1 Front cover By artist Erwin Hubert. 2 DOCTORAL THESIS 2017 DIVERSIFICATION PATTERNS AND PHYLOGEOGRAPHY IN A GENUS SUBTERRANEAN AMPHIPOD (PSEUDONIPHARGUS: MELITIDAE) Morten Stokkan 3 DOCTORAL THESIS 2017 Doctoral Programme of Marine Ecology DIVERSIFICATION PATTERNS AND PHYLOGEOGRAPHY IN A GENUS SUBTERRANEAN AMPHIPOD (PSEUDONIPHARGUS: MELITIDAE) Morten Stokkan Director/a: Damià Jaume Llabrés Director/a: Joan Pons Pons Tutor/a: Antoni Martínez Taberner Doctor by the Universitat de les Illes Balears 4 Acknowledgements There is a myriad of people that I would like to take this opportunity to thank for their inspiration and or influence in the process of this thesis. It has been a great part of my life and has been influenced in infinite different ways. First and foremost I would like to take this opportunity to thank my supervisors Damià Jaume Llabrés and Joan Pons Pons for their endless support and guidance. You guys nurtured my interests and encouraged me to continue. And for listening to me and lending me your finite time to my work. To José Antonio Jurado-Rivera for your friendship, showing me how to really work in a lab making me a better biologist and showing me how to be an honorary Spaniard. Also to my loving wife and daughter for being there with me, holding out with me on these long days of work and for showing me the endless love that I feel for them. Thank you for teaching me so much and being a part of my life. For my amazing parents for their unconditional support and care, as well as their help and kind words and the connection I feel with them.,and for my extended family both in Norway and Mexico for their love and support. I would also like to take this opportunity to thank Dr. Lars Podsiadlowski for allowing me to visit the University of Bonn, being interested in my subjects and showing me “the unknowns” of the field and being a great inspiration. For my friends and colleagues at the University of the Balearic Islands (UIB) and The Mediterranean Institute of Advanced Studies (IMEDEA) for the great and caring community we created, when most of us were far away from family we became our own family together. For my friends and colleagues back in Norway that I have shared over a decade of friendship, learning, laughter and love with. I thank you for always cheering me on and continuing to broaden my knowledge with fantastic and interesting conversations. Thanks to the Spanish National Research Council (CSIC) for realizing the FPI program and providing the funding, allowing me to embark on this wonderful, expanding and amazing journey. And finally I would like to thank Spain and more specifically Mallorca and its people for their warmth and acceptance, for showing me the culture and traditions and expanding my mind both in science and as a person. Thank you all 5 Published papers of this thesis: STOKKAN, M., JURADO-RIVERA, J. A., JUAN, C., JAUME, D. & PONS, J. 2016. Mitochondrial genome rearrangements at low taxonomic levels: three distinct mitogenome gene orders in the genus Pseudoniphargus (Crustacea: Amphipoda). Mitochondrial DNA, 1-11. (See Appendix 2) 6 Index Summary .................................................................................................................................... 8 Chapter 1. Introduction ............................................................................................................ 11 Chapter 2. Objectives ............................................................................................................... 35 Chapter 3. Materials and Methods .......................................................................................... 36 Specimen sampling and morphological methods ............................................................ 36 Molecular methods .......................................................................................................... 41 Chapter 4. Results and Discussion............................................................................................ 51 Chapter 4.1 Diversity, species delimitation and crypticism in a genus of subterranean water amphipod crustaceans (Pseudoniphargus: Pseudoniphargidae) .............................. 51 Results .............................................................................................................................. 51 Discussion ........................................................................................................................ 57 Chapter 4.2 Taxonomical description of new species .......................................................... 62 Taxonomy ......................................................................................................................... 62 Chapter 4.3 Mitogenome rearrangement of 3 species of the genus Pseudoniphargus ...... 80 Results and Discussion ..................................................................................................... 80 Chapter 4.4 Phylogeny based on 32 mitogenomes ............................................................. 96 Results .............................................................................................................................. 96 Discussion ....................................................................................................................... 114 Chapter 5. Conclusions ........................................................................................................... 121 Chapter 6. References ............................................................................................................ 123 Appendix ................................................................................................................................. 142 7 Summary Summary This thesis deals with the phylogeny and phylogeography of the aquatic crustacean amphipod genus Pseudoniphargus Chevereux, 1901, an obligate inhabitant of continental subterranean waters. Special emphasis is placed on the origin of the genus, its extremely disjunct distribution on both sides of the Atlantic as well as in the Mediterranean Sea, and the processes underlying this distribution. Both morphological and molecular approaches are used to investigate the species diversity and detect possible cases of morphological convergence or of recently formed species hidden under cryptisism. Additionally this thesis attempts to place the evolution of the genus in a space-temporal framework by using both fossils and palaeogeographical events as calibration dating points. An ample sampling and collecting effort across the entire known distribution of the genus was undertaken and together with rigorous morphological analysis, molecular data was collected. Sanger sequencing and Next Generation Sequencing (NGS) was utilized to obtain both individual short marker and entire mitochondrial genomes covering all the major clades of the genus. By using four different methods of species delimitation based on mitochondrial cox1sequences, as well as morphology and geographical location we were able to assess the diversity attained by Pseudoniphargus across the vast majority of its current distribution. Several potential cryptic species were encountered, highlighting both the presence of strong morphological convergence and the necessity of using a broad multiple methodological approach to fully investigate species diversification. Two new species are formally described P. morenoi and P. gevi, both from gypsum caves in southern Spain. We used NGS on 31 species covering the major clades and distribution of the genus to recover their mitochondrial genome. The thesis presents a strongly supported, fully resolved phylogeny of the genus Pseudoniphargus identifying four main clades within the genus. By using both fossil data and palaeogeographical events we were able to place the genus in a temporal framework enabling to identify both vicariance and dispersal as contributing processes to explain the current distribution of the genus. Age and origin of the genus is estimated at 55 Ma, consistent with previous investigations and correlates well with the ancient (Eocene) Tethys Sea configuration and its subsequent regression. The mitogenomes also revealed the occurrence of an unprecedented level of gene rearrangements within the genome of various species, up to five, whereupon two of them involved protein coding genesthat is rare event in metazoans, and particularly within a genus. A detailed analysis of the nucleotide and aminoacid composition, and the secondary structure of both RNA and the control region shed light on the molecular patterns of evolution in mitochondrial genomes that in turn are crucial to implement accurate phylogenetic models including mitochondrial sequences. The new mitochondrial genomes presented herein offer an important possibility to establish further comparisons with other amphipods from identical habitats and showing similar distribution patterns to Pseudoniphargus. 8 Summary Resumen Esta tesis investiga el origen y la diversificación del género de crustáceos anfípodos Pseudoniphargus, Chevereux, 1901, exclusivo de aguas subterráneas continentales que presenta una distribución disjunta extrema con representantes a ambos lados del Atlántico Norte. Para ello se realizó un amplio muestreo que ha abarcado el área de distribución completa del género, que ha permitido abordar un riguroso análisis morfológico y genético de 410 especímenes. Mediante el uso de cuatro criterios diferentes de diferenciación de especies basados en un único locus de ADN, combinados con