Systematics and Phylogeography of the Cape Parrot (Poicephalus robustus) by Willem G. Coetzer Submitted in fulfilment of the academic requirements of Doctor of Philosophy in Genetics School of Life Sciences College of Agriculture, Engineering and Science University of KwaZulu-Natal Pietermaritzburg South Africa Supervisor: Dr S. Willows-Munro Co-supervisor: Prof C.T. Downs Prof M.R. Perrin December 2015 i PREFACE The research contained in this thesis was completed by the candidate while based in the Discipline of Genetics, School of Life Sciences of the College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa. The research was financially supported by SANBI, NRF and the University of KwaZulu-Natal. The contents of this work have not been submitted in any form to another university and, except where the work of others is acknowledged in the text, the results reported are due to investigations by the candidate. ………………………….. Signed: Dr S. Willows-Munro Date: 3 December 2015 ii DECLARATION 1: PLAGIARISM I, Willem G. Coetzer, declare that: (i) the research reported in this dissertation, except where otherwise indicated or acknowledged, is my original work; (ii) this dissertation has not been submitted in full or in part for any degree or examination to any other university; (iii) this dissertation does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons; (iv) this dissertation does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a) their words have been re-written but the general information attributed to them has been referenced; b) where their exact words have been used, their writing has been placed inside quotation marks, and referenced; (v) where I have used material for which publications followed, I have indicated in detail my role in the work; (vi) this dissertation is primarily a collection of material, prepared by myself, published as journal articles or presented as a poster and oral presentations at conferences. In some cases, additional material has been included; (vii) this dissertation does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the dissertation and in the References sections. ………………………….. Signed: W.G. Coetzer Date: 3 December 2015 iii DECLARATION 2: PUBLICATIONS My role in each paper and presentation is indicated. The * indicates corresponding author. Chapter 2 1. *Coetzer, W.G., Downs, C.T., Perrin, M.R. and Willows-Munro, S. 2015. Molecular systematics of the Cape Parrot (Poicephalus robustus): implications for taxonomy and conservation. Paper presentation to the bi-annual Zoological Society of Southern Africa symposium, July 2015, Grahamstown, Eastern Cape, South Africa. Presented by W.G. Coetzer. 2. *Coetzer, W.G., Downs, C.T., Perrin, M.R. and Willows-Munro, S. 2015. Molecular systematics of the Cape Parrot (Poicephalus robustus): implications for taxonomy and conservation. Paper presentation to the Australasian Ornithological Congress, November 2015, Adelaide, Australia. Presented by M.R. Perrin. 3. Coetzer, W.G., Downs, C.T., Perrin, M.R. and *Willows-Munro, S. 2015. Molecular systematics of the Cape Parrot (Poicephalus robustus): implications for taxonomy and conservation. PloS ONE. 10(8): e0133376. doi: 10.1371/journal.pone.0133376. Conceived and designed the experiments: WGC CTD MRP SW-M. Performed the experiments: WGC. Analysed the data: WGC. Contributed reagents/materials/analysis tools: CTD MRP SW-M. Wrote the paper: WGC CTD MRP SW-M. Chapter 3 4. *Coetzer, W.G., Downs, C.T., Perrin, M.R. and Willows-Munro, S. 2015. Phylogeography of the South African endemic Cape Parrot (Poicephalus robustus). Paper presentation at the annual symposium of the Southern African Wildlife Management Association, September 2015, Kimberley, Northern Cape, South Africa. Presented by W.G. Coetzer. 5. *Coetzer, W.G., Downs, C.T., Perrin, M.R. and Willows-Munro, S. 2015. Phylogeography of the South African endemic Cape Parrot (Poicephalus robustus). Paper presentation to the Australasian Ornithological Congress, November 2015, Adelaide, Australia. Presented by M.R. Perrin. Conceived and designed the experiments: WGC CTD MRP SW-M. Performed the experiments: WGC. Analysed the data: WGC. Contributed reagents/materials/analysis tools: CTD MRP SW-M. Prepared the presentations: WGC. ………………………….. Signed: W.G. Coetzer Date: 3 December 2015 iv ABSTRACT The Cape Parrot (Poicephalus r. robustus) is the only endemic parrot found in South Africa. This subspecies is restricted to the mistbelt forests of the Eastern Cape, Kwa-Zulu Natal and Limpopo Provinces. Recent population census estimates suggest that there are fewer than a 1600 Cape Parrots left in the wild. Habitat loss and illegal harvesting for the pet trade are among the major drivers of these low population numbers. The current conservation status of P. robustus ssp. does not lend the Cape Parrot sufficient protection national and international conservation agencies, as it is internationally seen as a subspecies of Poicephalus robustus i.e. P. robustus robustus. A better understanding of the Cape Parrot’s taxonomic position within the genus is therefore urgently needed. The three data chapters presented in this thesis address the main aims of this study. The taxonomic status of the Cape Parrot (Poicephalus robustus robustus) has been the focus of much debate over the years. A number of authors have suggested that the Cape Parrot should be viewed as a distinct species separate from the other two P. robustus subspecies (P. r. fuscicollis and P. r. suahelicus). These recommendations were based on previously published morphological, ecological and behavioural assessments. In this chapter the validity of these recommendations were investigated using multilocus DNA analyses. A total of 138 specimens from five Poicephalus species (P. cryptoxanthus, P. gulielmi, P. meyeri, P. robustus and P. rueppellii) were genotyped using 11 microsatellite loci. Additionally, two mitochondrial (cytochrome oxidase I gene and 16S ribosomal RNA) markers and one nuclear intron (intron 7 of the β-fibrinogen gene) marker were amplified and sequenced. Bayesian clustering analysis and pairwise FST analysis of microsatellite data identified P. r. robustus as genetically distinct from the other P. robustus subspecies. Phylogenetic analysis on sequence data also supported the microsatellite analyses, placing P. r. robustus in a distinct clade separate from the other P. robustus subspecies. Molecular clock analysis places the most recent common ancestor of P. r. robustus and P. r. fuscicollis / P. r. suahelicus at 2.13 to 2.67 million years ago. These results all support previous recommendations to elevate the Cape Parrot to species level. In the second data chapter, an assessment of the historical and contemporary genetic structure of the Cape Parrot was performed. The effect of anthropogenic habitat fragmentation on species, which live in naturally patchy habitats, has rarely been examined in South Africa. The Cape Parrot is a habitat specialist, restricted to forest patches in the Eastern Cape, KwaZulu-Natal and Limpopo Provinces of South Africa. Although current overexploitation of forests in southern Africa is certainly an important driver of fragmentation, this is not solely v responsible for the relictual nature of South African forests. In the Pliocene, periods of climate change driven aridity and increased fire frequency, contributed towards the ‘natural’ fragmentation of the forests in southern Africa. In this chapter, 85 modern samples, collected from 1951 to 2014, and 29 historical samples, collected from 1870 to 1946, were used to investigate the historical and contemporary genetic structure of Cape Parrots using 16 microsatellite loci. Bayesian clustering analysis identified three geographically correlated genetic clusters: a southern group restricted to forest patches in the Eastern Cape, a central group including birds from KwaZulu-Natal and a genetically distinct northern Limpopo cluster. Results suggest that Cape Parrots have experienced at least two major population bottlenecks. An ancient decline during the mid-Holocene (~1800-3000 years before present) linked to climate change, and a more recent bottleneck, associated with logging of forests during the early 1900’s. This chapter highlights the effects of climate change and human activities on this endangered species. The third data chapter, deals with the use of molecular data in forensic analysis of Cape Parrots. The illicit harvesting of wild Cape Parrots for the pet trade is a significant threat faced by this endemic South African species. Illegal trade in rare wildlife species is a major threat to many parrot species around the world. Wildlife forensics plays an important role in the preservation of endangered or threatened wildlife species. Identification of illegally harvested or traded animals through DNA techniques is one of the many methods implemented during forensic investigations. In this study, 16 microsatellite markers specifically designed for the South African endemic Cape Parrot were assessed for their utility in forensic casework. In addition, this chapter evaluates the genetic diversity of the captive Cape Parrot population and compares this to the wild Cape Parrot population,