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Centro De Investigación Y Estudios Avanzados Del Instituto Politécnico Nacional CENTRO DE INVESTIGACIÓN Y ESTUDIOS AVANZADOS DEL INSTITUTO POLITÉCNICO NACIONAL. UNIDAD DE GENÓMICA AVANZADA “CARACTERIZACIÓN DEL MICROBIOMA FÚNGICO DE SALAMANDRAS CAVERNICOLAS DE MÉXICO” Tesis que presenta Lic. en Microbiología Emanuel Martínez Ugalde Para obtener el grado de Maestro en Ciencias En la especialidad de Biotecnología de plantas Director de tesis: Dr. Sean M. Rovito Irapauto, Gto. Este trabajo se realizó en el Laboratorio Nacional de Genómica para la Biodiversidad en el Laboratorio de Genómica y Diversidad de Vertebrados bajo la dirección del Dr. Sean M Rovito, gracias al apoyo del Consejo de Ciencia y Tecnología (CONACyT) a través del proyecto titulado “Desarrollo de una estrategia de conservación contra enfermedades infecciosas fúngicas a través de la caracterización y el uso del microbioma de la piel de salamandras” y de la beca 781100. Este trabajo se realizó del 1 de marzo de 2017 a 17 de diciembre de 2018. Miembros del comité: Sean M Rovito Eugenio Mancera Ramos Alfredo Heriberto Herrera Estrella Fecha de examen: 28 de febrero de 2019 i Agradecimientos Agradezco al Consejo Nacional de Ciencia y Tecnologia por el apoyo económico brindado para la realización de este proyecto. Al Dr. Sean M Rovito y a las personas del Laboratorio de Genómica y Diversidad de Vertebrados, por el apoyo el apoyo brindado durante el tiempo que conviví en el laboratorio con ellos. Especialmente al Dr. Rovito por su apoyo durante los momentos complicados que se presentaron durante la realización del proyecto. A los miembros del comité tutorial, Dr. Alfredo Herrera-Estrella, Dr. Eugenio Mancera Ramos por sus comentarios hacia el trabajo realizado. También a los antiguos miembros del comité. Dra. Laila Partida Martínez y Dr. Ruairidh Sawers, por los comentarios e ideas aportadas durante los exámenes tutórales. A mis amigos por su tiempo y consejos durante mi estancia en el CINVESTAV, han sido de gran ayuda para llegar hasta este momento. Aunque ya no estén muchas gracias por todo. A mi familia por el apoyo incondicional en todo momento. Gracias. ii Índice de contenido. Resumen. ................................................................................................................................ 3 Abstract. ................................................................................................................................. 5 Introducción. .......................................................................................................................... 7 Generalidades del microbioma. .......................................................................................... 7 Enfermedades emergentes y Quitridiomicosis................................................................. 10 Antecedentes. ....................................................................................................................... 13 Microbioma cutáneo de anfibios. ..................................................................................... 13 Resistencia a la quitridiomicosis mediada por el microbioma. ........................................ 15 Estrategias de estudio del microbioma. ........................................................................... 16 Hábitat cavernícola. .......................................................................................................... 20 Salamandras cavernícolas. ................................................................................................ 22 Justificación. ......................................................................................................................... 24 Objetivo ................................................................................................................................ 26 Objetivos específicos. ....................................................................................................... 26 Hipótesis. .............................................................................................................................. 27 Métodos................................................................................................................................ 28 Colecta de salamandras y obtención de frotis cutáneos. ................................................. 28 Análisis de los datos de secuenciación. ............................................................................ 30 Análisis de Diversidad. ...................................................................................................... 31 Diversidad alfa. .............................................................................................................. 31 Diversidad beta. ............................................................................................................ 31 Nucleó del micobioma cutáneo. ....................................................................................... 33 Resultados. ........................................................................................................................... 35 Clasificación taxonómica de los OTU fúngicos. ................................................................ 35 Nucleó del micobioma fúngico cutáneo. .......................................................................... 40 Presencia de Batrachochytrium dendrobatidis en elmicrobioma cutáneo. ..................... 42 Diversidad alfa .................................................................................................................. 46 Diversidad Beta ................................................................................................................. 48 Discusión. .............................................................................................................................. 61 iii Diversidad fúngica sobre el microbioma cutáneo de las salamandras cavernícolas. ...... 61 Presencia de Batrachocytrium dendrobatidis en el micobioma cutáneo......................... 68 Factores que influyen sobre la composición del micobioma cutáneo. ............................ 70 Conclusiones ......................................................................................................................... 75 Perspectivas. ......................................................................................................................... 76 Anexo 1. ................................................................................................................................ 78 Anexo 2. ................................................................................................................................ 80 Anexo 3. ................................................................................................................................ 85 Bibliografía. ........................................................................................................................... 87 iv Índice de Figuras. Figura 1. A) Mapa de las especies de salamandras cavernícolas de los géneros Chiropterotriton e Ixalotriton incluidas en el proyecto. ............................................... 28 Figura 2 Heatmap que muestra la asignación taxonómica a nivel de filo por especie. .. .... 36 Figura 3. Barplots que muestran la asignación taxonómica a nivel de filo. ......................... 37 Figura 4. Heatmap que muestra la asignación taxonómica a nivel de orden por especie. 39 Figura 5. Ordenes fúngicos en baja abundancia. Se muestran los ordenes fúngicos más abundantes pertenecientes a los filos de baja frecuencia relativa en cada una de las especies. ........................................................................................................................ 40 Figura 6. Núcleo del micobioma cutáneo de salamandras. ................................................. 41 Figura 7. Núcleo del micobioma cutáneo de salamandras en donde se detectó Bd. .......... 43 Figura 8. Abundancia de reads entre muestras con presencia y ausencia de Bd. .............. 44 Figura 9. Abundancia de reads entre muestras con presencia y ausencia de Bd de los cinco ordenes mas abundantes del filo Basidiomycota. ........................................................ 45 Figura 10. Gráficos de acumulación de especies. ................................................................. 48 Figura 11. Índices de diversidad. .......................................................................................... 49 Figura 12. PCA del set de datos completo (68 muestras) sin aplicar la transformación de Hellinger. ....................................................................................................................... 51 Figura 13. PCA del set de datos completo (68 muestras) después de aplicar la transformación de Hellinger. ........................................................................................ 52 Figura 14. PCA de las muestras pertenecientes al género Chiropterotriton. ....................... 53 Figura 15. PCA de las muestras ambientales dentro de las cuevas. .................................... 54 Figura 16. PCA de las muestras pertenecientes a la especie C. magnipes de dos localidades distintas. ........................................................................................................................ 55 Figura 17. PCA de las muestras pertenecientes a la especie C. multidentatus de tres localidades distintas y de origen tanto dentro (Grieta el cincuenta y ocho y Valle de los Fantasmas) y fuera de las cuevas (Bosque del repetidor). ...................................... 55 Figura 18. PCA de las muestras
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