Craniometrics is a very reliable and effective tool for studying the difference in animal morphology. Previously, traditional craniometrics were conducted with the aid of calipers in two dimensional format (2D). Such discounting of actual three-dimensional 3D form may result in loss of some relevant and critical information leading to compromised and unreliable results for studies such as population variation analysis of morphology. The employment of 3D photogrammetry allows a close to complete representation of the physical dimensions of a specimen. The use of photogrammetry in mammalogy concentrated on measuring of body size/mass, but little has been done on animal skull delineation through photogrammetry. This dissertation describes advances in morphometrics and 3D photogrammetry application in craniometrics, investigates the craniometric variation between closely related species (Arctocephalus gazella and A. tropicalis), and A. tropicalis interpopulation craniometrics between two geographically distinct populations, at Marion Island and Gough Island, using Photomodeler Scanner® (PMSc®) three-dimensional (3D) modelling software to produce accurate, high resolution 3D skull models. A total of 117 3D models were created from adult male fur seal crania, and 16 traditional measurements recorded, using specimens archived at the Port Elizabeth Museum, Bayworld, South Africa. Sixteen linear measurements, (8 caliper recordings and 8 3D recordings) were used for PMSc® methodology testing, 16 (A. gazella n= 8 and A. tropicalis n= 8) used for species cranial comparison and 85 (Marion Island n= 54 and Gough Island n= 31) used for interpopulation variation. The craniometric variations were analysed using the Statistica® v11 software package, StatSoft, Inc. The comparison between linear traditional caliper cranial measurements and 3D measurements produced significantly similar results, attesting to the accuracy of the PMSc® 3D model production. Photomodeler Scanner® therefore produces accurate and high resolution 3D models of skulls which allow 3D measurements. I predicted that PMSc® would detect the existing significant differences between the skulls of adult male A. gazella and A. tropicalis and modelled and compared their 3D models, and I further predicted that PMSc® would detect any existing differences between the skulls of A. tropicalis from Gough and Marion islands by comparison of their 3D models. The Gough Island and Marion Island A. tropicalis populations could not be discriminated based on linear 3D cranial I conclude that PMSc® is a reliable and effective tool for accurate and high resolution 3D modelling. The present study confirms previous findings and contributes additional evidence that suggests that adult A. tropicalis males from Gough Island and Marion Island cannot be discriminated based on linear measurements of craniometrics, and deviates from the Bergmanian rule as applied to large mammals. The present study, however, makes several noteworthy contributions to the use of PMSc® and 3D modelling in morphometrics. Taken together, these findings suggest a role for PMSc® 3D modelling in promoting accurate digitization of museum specimens and creation of online museum libraries. This research will serve as a baseline for future studies and usefulness of PMSc® in 3D morpho-volumetric measurements. / Dissertation (MSc (Zoology))--University of Pretoria, 2014. / Zoology and Entomology / MSc (Zoology) / Unrestricted

Bothrops bilineatus é uma das seis espécies presentes no grupo taeniatus, é reconhecida atualmente por duas sub-espécies: B. b. smaragdinus que se distribui pelo oeste Amazônico e B. b. bilineatus com distribuição disjunta pela Amazônia e Mata Atlântica. Este trabalho tem como objetivo testar a validade das duas sub-espécies com base em ferramentas moleculares, morfométricas, merísticas, hemipenianas e osteológicas, reconhecendo e delimitando as linhagens encontradas. Pretende-se ainda investigar as relações filogeográficas entre as populações ao longo da sua distribuição, analisando o tempo de divergência e os possíveis eventos associados a sua diversificação. Além disso, pretende-se comparar a variação molecular e hemipeniana de B. bilineatus com B. taeniatus (espécie filogeneticamente próxima) e as existentes nos outros grupos de espécies do gênero. Os resultados mostram que a variação genética e hemipeniana encontram se em diferentes níveis ao longo dos grupos de espécies em Bothrops, fornecendo informação relevante a nível intra e inter-específico. Foram recuperados seis clados estruturados para Bothrops bilineatus, representando quatro linhagens independentes e diagnosticáveis com base conjunta nos caracteres morfológicos, hemipenianos, osteológicos e moleculares: linhagem 1 - Amazônia central, basal as outras, linhagem 2 - Mata Atlântica+Escudo das Guianas externa às linhagens 3 e 4 Amazônia oeste parte sul e norte, respectivamente. Já para B. taeniatus quatro clados representando três linhagens foram recuperadas com bases nos caracteres moleculares e hemipenianos, porém se faz necessário buscar por mais caracteres morfológicos e osteológicos para melhor delimitar e diagnosticar as linhagens. As mudanças paleoclimáticas e geomorfológicas ocorridas na transição Terciário/Quaternário parecem ser os principais eventos a influenciar na diversificação das linhagens encontradas em Bothrops bilineatus e B. taeniatus / Bothrops bilineatus is one of six species in the taeniatus group, is now recognized by two subspecies: B. b. smaragdinus that distributes through western Amazon and B. b. bilineatus with disjunct distribution through Amazon and Atlantic Forest. This work aims to test the validity of these two sub-species, based on molecular, morphometric tools, meristic, osteological and hemipenial data, recognizing and delimiting lineages found. We also intend to investigate the phylogeographic relationships among populations throughout its distribution, analyzing the divergence time and possible events associated with it diversification. In addition, we intend to compare the molecular and hemipenial variation presented in B. bilineatus with B. taeniatus (phylogenetically close species) and those in the other groups of the genus. The genetic and hemipenial results show different levels of variation along the groups of Bothrops species, providing significant intra-and inter-specific information. Six clades structured to Bothrops bilineatus were recovered, representing four independent lineages diagnosable with joint based on morphology, hemipenial, osteological and molecular characters: lineage 1 - central Amazon, basal the others, lineage 2 - Atlantic Forest + Guayana Shield region recovered outside the sisters lineages 3 and 4 - western Amazon, southern part and western Amazon, northern part, respectively. As for B. taeniatus four clades representing three lineages were recovered with bases in molecular and hemipenial characters, however it is necessary to search for more morphological and osteological characters to better delimit and diagnose the lineages. Paleoclimatic and geomorphic changes in the transition Tertiary/Quaternary, probably are the major events influencing the diversification of lineages found in Bothrops bilineatus and B. taeniatus

Bothrops bilineatus é uma das seis espécies presentes no grupo taeniatus, é reconhecida atualmente por duas sub-espécies: B. b. smaragdinus que se distribui pelo oeste Amazônico e B. b. bilineatus com distribuição disjunta pela Amazônia e Mata Atlântica. Este trabalho tem como objetivo testar a validade das duas sub-espécies com base em ferramentas moleculares, morfométricas, merísticas, hemipenianas e osteológicas, reconhecendo e delimitando as linhagens encontradas. Pretende-se ainda investigar as relações filogeográficas entre as populações ao longo da sua distribuição, analisando o tempo de divergência e os possíveis eventos associados a sua diversificação. Além disso, pretende-se comparar a variação molecular e hemipeniana de B. bilineatus com B. taeniatus (espécie filogeneticamente próxima) e as existentes nos outros grupos de espécies do gênero. Os resultados mostram que a variação genética e hemipeniana encontram se em diferentes níveis ao longo dos grupos de espécies em Bothrops, fornecendo informação relevante a nível intra e inter-específico. Foram recuperados seis clados estruturados para Bothrops bilineatus, representando quatro linhagens independentes e diagnosticáveis com base conjunta nos caracteres morfológicos, hemipenianos, osteológicos e moleculares: linhagem 1 - Amazônia central, basal as outras, linhagem 2 - Mata Atlântica+Escudo das Guianas externa às linhagens 3 e 4 Amazônia oeste parte sul e norte, respectivamente. Já para B. taeniatus quatro clados representando três linhagens foram recuperadas com bases nos caracteres moleculares e hemipenianos, porém se faz necessário buscar por mais caracteres morfológicos e osteológicos para melhor delimitar e diagnosticar as linhagens. As mudanças paleoclimáticas e geomorfológicas ocorridas na transição Terciário/Quaternário parecem ser os principais eventos a influenciar na diversificação das linhagens encontradas em Bothrops bilineatus e B. taeniatus / Bothrops bilineatus is one of six species in the taeniatus group, is now recognized by two subspecies: B. b. smaragdinus that distributes through western Amazon and B. b. bilineatus with disjunct distribution through Amazon and Atlantic Forest. This work aims to test the validity of these two sub-species, based on molecular, morphometric tools, meristic, osteological and hemipenial data, recognizing and delimiting lineages found. We also intend to investigate the phylogeographic relationships among populations throughout its distribution, analyzing the divergence time and possible events associated with it diversification. In addition, we intend to compare the molecular and hemipenial variation presented in B. bilineatus with B. taeniatus (phylogenetically close species) and those in the other groups of the genus. The genetic and hemipenial results show different levels of variation along the groups of Bothrops species, providing significant intra-and inter-specific information. Six clades structured to Bothrops bilineatus were recovered, representing four independent lineages diagnosable with joint based on morphology, hemipenial, osteological and molecular characters: lineage 1 - central Amazon, basal the others, lineage 2 - Atlantic Forest + Guayana Shield region recovered outside the sisters lineages 3 and 4 - western Amazon, southern part and western Amazon, northern part, respectively. As for B. taeniatus four clades representing three lineages were recovered with bases in molecular and hemipenial characters, however it is necessary to search for more morphological and osteological characters to better delimit and diagnose the lineages. Paleoclimatic and geomorphic changes in the transition Tertiary/Quaternary, probably are the major events influencing the diversification of lineages found in Bothrops bilineatus and B. taeniatus

This body of work is concerned with the genetics of craniofacial morphology and specifically with that of the cranial sutures which form fibrous articulations between the calvarial bones. The premature fusion of these sutures, known as craniosynostosis, is a common developmental abnormality and has been extensively utilised here as a tool through which to study the genetics of suture morphogenesis and craniofacial diversity. Investigations began with a search for polymorphisms associated with normal variation in human craniofacial characteristics. Denaturing High-Performance Liquid chromatography was used to identify polymorphisms in two genes causative for craniosynostosis by analysing DNA from a large cohort of individuals from four ethnogeographic populations. A single nucleotide polymorphism in fibroblast growth factor receptor 1 was identified as being associated with variation in the cephalic index, a common measure of cranial shape. To further, and specifically, investigate the molecular processes of suture morphogenesis gene expression was compared between unfused and prematurely fusing/fused suture tissues isolated from patients with craniosynostosis. Two approaches, both utilising Affymetrix gene expression microarrays, were used to identify genes differentially expressed during premature suture fusion. The first was a novel method which utilised the observation that explant cells from both fused and unfused suture tissue, cultured in minimal medium, produce a gene expression profile characteristic of minimally differentiated osteoblastic cells. Consequently, gene expression was compared between prematurely fused suture tissues and their corresponding in vitro de-differentiated cells. In addition to those genes known to be involved in suture morphogenesis, a large number of novel genes were identified which were up-regulated in the differentiated in vivo state and are thus implicated in premature suture fusion and in vivo osteoblast differentiation. The second microarray study involved an extensive analysis of 16 suture tissues and compared gene expression between unfused (n=9) and fusing/fused sutures (n=7). Again, both known genes and a substantially large number of novel genes were identified as being differentially expressed. Some of these novel genes included retinol binding protein 4 (RBP4), glypican 3 (GPC3), C1q tumour necrosis factor 3 (C1QTNF3), and WNT inhibitory factor 1 (WIF1). The known functions of these genes are suggestive of potential roles in suture morphogenesis. Realtime quantitative RT PCR (QRT-PCR) was used to verify the differential expression patterns observed for 11 genes and Western blot analysis and confocal microscopy was used to investigate the protein expression for 3 genes of interest. RBP4 was found to be localised on the ectocranial surface of unfused sutures and in cells lining the osteogenic fronts while GPC3 was localised to suture mesenchyme of unfused sutures. A comparison between each unfused suture (coronal, sagittal, metopic, and lambdoid) demonstrated that gene expression profiles are suture-specific which, based on the identification of differentially expressed genes, suggests possible molecular bases for the differential timing of normal fusion and the response of each suture to different craniosynostosis mutations. One observation of particular interest was the presence of cartilage in unfused lambdoid sutures, suggesting a role for chondrogenesis in posterior skull sutures which have generally been thought to develop by intramembranous ossification without a cartilage precursor. Finally, the effects of common media supplements used in in vitro experiments to stimulate differentiation of calvarial suture-derived cells were investigated with respect to their ability to induce in vivo-like gene expression. The response to standard differentiation medium (ascorbic acid + β-glycerophosphate) with and without dexamethasone was measured by both mineralisation and matrix formation assays and QRT-PCR of genes identified in the above described microarray studies. Both media induced collagen matrix and bone nodule formation indicative of differentiating osteoblasts. However, the genes expression profiles induced by both media differed and neither recapitulated the levels and profiles of gene expression observed in vivo for cells isolated from both fused and unfused suture tissues. This study has implications for translating results from in vitro work to the in vivo situation. Significantly, the dedifferentiation microarray study identified differentially expressed genes whose products may be considered candidates as more appropriate osteogenic supplements that may be used during in vitro experiments to better induce in vivo-like osteoblast differentiation. This study has made a substantial contribution to the identification of novel genes and pathways involved in controlling human suture morphogenesis and craniofacial diversity. The results from this research will stimulate new areas of inquiry which will one day aid in the development of better diagnostics and therapeutics for craniosynostosis, and other craniofacial and more general skeletal abnormalities.