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Phylogeography and Ecology of New Zealand Freshwater Amphipoda (Paracalliope, Paraleptamphopus and Phreatogammarus) A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biological Sciences at the University of Waikato by Darin Lee Sutherland The University of Waikato 2006 ABSTRACT This thesis examines phylogenetic patterns in three New Zealand amphipod taxa in relation to current geographic distributions and historical climatic (e.g. glaciation, marine inundation) and geological (e.g. mountain building) events using DNA sequencing and distributional data. It also examines how recognition behaviour can be used to delineate potential species boundaries and to assess the role of sexual selection. The endemic genus Phreatogammarus has been found in only a limited number of sites and is not very abundant. An analysis of the genetic variation of two species within the genus using allozyme electrophoresis revealed high levels of genetic differentiation among populations but low levels within populations. This suggested that limited dispersal occurred among habitats with one population possibly representing a cryptic species. The endemic freshwater genus Paraleptamphopus is thought to contain a large number of undescribed species with a number of these existing in small waterbodies such as seepages. Examination of the phylogeographic patterns using both mtDNA (CO1) and nuclear DNA (28S) showed that a number of distinct genetic lineages exist, with CO1 revealing 21 haplotypes with genetic distance of over 20%. Using a molecular clock rate of 2.4%, most haplotypes diverged approximately 8-12 million years ago during the Miocene era, possibly as a result of greater land availability increasing habitat diversity or by allopatric speciation. Morphological and genetic differences were not congruent, with morphologically similar taxa appearing among highly genetically distinct lineages, and some morphologically distinct forms appearing within single lineages. ii The distribution and habitat variables of 419 sites were analysed to determine what was affecting the presence or absence of Paraleptamphopus. The presence of native vegetation in catchments had a positive affect on Paraleptamphopus distribution suggesting that large anthropogenic changes in catchment vegetation could have a negative effect on their abundance. I found smaller waterbodies to be more important than larger ones highlighting the need to study such sites as rare taxa may be ignored. A better understanding is needed on the role of small waterbodies in promoting overall species diversity in catchments. Examination of Paracalliope fluviatilis phylogenetic patterns using the mtDNA gene CO1 showed that a number of separate clades existed suggesting long term isolation and limited dispersal among catchments. Due to the large genetic divergences among some populations there was the possibility that cryptic species might exist. Species recognition experiments were conducted on seven populations to help determine whether cryptic species were present. For the three most genetically divergent crosses there was bias against inter-population pairings, suggesting that there were between two or three separate species. Using a combined field and laboratory approach, size assortative mating was examined in Paracalliope fluviatilis. The field study showed positive size assortative mating and that larger females carried more eggs, suggesting they were more fecund. A series of laboratory experiments examining four existing theories explaining the phenomenon found that none adequately explained positive size assortative mating in P. fluviatilis. I therefore presented two new explanations to explain size assortative mating: a combination of female resistance and size- related variation in a male’s capacity to amplex larger females or a form of indirect intra-sexual competition. iii ACKNOWLEDGEMENTS I wish to thank my supervisors Dr Ian Hogg and Dr Ann Chapman who have provided encouragement throughout my PhD and were always willing to point me in the right direction when I needed it. A special thanks has to go to Dr Joe Waas who provided valuable advice on the behavioural component of the PhD. My next big thanks goes to all those people whom have made comments on my manuscripts, Mark Stevens, Ian Duggan, Naomi Crawford, David West, Grant Tempero, and Angela McGaughran. No PhD can be completed without technical help so I would like to make special mention of the university technicians who have helped me over the years: Gavin Reynolds, Leigh Laboyrie, Barry O’Brien, Louise Hathaway, Lynne Baxter and Tracey Jones. I would also like to thank people who either accompanied me in the field, made collections on my behalf or provided me with samples: Graham Fenwick, Peter Sutherland, June Sutherland, Robert Torrens, Mark Winterbourn, Leanne O’Brien, Paul Leisnham, Mark Lay, Alton Perrie, Eloise Ryan, Martin Haase and Mark Stevens. Finally, I wish to thank my parents, Peter and June Sutherland whose support and encouragement over the years have enabled me to be where I am today. iv TABLE OF CONTENTS ABSTRACT.............................................................................................................ii ACKNOWLEDGEMENTS....................................................................................iv TABLE OF CONTENTS.........................................................................................v LIST OF TABLES..................................................................................................ix LIST OF FIGURES..................................................................................................x LIST OF APPENDICES.........................................................................................xi INTRODUCTION..................................................................................................1 The development of evolution and genetics..........................................1 Species Concepts...................................................................................2 Phylogeography.....................................................................................4 Cryptic species and Molecular Markers................................................5 Behaviour and species...........................................................................6 New Zealand geology and geography...................................................6 Chapter Introduction.............................................................................8 REFERENCES.............................................................................................12 CHAPTER I: Allozyme variability among populationsof Phreatogammarus (Crustacea: Amphipoda): Reflects restricted distribution and limited dispersal................................................................................................................16 ABSTRACT.................................................................................................17 INTRODUCTION........................................................................................18 MATERIALS AND METHODS.................................................................19 Field sites and sampling......................................................................19 Allozyme electrophoresis....................................................................21 Data analyses.......................................................................................22 RESULTS.....................................................................................................22 Allozyme variation and population genetic structure.........................24 DISCUSSION...............................................................................................28 ACKNOWLEDGEMENTS..........................................................................30 REFERENCES.............................................................................................31 v CHAPTER II: Phylogeography of the amphipod genus Paraleptamphopus (Eusiridae): Evidence of multiple Miocene- Pleistocene radiations instigated by dispersal and adaptive radiation...................................................................34 ABSTRACT.................................................................................................35 INTRODUCTION........................................................................................36 METHODS...................................................................................................38 Field sites and animal collections........................................................38