Rooting the tree of life by transition analyses

BACKGROUND: Despite great advances in clarifying the family tree of life, it is still not agreed where its root is or what properties the most ancient cells possessed – the most difficult problems in phylogeny. Protein paralogue trees can theoretically place the root, but are contradictory because of tree-reconstruction artefacts or poor resolution; ribosome-related and DNA-handling enzymes suggested one between neomura (eukaryotes plus archaebacteria) and eubacteria, whereas metabolic enzymes often place it within eubacteria but in contradictory places. Palaeontology shows that eubacteria are much more ancient than eukaryotes, and, together with phylogenetic evidence that archaebacteria are sisters not ancestral to eukaryotes, implies that the root is not within the neomura. Transition analysis, involving comparative/developmental and selective arguments, can polarize major transitions and thereby systematically exclude the root from major clades possessing derived characters and thus locate it; previously the 20 shared neomuran characters were thus argued to be derived, but whether the root was within eubacteria or between them and archaebacteria remained controversial. RESULTS: I analyze 13 major transitions within eubacteria, showing how they can all be congruently polarized. I infer the first fully resolved prokaryote tree, with a basal stem comprising the new infrakingdom Glidobacteria (Chlorobacteria, Hadobacteria, Cyanobacteria), which is entirely non-flagellate and probably ancestrally had gliding motility, and two derived branches (Gracilicutes and Unibacteria/Eurybacteria) that diverged immediately following the origin of flagella. Proteasome evolution shows that the universal root is outside a clade comprising neomura and Actinomycetales (proteates), and thus lies within other eubacteria, contrary to a widespread assumption that it is between eubacteria and neomura. Cell wall and flagellar evolution independently locate the root outside Posibacteria (Actinobacteria and Endobacteria), and thus among negibacteria with two membranes. Posibacteria are derived from Eurybacteria and ancestral to neomura. RNA polymerase and other insertions strongly favour the monophyly of Gracilicutes (Proteobacteria, Planctobacteria, Sphingobacteria, Spirochaetes). Evolution of the negibacterial outer membrane places the root within Eobacteria (Hadobacteria and Chlorobacteria, both primitively without lipopolysaccharide): as all phyla possessing the outer membrane β-barrel protein Omp85 are highly probably derived, the root lies between them and Chlorobacteria, the only negibacteria without Omp85, or possibly within Chlorobacteria. CONCLUSION: Chlorobacteria are probably the oldest and Archaebacteria the youngest bacteria, with Posibacteria of intermediate age, requiring radical reassessment of dominant views of bacterial evolution. The last ancestor of all life was a eubacterium with acyl-ester membrane lipids, large genome, murein peptidoglycan walls, and fully developed eubacterial molecular biology and cell division. It was a non-flagellate negibacterium with two membranes, probably a photosynthetic green non-sulphur bacterium with relatively primitive secretory machinery, not a heterotrophic posibacterium with one membrane. REVIEWERS: This article was reviewed by John Logsdon, Purificación López-García and Eric Bapteste (nominated by Simonetta Gribaldo)

Eukaryotic Organisms in Proterozoic Oceans

The geological record of protists begins well before the Ediacaran and Cambrian diversification of animals, but the antiquity of that history, its reliability as a chronicle of evolution and the causal inferences that can be drawn from it remain subjects of debate. Well-preserved protists are known from a relatively small number of Proterozoic formations, but taphonomic considerations suggest that they capture at least broad aspects of early eukaryotic evolution. A modest diversity of problematic, possibly stem group protists occurs in ca 1800–1300 Myr old rocks. 1300–720 Myr fossils document the divergence of major eukaryotic clades, but only with the Ediacaran–Cambrian radiation of animals did diversity increase within most clades with fossilizable members. While taxonomic placement of many Proterozoic eukaryotes may be arguable, the presence of characters used for that placement is not. Focus on character evolution permits inferences about the innovations in cell biology and development that underpin the taxonomic and morphological diversification of eukaryotic organisms.Organismic and Evolutionary Biolog

Discovery of catalases in members of the Chlamydiales order.

Catalase is an important virulence factor for survival in macrophages and other phagocytic cells. In Chlamydiaceae, no catalase had been described so far. With the sequencing and annotation of the full genomes of Chlamydia-related bacteria, the presence of different catalase-encoding genes has been documented. However, their distribution in the Chlamydiales order and the functionality of these catalases remain unknown. Phylogeny of chlamydial catalases was inferred using MrBayes, maximum likelihood, and maximum parsimony algorithms, allowing the description of three clade 3 and two clade 2 catalases. Only monofunctional catalases were found (no catalase-peroxidase or Mn-catalase). All presented a conserved catalytic domain and tertiary structure. Enzymatic activity of cloned chlamydial catalases was assessed by measuring hydrogen peroxide degradation. The catalases are enzymatically active with different efficiencies. The catalase of Parachlamydia acanthamoebae is the least efficient of all (its catalytic activity was 2 logs lower than that of Pseudomonas aeruginosa). Based on the phylogenetic analysis, we hypothesize that an ancestral class 2 catalase probably was present in the common ancestor of all current Chlamydiales but was retained only in Criblamydia sequanensis and Neochlamydia hartmannellae. The catalases of class 3, present in Estrella lausannensis and Parachlamydia acanthamoebae, probably were acquired by lateral gene transfer from Rhizobiales, whereas for Waddlia chondrophila they likely originated from Legionellales or Actinomycetales. The acquisition of catalases on several occasions in the Chlamydiales suggests the importance of this enzyme for the bacteria in their host environment

Molecular and Immunological Methods to Confirm Toxiginicity (Microcystin Production) of Westiellopsis Prolifica Isolated from Tigris River – Iraq

تنتج العديد من الطحالب الخضر المزرقة السم الكبدي المايكروسستين. ولكون هذا السم يشكل خطرا على الصحة والبيئة, لذا فأن فحص مصادر المياه لوجود هذه السموم بصوره متزايدة يعد من الإجراءات البيئية الموصي بها في العديد من بلدان العالم. أجريت هذه الدراسة لتقييم الطحلب الأخضر المزرق Westillopsis proloficaالمعزول من نهر دجلة والذي شكل في الآونة الأخيرة  ازدهارات في المياه العذبة العراقية وبيان قدرته على انتاج المايكروسستين عن طريق التحاليل الجزيئية والمناعية التاكيديه. وقد قورنت إنتاجية W.prolofica للسم عن طريق التجارب المختبرية، مع بعض الطحالب الخضر المزرقة السائدة في المياه العذبة العراقية والمعزولة من نهر دجله وهي Microcystis aeruginosa, Chroococcus turigidus, Nostoccarneum, and Lyngbya sp. وخلافا للمفهوم السائد القائل بان الطحلب الأخضرMicrocystis aeruginosa هو المنتج الرئيسي لسم الميكروسيتين في المياه العذبة في جميع انحاء العالم فأن انتاج طحلب W. prolofica في هذه الدراسة قد فاق إنتاجية M. aeruginosa.  اما الطحالب الأخرى المعزولة لم يلاحظ أي إنتاج لهذه الأنواع في جميع الفترات الزمنيه التي لُوحظت مختبريا. اما بالنسبة C. turigidus, N. carneum و Lyngbya sp فلم يكن لها تعبير جيني ملحوظ للجينmcyE  أو إنتاج ملحوظ لسم الميكروسيتين. بشكل عام فأن البيانات الناتجة من التعبيرالجيني لجين mcyE  بواسطة تقنية تفاعل سلسلة البلمرة اللحظي كانت متفقة مع تلك التي تم الحصول عليها من القياس بواسطة تقنية الامتزاز المناعي المرتب بالانزيم ELISA)). ومن المثير للاهتمام في هذه الدراسة، ان طحلب W. prolofica أظهرالقدرة الواضحة لانتاج سم الميكروسيتين، والتي لم يتم  الإشارة عنها من قبل في الدراسات السابقة فيما يخص انتاجه لهذا السم، ومن الممكن اضافته إلى قائمه الطحالب الخضر المزرقة كطحلب جديد منتج لسم الميكروسيتين.Several toxigenic cyanobacteria produce the cyanotoxin (microcystin). Being a health and environmental hazard, screening of water sources for the presence of microcystin is increasingly becoming a recommended environmental procedure in many countries of the world. This study was conducted to assess the ability of freshwater cyanobacterial species Westiellopsis prolifica to produce microcystins in Iraqi freshwaters via using molecular and immunological tools. The toxigenicity of W. prolifica was compared via laboratory experiments with other dominant bloom-forming cyanobacteria isolated from the Tigris River: Microcystis aeruginosa, Chroococcus turigidus, Nostoc carneum, and Lyngbya sp. significant expression of mcyE gene and microcystin production was most evident in W. prolifica. Contrary to the prevailing concept that M. aeruginosa is a main microcystin producer in freshwaters around the world, no significant microcystin production was observed with this species throughout the time points studied in our laboratory methods. As for C. turigidus, N. carneum and Lyngbya sp., neither mcyE expression nor microcystin production was significant. Data from mcyE expression by RT-qPCR were generally in agreement with those obtained from microcystin quantification by ELISA. Interestingly, W. prolifica, which showed clear microcystin-producing ability in this study and which was not reported before in the literature to produce microcystin, can be added as a new microcystin producer to the list of toxigenic cyanobacteria

PERTUMBUHAN CHROOCOCCUS DISPERSUS DALAM MEDIA LIMBAH CAIR PABRIK MINYAK KELAPA SAWIT

Research on the effect of palm oil mill effluent medium on Chroococcus dispersus growth had been done.  The research was an experimental study with complete random design with 8 treatment (sediment and anaerob wastewater with concentration 25%, 50%, 75% and 100%). The growth curve was made based on the value of optical density (OD) measured at a wavelength of 680 nm. Chroococcus dispersus best growth seen at sediment wastewater medium with concentration of 100% and at anaerob wastewater medium with concentration of 75%.Penelitian ini dilakukan untuk mengetahui pengaruh media limbah cair pabrik minyak kelapa sawit terhadap pertumbuhan Chroococcus dispersus. Penelitian ini adalah penelitian eksperimen dengan 8 perlakuan (limbah sedimen dan limbah anaerob dengan konsentrasi 25%, 50%, 75%, dan 100%). Kurva pertumbuhan dibuat berdasarkan nilai optical density (OD) pada panjang gelombang 680 nm. Pertumbuhan Chroococcus dispersus yang terbaik terlihat pada limbah sedimen dengan konsentrasi 100% dan limbah anaerob pada konsentrasi 75%

AMR - An R Package for Working with Antimicrobial Resistance Data

Antimicrobial resistance is an increasing threat to global health. Evidence for this trend is generated in microbiological laboratories through testing microorganisms for resistance against antimicrobial agents. International standards and guidelines are in place for this process as well as for reporting data on (inter-)national levels. However, there is a gap in the availability of standardized and reproducible tools for working with laboratory data to produce the required reports. It is known that extensive efforts in data cleaning and validation are required when working with data from laboratory information systems. Furthermore, the global spread and relevance of antimicrobial resistance demands to incorporate international reference data in the analysis process.In this paper, we introduce the AMR package for R that aims at closing this gap by providing tools to simplify antimicrobial resistance data cleaning and analysis, while incorporating international guidelines and scientifically reliable reference data. The AMR package enables standardized and reproducible antimicrobial resistance analyses, including the application of evidence-based rules, determination of first isolates, translation of various codes for microorganisms and antimicrobial agents, determination of (multi-drug) resistant microorganisms, and calculation of antimicrobial resistance, prevalence and future trends. The AMR package works independently of any laboratory information system and provides several functions to integrate into international workflows (e.g. WHONET software provided by the World Health Organization)

Isolation and Identification of Foodborne Pathogens of Special Interest in Food Safety

[ES] La seguridad alimentaria es una prioridad para la población y en la actualidad cobra mayor importancia por ciertas tendencias alimentarias como el consumo de alimentos crudos y la distribución generalizada de alimentos orgánicos, que pueden ser la causa de enfermedades transmitidas por alimentos. Para garantizar la seguridad alimentaria, la detección de estos microorganismos debe realizarse de manera rápida y eficiente. Par eso, el método de cultivo microbiológico se considera el oficial para la detección de estos patógenos. Sin embargo, adolece de importantes inconvenientes, ya que no solo requiere mucho tiempo, sino que también es laborioso y consume muchos recursos. Además, puede ser limitado con respecto a la detección de bacterias fisiológicamente alteradas y/o estresadas durante el almacenamiento y la conservación. En este trabajo se ha desarrollado un protocolo sencillo y rápido para la detección simultánea de E. coli, L. monocytogenes, S. aureus y S. enterica en alimentos, mediante la combinación de una etapa de co-cultivo en medio líquido y la detección por PCR múltiple. Se ha evaluado la eficiencia de varios medios de enriquecimiento y se seleccionó el agua de peptona tamponada como el medio óptimo para el co-cultivo de las cuatro bacterias diana. También se optimizaron las condiciones de PCR múltiple y se aplicaron tanto a co-cultivos como a muestras de alimentos inoculados artificialmente, lechuga orgánica y carne picada. Después de la optimización, la PCR múltiple desarrollada fue capaz de detectar las cuatro bacterias simultáneamente, hasta con una inoculación inicial de 10^0 UFC/mL. En presencia de ambas matrices alimentarias inoculadas, tras la etapa de co-cultivo, la PCR múltiple pudo detectar simultáneamente las 3 bacterias E. coli, S. enterica y L. monocytogenes, mientras que S. aureus se ha detectado por PCR simplex, a partir del mismo extracto de ADN del co-cultivo. Los resultados obtenidos permiten concluir que el uso de un paso de co-cultivo en Agua peptona tamponada, antes de la detección por PCR simple y múltiple, puede facilitar la detección simultánea de las cuatro bacterias potencialmente presentes en las matrices alimentarias. La presencia o ausencia de la bacteria diana en los alimentos se confirma en unas 30 horas, lo que reduce el tiempo requerido para la detección en comparación con el tiempo mínimo de 7 días por método cultural. Asimismo, permite reducir el número de medios de cultivo y reactivos, para el aislamiento e identificación de bacterias que no son detectadas por PCR y que no están presentes en las matrices alimentarias, lo que supone un importante ahorro económico.[CA] La seguretat alimentària sempre és una prioritat per a la població i en l' actualitat cobra major importància per certes tendències alimentàries, com el consum d' aliments crus i la distribució generalitzada d' aliments orgànics, que poden ser la causa de malalties transmeses per aliments. Per garantir la seguretat alimentària, la detecció d' aquests microorganismes s' ha de realitzar de manera ràpida i eficient. Per a això, el mètode de cultiu microbiològic es considera l' oficial per a la detecció d' aquests patògens. Però, hi ha importants inconvenients, ja que no només requereix més temps, sinó que també és laboriós i consumeix molts recursos. A més, pot ser limitat pel que fa a la detecció de bacteris fisiològicament alterats i/o estressats durant l'emmagatzematge i la conservació. En aquest treball s'ha desenvolupat un protocol senzill i ràpid per a la detecció simultània d' E. coli, L. monocytogenes, S. aureus i S. enterica en aliments, mitjançant la combinació d' una etapa de co-cultiu en medi líquid i la detecció per PCR múltiple. S'ha avaluat l'eficiència de diversos mitjans d'enriquiment i s'ha seleccionat l'aigua de peptona tamponada com el medi òptim per al co-cultiu dels quatre bacteris diana. També es van optimitzar les condicions de PCR múltiple i es van aplicar tant a co-cultius com a mostres d'aliments inoculats artificialment, enciam orgànic i carn picada. Després de l'optimització, la PCR múltiple desenvolupada va ser capaç de detectar els quatre bacteris simultàniament, fins a una inoculació inicial de 10^0 UFC/mL. En presència d' ambdues matrius alimentàries inoculades, després l' etapa de co-cultiu, la PCR múltiple va poder detectar simultàniament els 3 bacteris: E. coli, S. enterica i L. monocytogenes, mentre que S. aureus s' ha detectat per PCR simple, a partir del mateix extracte d' ADN del co-cultiu. Els resultats obtinguts permeten concloure que l' ús d' un pas de co-cultiu en Aigua de peptona tamponada, abans de la detecció per PCR simple i múltiple, pot facilitar la detecció simultània dels quatre bacteris potencialment presents en les matrius alimentàries. La presència o absència del bacteri diana en els aliments es confirma en unes 30 hores, la qual cosa redueix el temps requerit per a la detecció en comparació amb el temps mínim de 7 dies per mètode cultural. Així mateix, permet reduir el nombre de mitjans de cultiu i reactius, per a l' aïllament i identificació de bacteris que no són detectats per PCR i que no estan presents en les matrius alimentàries, la qual cosa suposa un important estalvi econòmic.[EN] Food safety is a priority for the population and is nowadays more important than ever due to certain dietary trends such as the consumption of raw foods and the widespread distribution of organic foods, which may be the cause of foodborne diseases. To ensure food safety, the detection of these microorganisms must be done quickly and efficiently. Although, the microbiological culture method is considered to be the official method for the detection of these food-borne pathogens, it suffers from significant drawbacks, such as time-consuming, laborious and expensive, in addition it may be limited regarding the detection of physiologically altered and/or stressed bacteria, during storage and preservation. In this work has been developed a simple and rapid protocol for the simultaneous detection of E. coli, L. monocytogenes, S. aureus and S. enterica in food, by combining a liquid co-culture step and detection by multiplex PCR. The efficiency of several enrichment media was evaluated and buffered peptone water was chosen as the optimal medium for the co-culture of the four target bacteria. Then, optimized multiplex PCR conditions were applied to both the co-cultures and the samples of artificially inoculated foods, organic lettuce and ground meat. After optimization, the developed multiplex PCR was able to simultaneously detect the four bacteria, up to an initial inoculation of 10^0 CFU/mL. In the presence of the two inoculated food matrices, after a co-culture step, the multiplex PCR could simultaneously detect the 3 bacteria: E. coli, S. enterica and L. monocytogenes, whereas, S. aureus has been detected by simplex PCR, from the same co-culture DNA template. The results obtained allow conclusion that the use of a co-culture step in Buffered Peptone Water, before detection by simplex and multiplex PCR, can facilitate the simultaneous detection of the four bacteria potentially present in the food matrices. The presence or the absence of the target bacteria in food is confirmed in approximately 30 hours, which reduce the time required for the detection compared to the minimum time of 7 days by cultural method. Also, it allows to reduce the number of culture media and reagents, for the isolation and identification of bacteria that are not detected by PCR and which are not initially present in the food matrices, which represents a significant economic savings.Boukharouba, A. (2022). Isolation and Identification of Foodborne Pathogens of Special Interest in Food Safety [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182828TESI

Phylogenetic relationships of the most common pathogenic Candida species inferred by sequence analysis of nuclear genes

Dissertação de mestrado em Genética MolecularAs regiões genómicas codificantes são as mais usadas em estudos filogenéticos, quer através de análises multigénicas, quer filogenómicas. Contudo, a maioria destas análises usa apenas as regiões com elevada similaridade entre as sequências ortólogas, não considerando as restantes regiões, as quais nos podem dar informações muito úteis. Deste modo, o principal objectivo desta tese foi avaliar o uso dos genes da família MADS-box, RLM1 e MCM1, assim como o gene IFF8, que codifica para uma proteína GPI, em estudos de filogenia de fungos. Foram obtidas setenta e seis sequências ortólogas para RLM1 e MCM1 e oito para IFF8 através da pesquisa em várias bases de dados. O alinhamento das sequências foi realizado mediante o uso dos programas CLUSTALW e MUSCLE e a análise filogenética, utilizando os programas PHYML 3.0 e MrBayes 3.2. Os resultados obtidos usando o factor de transcrição RLM1 indicaram que este apresenta condições favoráveis para ser incluído em estudos filogenéticos de fungos, uma vez que a topologia obtida está muito próxima das estabelecidas em outras análises multigénicas e filogenómicas. O factor de transcrição MCM1 demonstrou limitações para ser utilizado em estudos filogenéticos a nível do reino, visto que as sequências obtidas apresentam uma grande variabilidade de tamanho, originando problemas nos alinhamentos. Apesar desta limitação este gene pode ser utilizado, quer independentemente quer combinado com outros genes, para resolver filogenias a nível do Filo ou de grupos de categoria inferior, uma vez que os resultados obtidos dentro do subfilo Saccharomycotina estão de acordo com os estudos publicados. A utilização do gene IFF8 para inferir filogenia apresentou grandes limitações uma vez que foram identificados ortólogos deste gene apenas no grupo CUG e além disso a filogenia obtida não estava de acordo com outros estudos publicados, em particular no respeitante à posição de C. tropicalis na filogenia de Candida spp. É presentemente aceite que no Subfilo Saccharomycotina ocorreu um processo de duplicação completa do genoma, nos grupos sensu stricto e sensu lato do ‗Complexo Saccharomyces‘ com conservação de vários genes duplicados. A pesquisa inicial por sequências ortólogas revelou que os factores de transcrição estudados neste trabalho estão dentro dos genes duplicados que foram mantidos. Assim, outro objectivo deste trabalho foi determinar se o gene RLM1 esteve sob selecção positiva no Subfilo Saccharomycotina. Esta análise identificou várias posições onde os aminoácidos estão possivelmente sob selecção positiva e embora estas substituições tenham sido observadas em diferentes locais da proteína, não se detectou nenhuma nas regiões conservadas. Esta observação sugere que a proteína executa uma função importante na célula que foi mantida durante o processo de divergência de espécies. A presença de aminoácidos sob selecção positiva, no inicio da região repetitiva do terminal carboxílico da proteína, bem como a diferença no aminoácido repetido entre as espécies com o genoma duplicado e as espécies com genoma não duplicado sugeriam que uma mutação de frameshift seria a responsável pelas alterações observadas. Esta hipótese foi então testada no Subfilo Saccharomycotina, desenhando as três grelhas de leitura e reconstruindo a sequência ancestral. Os resultados desta análise confirmaram que uma mutação de frameshift foi de facto responsável pelas alterações observadas na região repetitiva com substituição de aminoácidos, diversificando a função deste gene nas espécies que duplicaram o genoma. Os principais resultados desta tese foram: (i) a identificação do potencial do gene RLM1 para estudos de filogenia do reino Fungi, com especial ênfase na filogenia das espécies do género Candida spp.; e (ii) a identificação do mecanismo molecular responsável pela alteração da região repetitiva do terminal carboxílico da proteína, ocorrida no Saccharomyces sensu stricto durante a divergência das espécies após duplicação do genoma.Coding regions are used to resolve phylogenetic relationships through multigenic and phylogenomic analyses. However, the majority of these analyses uses regions with similarity only among orthologue gene sequences and do not take into account other regions which could give useful information. Thus, the main purpose of this thesis was to evaluate the use of RLM1 and MCM1 MADS-box transcription factors, and IFF8, a GPI-anchor protein-coding gene, in fungi phylogeny. Seventy six putative orthologue sequences for RLM1 and MCM1 and eight for IFF8 were obtained from different fungal databases. Sequence alignments were performed by using CLUSTALW and MUSCLE and phylogeny was inferred by using PHYML 3.0 and Mr Bayes 3.2. Results obtained from the phylogenetic analysis, using the transcription factor RLM1, indicated that it presents conditions to be considered within a multigene analysis, since the obtained fungal phylogeny is closer to the ones established by multigene and phylogenomic analyses. The transcription factor MCM1 presented limitations to be used in phylogeny at the kingdom level, because of its variable sequence sizes. However, despite this limitation this gene can be used to resolve phylogeny at the phylum or lower clade levels since the results obtained, independently and/or concatenated with the other genes used in this study, within the subphylum Saccharomycotina were in agreement with published studies. On the other hand, the use of IFF8 gene to infer phylogeny is limited and restricted to the CUG group, since other orthologues were not found within the kingdom Fungi and the results obtained in the CUG group phylogeny presented conflicts, particularly in the position of Candida tropicalis which is not in agreement with previously determined relationship in Candida phylogeny. It is known that within the subphylum Saccharomycotina the process of genome duplication has occurred in the ‗Saccharomyces complex‘, groups sensu stricto and sensu lato, resulting in the duplication of some genes. The initial search for orthologue sequences showed that the transcription factors studied in this work are within the duplicated genes that were maintained. Thus, another objective of this work was to determine if RLM1 was under positive selection to search for an alternative explanation for the persistence and diversification of gene duplicates. These analyses identified several amino acid sites under positive selection within the subphylum Saccharomycotina and although these substitutions were present in different positions, they were not inside conserved regions, suggesting that the protein plays an important role in fungi that was maintained during the evolution process of divergence of species. The presence of amino acids under positive selection at the beginning of Rlm1 Cterminal repetitive region and the differences in the amino acid under repetition between species that presented the duplicated genome (WGD) and species with non duplicated genome was indicative of a possible frameshift mutation. This hypothesis was tested in Saccharomycotina group by designing the three open reading frames and reconstructing the ancestral sequence. Results from this analysis showed that amino acid substitution that occurred during the divergence of species changed this repetitive region and diversified gene function in WGD species avoiding the loss of the protein function. The major findings of this work were (i) the identification of the potential use of RLM1 gene for inferring phylogeny in the kingdom Fungi with special emphasis to Candida species, and (ii) the observation that this gene evolved within the Saccharomyces sensu stricto after genome duplication, being the molecular mechanism responsible for the change observed in the C-terminal of this protein most probably a frameshift mutation.Las regiones genómicas codificantes son usadas en estudios filogenéticos através de análisis a niveles multigenicos y filogenómicos. Pero la mayoría de estos análisis usan solo regiones con similaridad entre secuencias de genes ortólogos y no toma en cuenta otras regiones las cuales podrían darnos informaciones útiles. Por lo tanto, el objetivo principal de esta tesis fue evaluar el uso de los genes MADS-box, RLM1 y MCM1, así como IFF8, un gen codificante de proteína de anclaje GPI, en la filogenia de hongos. Se obtuvieron setenta y seis secuencias putativas ortólogas para RLM1 y MCM1, y ocho para IFF8 a partir de las diferentes bases de datos de hongos. Los alineamientos de secuencias se realizaron mediante el uso de los programas CLUSTALW y MUSCLE, y la filogenia fue inferida por medio de los programas PHYML 3.0 y MrBayes 3.2. Los resultados obtenidos de los análisis filogenéticos, utilizando el factor de transcripción RLM1 indicaron que este presenta condiciones para ser considerado dentro de análisis multigénico, ya que la filogenia obtenida para hongos está muy próxima a las establecidas por otros análisis multigénicos y filogenómicos. El factor de transcripción MCM1 presentan limitaciones para ser utilizado en filogenia a nivel de reino, debido a sus secuencias de tamaño variables, dando lugar a problemas en el alineamiento. A pesar de esta limitación este gen se puede utilizar para resolver filogenias a nivel de Filo o clados inferiores debido a los resultados obtenidos con su uso, de manera independiente y/o combinado con el resto de genes utilizados en este estudio, ya que dentro del Subfilo Saccharomycotina estuvieron de acuerdo con estudios publicados. Por otro lado, el uso de IFF8 gen para inferir filogenia es limitado y restringido al grupo CUG, ya que otros ortólogos no se han encontrado en el reino Fungi y los resultados obtenidos en la filogenia del grupo CUG presentaron conflictos, en particular en la posición de C. tropicalis que no está de acuerdo con lo que ya se ha determinado en la filogenia de Candida spp. Es actualmente aceptado que dentro del subfilo Saccharomycotina el proceso de duplicación del genoma se ha producido en los grupos sensu stricto y sensu lato del 'Complejo Saccharomyces‘, resultando en la manutención de algunos genes. La búsqueda inicial de secuencias ortólogas mostró que los factores de transcripción estudiados en este trabajo están dentro de los genes duplicados que han sido mantenidos. Así pues, otro objetivo de este trabajo fue determinar si RLM1 estuvo bajo selección positiva. Estos análisis identificaron varios sitios donde los aminoácidos estuvieron posiblemente bajo selección positiva dentro del Subfilo Saccharomycotina y aunque estas sustituciones estaban presentes en diferentes posiciones, no estuvieron presentes en las regiones conservadas, lo que sugiere que la proteína ejecuta una función importante en la célula que fue mantenida durante el proceso de divergencia de especies. La presencia de aminoácidos bajo selección positiva en el inicio de la region repetitiva del C-terminal en Rlm1 y la diferencia en el aminoácido bajo repetición entre las especies con el genoma duplicado y las especies con genoma no duplicado indicaba un posible frameshift mutation. Asi, esta hipótesis fue testada en el subfilo Saccharomycotina diseñando tres open reading frames y reconstruyendo la secuencia ancestral. Los resultados de este análisis mostraron que la substitución de aminoácidos ocurrida durante la divergencia de especies que alteró esa región fue mediante una frameshift mutation diversificando la función de este gen en las especies que duplicaron su genoma. Los principales resultados de esta tesis son: (i) la identificación del potencial del gen RLM1 para estudios de filogenia en el reino Fungi, con especial énfasis en la filogenia de las especies de Candida spp y (ii) la identificación del mecanismo molecular responsable por el cambio observado en el C-terminal de esta proteína en el grupo Saccharomyces sensu stricto que alteró el gen durante la divergencia de especies después del proceso de duplicación de genoma.This Thesis was supported by the Programme ALβAN, the European Union Programme of High Level Scholarships for Latin America, scholarship No E06M103915PE