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Universidad Nacional Autónoma De México Posgrado En Ciencias Del Mar Y Limnología (Biología Marina) UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO POSGRADO EN CIENCIAS DEL MAR Y LIMNOLOGÍA (BIOLOGÍA MARINA) “ESTUDIO DE LA ABUNDANCIA DE CUATRO CLADOS DE DINOFLAGELADOS SIMBIONTES DEL GÉNERO Symbiodinium PRESENTES EN EL CORAL Montastraea faveolata DEL ARRECIFE DE PUERTO MORELOS, QROO.” TESIS QUE PARA OPTAR POR EL GRADO DE MAESTRO EN CIENCIAS PRESENTA: ELISA LÓPEZ GARCÍA TUTOR: Dr. ROBERTO IGLESIAS PRIETO INSTITUTO DE CIENCIAS DEL MAR Y LIMNOLOGÍA (UNIDAD ACADÉMICA DE PUERTO MORELOS) COMITÉ TUTOR: Dr. AXAYACATL ROCHA OLIVARES POSGRADO EN CIENCIAS DEL MAR Dra. ROSALBA ALONSO RODRÍGUEZ INSTITUTO DE CIENCIAS DEL MAR Y LIMNOLOGÍA (UNIDAD ACADÉMICA DE MAZATLÁN) Dr. HÉCTOR REYES BONILLA POSGRADO EN CIENCIAS DEL MAR Dr. MARCO ANTONIO VILLANUEVA INSTITUTO DE CIENCIAS DEL MAR Y LIMNOLOGÍA (UNIDAD ACADÉMICA DE PUERTO MORELOS) MÉXICO, D.F. ENERO 2013 “ESTUDIO DE LA ABUNDANCIA DE CUATRO CLADOS DE DINOFLAGELADOS SIMBIONTES DEL GÉNERO Symbiodinium PRESENTES EN EL CORAL Montastraea faveolata DEL ARRECIFE DE PUERTO MORELOS, QROO.” . T E S I S que para obtener el grado académico de MAESTRA EN CIENCIAS (BIOLOGÍA MARINA) p r e s e n t a ELISA LÓPEZ GARCÍA Director de Tesis: Dr. Roberto Iglesias-Prieto Comité Tutoral: Dr. Axayacatl Rocha Olivares Dra. Rosalba Alonso Rodríguez Dr. Héctor Reyes Bonilla Dr. Marco Antonio Villanueva MÉXICO, D.F. ENERO 2013 Agradecimientos En primer lugar me gustaría mostrar mi agradecimiento al Dr. Roberto Iglesias Prieto, por abrirme las puertas de su laboratorio y aceptarme como estudiante muchos años antes de llegar a Puerto Morelos; por su confianza, su apoyo y su entusiasmo. Espero algún día poder devolverte parte de todo lo que me has ayudado. Gracias al Posgrado de Ciencias del Mar y Limnología por darme la oportunidad de estudiar esta Maestría, en especial a la Dra. Gloria Vilaclara, que siempre creyó en mis posibilidades; al personal de la sede en Ciudad Universitaria por tantos emails, llamadas, valijas y documentos; así como al personal administrativo de la Unidad Académica de Puerto Morelos, siempre dispuestos a ayudar. Al Consejo Nacional de Ciencia y Tecnología (CONACYT) por otorgarme la beca con número de registro 391636 para realizar mis estudios de Maestría. A los integrantes de mi Comité Tutoral: Dr. Axayacatl Rocha Olivares, Dra. Rosalba Alonso Rodríguez, Dr. Héctor Reyes Bonilla y Dr. Marco Antonio Villanueva, por estar presentes en mis exámenes y tener la paciencia de leer una y otra vez mis manuscritos. Por ayudarme a aceptar mis faltas y luchar por mis aciertos. Al Profesor Todd LaJeunesse por aceptarme en su laboratorio durante mi estancia de dos meses en la Pennsylvania State University, por su generosidad, por compartir su “buena ciencia” conmigo y hacerme sentir como en casa; y a sus estudiantes, los Candidatos a Doctor Allison Lewis y Francis Wham (Drew), por compartir su espacio de trabajo y ayudarme siempre que pudieron; gracias por su amistad, sin ustedes nunca hubiera terminado esta tesis. A la Profesora Deborah Grove, directora de Análisis Genéticos en las Instalaciones de Genómica de University Park, por escuchar mis desvaríos sobre genética y ayudarme a resolver el último clado. A la Dra. Patricia Thomé, por el apoyo en el contexto molecular de esta tesis, por estar horas y horas escuchándome, por su guía, su paciencia y sus sonrisas. A la Dra. Michele Weber por sus estupendos consejos. Me gustaría también agradecer al resto de estudiantes y en especial a Luis, Tati e Inti por su paciencia conmigo y tantas horas compartidas. A Tim por ser mi ángel de la guarda y amigo, a Sergio por molestarme todas las mañanas con una sonrisa, a Mac y Parmenio por estar siempre ahí, a mi queridísimo Rumi por quejarse conmigo y por cada café de buenos días, y a Mélina, siempre tan empática con mis problemas. En general, a los que se fueron porque nunca les olvidaré y a los que se quedan porque nunca me olviden. Estudio de la abundancia de cuatro clados de dinoflagelados simbiontes del género Symbiodinium presentes en el coral Montastraea faveolata del arrecife de Puerto Morelos, QRoo. Por último, hay tres personas sin las que esta tesis no hubiera sido más que un bonito sueño. Mis padres, por ser los mejores y más eficientes secretarios que se puede pedir, por todo el drama y frustración, por la distancia, por las lágrimas de alegría compartida, por su apoyo incondicional y sus recientemente adquiridas nociones sobre genética molecular, fotosíntesis y simbiosis; esta tesis es también vuestra, no lo olvidéis. Y a Román, por levantarme cuando me caía y por acostarse conmigo cuando no me podía levantar. ¡A por la siguiente! Elisa López García 3 Estudio de la abundancia de cuatro clados de dinoflagelados simbiontes del género Symbiodinium presentes en el coral Montastraea faveolata del arrecife de Puerto Morelos, QRoo. Lista de figuras FIGURA 1. CICLO DE VIDA DE SYMBIODINIUM. A, QUISTE VEGETATIVO. B, QUISTE VEGETATIVO EN DIVISIÓN.C, ZOOSPORA EN DESARROLLO. D, ZOOSPORA (MASTIGOTE). CL, CLOROPLASTO. N, NÚCLEO. PA, PRODUCTOS DE ACUMULACIÓN. FL, FLAGELO LONGITUDINAL. FT, FLAGELO TRANSVERSAL (STAT ET AL. 2006)...................................................................................................................................9 FIGURA 2. ORGANIZACIÓN ESQUEMÁTICA DE LOS GENOMAS NUCLEAR Y ORGANELAR PRESENTES EN EL DINOFLAGELADO SIMBIÓTICO DEL GÉNERO SYMBIODINIUM, ASÍ COMO LAS TÉCNICAS EMPLEADAS PARA SU ANÁLISIS (MODFICADO POR T.C. LAJEUNESSE DE SAMPAYO ET AL. 2007)............................................ 11 FIGURA 3. RELACIONES FILOGENÉTICAS ENTRE LOS CLADOS DOMINANTES DEL GÉNERO SYMBIODINIUM. LAS POSICIONES DE LOS CLADOS B, C, F Y H VARÍAN DEPENDIENDO DEL MÉTODO Y REGIONES ANALIZADAS (NR28S-RDNA Y CP23S-RDNA) (POCHON Y GATES 2010). ................................................... 12 FIGURA 4. OCURRENCIAS RELATIVAS DE DISTINTOS FILOTIPOS DE “ZOOXANTELA” EXTRAÍDAS DE MUESTRAS INDIVIDUALES DE M. ANNULARIS Y M. FAVEOLATA A LO LARGO DE UN GRADIENTE DE PROFUNDIDAD (ROWAN Y KNOWLTON 1995).......................................................................................................................................... 14 FIGURA 5. REPRESENTACIÓN GRÁFICA DE UN HAZ DE LUZ DIRECTA Y SU ÁREA PROYECTADA A LO LARGO DE UNA SUPERFICIE HORIZONTAL (B) Y OTRA A Α GRADOS DE INCLINACIÓN (A). LA ECUACIÓN E(A) = E(B) COSΑ DESCRIBE LA IRRADIANCIA EN AMBAS SUPERFICIES DE ACUERDO CON LA LEY DEL COSENO DE LAMBERT. ................................................................................................................................................................................ 16 FIGURA 6. RELACIÓN DE LAS IRRADIANCIAS CALCULADAS PARA SEIS PROFUNDIDADES A LO LARGO DE UN GRADIENTE DE INCLINACIÓN. LAS ÁREAS SOMBREADAS REPRESENTAN LOS NICHOS OCUPADOS POR LOS CLADOS A, B Y C EN M. ANNULARIS A LO LARGO DE UN GRADIENTE DE IRRADIANCIAS (MODIFICADO DE ROWAN ET AL. [1997]). ................................................................................................................... 17 FIGURA 7. PATRÓN DE ZONACIÓN DE TRES TIPOS DE SYMBIODINIUM EN UNA COLONIA DE M. FAVEOLATA DEL ARRECIFE DE PUERTO MORELOS, A LO LARGO DE TRES TRANSECTOS VERTICALES CON ORIENTACIONES NORTE, NORESTE, NOROESTE Y ESTE. (KEMP ET AL. 2008).............................................. 18 FIGURA 8. SEPARACIÓN DE FASES DURANTE LA EXTRACCIÓN POR FENOL:CLOROFORMO Y SUBSECUENTE PRECIPITACIÓN CON ISOPROPANOL............................................................................................................................... 20 FIGURA 9. AMPLIFICACIÓN DEL ADN MOLDE POR UN PAR DE CEBADORES CON LA SONDA TAQMAN CORRESPONDIENTE. A) FASE DE ALINEAMIENTO: LA SONDA SE UNE A LA HEBRA, B) FASE DE ALINEAMIENTO: LOS CEBADORES SE UNEN A LA HEBRA, C) FASE DE ELONGACIÓN: LA ENZIMA COMIENZA A POLIMERIZAR EN SENTIDO 5´-3´, D) HIBRIDACIÓN DE LA SONDA DEBIDO A LA ACCIÓN EXONUCLEASA DE LA ENZIMA Y EMISIÓN DE FLUORESCENCIA POR LA DISOCIACIÓN FLUORÓFORO (F)- QUENCHER(Q)....................................................................................................................................................................... 23 FIGURA 10. CURVA DE AMPLIFICACIÓN TÍPICA DE PCR EN TIEMPO REAL CON LAS TRES FASES: EXPONENCIAL, LINEAL Y PLATEAU, LOS PARÁMETROS LÍNEA UMBRAL, LÍNEA DE BASE Y CICLO UMBRAL, Y LA CURVA DEL CONTROL NEGATIVO........................................................................................................................... 25 FIGURA 11. REPRESENTACIÓN GRÁFICA DELTA RN VS. CICLO DE CUATRO CURVAS DE AMPLIFICACIÓN EN QPCR DE DILUCIONES SERIADAS CON EL UMBRAL CONSTANTE A 0.1. EN EL CUADRO SUPERIOR APARECE LA RECTA DE CALIBRACIÓN QUE ENFRENTA CADA CICLO UMBRAL CON EL LOGARITMO DE LA CONCENTRACIÓN DE ADN INICIAL EN CADA DILUCIÓN. .......................................................................................... 26 FIGURA 12. PERFILES DE PCR-DGGE O HUELLA GENÉTICA. EN EL MARGEN SUPERIOR APARECE EL NOMBRE DE LA MUESTRA (CUADRO 2) CORRESPONDIENTE A CADA CARRIL, MIENTRAS QUE EN EL MARGEN INFERIOR SE IDENTIFICA SU GENOTIPO. LOS RECUADROS EN BLANCO SIN BORDE IDENTIFICAN LOS CINCO POSIBLES GENOTIPOS ENCONTRADOS, LOS RECUADROS CON BORDE NEGRO CORRESPONDEN AL MARCADOR (MKR) DE GENOTIPO CONOCIDO Y QUE ACTÚAN COMO ESTÁNDARES PARA LA IDENTIFICACIÓN.................................................................................................................................................... 28 FIGURA 13. EJEMPLO DE HUELLAS GENÉTICAS Y SU INTERPRETACIÓN. EN LA PARTE SUPERIOR APARECE EL NOMBRE DE LAS MUESTRAS CORRESPONDIENTES
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