested. The first one, James’Rule (JR), states that individuals occurring in hot humid environments generally have smaller body sizes than conspecifics that occur in cooler, dryer environments, and the largest are expected to occur in cool, dry areas. On this basis and because of the known relationship between body size and RF, it was predicted that there should be a correlation between body size and climatic factors and between body size and RF. The second hypothesis was Isolation by Environment (IbE) mediated through sensory drive, which proposes that diversification of lineage may be driven by environmentally-mediated differences in sensory systems. Under this hypothesis, it was predicted that call frequency variation should be correlated with climatic variables. The third hypothesis was that Isolation by Distance (IbD) can influence phenotypic trait variation by restricting gene flow between populations. Under the Isolation by Distance (IbD) Hypothesis, it was predicted that call frequency variation should be partitioned in accordance with geographic distance between populations. To investigate the probability of the JR, IbE and IbD, the Akaike’s information criterion AICc candidate models were evaluated with different combinations of environmental (annual mean temperature and relative humidity), spatial (latitude and region) and biological (forearm as a proxy for body size) predictor variables to determine their influence on resting frequency (RF) across the distributional range of R. damarensis. Linear mixed effects models (LMEs) were employed to analyse the relationship between the response variable (RF) and the environmental, spatial and biological predictor variables. The influence of prey detection range and atmospheric attenuation was also investigated. The results showed no evidence for JR or for random genetic drift. Body size was neither correlated with RF nor environmental variables, suggesting that variation in RF was not the result of concomitant variation in body size as proposed by JR. Similarly, the Mantel test showed no IbD effect and there was therefore no evidence that genetic drift was responsible for the variation in RFs. In contrast, the LMEs showed that there was support for IbE in the form of an association between RF and region (in the form of the variable “Reg”) which was based on the two geographically separated genetic lineages. Furthermore, RF variation was also associated with the climatic variable AMT. The taxonomic status of R. damarensis was investigated using ecological traits and phenotypic characters including skulls, wings and echolocation (Chapter 3) and three dimensional (3D) scanned skulls and mandibles (Chapter 4). The main objective (Chapter 3 and Chapter 4) was to test whether previously reported genetic divergence between the two R. damarensis lineages was associated with phenotypic divergence. Morphometric and echolocation measurements were taken from hand held individual bats in the field, and skull measurements were taken from field collected voucher specimens as well as museum specimens. Discriminant Function Analyses (DFA) revealed that there was geographic variation among populations and lineages of R. damarensis. Multivariate Linear Regressions (MLV) and Linear models (LM) on the basal parts of bacula revealed significant differences between the southern and northern lineages of R. damarenis. The bacula of the two lineages of R. damarensis appear to have different shapes. Diversification through shape analyses (Chapter 4) was investigated using three dimensional (3D) geometric morphometric analyses based on X-ray microcomputed tomography (µCT) scanning of dried skulls and mandibles of R. damarensis. Procrustes Anova results of both mandibles and skulls indicated that there were no significant differences between sexes but that the shape of skulls and mandibles varied across different localities (Chapter 4). Canonical Variate Analysis (CVA) suggested that geographic variation in R. damarensis mandibles was based on the shape and thickness of the alveolar bone. Geographic variation in the skulls was based on changes in the rostrum, anterior medial swelling and brain case. Some populations had slightly deeper rostra, slightly larger anterior medial swellings and smaller braincases, whilst others had slightly shallower rostra, slightly smaller anterior medial swellings and larger braincases. The northern lineage was found to be separated from the southern lineage based on the changes in skull and mandible shape. Therefore, separation of lineages within R. damarensis (Chapter 4) could be associated with the foraging and feeding behaviour of the species under different ecological conditions due to ecological opportunity. Overall, differences in the RF were found to be associated with Isolation by Environment mediated through sensory drive and this has led to the formation of two regional (northern and southern) groupings in RF (Chapter 2). The two lineages were supported by both the phenotypic divergence (Chapter 3) and shape variation within R. damarensis skulls and mandibles (Chapter 4). Thus, phenotypic differences corresponded to genetic differences between the two lineages and provide support for IbE.

Bats (Order Chiroptera) are the only mammals capable of power flight. The flight membranes of bats are not only essential for locomotion, but also play vital roles in homeostasis. Although understanding wound healing in the flight membranes of bats is important because injuries in the wild are common, with the recent emergence of white-nose syndrome, understanding wound healing in bat flight membranes has become even more important. In order to conduct my studies on wound healing in the flight membranes of bats, it was necessary to manually restrain bats. In this thesis I present a novel bat restrainer that I designed and that reduces stress experienced by restrained bats during experimentation and data collection. Wound healing is an energy dependent process, as such it is expected that wound healing times will vary during periods of energy constraint (i.e. hibernation) and/or at times of peak demand (i.e. lactation). However, previous studies on wound healing have only looked at healing at times when there are no energy constraints. In thesis I aimed to better understand the effects of seasonality and reproduction on wound healing. Using an 8 mm circular punch, I inflicted biopsy wounds to the chiropatagium of healthy captive big brown bats, Eptesicus fuscus. I compared wound healing times between winter and summer seasons, and between reproductive (i.e. lactating) and non-reproductive females. As expected, wound healing times were longer during the winter months when bats are conserving energy. On the other hand, reproductive status did not have an effect on wound healing times. Although most bats heal, I observed impaired wound healing. This finding is important because it is the first time that impaired wound healing is reported in healthy bats. / Thesis / Master of Science (MSc)

Foliage is very common feature in the habitats of echolocation bats and thus its echoes constitute the major input of bats' sensory systems. Acquiring useful information from vegetation echoes facilitates the bats significantly in the navigation and foraging behaviors. To better understand the foliage echoes, in this dissertation, a computer model was constructed to simulate foliage echoes with following simplifications: approximating leaves as circular disks, leaving out shading effects between leaves, and distributing leaves uniformly in the space. Then one tree can be described with three parameters in the model, leaf radius, orientation, and leaf density, where the first two determine the beampattern of each leaf. Compared with echoes collected from real trees, the simulation echoes are qualitatively accurate, i.e., they match in waveforms and also first-order statistics. Since the ground truth is known in the model, the three parameters were estimated with lasso model by selecting 40 features from each echo. The results have shown that estimation of one parameter with the other two known is usually successful with coefficient of determination close to one, and the classification still has reasonable accuracy when the number of known parameter is reduced to one. Besides, the three simplifications were examined with both experimental and simulation approaches. To assess the acoustic impact of leaf geometry on individual leaves, experiments were carried out by ensonifying leaves from both a single and different species. How the leaves' impulse responses change according to their equivalent radii was investigated. The simulation model of disks fits the experiments done with real leaves within one species and across species reasonably well. Shading effect is found to exist locally when two disks were 25 cm apart and were both in pulse direction. In addition, the inhomogeneous distribution of leaves was introduced by using the branching patterns of L-system. The evaluation of inhomogeneity in echoes produced with two distributions shows that there is always inhomogeneity in echoes, and L-system model does bring more inhomogeneity but not to the same extent as changes in the relative orientation between sonar beam and foliage do. / Ph. D.

Bats using their biosonar while flying in dense swarms may face significant bioacoustic challenges, in particular mutual sonar jamming. While possible solutions to the jamming problem have been investigated multiple times in literature, the severity of this problem has received far less attention. To characterize the acoustics of bat swarms, a simple model of the acoustically relevant properties of a bat swarm has been set up and evaluated. The model contains only four parameters: bat spacial density, biosonar beamwidth, duty cycle, and a scalar measure for the smoothness of the flight trajectories. In addition, a threshold to define substantial jamming was set relative to the emission level. The simulations results show that all four model parameters can have a major impact on jamming probability. Depending on the combination of parameter values, situations with or without substantial jamming probabilities could be produced within reasonable ranges of all model parameters. Hence, the model suggests that not every bat swarm does necessarily impose grave jamming problem. A fitting process was introduced to describe the relationship between the four parameters and jamming probability, hence produce a function with jamming probability as output and four parameters as input. Since the model parameters should be comparatively easy to estimate for actual bat swarms, the simulation results could give researchers a way to assess the acoustic environment of actual bat swarms and determine cases where a study of biosonar jamming could be worthwhile. / Master of Science

The Leptonycteris yerbabuenae is found in southern Arizona, Mexico, Guatemala, and El Salvador. Some females are migratory, mating in southern Mexico, and migrating to maternity roosts in northern Mexico and southern Arizona to give birth. Twelve microsatellite loci markers and the Mitochondrial DNA Control Region (CR) were amplified to examine population structure and phylogenetic relationships among roosts. Twelve polymorphic microsatellite loci were isolated from L. yerbabuenae. A total of sixteen localities in AZ and Mexico were sampled. The mtDNA CR fragment resulted in 102 haplotypes. The phylogenetic analyses resulted in two clades, but no observable geographic structuring. The average FST value across all loci and all sampled localities was 0.022. Program STRUCTURE analyses indicate one population (K=1) throughout the sampling area. These results suggest movement between maternity colonies and transient roosts in Arizona, Sonora, and Chamela, Management recommendations based on these results would be to manage as a single population.

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