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DNA Metabarcoding Derived Biotic Indices for Marine Monitoring and Assessment

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DNA Metabarcoding Derived Biotic Indices for Marine Monitoring and Assessment DNA metabarcoding derived biotic indices for marine monitoring and assessment EVA AYLAGAS MARTÍNEZ PhD Thesis, 2017 PhD Thesis DNA metabarcoding derived biotic indices for marine monitoring and assessment Presented by Eva Aylagas Martínez Thesis Supervisors Dr. Naiara Rodríguez-Ezpeleta Dr. Ángel Borja Yerro Department of Zoology and Cellular Biology Year 2017 (c)2017 EVA AYLAGAS MARTINEZ “Whatever you do will be insignificant, but it is very important that you do it.” Mahatma Gandhi A Marcos y Hugo ACKNOWLEDGEMENTS No puedo empezar de otra manera sino dando las gracias a mis supervisores, Ángel y Naiara, por haber confiado en mí para llevar este trabajo adelante. Esta tesis no hubiera sido posible sin vuestra ayuda, paciencia y apoyo durante estos cuatro años. Ángel, gracias por transmitirme tu confianza, por ayudarme a crecer como investigadora y por tu inmensa paciencia. Naiara, sin tu constancia y perseverancia no estaría donde estoy. Gracias por todo el tiempo que le has dedicado a esta tesis y por la ilusión que has puedo en ella (¡casi tanta como yo!). Me gustaría agradecer a las instituciones, organizaciones y proyectos que han financiado esta tesis: AZTI, Fundación Centros Tecnológicos – Iñaki Goenaga, DEVOTES (DEVelopment Of innovative Tools for understanding marine biodiversity and assessing good Environmental Status - http://www.devotes- project.eu) financiado por la Union Europea (7th Framework Program ‘The Ocean of Tomorrow’ Theme, grant agreement no. 308392), Agencia Vasca del Agua (URA) y Convenio con la Dirección de Pesca y Acuicultura (Convenio Marco). También a Iñaki Saiz Salinas, del departamento de Zoología y Biología Celular Animal de la UPV, gracias por aceptar la tutorización de esta tesis y por tu enorme ayuda con todos los trámites administrativos. Quiero agradecer a toda la gente de AZTI que en algún momento me ha echado una mano durante este tiempo, en especial a Xabier, Javi, María Uyarra, Laura Alonso, Guillem, Iñigo Muxika, Germán, Ibon y Luis. Y las palabras de ánimo constantes de Iratxe, Mikel, Imanol e Irati. De una manera muy especial a Iñaki Mendibil. Millones de gracias por tratar con tanta delicadeza mis muestras y por las horas compartidas en el laboratorio; aquellos tiempos de contaminaciones y risas han marcado el rumbo de esta tesis. A mis compañeros de guerra que empezaron conmigo a darlo todo: Jon, Ernesto, Maddi, Iñigo y Jorge: nadie nos dijo que esto iba a ser fácil! Y a todos los demás doctorandos que me han acompañado durante los primeros años (Natalia, Mireia, Jon López, Jon Uranga, Nagore Cuevas, Nagore Zaldua eta Aizokorri) o los últimos (Itxaso, Oihane, Natalia, Ane, Anaïs, Blanca, Sarai, Isa, Kemal, Maite y Unai) de todo este camino. Y de una manera muy especial a Nerea y Lohitzune por todo el apoyo que me habéis dado hasta el ultimísimo momento, gracias, gracias, gracias!! I would like also to thank to all the colleges I have met during the different travels I made during these four years. Matt and Laetitia, thank you for the great time we spent in Bocas del Toro (Panama) and all the advices you gave me when I was just starting this trip. Also, Susana and John, thanks for showing 1 me the nicest part of KAUST, and especially for all the help you have provided me with this thesis. Finally, special thanks to Danovaro for your support during my time in Ancona and to Cinzia, Mike and Antonio. It was a pleasure working with you. A vosotros, Marta, Juan, César, Victor, Carlos, Marina y Carmen, que me hacéis sentir el calor del Mediterráneo de una manera tan especial. A vosotras, Donostiarras, que sin saber muy bien de qué va todo esto no dejáis de decirme lo mucho que valgo. Y a mis compañeras de viajes y aventuras Oihane, Lucía y Tuca, la distancia sigue sin poder con nuestra amistad. A todos vosotros gracias por estar siempre ahí. No puedo olvidar a la gente que me he encontrado por el camino y ha llegado a convertirse en mi familia. Esti y Unai, habéis sido un gran apoyo en mis idas y venidas a Sukarrieta. Gracias por ser simplemente como sois! Y como no, a mi a mis “forasteras”, Andrea y Ángela. Encontrar almas gemelas no es fácil y menos con tanta paciencia como vosotras. Eneko, no te escapas de esta! Eskerrikasko benetan. Gracias, muy especialmente, a mi familia, por vuestro apoyo incondicional, vuestra cercanía a pesar de la distancia, vuestra comprensión y vuestra generosidad absoluta. No tengo palabras para agradeceros todo lo que habéis hecho por mí. Diego, esta tesis es en parte tuya; gracias, una vez más, por creer en mí. Gracias Vanessa, Marcos y Hugo por los momentos de felicidad que hemos compartido juntos (algunos desde hace muy muy poquito tiempo) y los que nos quedan. Eskerrak eman baita ere Urnietako nire familiari, Maddi eta Patxi, gracias por hacerme sentir una más en casa, sabéis que sois muy especiales para mí. Gracias, a ti, Xabi, porque te mereces todo y porque cada día a tu lado es una caja de sorpresas que no cambiaría por nada del mundo. Gracias por tu cariño, paciencia y positividad. Ya hemos terminado la tesis! 2 CONTENTS Aknowledgements…………………………………………………………………………………… 1 Contents…………………………………………………………………………………………………... 3 Summary………………………………………………………………………………………………….. 5 Resumen………………………………………………………………………………………………….. 9 Introduction……………………………………………………………………………………………. 15 Hypothesis and Objectives……………………….…………………………………………….. 35 Thesis Structure………………………………………………………………………………………. 39 Chapter 1: Ecological status assessment using DNA metabarcoding: towards a genetics-based Marine Biotic Index (gAMBI)……………………………. 41 Chapter 2: Marine Sediment Sample Pre-processing for Macroinvertebrates Metabarcoding: Mechanical Enrichment and Homogenization……. 63 Chapter 3: Analysis of Illumina MiSeq amplicon reads: application to benthic indices for environmental monitoring............................................ 79 Chapter 4: Benchmarking DNA metabarcoding for biodiversity-based monitoring and assessment............................................................ 97 Chapter 5: Metabarcoding-based marine biomonitoring and assessment: from scientific concepts to management directives…………………… 121 Chapter 6: A bacterial community-based index to assess the ecological status of estuarine and coastal environments.............................. 137 General Discussion………………………………………………………………………………… 165 Further Recommendations……………………………………………………………………. 173 Conclusions and Thesis ………………………………………………………………………… 175 References……………………………………………………………………………………………. 179 Supplementary Material – Figures………………………………………………………… 201 Supplementary Material – Tables…………………………………………………………. 211 References for Supplementary Material – Tables………………………………….. 235 3 4 SUMMARY The Water Framework and Marine Strategy Framework Directives have been developed within European legislation in order to protect and restore aquatic ecosystems. For this purposes, performing environmental status assessments that allow an integrated ecosystem management is required. Environmental status assessment is based on monitoring indicators; however, there is a demand of developing innovative monitoring tools and including new indicators within the assessments in order to increase speed, accuracy and cost-efficiency of monitoring programs. Among the promising innovative tools that can ease monitoring and potentially lead to new indicators are those based on genomic techniques, and in particular, on metabarcoding, a method that allows the accurate and cost-efficient taxonomic identification of multiple environmental samples even when including early developmental stages, cryptic species or degraded specimens. As such, this technique has been applied as a powerful alternative to traditional methodologies to detect toxic species, understand trophic interactions by analysing faeces or stomach contents or monitor early introduction of non-indigenous species, among others. However, before being included in current European legislation, metabarcoding needs to be benchmarked against morphology-based species identification, and proved capable of improving current monitoring by being more cost- effective and/or of producing new indicators that can be included in the mentioned directives. This Thesis explores the application of metabarcoding for responding to the need of new monitoring tools and indicators that allow more cost-effective and integrated environmental status assessments required for application in European directives. The specific aims of this Thesis are presented in six different Chapters. Chapter 1 sets the ground for the implementation of a metabarcoding- based macroinvertebrate biotic index. The AZTI’s Marine Biotic Index (AMBI) is one of the most successful and worldwide applied biotic indices for assessing seafloor integrity and derive health status of marine environments. Yet, this index is based on the benthic macroinvertebrate diversity, which is traditionally inferred by morphological taxonomic identification of the specimens present in each sample, which is time 5 consuming and requires not always available specialized taxonomical expertise. This chapter assesses the potential of metabarcoding as a cost- effective alternative to visual taxonomy by evaluating the requirements for the implementation of a genetics-based AMBI (gAMBI). The in silico analyses performed using available DNA sequence data showed that information about presence/absence of the most frequently occurring species provides accurate AMBI values and revealed the most suitable genetic fragment for metabarcoding marine macroinvertebrates.
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