Phylogenomics of the Pulmonate Land Snails
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PHYLOGENOMICS OF THE PULMONATE LAND SNAILS Luisa Cinzia Teasdale Submitted in total fulfilment of the requirements of the degree of DOCTOR OF PHILOSOPHY January 2017 School of BioSciences The University of Melbourne 1 Phylogenomics of the Pulmonate Land Snails by Luisa C. Teasdale © 2017 Supervisors: Adnan Moussalli and Devi Stuart-Fox Cover Image: Montidelos orcardis taken by Luisa C. Teasdale 2 For Kevin, and my parents: Maria and Stephen. 3 Abstract The pulmonates are the most speciose gastropod lineage and are highly diverse in morphological form and habitat. The evolutionary relationships among the pulmonates have remained controversial despite a long history of scientific study. Recent molecular studies have placed traditionally pulmonate (air-breathing) and non-pulmonate taxa into Panpulmonata; however, the relationships within this new group are still poorly understood. Incongruence between molecular studies has generally resulted from a lack of informative loci but the advent of next generation sequencing technologies means it is now feasible to produce large genetic datasets for non-model organisms. The main aim of my thesis was to investigate the timing and pattern of evolutionary relationships within the Panpulmonata, at multiple taxonomic scales, using phylogenomic datasets. The qualification of orthology is a significant challenge when developing large, multi- locus datasets for phylogenetics from transcriptome assemblies. In Chapter 2, I identified 500 orthologous single-copy genes from 21 transcriptome assemblies across the Eupulmonata (mostly terrestrial land snails and slugs) using a thorough approach to orthology determination, involving manual alignment curation and gene tree assessment. I further qualified orthology by sequencing the genes from the genomic DNA of 22 representatives of the Australian land snail family Camaenidae using exon capture. Through comparison, I also found that automated orthology determination approaches can be susceptible to transcriptome assembly errors. I then used the orthologous genes identified in Chapter 2 to investigate the pattern and timing of evolution across Panpulmonata in Chapter 3. My dataset included representatives of all major clades within Panpulmonata including a wide representation of the stylommatophoran land snails, the most successful lineage of terrestrial molluscs. Maximum likelihood and Bayesian analyses confirm that Panpulmonata is monophyletic, and that Pulmonata is not monophyletic, implying that air-breathing has likely evolved more than once. Within Panpulmonata I show strong support for relationships previously unsupported or weakly supported in molecular analyses, including the Geophila, and the Pylopulmonata, a clade that unites the operculate panpulmonates. Molecular dating suggests a Permian or Early Triassic origin for Panpulmonata and a Triassic/Jurassic boundary origin for Eupulmonata and the freshwater Hygrophila. 4 In addition to investigating deep relationships within Panpulmonata, I used a similar approach to investigate phylogenetic relationships on a shallower scale within a land snail family, the Rhytididae. Australia has the highest taxonomic diversity of the Rhytididae, a carnivorous family of land snails with a Gondwanan distribution. Previous higher classifications of the Australian Rhytididae are based on limited morphological characters and have not been assessed with molecular evidence. I present a molecular phylogeny of the Australian Rhytididae based on a large multi-locus dataset comprising nuclear exons sequenced using exon capture. I identified four major monophyletic lineages within the Australian Rhytididae. I also show that there is a high amount of unrecognised diversity, particularly in the smaller rhytidids. Contrary to shell morphology, on which the current taxonomy is based, a number of currently recognised genera are either polyphyletic or paraphyletic. The Australian lineages all resulted from an apparent pulse of diversification approx. 45-30 Ma. Given the South African Nata and the New Zealand Delos and Schizoglossa also belong to this clade, this date suggests that cross-water dispersal has played a role in the evolution of this group. 5 Declaration This is to certify that: i. The thesis comprises only my original work towards the PhD except where indicated in the preface, ii. Due acknowledgement has been made in the text to all other material used, iii. The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Signature: _______________________________ Date: ________6/01/2017________ Luisa C. Teasdale 6 Preface This thesis includes a series of independent publications; consequently there may be some repetition between chapters. Chapters 2 – 4 comprise co-authored manuscripts that have either been published or will be submitted for publication. This thesis includes the largely unchanged reprint of the following previously published journal article, which has been included as a chapter with the permission of all co-authors: Teasdale, L.C., Koehler, F., Murray, K.D., O’Hara, T. and Moussalli, A. 2016. Identification and qualification of 500 nuclear, single-copy, orthologous genes for the Eupulmonata (Gastropoda) using transcriptome sequencing and exon-capture. Molecular Ecology Resources, DOI: http://dx.doi.org/10.1101/035543 (Chapter 2) I am the primary author of this work and was responsible for experimental design, performing the research, analysing the data, and writing the manuscript. Adnan Moussalli contributed to experimental design, specimen collection, and data analysis. Frank Köhler assisted with specimen collection, and Kevin Murray and Tim O’Hara provided bioinformatics assistance. All co-authors provided feedback on the manuscript. Additionally the following manuscripts, for which I am also primary author, are in preparation for publication: Teasdale, L.C., Hugall, A., Köhler, F., Herbert, D., Barker, G., O’Hara, T. and Moussalli, A. In prep. The pace and pattern of pulmonate evolution. Molecular Biology and Evolution. (Chapter 3) Teasdale, L.C. and Moussalli, A. In prep. Phylogenetic investigation of the Australian Rhytididae using exon capture. Molecular Phylogenetics and Evolution. (Chapter 4) Adnan Moussalli contributed to experimental design, specimen collection, and data analysis for Chapters 3 and 4. For Chapter 3, Frank assisted with specimen collection, Tim O’Hara assisted with the bioinformatics, Andrew Hugall provided assistance with the phylogenetic analyses, Gary Barker provided the morphological data, and David Herbert assisted with the biological interpretation. All co-authors provided feedback on the manuscripts. 7 Acknowledgements First and foremost I would like to thank my supervisor Adnan Moussalli for all his help, patience, and support throughout my PhD. I have valued our rigorous scientific discussions and without his help this PhD would not have been possible. I also thank my co- supervisor, Devi Stuart-Fox, for her support, mentorship, and guidance. I thank the members of my advisory committee, Michael Keogh and Ary Hoffman for their time and expertise. I thank my collaborators for their contribution to this work. Specifically, I thank Andrew Hugall for invaluable discussions as well as mentoring me through the tangled world of phylogenetic analysis. I thank Frank Köhler for his assistance with field work and the biological interpretation of my results, but perhaps not for his sense of direction. I also thank Gary Barker for his guidance through the complexities of snail morphology. I am gratefully for Kevin Bonham, Francesco Criscione, and Winston Ponder who generously collected and provided samples. I would also like to thank the service providers who conducted a lot of the lab work and assisted with troubleshooting, namely Roger Neilsen and Travis Glenn at the Georgia Genomics Facility, and Jake Enk and Alison Devault at MYcroarray. I would also like to thank a number of people at Museum Victoria who have provided assistance, support, and friendship during my PhD, including Stella Claudius, Maggie Haines, Melanie Mackenzie, Claire McLean, Michela Mitchell, Tim O’Hara, Chris Rowley, Jo Sumner, and Jo Taylor. I would particular like to thank Martin Gormon for his mentorship, which I have valued more than I think he realises. I would also like to thank Craig Moritz and his lab at ANU for adopting me while my supervisors were on sabbatical. I thank Maddie and Tom for their friendship and support at Moor St and my Canberra friends Panda, Brian, and Claire. I thank my best friend Kim for her support, friendship, and ability to distract. I would also like to thank my family for their support and patience when listening to explanations of why snails are interesting. Most of all, I would like to thank Kevin for his patience, support, assistance, and friendship. Lastly, I would like to thank the snails for being so interesting and David Attenborough and Quantum for igniting my interest in zoology in the first place. 8 The fieldwork conducted in this thesis was carried out under a permit to Take, Use, Keep or interfere with Cultural or Natural Resources (ScientificPurpose) from the Queensland government (WITK12586613) and a Scientific License from the New South Wales government (SL101119). Financial support for this research was provided by the Holsworth Wildlife Research Endowment, the National Environmental Research Program (NERP), Museum Victoria (1854 Student Scholarship) and the University of Melbourne (Drummond Award, MacBain