Phylogeographic and Morphometric Studies on the Eurasian Pygmy Shrew Sorex Minutus : Insights Into Its Evolutionary History
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Phylogeographic and morphometric studies on the Eurasian pygmy shrew Sorex minutus : insights into its evolutionary history and postglacial colonisation in Europe Rodrigo Rafael Vega Bernal PhD Thesis University of York Department of Biology July 2010 Abstract Here, I investigate the phylogeography and morphology of the Eurasian pygmy shrew Sorex minutus , searching for significantly differentiated lineages, colonisation routes and demographic parameters that would explain the effects of the Quaternary glaciations on the current distribution of the species. I also explore the genetic and morphological diversity and origin of pygmy shrew populations in the British Isles, particularly focusing on Ireland and the Orkney islands. Mitochondrial and nuclear DNA markers were used for the phylogeographic analyses, and a geometric morphometrics approach was implemented on mandible and skull samples. There was an evident phylogeographic structure across Eurasia consistent with occurrence of southern glacial refugia, and there were two distinct lineages in Northern-Central Europe and near the Pyrenees supporting the existence of northern glacial refugia through the characteristics of their distribution and population expansion. Haplotypes from Britain belonged to these two northern lineages, with the Pyrenean lineage forming a peripheral ‘Celtic fringe’. I show that it is most likely that pygmy shrews on both Ireland and Orkney were introduced by humans from mainland British Celtic fringe rather than further afield, even though there is a haplotype found in Northern Spain identical to one in Ireland. Mandible size increased noticeably with decreasing latitude, but skulls showed no evident trend in size variation. Shape variation was significant but modest when analysing the sample divided into phylogeographical groups. However, the samples from different islands within the British Isles show that island evolution played an important role in morphological diversity, with mandible and skull shape divergence on small islands and low genetic diversity. These results notably expanded previous findings and indicate that S. minutus is an excellent model for understanding the effects of climate change on biological diversity, colonisation and differentiation in i refugia, and island evolution, useful for the conservation of genetic and morphological diversity. ii List of contents Abstract ......................................................................................................................... i List of contents ........................................................................................................... iii List of tables .............................................................................................................. vii List of figures .............................................................................................................. ix List of appendices . .................................................................................................... xv Dedication ................................................................................................................ xvii Acknowledgements ................................................................................................. xviii Author declaration ..................................................................................................... xx Chapter 1 General introduction ................................................................................. 1 1.1 Structure of the thesis .................................................................................... 1 1.2 The European peninsula as a geographical setting ........................................ 2 1.3 Shrews including Sorex minutus .................................................................... 3 1.4 The Quaternary glaciations ............................................................................ 6 1.5 Phylogeography defined ................................................................................ 8 1.6 Geometric morphometrics defined ................................................................10 1.7 Aims .............................................................................................................11 Chapter 2 Phylogeography of Sorex minutus in Europe ........................................13 2.1 Introduction ...................................................................................................13 2.2 Materials and methods ..................................................................................17 2.2.1 Samples and GenBank sequences for molecular analysis ......................17 2.2.2 DNA extraction, gel electrophoresis and visualization .............................18 2.2.3 PCR amplification, purification and sequencing ......................................19 2.2.4 Analysis of sequence polymorphism .......................................................20 2.2.5 Phylogenetic analyses ............................................................................21 2.2.6 Analysis of sequence diversity ................................................................26 iii 2.2.7 Divergence and genetic structure of populations .....................................27 2.2.8 Estimation of historical demographic parameters ....................................29 2.3 Results .........................................................................................................33 2.3.1 Sequence polymorphism.........................................................................33 2.3.2 Phylogenetic analyses ............................................................................34 2.3.3 Sequence diversity ..................................................................................45 2.3.4 Divergence and genetic structure of populations .....................................46 2.3.5 Historical demography ............................................................................53 2.4 Discussion and conclusions ..........................................................................58 2.4.1 Phylogenetic findings and genetic population structure ...........................58 2.4.2 Insights on the colonisation routes into Europe .......................................60 2.4.3 Colonisation and historical demography of southern peninsulas .............61 2.4.4 Northern refugia of the North-Central European phylogroup ...................64 2.4.5 Colonisation history of Northern Europe and Britain ................................66 2.4.6 Northern refugium of the Pyrenean phylogroup? .....................................67 2.4.7 Origins of the Irish pygmy shrew .............................................................67 2.4.8 Other considerations ...............................................................................69 Chapter 3 Morphological diversity of Sorex minutus across Europe ....................71 3.1 Introduction ...................................................................................................71 3.2 Materials and methods ..................................................................................73 3.2.1 Collection and preparation of samples ....................................................73 3.2.2 Digitization of mandibles and skulls ........................................................75 3.2.3 Statistical analysis of landmark coordinates data ....................................77 3.3 Results .........................................................................................................82 3.3.1 Landmark placing error ...........................................................................82 3.3.2 Asymmetry and sex differences ..............................................................82 3.3.3 Morphometric analysis of mandibles .......................................................83 iv 3.3.4 Morphometric analysis of skulls ..............................................................94 3.4 Discussion and conclusions. ....................................................................... 104 3.4.1 Mandible and skull size variation in Sorex minutus ............................... 104 3.4.2 Mandible and skull shape variation in Sorex minutus ............................ 105 3.4.3 Taxonomic implications ......................................................................... 107 Chapter 4 Island phylogeography and morphometrics of Sorex minutus ........... 109 4.1 Introduction ................................................................................................. 109 4.2 Materials and methods ................................................................................ 111 4.2.1 Samples for genetic and morphological analysis ................................... 111 4.2.2 Sequence diversity, divergence and differentiation ............................... 113 4.2.3 Estimation of migration routes ............................................................... 114 4.2.4 Geometric morphometric analysis ......................................................... 115 4.3 Results ....................................................................................................... 116 4.3.1 Genetic diversity and structure .............................................................. 116 4.3.2 Migration routes .................................................................................... 120 4.3.3 Geometric morphometrics findings ........................................................ 122 4.4 Discussion