Philosophiae Doctor - PhD / The Ruschieae is the most diverse and speciose tribe within the large subfamily Ruschioideae (Aizoaceae), with approximately 71 genera and a distribution centred in the arid parts of the Greater Cape Floristic Region (GCFR) of South Africa. Recent phylogenetic analyses provided the first insights into generic relationships within the tribe, with a number of novel generic relationships discovered. The tribal phylogeny recovered 12 large clades, of which the Conophytum-clade was one the most morphologically diverse based on leaf and capsule characters. The Conophytum-clade is an early-diverging lineage of the Ruschieae and includes the following 10 genera: Cheiridopsis N.E.Br., Conophytum N.E.Br., Enarganthe N.E.Br., Ihlenfeldtia H.E.K.Hartmann, Jensenobotrya A.G.J.Herre, Namaquanthus L.Bolus, Octopoma N.E.Br., Odontophorus N.E.Br., Ruschianthus L.Bolus and Schlechteranthus Schwantes. The present study presents an expanded phylogenetic analysis of the Conophytum-clade, with the sampling of the majority of species in the genera and a representative sampling (56% of species) of the speciose genus Conophytum. Phylogenetic data for up to nine plastid gene regions (atpB–rbcL, matK, psbJ–petA, rpl16, rps16, trnD– trnT, trnL–F, trnQᶷᶷᶢ–rps16, trnS–trnG) were produced for each of the sampled species. The produced plastid data was analyses using maximum parsimony, maximum likelihood and Bayesian inference. The combined plastid phylogenetic analyses were used in combination with morphological, anatomical and palynological data to assess generic and subgeneric circumscriptions within the clade. Upon assessment of generic circumscriptions in the Conophytum-clade, the number of recognised genera in the clade decreased from ten to seven. Arenifera A.G.J.Herre, which had not been sampled in any phylogeny of the Ruschieae, and Octopoma were recovered as polyphyletic, with species placed in the Conophytum-clade, while the type species was placed in the xeromorphic clade of the tribal phylogeny. The species of Arenifera and Octopoma placed in the Conophytum-clade were subsequently included in Schlechteranthus upon assessment of generic circumscriptions between the taxa. Two morphological groupings were recognised within Schlechteranthus, one including the species of Schlechteranthus and the other including species previously recognised as Arenifera and Octopoma. These two morphological groupings were treated as subgenera, with the erection of the new subgenus Microphyllus R.F.Powell. A detailed taxonomic revision of subgenus Microphyllus is presented with a key to species, descriptions of the species (including a new species: S. parvus R.F.Powell & Klak), known geographical distributions and illustrations of the species. In addition to the changes mentioned above, the expanded sampling and phylogenetic analyses of the Conophytum-clade recovered Ihlenfeldtia and Odontophorus embedded in Cheiridopsis. The species of Ihlenfeldtia were recovered with species of heiridopsis subgenus Aequifoliae H.E.K.Hartmann, while the species of Odontophorus were recovered as polyphyletic within the Cheiridopsis subgenus Odontophoroides H.E.K.Hartmann clade. Cheiridopsis was subsequently expanded to include the species of Ihlenfeldtia and Odontophorus, with these species accommodated in the subgenera of Cheiridopsis. The phylogenetic placement and relationship of these species was supported by the shared capsule morphology. The expanded sampling of the clade did not resolve the phylogenetic relationship of the monotypic genera Enarganthe, Jensenobotrya, Namaquanthus and Ruschianthus, with these genera unresolved in the Conophytum-clade. These genera however, exhibit a unique combination of morphological characters, such as a glabrous leaf epidermis and variation in pollen exine and colpi structure, in contrast to the other genera of the clade. The assessment of the generic circumscription of these genera, based on the molecular, morphological, anatomical and palynological data suggested that the generic statuses of these monotypic genera should be maintained. The expanded phylogenetic sampling of the morphologically diverse and speciose genus Conophytum recovered the genus as monophyletic. This monophyly was supported by the unique floral type in Conophytum, with the fused petaloid staminodes forming a tube. None of the sectional classifications were recovered as monophyletic but the phylogenetic analyses did recover a few clades which more or less corresponded to the current sectional classification of the genus. A number of clades were also recovered which included species from a range of different sections. Diverse leaf and floral traits were shown to have evolved numerous times across the genus. This was particularly interesting with regards to the selected floral traits, as the phylogeny indicated a number of switches in floral morphologies across the genus. The floral diversity was assessed in complex species communities of Conophytum across the GCFR, where up to 11 species of Conophytum are found occurring sympatrically, and floral traits were shown to be different across the species within the communities. Pollination competition and adaptation were suggested as possible drivers of floral diversity in the genus, with differences in phenology, anthesis and floral morphology within the species complex communities. The unique floral type of Conophytum has enabled the species to develop a diverse range of specialised flowers, with a variety of structures, scents and colours, resulting in the diverse floral morphologies found across the genus. The complex Conophytum species communities included both closely as well as distantly related species, suggesting the soft papery capsules of Conophytum are wind dispersed. This adaptation to long distance seed dispersal resulted in a significantly higher phylogenetic diversity in Conophytum when compared to its sister genus, Cheiridopsis. A population genetics study of Conophytum also suggested that the capsules may be wind dispersed, with an indication of genetic connectivity between the geographically isolated populations of C. marginatum Lavis across the Bushmanland Inselberg Region. Although the capsules are dispersed by wind, the seeds are released from the hygrochastic capsules by runoff during rainfall events. The relationship between seed dispersal and runoff is evident from the genetic structure of populations of C. maughanii N.E.Br. and C. ratum S.A.Hammer that occur on the tops and the surrounding bases of the inselbergs, as the drainage pattern was found to directly influence population structure in these species. In addition, the AFLP analyses provided insight into the conservation of the flagship species C. ratum. The summit populations of this species were shown to sustain the populations at the base of the Gamsberg. This finding is especially important, as the distribution of the species is restricted to the Gamsberg inselberg, where mining has already commenced as of this year. / National Research Foundation (NRF)

Abstract A mitochondrial disorder may be one of the rare aetiologies of occipital stroke. Clinical and molecular analysis has suggested that 10% of young patients with occipital stroke have a mitochondrial disorder and 6% harbour the mutation 3243A>G in mitochondrial DNA (mtDNA), causing the MELAS syndrome. To identify other possible mtDNA mutations involved, we studied mtDNA genotypes in patients who had suffered an occipital stroke and in whom the common pathogenic mutations in mtDNA had been excluded. Since one systematic way of comparing mtDNA sequences is through phylogenetic analysis, a phylogenetic network for the Finnish mtDNA haplogroup U was constructed and used to identify differences in mtDNA between patients and controls. The usefulness of conformation sensitive gel electrophoresis (CSGE) for analysing differences within the coding sequence of mtDNA was also estimated. We studied mtDNA genotypes of 29 patients with occipital stroke. The aetiology of the stroke was assessed using the criteria of the Baltimore-Washington Cooperative Young Stroke Study, and migraine was diagnosed in 18 patients according to the International Headache Society criteria. Moreover, we studied the mtDNA genotypes of 42 patients with migraine and a total of 480 population controls who reported that they themselves and their mothers were healthy with respect to common clinical manifestations of mtDNA disease. The mtDNA haplogroups were detected by restriction fragment analysis and the mtDNA structures of 14 patients with occipital stroke and 43 subjects belonging to haplogroup U were examined by CSGE. The data acquired by CSGE were then used to construct a phylogenetic network for the Finnish mtDNA haplogroup U. We found CSGE to be a highly sensitive and specific method for screening mutations and polymorphisms in mtDNA. The sequence data on the 43 subjects belonging to the mtDNA haplogroup U were used to construct a phylogenetic network, which was found to be an unambiguous tree with few homoplasies that pointed to several previously unidentified common polymorphisms. The major branch of the network was U5, which seemed to be quite specific to the Finns. Branches representing haplogroups U2, U4, U7 and K could also be detected. Restriction fragment analysis of the patients with occipital stroke revealed that all those with migraine as a probable aetiology belonged to the mtDNA haplogroup U, suggesting that this genotype confers a risk of occipital stroke. In addition to the five patients with migrainous stroke, we analyzed the complete mtDNA coding sequences of nine other patients with occipital stroke belonging to haplogroup U by CSGE. Analysis of the phylogenetic network revealed an association of migrainous stroke with mtDNA haplogroup U5. Furthermore, the distribution of the mtDNA genotypes in the patients with stroke differed from that found in the controls. Four patients harboured potentially pathogenic mutations. CSGE proved to be an effective method for use in mitochondrial genetics, enabling us to construct an unambiguous network for the Finnish haplogroup U. Similar phylogenetic networks are required for the purposes of both medical genetics and population genetics. Such networks were found to be helpful in deciding between a rare polymorphism and a pathogenic mutation in patients with occipital stroke. Likewise, they enabled more detailed comparisons to be made between and within populations and allowed more accurate phylogenetic relationships to be determined.

The fossil record yields information on macroevolutionary patterns that remains inaccessible from the study of extant organisms alone, presenting a natural laboratory for us to test hypotheses about the long-term drivers and processes of evolution. Fossil data are therefore increasingly incorporated into evolutionary analyses, both on their own and in combination with neontological data. Phylogeny (an explicit hypothesis of the evolutionary relationships between taxa) can be used as a framework to enable direct comparison of results of comparative methods across many different timescales and taxa, and is now commonly used in investigations of fossil data. This represents an important step towards a unified approach, however, it is not yet fully understood what the effect of using fossil data is on the results of downstream phylogenetic comparative methods, which were originally developed with only living taxa in mind. In this thesis I explore the validity of phylogenetic interpretations of fossil record data. I begin with only taxonomic classification and show that this can in some cases substitute for a cladistically inferred phylogeny in phylogenetic comparative methods, without biasing results. Moving on to scenarios where a timescaled phylogeny is available I investigate the relationship between phylogeny and extinction in the geological past, show that phylogenetic clustering of extinction was common in tetrapods, and present a summary of the ways in which fossil data biases this measurement. Finally, with timescaled phylogenies and a detailed continuous trait dataset available, I interrogate the fossil record of Sauropterygia to uncover the processes of evolutionary change in this highly labile clade. By comparing the results of a suite of phylogenetic comparative methods I demonstrate that neck length evolved through changing vertebral counts rather than somite growth; that the clade experienced a release in evolutionary constraint at the Triassic-Jurassic boundary; and that evidence does not support evolution towards a stationary adaptive peak as a suitable model for phenotypic change in the clade.

While there is increasing interest in the evolutionary consequences of species invasions on native plant communities, the connection between the phylogenetic relatedness of invaders and natives, and its evolutionary consequences, has not yet received much attention. One way to examine the role of relatedness on the impact of species invasions is through the use of meta-analytical techniques combined with a phylogenetic framework. I apply this technique and expand on a prior meta-analysis by Oduor (2013), in order to compare how native plant species that either have prior experience coexisting with an invasive (‘experienced’) or do not (‘naïve'), differ in terms of their growth and reproduction in the presence of the invasive for evidence of a phylogenetic signal in their response to plan invasions. My results suggest that the effects of a species invasion on native growth and reproductive traits may be greater when the native and invasive species are distantly related, and/or when the invasive species is allelopathic, but only in the presence on the invader. I also found that the negative effect of competition with an invader on native growth traits tended to be greatest when the native and invasive species were closely related, suggesting that the strength of competition and the evolutionary response of native plants to invasive plants may not be as closely associated as it is often assumed. Overall, my analysis suggests that future studies may benefit from integrating phylogenetic relatedness when exploring native evolutionary responses to invasions, but that considerable work need sot be done to tease apart the roles of relatedness and competition.

Notre travail a consisté à rechercher des éléments génétiques nouveaux associés aux souchesde Streptococcus agalactiae (SGB) responsables de méningites néonatales (MNN).L'amplification au hasard des ADN génomiques a identifié 3 fragments prophagiquesassociés aux souches à haut risque de MNN. Trente-six phages ont été isolés à partir desouches invasives et non invasives de SGB. La caractérisation moléculaire a défini sixgroupes moléculaires. L'étude du spectre lytique a montré des particularités pour chacun dessix groupes moléculaires. L'étude des capacités métaboliques de souches de SGB a montréque les souches lysogènes appartenant aux lignées phylogénétiques impliquées dans les MNNet présentent des pertes cataboliques. Les transferts génétiques horizontaux qui marquent lesclones des MNN ne semblent pas jouer un rôle dans la résistance aux macrolides. Desmécanismes de transduction ont joué un rôle important dans l'émergence de lignéesphylogénétiques impliquées dans les MNN. / We tried to identify new genetic elements in the genome of Streptotoccus agalactiae (SGB)strains responsible for neonatal meningitis (MNN). Three prophage-related DNA elementswere identified by randomly amplified polymorphie DNA analysis significantly associatedwith SGB strains presented a high risk of causing MNN. Thirty-six phages were isolated from invasive and non invasive SGB strains. Molecular characterization of phage DNA identified six distantly related molecular groups. The various phage groups had specifie lytic activities.The lysogenic strains belonging to phylogenetic lineages most involved in MNN presented catabolic losses. The horizontal gene transfers that mediate mobile genetic elementsrecognized as markers of highly virulent clones do not seem to be have played a role in theemergence of macrolide resistance. Ali our results indicate that transduction may have play arole in the emergence of phylogenetic lineages implicated in MNN.

A striking variation in the behaviour of pollinating fig wasps (Agonidae) is the occurrence of male fighting in some species while in others it is completely absent. Fighting behaviour was investigated at two levels. Firstly, the variation in fighting behaviour between the species was used to examine factors that might cause the evolution thereof. Comparisons across species were done using phylogenetic regression. This method takes similarity due to phylogenetic constraints into account when data are compared. Kin selection theory implies that fighting is barred by the high degree of relatedness in competing males. We however find that the relatedness of the males do not have an influence on the evolution of fighting and this finding supports models suggesting that high LMC cancels benefits due to relatedness. Rather, that the only factor having a significant correlation with fighting is the release sex ratio. The release sex ratio and dispersal is also associated. Fighting and dispersal are not expected to have direct influence on each other and the association of both with the release sex ratio imply that this may be an indirect link between these two behaviours. A syndrome where fighting and dispersal is found together is in part explained by the release sex ratio. We conclude that the release sex ratio is the most likely cause of the evolution of fighting behaviour in pollinating fig wasps. The second part of this study deals with the proximal determinants causing fighting, in the males of the species Platyscapa awekei. We show that the sex ratio which, is less female biased than non-fighting pollinator species, rapidly becomes even less female biased as soon as both sexes becomes active. Numerous fights are fought by the males in the female limited environment. The activity of the wasps is shown to be regulated by the gaseous environment, which change from a high to a low CO2 concentration with the construction of an exit hole from the fig. The males of the species P. awekei are inactive, and do not engage in mating or fighting activities, in high CO2, contrasted to males of other species, which are active in this environment. P. awekei females rapidly release once the CO2 level is lowered and mating behaviour is only observed in this environment. The number of female to male encounters of every male decrease as the operational sex ratio becomes less female biased. Male fighting in this species is therefore expected due to the high sex ratio, which is enforced by the increase thereof. We conclude that the physical environment, in this species, affects the mating environment. The resultant reduction in the number of potential mating opportunities therefore escalates fighting between the males. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

Na pesquisa evolutiva do grupo Hydrozoa, um dos maiores desafios continua sendo a proposta de relações históricas entre grandes grupos. A partir de um ciclo de vida \"tradicional\" nos Medusozoa, determinado pela metagênese (a geração da fase adulta pelágica de medusa a partir de um pólipo bentônico), as linhagens apresentam espécies com formas alternativas de ciclo de vida e uma riqueza de espécies diversa, sendo destacado o grupo mais rico em espécies, os Leptothecata. Novos dados moleculares para diversas espécies de Hydrozoa, e principalmente de Leptothecata, foram gerados para combinar com informação genética já disponível. A partir de matrizes multilocus e individuais gênicas de marcadores ribossomais, foram realizadas diferentes análises para inferir e avaliar filogenias, assim como também para definir e estabelecer o impacto dos métodos utilizados na qualidade dos estudos filogenéticos desenvolvidos para Hydrozoa em geral, e Leptothecata de forma específica. Os resultados permitem propor novas relações sistemáticas básicas nos Hydroidolina, assim como uma nova classificação taxonômica para uma das suas linhagens principais, os Leptothecata. A partir do cenário filogenético obtido, são discutidos fenômenos que podem ter promovido o padrão macroevolutivo do grupo dos leptotecados. Desafios e questões de genética evolutiva, que podem ser de grande relevância na evolução do grupo, são apresentados como vínculo temático para futuros estudos no capítulo final / In modern evolutionary research considering lineages such as the Hydrozoa, one of the major challenges is the proposal of historical relationships between their main groups. From a \"traditional\" life cycle in Medusozoa, defined by a metagenesis process (the generation of a pelagic adult phase from a benthic polyp), the lineages exhibit a diverse array of life cycles and species richness, with Leptothecata being considered the richest group in number of species. New molecular data for diverse Hydrozoa species, particularly from Leptothecata, were generated to combine with available genetic information. From new multilocus and single gene matrices of ribosomal markers, different analyses were created to infer and evaluate phylogenies, as well as to define and establish the impact of used methods on data treatment and nodal support to Hydrozoa, and more specifically, Leptothecata.The results allow proposing new basic systematic relationships in Hydroidolina, as well as a new taxonomic classification for Leptothecata. From this phylogenetic scenario, potential phenomena that may have promoted the macroevolutionary pattern of the group are presented and discussed. Questions and challenges from evolutionary genetics, which may be relevant in the evolution of the group, are presented as nexus for future studies in the final chapter

Os loricariíneos são Siluriformes altamente derivados e contabilizam cerca de 210 espécies, apresentando uma irradiação adaptativa bem sucedida, tendo representantes na Costa Rica, no Panamá e em toda a América do Sul, e ocorrendo também em ambos os lados dos Andes. Discussões taxonômicas conflituosas são encontradas sobre esta subfamília, envolvendo muitos de seus gêneros, principalmente os mais numerosos, como é o caso de Rineloricaria. O elevado número de espécies (64), a ampla distribuição geográfica e a presença de características diagnósticas válidas para todos os gêneros da subfamília, levam à discussão a proposta de uma subdivisão no gênero Rineloricaria, além de indicar as dificuldades de identificação apresentadas pelo gênero. Em relação às características citogenéticas, Loricariinae apresenta-se como uma subfamília heterogênea e diversificada, com o número diplóide variando de 2n=36 cromossomos a 2n=74 cromossomos. O gênero Rineloricaria contribui intensamente para essa variação, com números cromossômicos variando de 2n=36 a 2n=70 cromossomos. A participação de rearranjos Robertsonianos na evolução cariotípica de Rineloricaria é algo claro, mas evidências que confirmem a ordenação desses eventos não existem. No presente trabalho, técnicas citogenéticas e moleculares foram utilizadas com a finalidade de estabelecer relações evolutivas entre os integrantes da subfamília Loricariinae, em especial do gênero Rineloricaria, entender a evolução cariotípica dessa subfamília e identificar marcadores citogenéticos e moleculares úteis na identificação e descrição de espécies de Rineloricaria. Foram analisados os cromossomos de doze espécies de Loricariinae, pertencentes aos gêneros Harttia, Loricariia, Loricariichthys e Rineloricaria, coletadas na Bacia do Alto Paraná e nas drenagens do Leste brasileiro. Três regiões do genoma mitocondrial (ATPase 6 e 8, Citocromo b e Citocromo c Oxidase I) foram investigadas nessas espécies no presente trabalho. As análises filogenéticas recuperaram o gênero Rineloricaria como um grupo monofilético. Os dados citogenéticos permitiram o estabelecimento de tendências de evolução cromossômica em Loricariinae e no gênero Rineloricaria. O presente trabalho reforça a importância da utilização de diferentes abordagens na realização de estudos taxonômicos e evolutivos, sugerindo uma nova revisão do gênero Rineloricaria que leve em consideração os dados genéticos obtidos. / Loricariinae are Siluriformes highly derivative and account for about 210 species, with a successful adaptive radiation and representatives can be seen in Costa Rica, Panama and throughout South America, and also occurring on both sides of the Andes. Conflicting taxonomic discussions are found on this subfamily, involving many of its genera, especially the most numerous, as Rineloricaria. The high number of species (64), the wide geographic distribution and the presence of valid diagnostic features for all genera of the subfamily, leads to a discussion of a proposed subdivision in the genus Rineloricaria, besides indicating the difficulties of identification presented by the genus. With respect to cytogenetic characteristics, Loricariinae is presented as a heterogeneous and diverse subfamily, varying diploid number of 2n = 36 chromosomes to 2n = 74 chromosomes. The genus Rineloricaria contributes strongly to this variation, with chromosome numbers ranging from 2n = 36 to 2n = 70 chromosomes. The involvement of Robertsonian rearrangements in the karyotype evolution of Rineloricaria is something unclear, but evidences that confirm the ordination of these events does not exist. In this study, cytogenetic and molecular techniques were used in order to establish evolutionary relationships among members of the subfamily Loricariinae, especially the genus Rineloricaria, and also to understand the karyotype evolution of this subfamily and to identify cytogenetic and molecular markers useful in identifying and describing species of Rineloricaria. We analyzed the chromosomes of twelve species of Loricariinae from genera Harttia, Loricariia, Loricariichthys Rineloricaria from the Upper Paraná Basin and the drainages of eastern Brazil. Three regions of the mitochondrial genome (ATPase 6 and 8, Cytochrome b and Cytochrome c Oxidase I) were investigated in these species of this study. The phylogenetic analysis recovered the genus Rineloricaria as a monophyletic group. The cytogenetic data allowed us to establish trends in chromosomal evolution in Loricariinae and in the genus Rineloricaria. The present study underscores the importance of using different approaches in carrying out taxonomic and evolutionary studies, suggesting a further revision of the genus Rineloricaria that takes into account the genetic data obtained.

Orientador: Vera Lúcia Mores Rall / Resumo: A mastite bovina é uma inflamação da glândula mamária, geralmente ocasionada por micro-organismos, que causa grandes prejuízos às fazendas leiteiras, devido ao uso de medicamentos, pagamento aos médicos veterinários, descarte de leite e até do animal e a Escherichia coli é o principal agente etiológico causador da forma clínica dessa doença. Neste trabalho, foram pesquisados genes para a classificação de E. coli diarreiogênica (DEC) ou de E. coli patogênica extra-intestinal (ExPEC), dentro dos grupos filogenéticos, além de genes que codificam fatores de virulência e resistência aos antimicrobianos. Também foram realizados testes de quantificação de produção de biofilme e produção de beta-lactamase de espectro estendido (ESBL). Entre os 161 isolados, dois (1,2%) foram classificados como DEC. Considerando-se os genes pesquisados, os mais frequentes foram fimH (160 = 99,3%), traT (126 = 78,3%), ecpA (108 = 67%) e ompT (60 = 37,3%). Em relação aos grupos filogenéticos, a maioria foi classificada nos grupos B1 (52,8%) e A (36,6%). Quanto à produção de biofilme, 68 (42,2%) foram classificadas como não produtoras, 80 (49,7%) como fracas, 11 (6,8%) como moderadas e apenas duas (1,2%) foram fortes produtoras. Apenas dois (1,2%) isolados apresentaram positividade no teste fenotípico de ESBL, com a presença do gene blaCTX-M-15. Considerando-se outros genes de resistência, blaTEM foi encontrado em sete (4,3%) isolados e blaSHV em um (0,6%). Os dois (1,2%) isolados escN+ apresentaram padr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bovine mastitis is an inflammation of the mammary gland, usually caused by microorganisms, which causes great damage to dairy farms, due to the use of medicines, payment to veterinarians, disposal of milk and even the animal and Escherichia coli is the main etiologic agent that causes the clinical form of this disease. In this study, genes were searched for the classification of diarrhogenic E. coli (DEC) or extra-intestinal pathogenic E. coli (ExPEC), within the phylogenetic groups, in addition to genes that encode virulence factors and resistance to antimicrobials. Quantification tests of biofilm production and production of extended spectrum beta-lactamase (ESBL) were also performed. Among the 161 isolates, two (1.2%) were classified as DEC. Considering the researched genes, the most frequent were fimH (160 = 99.3%), traT (126 = 78.3%), ecpA (108 = 67%) and ompT (60 = 37.3%). Regarding phylogenetic groups, most were classified in groups B1 (52.8%) and A (36.6%). As for the production of biofilm, 68 (42.2%) were classified as non-producing, 80 (49.7%) as weak, 11 (6.8%) as moderate and only two (1.2%) were strong producers. Only two (1.2%) isolates were positive in the ESBL phenotypic test, with the presence of the blaCTX-M-15 gene. Considering other resistance genes, blaTEM was found in seven (4.3%) isolates and blaSHV in one (0.6%). The two (1.2%) escN+ isolates showed a “localized-like” adhesion pattern in HeLa cells and their potential to induce attaching/effacing lesio... (Complete abstract click electronic access below) / Mestre

abstract: Cyanobacteria and algae living inside carbonate rocks (endoliths) have long been considered major contributors to bioerosion. Some bore into carbonates actively (euendoliths); others simply inhabit pre-existing pore spaces (cryptoendoliths). While naturalistic descriptions based on morphological identification have traditionally driven the field, modern microbial ecology has shown that this approach is insufficient to assess microbial diversity or make functional inferences. I examined endolithic microbiomes using 16S rRNA genes and lipid-soluble photosynthetic pigments as biomarkers, with the goal of reassessing endolith diversity by contrasting traditional and molecular approaches. This led to the unexpected finding that in all 41 littoral carbonate microbiomes investigated around Isla de Mona (Puerto Rico) and Menorca (Spain) populations of anoxygenic phototrophic bacteria (APBs) in the phyla Chloroflexi and Proteobacteria, were abundant, even sometimes dominant over cyanobacteria. This was not only novel, but it suggested that APBs may have been previously misidentified as morphologically similar cyanobacteria, and opened questions about their potential role as euendoliths. To test the euendolithic role of photosynthetic microbes, I set a time-course experiment exposing virgin non-porous carbonate substrate in situ, under the hypothesis that only euendoliths would be able to initially colonize it. This revealed that endolithic microbiomes, similar in biomass to those of mature natural communities, developed within nine months of exposure. And yet, APB populations were still marginal after this period, suggesting that they are secondary colonizers and not euendolithic. However, elucidating colonization dynamics to a sufficiently accurate level of molecular identification among cyanobacteria required the development of a curated cyanobacterial 16S rRNA gene reference database and web tool, Cydrasil. I could then detect that the pioneer euendoliths were in a novel cyanobacterial clade (named UBC), immediately followed by cyanobacteria assignable to known euendoliths. However, as bioerosion proceeded, a diverse set of likely cryptoendolithic cyanobacteria colonized the resulting pore spaces, displacing euendoliths. Endolithic colonization dynamics are thus swift but complex, and involve functionally diverse agents, only some of which are euendoliths. My work contributes a phylogenetically sound, functionally more defined understanding of the carbonate endolithic microbiome, and more specifically, Cydrasil provides a user-friendly framework to routinely move beyond morphology-based cyanobacterial systematics. / Dissertation/Thesis / APPENDIX A / APPENDIX B / APPENDIX C / Doctoral Dissertation Microbiology 2020