A Phylogenetic Study of Vulnerable Batoid Species from the North Atlantic Submitted by Maisie Bache-Jeffreys to the University of Exeter as a thesis for the degree of Masters by Research in Biological Sciences, September 2019. This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. (Signature) ............................................................................................. 1 Contents Contents of Figures ........................................................................................................................ 3 Contents of Tables ......................................................................................................................... 6 Abstract ........................................................................................................................................... 8 Chapter 1: General Introduction .................................................................................................. 10 1.1 Elasmobranchs: what are they and why are they at risk? ............................................... 10 1.2 Taxonomic confusion ........................................................................................................ 12 1.3 Genetic markers in elasmobranch phylogenomics ......................................................................... 19 1.4 Aims of this thesis: answering phylogenetic questions in North Atlantic batoids ....................... 26 1.5 References ............................................................................................................................................ 29 Chapter 2: Resolving the spatial distributions of Dipturus intermedius and Dipturus batis - the two novel taxa formerly known as the common skate. ........................................................ 58 2.1 Abstract .................................................................................................................................................. 58 2.2 Introduction ........................................................................................................................................... 60 2.3 Materials and methods ........................................................................................................................ 62 2.4 Results ................................................................................................................................................... 67 2.5 Discussion ............................................................................................................................................. 73 2.6 Acknowledgements .............................................................................................................................. 77 2.7 References ............................................................................................................................................ 78 2.8 Supplementary materials: Resolving the spatial distributions of Dipturus intermedius and Dipturus batis - the two novel taxa formerly known as the common skate. ....................................... 87 Chapter 3: NextRAD and mitochondrial DNA sequencing reveal hidden diversity within vulnerable species of batoid fish. .............................................................................................. 103 3.1 Abstract ................................................................................................................................................ 103 3.2 Introduction ......................................................................................................................................... 104 3.3 Methods ............................................................................................................................................... 107 3.4 Results ................................................................................................................................................. 112 3.5 Discussion ........................................................................................................................................... 121 3.6 References .......................................................................................................................................... 139 3.7 Supplementary materials: NextRAD and mitochondrial DNA sequencing reveal hidden diversity within vulnerable species of batoid fish. ................................................................................ 164 Chapter 4: General discussion .................................................................................................. 174 4.1 Conclusions and future directions ................................................................................................... 174 4.2 Conclusion………………………….………………………………………………………………181 4.3 References .......................................................................................................................................... 182 2 Contents of Figures Chapter 1 Figure 1. A simplified diagram of the taxonomic structure of Chondrichthyans and the Batoidea. .. 13 Figure 2. Two examples of morphological conservatism in batoid species. The reef manta, Manta alfredi (A; Marshall et al., 2018a) and the oceanic manta, Manta birostris (B; Marshall et al., 2018a) exhibit striking levels of morphological conservatism, despite being distinct species. The same is true for the blonde ray, Raja brachyura (C; Ellis et al., 2009) and the spotted ray, Raja montagui (D; Ellis et al., 2007)........................................................................................................................................16 Figure 3. Number of ray species described each decade from Linnaeus in 1758 until 2016 (Last et al., 2016)......................................................................................................................................... 18 Figure 4. Mitogenome map of the Kwangtung skate, Dipturus kwangtungensis. The innermost circle represents GC% per every 5bp of the mitogenome; the darker the line, the higher the GC%. (Jeong et al., 2015) ..................................................................................................................................... 22 Chapter 2 Figure 1. (A) Map detailing the range of the ‘common skate’ in 2002 (adapted from Dulvy et al., 2002); (B) The geographical locations of all blue skate (Dipturus batis) specimens used in phylogenetic analysis, including ones representing CR sequences downloaded from Genbank; (C). The geographical locations of all flapper skate (Dipturus intermedius) specimens used in phylogenetic analysis, including ones representing CR sequences downloaded from Genbank. * indicates that the flapper skate specimen from Portugal did not have exact latitude and longitude 3 information available, and so the proximate location has been indicated. Because this is the only specimen that represents a COI sequence, it was not included in the CR haplotype network. The size of a point is proportional to the number of individuals it represents………………………...…….66 Figure 2. PhyML tree built from COI data (A); Mr Bayes tree built from COI data (B). Numbers above branches represent bootstrap support values. Blue numbers below branches correspond to accession numbers and location infomration in Appendix two and four, supplementary materials. .................. 68 Figure 3. PhyML tree built from CR data (A); Mr Bayes tree built from CR data (B). Numbers above branches represent bootstrap support values. Blue numbers below branches correspond to accession numbers and location infomration in Appendix two and three, supplementary materials.69 Figure 4. Control region (CR) haplotype network of D. intermedius and D. batis samples collected from across the North Atlantic and downloaded from Genbank………………………………..………71 Appendix 1. Image of the flapper skate (Dipturus intermedius) specimen 'AZP90' from the Azores………………………………………………………………………………………………………...88 Chapter 3 Figure 1. Map detailing locations specimens sampled in the current study. * indicates the exact latitude and longitude of the sample was unknown, in this instance the point shows an approximate location. All points represent one specimen……………………………………………………………..109 Figure 2. (A) RAxML tree built from concatenated (CR and COI) mtDNA data (ms4); (B) RAxML tree built from nextRAD-seq data. Numbers below branches represent bootstrap support values. Scale bars refer to the number of substitutions per site .......................................................................... 119 Figure 3. SVDquartet tree built from nextRAD-seq data (ms4 dataset). Support from 1000 bootstrap replicates are shown for branches with support >50%. Branch lengths do not reflect divergence…………………………………………………………………………………………………..120 4 Figure 4. Figure detailing conlusions drawn from the present study for key species. Appendix 1. Workflow of NextRAD Nextera-tagmented reductively-amplified DNA) sequencing. A small amount of DNA (~10 ng) is mixed with two engineered transposomes of Nextera reagents which tag as well as add short adaptors to genomic DNA. Sequencing primers Read one (R1, light blue bars) and Read two (R2,