The Radiation and Biogeography of Snakes Across South-Eastern Africa with Respect to the Evolution of the Savanna Biome

Home , Crotaphopeltis, Crotaphopeltis tornieri, Dipsadoboa, Eirenis levantinus, Hapsidophrys, Meizodon

The radiation and biogeography of snakes across south-eastern

Africa with respect to the evolution of the savanna biome

Hanlie M. Engelbrecht

Dissertation presented for the degree of Doctor of Philosophy (Zoology) in the Faculty of Science at Stellenbosch University

Supervisor: Professor Krystal A. Tolley1,2 Co-supervisor: Professor P. Le Fras N. Mouton2

1Biodiversity Research Information Monitoring, South African National Biodiversity Institute, Private Bag X7, Claremont, 7735, South Africa 2Department of Botany and Zoology, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa

December 2017

Declaration

By submitting this dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third-party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification.

December 2017

Abstract

The heterogeneous landscape of the African continent was preceded by a pan-African forest that has been transformed by concomitant contractions and expansions of this biome since the Oligocene epoch, to primarily savanna at present. As such, faunal groups that emerged during the Paleogene/Neogene period and have species distributed in both forest and savanna habitat should show a genetic signature of the possible evolutionary impact of these biome developments. Crotaphopeltis and Philothamnus were ideal candidate taxa to investigate the evolutionary impact of these biome developments on widespread African colubrid snakes. Species in these two genera occur throughout sub-Saharan Africa and are associated with either closed (forest), open (e.g. savanna), as well as both habitat types. The main research aim was to investigate synchronised timing and patterns of radiation for Crotaphopeltis and Philothamnus with respect to these African biome developments. Knowledge of the evolutionary relationships for the two genera was needed to construct their biogeographic histories reliably. Therefore, described species of Crotaphopeltis and Philothamnus were investigated for their validity under the Evolutionary Species Concept with an aim to identify cryptic species that are otherwise masked by phenotypic and/or ecological parallels. Newly identified species were used in subsequent analysis of timing of lineage diversification and the reconstruction of ancestral habitat states. The influence of historical environmental conditions on the evolution of the climatic niche of two wide-ranging species in each of the genera, C. hotamboeia and P. angolensis were additionally studied. The climatic distributions of the two species were modelled for contrasting climatic conditions during the Pleistocene period and their ancestral climatic states were reconstructed. Results show that both genera are monophyletic. Up to seven candidate species are recognised within Philothamnus and two within Crotaphopeltis, which provide a basis for future taxonomic revisions. Timing of lineage diversification and associated shifts of species into novel habitats correspond to the historical developments of the forest and savanna biomes, since the Oligocene. Philothamnus showed an ancestral preference toward closed habitat, while the ancestral habitat type for Crotaphopeltis was equivocal between closed and open habitat types. The Miocene epoch signifies a period of increased diversity within both genera. It specifically appears that the climatic oscillations during Early/Mid-Miocene facilitated the evolution of C. hotamboeia as a climatic generalist, whilst the Late-Miocene climatic conditions induced specialisation of P. angolensis in subtropical climate. The research presented here demonstrates that development of habitat throughout the Late Oligocene influenced the radiation patterns of the colubrid snake genera, Crotaphopeltis and Philothamnus.

Opsomming

Die veranderlike landskap van die Afrika-vasteland is voorafgegaan deur 'n pan-Afrika-woud wat deur die aaneenlopende en ossilerende inkrimping en uitbreidings van hierdie biome verander is sedert die Oligoseen-epog, tot hoofsaaklik die huidige savanna. Sodanig kan die diere groepe wat tydens die Paleogene/Neogene periode onstaan het met spesies wat in beide die woud- en savanna habitatte voorkom genetiese kenmerke toon van die moontlike evolusionêre impak van hierdie biome se ontwikkelings. Crotaphopeltis en Philothamnus was ideale kandidaat-taxa om die evolusionêre impak van hierdie verskeie bioom-ontwikkelings op wydverspreide Afrika colubrid-slange te ondersoek. Spesies in hierdie twee genera kom wyd-verspreid in sub-Sahara Afrika voor en word geassosieer met óf beskutte (woud) óf oop (bv. savanna) sowel as albei habitat soorte. Die hoof navorsingsdoel was om gesinkroniseerde tydsberekening en patrone van radiasie vir Crotaphopeltis en Philothamnus met betrekking tot die ontwikkelingsgang van hierdie Afrika-biome te ondersoek. Inligting rakend die evolusionêre verhoudings vir die twee genera was nodig om hul biogeografiese geskiedenisse betroubaar saam te stel. Daarom is reeds beskryfde spesies van Crotaphopeltis en Philothamnus, ondersoek vir hul geldigheid onder die Evolusionêre Spesies Konsep met die doel om kriptiese spesies te identifiseer wat andersins deur fenotipiese en/of ekologiese parallelle verdoesel kan word. Nuut geïdentifiseerde spesies is gebruik in die daaropeenvolgende analise van die tydsberekening van diversifisering en die heropbou van voorouer-habitat toestande. Die invloed van historiese omgewingstoestande op die evolusie van die klimaatsnisse van twee wydverspreide spesies in elk van die genera, C. hotamboeia en P. angolensis, is verder bestudeer. Die klimaatsverspreiding van die twee spesies is gemodelleer vir kontrasterende klimaatstoestande gedurende die Pleistoseen- tydperk en hul voorouer klimaatstoestande is herbou. Resultate toon dat beide genera monofileties is. Soveel as sewe kandidaat spesies word herken binne Philothamnus en twee binne Crotaphopeltis wat 'n basis vorm vir toekomstige taksonomiese hersienings. Die tydsberekeninge van diversifisering en die gepaardgaande verskuiwings van spesies na nuwe habitatte stem ooreen met die historiese ontwikkelinge van die woud en savanna biome, sedert die Oligoseen. Philotamnus toon ‘n voorouer voorkeur vir beskutte habitat tipes, terwyl die voorouer habitat tipe vir Crotaphopeltis ewekansig was tussen beskutte en oop habitatsoorte. Die Mioseen-epog dui op 'n tydperk van toenemende diversiteit binne beide genera. Dit blyk spesifiek dat die klimaat-ossillasies tydens die Vroeë/Middel-Mioseen die evolusie van klimaats-veralgemening fasiliteer het vir C. hotamboeia , terwyl die Laat-Mioseen klimaat spesialisasie van P. angolensis in subtropiese klimaat veroorsaak het. Die navorsing wat hier aangebied word toon dat die habitatsontwikkeling van die die Laat Oligoseen die radiasie patrone van die colubrid-slanggenera, Crotaphopeltis en Philothamnus beïnvloed het.

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Acknowledgements

This research was funded by the Andrew W. Mellon Foundation and partially funded by a National Geographic Society grant to Krystal Tolley. I am grateful for the student financial support that I received from the National Research Foundation (NRF) and Deutscher Akademischer Austausch Dienst (DAAD), which was supplemented by bursaries from Jordan Wines and the South African National Biodiversity Institute (SANBI), Kirstenbosch, Cape Town. I want to extend my thanks to DAAD for a short-term traveling scholarship under the In-region Scholarship Programme (ICIPE). Ethic clearance for research protocols were received from the South African National Biodiversity Institute (001/2013) as well as Stellenbosch University (SU-ACM14-00040). I thank the South African National Parks (SANPARKS) for their permission to conduct research on their properties, as well as Reserve Managers at Andover Nature Reserve (N.R.), Barberton N.R., Goegap N.R., Lajuma Mountain Resort, Lekgalameetse N.R., Manyeleti Game Reserve, Mokolo Dam N.R., Moyo Safari Lodge, and Songimvelo N.R. Research was conducted under national and provincial collection and export permits: Namaqua National Park (CRC/2015/011—2015), Northern Cape (FAUNA 265/2015; FAUNA/1304/2015; FAUNA 0088/2016; FAUNA 0089/2016), Mpumalanga (MPB. 537), and Limpopo (0092-CPM401-00006). The following people are thanked for fieldwork assistance: André Burger, Anrich Sunkel, Buyisile Makhubo, Chris Hobkrik, Cristóvão Nanvonamuquitxo, David van der Meer, Frans Nyathi, Gabriela Bittencourt-Silva, Jiří Šmíd, Jody Taft, Krystal Tolley, Michele Menegon, Nicolas Telford, Phuti Kwanate, Rupert Ndhlovu, Simon Loader, Sizwe Khanyile, Sonia Fischer, Themba Mathonsi, Tony Rikhoso, Tsepho Ramatseba, and Werner Conradie. A special thanks to those who donated tissue samples: John Measey, Johan Marais, Kate Jackson, Eli Greenbaum, Harith Farooq, Luke Verburgt, Marius Burger, Mark-Oliver Rödel, Michele Menegon, Simon Loader, Thomáš Mazuch, Tommy Collard, Tyron Ping and Werner Conradie. I also want to take this opportunity to thank my research collaborators: Eli Greenbaum, Marius Burger, Kate Jackson, Stefan Lötters, Stephan Fieldmeier, and Werner Conradie for making themselves available for discussions and sharing their expertise. Many thanks to SANBI, Kirstenbosch, for use of the Leslie Hill Molecular Ecology laboratory and office facilities. I would also like to thank the administrative personnel at SANBI: Gail Van Aswegen, Ferozah Conrad, Rowena Siebritz and René Du Toit for their logistical support. To my colleagues at SANBI: Jiří Šmíd, Jessica da Silva, Jody Taft, Paula Strauss, Ryan Daniels, Sarah Wishart, Shelley Edwards, Tesray Linvee, and Tlou Manyelo, thank you all for useful discussions and creating a pleasant working environment with your contagious sense of humour.

Roselind, Rhoderick and Heinrich Engelbrecht, I thank you for your endless love, support and encouragement.

Keshia Myburgh, thank you for your love and motivation. Above all, thank you for your patience.

Professor Le Fras Mouton, thank you so much for your guidance and advice over the past years. Most of all, thank you for introducing me to the research field of herpetology.

Professor Bill Branch, thank you for your recommendations and sharing your invaluable knowledge.

Professor Krystal Tolley You have set an example of excellence as a researcher, mentor, advisor, and role model. I would like to express my deepest appreciation for your patience in reading numerous drafts, and responding promptly with valuable feedback. I would also like to thank you for expanding my collaborative network. Lastly, thanks for always sharing my enthusiasm for this research from the very beginning to end.

The radiation and biogeography of snakes across south-eastern Africa with respect to the evolution of the savanna biome

Contents

Chapter One: General introduction ...... 1 Species concepts and species delimitations ...... 1 Systematic comparisons and inferences through time and space ...... 4 Hypothesis testing in historical biogeography ...... 5 The radiation and biogeography of Crotaphopeltis and Philothamnus species across sub-Saharan Africa with respect to the evolution of the savanna biome ...... 5 Research rationale ...... 5 Research aim and objectives ...... 8 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae) ...... 9 Introduction ...... 9 Materials & Methods ...... 11 Sample collection ...... 11 DNA extraction, amplification & sequencing ...... 12 Phylogenetic analyses ...... 12 Genetic species delimitations ...... 13 Results ...... 15 Phylogenetic analyses ...... 15 Genetic species delimitation ...... 15 Discussion ...... 18 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia...... 22 Introduction ...... 22 Materials & Methods ...... 25 Sample collection ...... 25 DNA extraction, amplification & sequencing ...... 26 Phylogenetic analyses ...... 26 Genetic species delimitations ...... 27 Results ...... 29 Phylogenetic analyses ...... 32 Genetic species delimitation ...... 29

vi The radiation and biogeography of snakes across south-eastern Africa with respect to the evolution of the savanna biome

Discussion ...... 32 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae) ...... 35 Introduction ...... 35 Material and Methods ...... 37 Species background ...... 37 Lineage divergence estimates ...... 38 Ancestral habitat state reconstruction ...... 39 Results ...... 41 Discussion ...... 44 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis ...... 47 Introduction ...... 47 Materials and Methods ...... 51 Ancestral climatic state reconstruction ...... 51 Species distribution modelling ...... 51 Results ...... 53 Ancestral climatic state reconstruction ...... 53 Species distribution modelling ...... 55 Discussion ...... 57 Chapter Six General conclusion and summary of dissertation ...... 60 Taxonomic considerations ...... 60 Philothamnus ...... 60 Philothamnus dorsalis and P. girardi ...... 61 Philothamnus carinatus ...... 61 Philothamnus semivariegatus ...... 62 Philothamnus natalensis ...... 62 Phliothamnus nitidus ...... 62 Crotaphopeltis ...... 63 Crotaphopeltis tornieri ...... 63 Radiation patterns within Crotaphopeltis and Philothamnus ...... 63 Directions for future research ...... 64 Cranial and feeding adaptations ...... 64

vii The radiation and biogeography of snakes across south-eastern Africa with respect to the evolution of the savanna biome

Functional morphology for climbing behaviour ...... 65 References ...... 66

viii The radiation and biogeography of snakes across south-eastern Africa with respect to the evolution of the savanna biome

List of Figures

Figure 1.1. Hypothetical speciation process. Different criteria (left), which fulfil different species concepts (right), accrue during the speciation process. Thus, alternative biological evidence will lead to varying conclusions in species recognition, hence a more general approach should take all available evidence into account (reproduced from de Queiroz, 1998; 2007) ...... 2

Figure 1.2. Time-calibrated phylogeny of Colubridae and its sister families, Lamprophiidae+ Elapidae, adapted from Zheng & Wiens (2016)...... 6

Figure 2.1. Sampling localities throughout sub-Saharan Africa for 12 species and four subspecies of Philothamnus included in this study...... 11

Figure 2.2. Maximum Likelihood topology for the total evidence DNA data (16S rRNA, cyt–b, ND4, c–mos and RAG1) for Philothamnus. Support values for both posterior probability (> 0.95 pp) and bootstrap (> 70%) are indicated by half black circles (BI on the left, ML on the right) at the nodes. Colours match symbol colours in Figure 2.1. Inferred species are indicated by vertical side bars: white (bGMYC), grey (mPTP), and black (barcoding)...... 16

Figure 2.3. Frequency distribution of intraspecific (black bars) and interspecific (white bars) sequence divergence amongst species and subspecies of Philothamnus for 16S, with the barcoding gap in grey (transition between intra- and inter-specific divergence). Numbers: 1-3 indicate species that show marginal interspecific pairwise sequence divergence. Numbers: 4-6 indicate species and subspecies that show intraspecific pairwise sequence divergence ...... 17

Figure 3.1. Sampling localities throughout sub-Saharan Africa for four species of Crotaphopeltis investigated during this study. Samples of C. hotamboeia are shown as circles...... 25

Figure 3.2. Maximum Likelihood topology for the total evidence DNA data (16S rRNA, cyt–b, ND4, c–mos and RAG1) for Crotaphopeltis. Support values for both posterior probability (> 0.95 pp) and bootstrap (> 70%) are indicated by half black circles (BI on the left, ML on the right) at the nodes. Colours match symbol colours in Figure 3.1. Inferred species are indicated by vertical side bars: white (bGMYC), grey (mPTP), and black (barcoding)...... 30

Figure 3.3. Frequency distribution of intraspecific (black bars) and interspecific (white bars) sequence divergence amongst species of Crotaphopeltis for cyt – b, with the barcoding gap in Grey (transition between intra- and inter-specific divergence). Black bars on the right of the frequency distribution represent the sequence divergence between C. tornieri 1 and C. tornieri 2 and are within the interspecific range (arrow)...... 31

ix The radiation and biogeography of snakes across south-eastern Africa with respect to the evolution of the savanna biome

Figure 4.1. Habitat classification for genetic samples of Crotaphopeltis and Philothamnus species. The map shows habitat types of Africa, adapted from the Terrestrial Ecoregions of the World (Olson et al., 2001). Closed habitat (forest classes) is indicated by grey shades. Open habitat (including savanna) is shaded white ...... 40

Figure 4.2. Chronograms for Crotaphopeltis and Philothamnus, with support values from the BEAST analysis of the unconstrained phylogeny (Appendix A4). Supported nodes (≥0.95) posterior probability) are indicated by black dots. Within Philothamnus, clade 1 denotes a group of species that comprise of predominantly forest species in the northern Afrotropical realm, and clade 2 a group of species that comprise open habitat specialists in southern Africa, as well as widespread habitat generalists in the rest of sub-Sharan Africa. Habitat reconstructions and terminal taxa are colour-coded according habitat classes; green (closed habitat), brown (open habitat) and green-orange gradient (both open and closed habitats), with likelihood values for each state (Table 4.1) shown as a proportion in the pie charts at each node...... 42

Figure 5.1. Chronograms and ancestral climatic state reconstruction for a) Crotaphopeltis hotamboeia and b) Philothamnus angolensis. Tree terminals are colour-coded according to the Köppen-Geiger climatic classifications. Black symbols (tree terminals and map) distinguish intraspecific clades within each species. Likelihood values for each reconstructed Köppen- Geiger climate class are represented by pie charts at each numbered node. The map presents the recent Köppen-Geiger climate classes: Af (Equatorial climate), Am (Monsoon climate), Aw (tropical savanna climate), BSh (Warm semi-arid climate), BSk (Cold semi-arid climate), BWh (warm desert climate), Cfb (temperate oceanic climate), Csb (temperate mediterranean climate), Cwa (humid subtropical climate and Cwb, humid subtropical climate/subtropical oceanic highland climate), adapted from Peel et al. (2007). Time scale in millions of years (Ma)...... 54

Figure 5.2. Projected current presence-absence maps a) Crotaphopeltis hotamboeia and b) Philothamnus angolensis. Black circles indicate locality records, and grey circles denote locality records for genetic samples. Grey shading indicate the predicted presence of the species in climatically suitable areas...... 55

Figure 5.3. Projected presence-absence maps for a) Crotaphopeltis hotamboeia and b) Philothamnus angolensis through the climatic extremes of the Pleistocene period. Maps show the sum of presence-absence maps of the LIG and the LGM. White areas indicate unsuitable climate during both periods, light grey indicate areas with low suitability, i.e. suitable in one of

x The radiation and biogeography of snakes across south-eastern Africa with respect to the evolution of the savanna biome

the two periods, and dark grey indicate areas of high climate suitability, i.e. suitable at both periods...... 56

Appendix A1. Maximum Likelihood topology for the total evidence DNA data (16S rRNA, cyt–b, ND4, c–mos and RAG1) for Philothamnus labelled with all samples from 14 countries. Support values for both posterior probability (> 0.95 pp) and bootstrap (> 70%) are indicated by half black circles (BI on the left, ML on the right) at the nodes...... 74

Appendix A2. Maximum likelihood topology for constrained relationship of Hapsidophrys as sister to (Philothamnus natalensis + P. hoplogaster)...... 75

Appendix A3. Maximum Likelihood topology for the total evidence DNA data (16S rRNA, cyt–b, ND4, c–mos and RAG1) for Crotaphopeltis labelled with all samples from 13 countries. Support values for both posterior probability (> 0.95 pp) and bootstrap (> 70%) are indicated by half black circles (BI on the left, ML on the right) at the nodes...... 76

Appendix A4. Time calibrated Bayesian Inference tree for Crotaphopeltis and Philothamnus species. The tree was inferred from the concatenated dataset of cyt–b, ND4 and c–mos gene fragments, based on three calibration points (see Material and Methods). Mean age estimates are provided near the nodes with bars indicating the 95% highest posterior densities (HPD). Black circles denote posterior probability values ≥ 0.95. Sample codes correlate to specimens in Appendix B4...... 77

Appendix A5. Projected presence-absence distribution maps for a) Crotaphopeltis hotamboeia and b) Philothamnus angolensis during the Last Intergalcial, and c) Crotaphopeltis hotamboeia, d) Philothamnus angolensis during the Last Glacial Maximum...... 78

xi The radiation and biogeography of snakes across south-eastern Africa with respect to the evolution of the savanna biome

List of Tables

Table 4.1. Results from ancestral habitat reconstruction for Crotaphopeltis and Philothamnus. Likelihood values are indicated for closed and open habitat states at the respective nodes. Node numbers refer to Figure 4.2...... 43

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Chapter One: General introduction

Earth and life evolve together (Croizat, 1958), thus patterns of biotic distribution and the connection between biotas are indisputable outputs of the evolutionary process (Santos & Amorim, 2007). That is to say, the natural world has an inherent hierarchy provided by descent with modification, and such biological hierarchical patterns can be used to recognise historical spatial relationships (Santos & Amorim, 2007). Species phylogenies are essentially biological hierarchical patterns resulting from descent with modification through time (Hennig, 1966). Therefore, genealogical relationships in phylogenies can reveal the relationships between areas in which species occur (Ficetola et al., 2017). As such, inaccurate species classification hamper our understanding of evolutionary relationships, which becomes a major constraint for the reliable recovery of biogeographical history (Santos & Amorim, 2007).

Species concepts and species delimitations

Although “species” is one of the most fundamental units in biology, the definition of the term is still controversial (Hausdorf, 2011). Numerous definitions, i.e. species concepts, have been proposed each specifying conditions that are necessary and sufficient to identify a group of individuals as a species, i.e. species criteria. Some of the basic species concepts are for example: biological species (reproductively isolated sexual species), ecological species (populations that are adapted to certain ecological niches), evolutionary species (evolving lineages), genetic species (common gene pool), and morphological species (defined by their form, or phenotypes) (Wilkins, 2011). In total more than twenty species concepts exist, of which many are not mutually exclusive in their species-recognition criteria (Hausdorf, 2011; Wilkins, 2011; Carstens et al., 2013). In fact, de Queiroz (1998) noted that species concepts often refer to stages of speciation (Fig. 1.1), and therefore an application of several different species criteria was warranted for species identification. For that reason, the General Lineage Concept (GLC) was proposed as a means to use traditional species criteria (e.g. morphological differences, monophyly or reproductive isolation) collectively in species identification. The GLC is further explained by the fact that each of the recognised species concepts directly or indirectly infer that species are “separately evolving (segments of) metapopulation lineages” (de Queiroz, 1998). The GLC has been widely adopted in recent approaches to species delimitation (Carstens et al., 2013).

1 Chapter One: General introduction

Under the GLC, phylogenies can be used to identify cryptic taxa that are morphologically and ecologically similar or alternatively identify species that show morphological and ecological variation across large geographical scales (for e.g. Bryson et al., 2007; Glaw et al., 2013; Mezzasalma et al., 2015). Amongst the available methods for phylogenetic species delimitation, distance and tree-based applications are frequently used. The distance-based methods, for example, DNA-barcoding applications, considers an a priori nucleotide distance threshold, below which specimens are considered conspecific, and above which they are considered to belong to different species (Fontaneto et al., 2015). The basis for this method is that intraspecific and interspecific variations do not overlap, which is known as the “barcode gap”. The barcode gap presumably exists because individuals of a given species are genetically more similar than individuals from different species. A sequence divergence threshold is therefore placed at the barcode-gap that can be used to delineate species (Lefébure et al., 2006). However, distance-based methods have been criticised, because they do not account for the evolutionary process. Moreover, sequence divergence thresholds are dependent on the genetic variation incorporated for the given taxon. Therefore intra- and interspecific thresholds may shift, given the completeness of the dataset under investigation; as it relies on an observable ‘barcoding-gap’ (Meyer & Paulay, 2005; Wiemers & Fiedler, 2007; Meier et al., 2008).

2 Chapter One: General introduction

Several different applications are available for phylogenetic tree-based species delimitation methods, that use either single-locus (e.g. Reid & Carstens, 2012; Kapli et al., 2016) or multi-locus data (Yang & Rannala, 2010; Ence & Carsterns, 2011; Rannala & Yang, 2013). The most commonly used applications have been designed for single-locus datasets (Fontaneto et al., 2015), for example, applications that use coalescent theory to distinguish between population–level and species-level processes, based on the generalized mixed Yule-coalescent model (GMYC: Pons et al., 2006; Fujisawa & Barraclough, 2013). The GMYC application sets a threshold between coalescent and species-level processes in order to delineate evolutionary units (EUs) akin to species (Simpson, 1951). This approach relies on the expectation that intraspecific coalescent branching proceeds discernibly quicker than speciation, which is modelled as a Yule process. Therefore, species can be identified in a gene tree as clusters of terminals separated by longer internal branches. The GMYC application is specifically employed on an ultrametric tree (i.e. a tree with branch lengths proportional to time), where changes in branching rates indicative of a shift from species-level processes (i.e. Yule) to population-level processes (i.e. coalescent) can be used to delimit EUs (Fontaneto et al., 2015). Another commonly used application is the Poisson Tree Process model (PTP; Zhang et al., 2013), which also uses coalescent theory to distinguish between population-level and species-level processes. It assumes that intraspecific and interspecific substitutions follow two distinct Poison processes, and that intraspecific substitutions are discernibly fewer than interspecific substitutions because they have less time to accumulate. This method uses substitutions directly to represent time rather than via a method that corrects for rate variation, such as GMYC (Fontaneto et al., 2015). Certain tree-based applications are however unable to properly delineate species on species-poor datasets, whereas other applications produce reliable results (e.g. Bayesian GMYC (bGMYC: Reid & Carstens, 2012) on datasets made up of a very few number of species (Flot, 2015). Single-locus species delimitation, in turn, has been criticised for not being able to account for non-monophyletic species, discordance in gene trees, incomplete lineage sorting, gene flow after divergence, and other confounding factors that may create problems in single-locus molecular species delimitations (Fujita et al., 2012; Fontaneto et al., 2015). While multi-locus molecular species delimitations could be more informative than single-locus approaches, the importance of integrating these applications with ecological as well as historical biogeography has been highlighted (Pelletier et al., 2015; Flot, 2015).

In general, each type of species delimitation method has caveats and pitfalls, and care should be taken when interpreting the results (Carstens et al., 2013; Fontaneto et al., 2015; Flot, 2015). The use of multiple delimitation approaches together with other lines of evidence (e.g. morphology, ecology, behaviour, geography, historical biogeography) allows for more defensible conclusions than would

3 Chapter One: General introduction any one method (Carstens et al., 2013; Fontaneto et al., 2015; Puillandre et al., 2012; Pante et al., 2015; Padial et al., 2010; Schlick-Steiner et al., 2010).

Systematic comparisons and inferences through time and space

The ultimate goal in historical biogeography (Ball, 1975; Crisp et al., 2011) is to study the systematics and geographical variation of species through time, in an attempt to identify possible causes and consequences of species radiations (sensu Gittenberger (1991). Therefore, historical biography serves as the interface among systematics, geography, geology and ecology (Crovello, 1981). Consequently, the progression of this discipline has been influenced by the developing paradigms of these related fields (Posadas et al., 2006). Specific advances include the emergence of plate tectonics as the dominant paradigm in geosciences. Moreover, the development of molecular phylogenetic systematics (Posadas et al., 2006) allows for robust time-calibrated phylogenies (e.g. Drummond & Rambaut, 2007) and reconstruction of character states that leave little or no trace in the fossil record (e.g. Maddison & Maddison, 2015). Additionally, geographic information systems (GIS) for spatial analyses (Rosauer et al., 2009) and predictive models of species geographic distributions (e.g. Phillips & Dudík, 2009) has had major impacts on the growing field of biogeography.

Furthermore, available global databases for distributional information (e.g. the Global Biodiversity Information Facility, 2017, available from http://gbif.org/), climatic data (e.g. WorldClim- Global Climate Data, Hijmans et al., 2005), vegetation classifications (e.g. Terrestrial Ecoregions of the World, Olson et al., 2001), and the relation between climate and vegetation classifications (e.g. the Köppen-Geiger Climatic Zones, Peel et al., 2007) have made data assimilation more convenient for biogeographical investigations. In combination, these accessible data sources and freely available software for data analyses makes for an effective integration of different research fields and rigorous hypothesis testing in biogeography.

4 Chapter One: General introduction

Hypothesis testing in historical biogeography

In biogeography, historical events can neither be observed directly nor manipulated experimentally. These limitations of biogeographical investigations have been used to justify a research approach of “pattern before process” (Crisp et al., 2011). This research approach necessitates initial observation and analyses of present-day patterns, followed by explanations in terms of historical processes (Andersson, 1996; Humphries, 2004). For example, biogeographical methods that are typically employed to describe a sequence of distributional change through time and subsequently find correlates between the distributional changes, that are inferred as causative (Crips et al., 2011). However, this approach lacks rigour, given that indefinite explanations can be proposed for a specific set of observations (Popper, 1959; Ball, 1975; McDowall, 2004; Waters, Craw & Paterson, 2006). The pattern before process approach has been criticised as storytelling and unscientific, because it tends to generate, rather than test hypotheses (Ball, 1975; Humphries, 2004). Restrictive propositions and testing specific predictions (e.g. pattern or timing) that can rule out other alternatives are proposed instead (Ball, 1975; Penny & Phillips, 2004; Crisp et al., 2011). At the same time, hypotheses should be tested with independent data to avoid circularity (Penny & Phillips, 2004; Waters, Craw & Paterson, 2006).

The radiation and biogeography of Crotaphopeltis and Philothamnus species across sub-Saharan Africa with respect to the evolution of the savanna biome

Research rationale

Biogeographical studies of African reptile taxa shows that the historical climate and tectonic events that are associated with environmental changes, played a key role in lineage diversification of African reptile taxa, especially during the Miocene period (Wüster et al., 2007; Tolley et al., 2008; Ceccarelli et al., 2014; Menegon et al., 2014; Dowell et al., 2016; Medina et al., 2016). Climatic and environmental changes during the Miocene affected different regions across the African continent at different stages and resulted in more heterogeneous habitats across the African landscape (e.g. Axelrod & Raven, 1978; Hewitt, 2004; Senut et al., 2009), and possibly contributed to a noticeable increase of diversity of African reptiles. The heterogeneous landscape of the African continent was preceded by a pan-African forest that has been transformed by concomitant contractions and expansions of these biomes since the Oligocene (Bobe, 2006; Couvreur et al., 2008; Kissling et al., 2012), to primarily savanna at present (Edwards et al., 2010).

5 Chapter One: General introduction

The progression, therefore, has been from a continent dominated by closed forest habitats, toward open habitat. Accordingly, it is expected that African reptile genera that emerged prior to the Miocene period and have species distributed in both forest and savanna habitat should show a genetic signature of the possible evolutionary impact of these biome developments. The Colubridae appear to have diverged from their sister clade, the Elapoidea (Elapidae+Lamprophiidae, Kelly et al., 2009), during the Eocene (Fig. 1.2), suggesting that genera within Colubridae are younger than 50 Mya (Zheng & Wiens, 2016). Therefore, African snake genera within the Colubridae family could have diverged from their forest relatives and radiated into open habitat since the Oligocene/Miocene transition, with the emergence of the savanna biome. Two specific African colubrid snake genera, Crotaphopeltis and Philothamnus are ideal candidate taxa to investigate the evolutionary impact of the forest-savanna developments on widespread African colubrid snakes. Species in these two genera are distributed throughout most parts of Sub-Saharan Africa and comprise species that are associated with either closed or open habitats, as well as species that are widespread throughout both habitat types (Loveridge, 1958; Broadley, 1983; Hughes, 1985; Rasmussen, 1993, 1997; 2000).

Figure 1.2. Time-calibrated phylogeny of Colubridae and its sister families, Lamprophiidae + Elapidae, adapted from Zheng & Wiens (2016). Overall, I hypothesised that that lineage diversification amongst species within Crotaphopeltis and Philothamnus correspond to the documented contraction and expansions events of the African forest

6 Chapter One: General introduction and savanna biomes since the Oligocene period. Furthermore, I anticipated that savanna specialist and generalist species of both genera recently radiated into open habitats.

As a research approach, Crotaphopeltis and Philothamnus species were placed in a phylogenetic framework to ensure reliable recovery of species radiation patterns and timing within these two genera. Species boundaries and the presence of cryptic taxa were examined within each of the genera. The identified evolutionary lineages were used in subsequent biogeographical analyses of divergence estimations to infer if timing of lineage diversification corresponds with concomitant contraction and expansion events of the forest and savanna biomes. The ancestral habitat character state, i.e. closed habitat (forest) or open habitat (e.g. savanna) was additionally reconstructed for each snake genus. Ancestral habitat reconstruction was used to investigate if Crotaphopeltis and Philothamnus had an ancestral closed habitat preference and recently radiated into open habitat. Subsequently, I investigated how the historical environmental conditions influenced the evolution of the climatic niche and distribution patterns of wide-ranging species in each genus (Crotaphopeltis hotamboeia and Philothamnus angolensis).

7 Chapter One: General introduction

Research aim and objectives

Overall, the main research aim was to investigate synchronised timing and patterns of radiation for Crotaphopeltis and Philothamnus in response to the concomitant development of the African forest and savanna biomes. This required examination of phylogenetic relationships within both genera to investigate the existence of possible cryptic taxa that may affect the reconstruction of biogeographic patterns.

These studies are presented in the following chapters.

Overarching objective: Chapter Two

Described species of Philothamnus were investigated for their validity under the Evolutionary Species Concept with an aim to identify cryptic species that are otherwise masked by phenotypic and/or ecological parallels.

Overarching objective: Chapter Three

The phylogenetic relationships between Crotaphopeltis species were investigated, with a special focus on C. hotamboeia, to evaluate if this widespread taxon is a valid species under the Evolutionary Species Concept.

Overarching objective: Chapter Four

Timing of lineage divergence within Crotaphopeltis and Philothamnus were investigated with respect to the historical contraction and expansion events of the African forest and savanna biomes. Additionally, the ancestral habitat state, i.e. closed (forest) or open (e.g. savanna) were reconstructed for each genus to infer if species recently radiated into open habitat.

Overarching objective: Chapter Five

The influence of historical environmental conditions on the evolution of the climatic niche of C. hotamboeia and P. angolensis were studied. The sub-Saharan distributions of these species were modelled for contrasting climatic conditions and their ancestral climatic preference were reconstructed.

Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

Introduction

Colubridae, with 1876 species (Uetz & Hošek, 2017), is the most specious snake family globally and is found on every continent, except Antarctica (Cogger et al., 1998). The monophyly of the family and respective subfamilies, as well as intergeneric relationships within subfamilies however, has been called into question (for e.g. Lawson et al., 2005; Vidal et al., 2007; Pyron et al., 2011; 2013; 2014; Chen et al., 2013; Zheng & Wiens 2016). Characteristically, traditional taxonomy is dominated by the morphological species concept, even though numerous other species concepts exist. Over the last past decades, molecular phylogenetic methods have proven to be practical for identifying independent evolutionary lineages, thus satisfying the General Lineage Concept, which encompasses several species concepts (such as the Evolutionary Species Concept, Queiroz, 1998; Fujita et al., 2012). Under the Evolutionary Species Concept, phylogenies can be used to identify cryptic taxa that are morphologically and ecologically similar or alternatively identify species that show morphological and ecological variation across large geographical scales. Several studies provide evidence of masked genetic divergence linked to taxonomic discrepancies for several snake taxa and highlight the necessity of comprehensive molecular phylogenetic studies to validate species and further uncover cryptic diversity, thus targeting more holistic approaches for taxonomic revisions (for e.g. Bryson et al., 2007; Glaw et al., 2013; Mezzasalma et al., 2015 ).

The African green and bush snakes of the genus Philothamnus are widely distributed across the entire sub-Saharan region. This species-rich genus is comprised of 20 species and four subspecies (Hughes, 1985; Trape & Roux-Esteve, 1990; Chippaux 2006; Wallach 2014; Trape & Baldé 2014). Members of this genus are predominantly arboreal, slender, with elongated distinct heads, proportionally large eyes and feed mainly on frogs and lizards. A third of the species in the genus are considered habitat specialists, occurring predominantly in either forest or savanna habitats, where most of them have relatively small distributions (e.g. P. girardi, P. carinatus, P. thomensis, P. ruandae, P. n. nitidus, P. n. loveridgei and, P. ornatus). The remainder of species are generalists occurring in both forest and savanna biomes and most have wide distributions, e.g. P. dorsalis, P. n. occidentalis, P. hoplogaster, P. angolensis and P. semivariegatus, the latter two with almost sub-Saharan distributions (Wallach 2014).

9 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

The Green and Bush snakes share a number of key morphological features, for e.g. lepidosis, dentition, ventral keeling, pigmentation, and have subtle differences in their habitus and ecology. The last revision of the genus (Loveridge 1958) and subsequent amendments have been primarily based on traditional morphological characters, although the considerable morphological overlap between species has resulted in taxonomic confusion. For example, prior to Hughes (1985) P. ruandae and P. carinatus were treated as subspecies of P. heterodermus, and P. dorsalis, P. girardi, P. thomensis and P. nitidus as subspecies of P. semivariegatus (Loveridge 1958). Similarly, P. punctatus was easily confused with P. semivariegatus (e.g. Barbour & Loveridge, 1928; Bogert, 1940; Loveridge, 1951; Mertens, 1955, Laurent1956; 1960), but was later demonstrated to have a closer resemblance to P. nitidus loveridgei, and thought to form part of the latter species complex (Hughes, 1985). In contrast, subspecies of P. natalensis (P. n. natalensis and P. n. occidentalis) and P. nitidus (P. n. nitidus and P. n. loveridgei) are morphologically and ecologically distinct and genetic divergence may confirm their specific status. Trape & Baldé (2014) have recently revived West African P. belli and P. pobeguini from the synonymy of P. heterodermus.

Few Philothamnus species have been included in higher-level phylogenies, and only one study focused on species occurring on islands in the Gulf of Guinea (Jesus et al., 2009). In all phylogenetic studies which included Philothamnus, few mainland species were represented. These studies showed discordant results for interspecific relationships and the monophyly of Philothamnus (Jesus et al., 2009; Pyron et al., 2013; Tonini et al., 2016; Zheng & Wiens, 2016). The phylogenetic placement of Hapsidophrys as the sister taxon of Philothamnus, as opposed to Hapsidophrys rendering Philothamnus paraphyletic, has been contested (Pyron et al., 2013; Zheng & Wiens, 2016), and resolution may require more inclusive taxon and intraspecific sampling for Philothamnus. Here, I investigated phylogenetic relationships within Philothamnus with improved taxon sampling at the species level as well as multiple samples within species with wide geographical sampling. The aim was to investigate if the present morphologically-defined species are supported in a phylogenetic context, and whether cryptic putative species exist and are masked by phenotypic and/or ecological parallels or variation. I hypothesise that Philothamnus is monophyletic with respect to Hapsidophrys, and that the currently recognised morphological species of Philothamnus are valid under coalescent- based species delimitation. Moreover, I hypothesise that the widespread generalist taxa P. angolensis and P. semivariegatus comprise multiple cryptic evolutionary lineages.

10 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

Materials & Methods

Sample collection

Phylogenetic analyses of Philothamnus species were carried out on samples from 14 countries, and island populations (Inhaca, Mozambique; São Tome and Annobón, Gulf of Guinea; Fig. 2.1 and Appendix B1). To test the monophyly of Philothamnus species of Hapsidophrys were included. Taxonomic sampling included 119 individuals from 12 species and four subspecies (P. angolensis, P. carinatus, P. dorsalis, P. heterodermus, P. hoplogaster, P. macrops, P. natalensis natalensis, P. natalensis occidentalis, P. nitidus nitidus, P. nitidus loveridgei, P. ornatus, P. punctatus, P. ruandae and P. semivariegatus, H. lineatus, H. smaragdinus). Sixteen additional sequences were acquired from GenBank, which included two additional Philothamnus species (P. girardi and P. thomensis) as well as H. principis (Appendix B1). Samples of P. belli, P. bequaerti, P. battersbyi, P. heterolepidotus, P. hughesi, P. irregularis, P. pobeguini and P. smithi were not available, thus a total of 12 species and four subspecies out of the 24 currently recognised Philothamnus species and subspecies were included. Macroprotodon cucullatus, Dispholidus typus, Thelotornis kirtlandii and Thrasops jacksonii were included as outgroup taxa of which, the latter two were additionally sequenced and the former downloaded from Genbank.

Figure 2.1. Sampling localities throughout sub-Saharan Africa for 12 species and four subspecies of Philothamnus included in this study.

11 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

DNA extraction, amplification & sequencing

A total of three mitochondrial and two nuclear markers were used in this study. The mitochondrial markers were: 16S ribosomal RNA (16S: 16Sa and 16Sb, Palumbi, 1996), protein-coding cytochrome b (cyt–b: CB3, L14910, and H16064, Palumbi, 1996; Burbrink et al., 2000) and nicotinamide adenine dehydrogenase subunit four (ND4: NADH4 and HIS12763V, Arevalo et al., 1994; Pook et al., 2009). Nuclear markers used were: oocyte maturation factor Mos (c – mos: S77 and S78, Lawson et al., 2005) and recombination-activating gene one (RAG1: R13 and R18, Groth & Barrowclough 1999). Genomic DNA was isolated from ethanol preserved tail, liver and muscle tissue, using a salt extraction protocol (Aljanabi & Martinez, 1997). Gene fragments were amplified in 25 µL reaction volumes containing 30-60 ng/µL genomic DNA, 10 x thermophilic buffer (20 mM Tris-HCL (pH

8.0), 100 Mm NaCl, 0.1 mM EDTA, 1 Mm DTT), 2.5 mM MgCl2, 0.25 µM primer, 0.2 mM dNTPs, and 0.2 U Super-Therm Taq DNA polymerase. The polymerase chain reaction (PCR) profile was 95 ˚C for 4 mins, followed by 38 cycles of 94 ˚C for 45 s, 48 – 60 ˚C for 45 s, 72 ˚C for 1min, 72 ˚C for 10 min, although annealing temperatures varied; 48 ˚C for 16S, 50 ˚C for cyt–b, 52 ˚C for ND4, 55 – 60 ˚C for c– mos and 62˚C for RAG1. Amplified PCR products were sequenced at Macrogen Inc. (Seoul, Korea).

Phylogenetic analyses

Sequences were aligned in Geneious v. 4. 8. 5 (Kearse et al., 2012) and screened for base ambiguities and stop codons (in coding genes). A total of 106 individuals were sequenced for 16S (492 bp), 58 individuals and for cyt–b 623 bp), 103 individuals for ND4 (650 bp) and tRNA (25 bp), 93 individuals for c–mos (584 bp) and 55 individuals for RAG1 (961 bp) (Appendix B1 The ribosomal 16S was independently aligned in Block Mapping and Gathering with Entropy alignments Mobyle portal (BMGE: http://mobyle.pasteur.fr/cgi-bin/portal.py) to remove ambiguously aligned regions (Criscuolo & Gribaldo, 2010). PartitionFinder was used to identify the best fit dataset partitioning scheme and accompanying substitutional models (Lanfear et al., 2014). A user defined search was conducted for pre-defined partitions into firstly genes, and secondly 16S and by codon position for the four remaining genes with pos 1 + pos2, pos 3 and the trRNA that follows ND4. As a third option, a greedy search was conducted on 14 data blocks that included 16S separately, trRNA separately, and all codon positions for the remaining four genes separately. Settings applied for both AIC and BIC criteria were: branch lengths linked and unlinked, searching for all models. The AIC selection criteria were recovered as the best, with branch lengths linked for the codon (pos 1 + pos2 and pos 3) partitioning scheme.

12 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

Phylogenetic trees were constructed in both Maximum Likelihood (ML) and Bayesian Inference (BI) frameworks (RAxML-VI-HPC and v. 3.2 MrBayes; Stamatakis 2006; Ronquist et al., 2012) for two datasets: mitochondrial only, and mitochondrial and nuclear genomes combined. Both analyses were run at the CIPRES Science Gateway v. 3.3 (Miller et al., 2010). For Bayesian Inference, four Markov chains were run in parallel, with each chain starting from random trees, running for 10 million generations, sampling each tree every 1,000 generations. The analyses were run according to the substitution models and partitioning scheme inferred by PartitionFinder: 16s rRNA (GTR + I+ G), cyt–b pos1 + pos2(TRN + I +G), cyt- b pos 3 (GTR + I+ G), ND4 pos1 + pos2(GTR + I +G), ND4 pos 3 (GTR + G), ND4 tRNA (SYM + I + G), cmos pos1 + pos2 (HKY + I +G), cmos pos 3 (HKY + I +G), RAG1 pos1 + pos2(TrN + I) and RAG1 pos 3 (TVM + I +G). Ten percent of the trees were discarded as burn-in, determined using Tracer v 1.6 (Rambaut et al., 2014) by examining the number of generations at which the effective samples size exceeded 200 for all parameters. A 50% majority- rule consensus tree was generated from the trees retained and nodes with posterior probabilities (pp) ≥ 0.95 were considered well supported. A partitioned Maximum Likelihood analysis was run with the same partition as Bayesian Inference, with the GTR + G model of evolution as implemented in RAxML and automatic halting bootstrapping. Bootstrap values ≥ 70 % were considered well supported (Wilcox et al., 2002). Competing phylogenetic hypotheses of monophyly for Philothamnus relative to Hapsidophrys were investigated by conducting a constrained RAxML analysis with Hapsidophrys spp. (Hapsidophrys lineatus, H. principis and H. smaragdinus) sister to P. natalensis + P. hoplogaster as previously found by Pyron et al. (2013) and Zheng & Wiens (2016), followed by a Shimodaira–Hasegawa (SH) test (Shimodaira & Hasegawa, 1999) conducted in phangorn package in R (Schliep, 2011). The S-H test was used to test which tree topologies were significantly more likely (p >0.05), with and without optimizing the rate matric and base frequencies.

Genetic species delimitations

To investigate putative taxonomic units within Philothamnus three species delimitation methods of which two were coalescent-based species delimitation methods, 1) Bayesian General Mixed Yule- Coalescent Model bGMYC), 2) a multi-rate Poisson Tree Processes (mPTP) and 3) a distance-based method barcoding approach were used. Bayesian General Mixed Yule-Coalescent Model (bGMYC) was implemented in R using the package bGMYC v. 3.0.1(Reid & Carstens, 2012; R Core Team 2013). The traditional GMYC employs speciation and neutral coalescent models and maximizes likelihood score by identifying the transition point between intraspecific (coalescent) and interspecific (speciational/divergence) branching processes on an ultrametric tree derived from a single locus (Yule, 1925; Hudson, 1990; Pons et al., 2006). The bGMYC overcomes two limitations of GMYC,

13 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae) in that the Bayesian implementation provide flexible prior distributions and accounts for error in phylogenetic estimation and uncertainty in model parameters by integrating over uncertainty in tree topology and branch lengths in parameters of the model via MCMC. Bayesian GMYC was conducted with the single tree method for 10, 000 generations, discarding the first 1000 trees as burn-in and sampling every 100 generations. The best ML tree from RAxML was converted to an ultrametric tree, using package APE in R (Paradis et al., 2004).

The multi-rate Poisson Tree Processes (mPTP) was conducted using the web server at http://mptp.h- its.org/#/tree. Compared to GMYC the Poisson Tree Processes (PTP) models the branching processes based on the number of accumulated expected substitutions between subsequent speciation events, determining the transition point from within and between species by assuming a two parameter model one parameter for speciation and a second parameter for the coalescent process that best fits the data. In comparison, mPTP incorporates different levels of intraspecific genetic diversity deriving from differences in either the evolutionary history or sampling of each species. Additionally, the mPTP fits the branching events of each species to a distinct exponential distribution, instead of one exponential distribution across all species (Kapli et al., 2017). The total evidence (mtDNA + nuDNA) ML tree was used for the two coalescent species delimitation methods (bGMYC and mPTP).

Distance-based methods, such as DNA-barcoding applications, considers a nucleotide distance threshold below which specimens are considered conspecific and above which they are considered to belong to different species (Fontaneto et al., 2015). The basis for this method is that intraspecific and interspecific variations should not overlap, which is known as the “barcode gap”. The barcode gap presumably exists because individuals of a given species are genetically more similar than individuals from different species. A sequence divergence threshold is therefore placed at the barcode-gap that can be used to delineate species (Lefébure et al., 2006). SpeciesIdentifier v1.8 (Meier et al., 2006) was used to compare frequency distributions between intra- and interspecific sequence divergence. Sequence divergence thresholds are dependent on the genetic variation incorporated for the given taxon and markers, therefore intra- and interspecific thresholds will shift given the taxon and dataset under investigation (Meyer & Paulay, 2005; Wiemers & Fiedler, 2007; Meier et al., 2008). Thus, the barcoding analysis was carried out on the 16S dataset as this was the most complete dataset.

14 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

Results

Phylogenetic analyses

A total of 3252 bp from four mitochondrial and two nuclear markers were suitable for analysis. Both phylogenetic methods (BI, ML) recovered identical topologies for both mtDNA and total evidence datasets (Fig. 2.2). The monophyly of Philothamnus was well supported (100 % ML bootstrap and 1.00 Bayesian pp) relative to Macroprotodon cucullatus, Thrasops jacksonii, Thelotornis kirtlandii, Dispholidus typus, Hapsidophrys lineatus, H. principis and H. smaragdinus. The Shimodaira- Hasegawa test indicates that the constrained topology (Hapsidophrys spp. sister to P. natalensis + P. hoplogaster, Appendix A2) is rejected, and produced a significantly worse fit (p <0.05) when compared with the unconstrained topology (Table 2.1). Philothamnus semivariegatus was found to be paraphyletic with four clades spanning central Africa (P. semivariegatus 4), southeastern Africa (P. semivariegatus 3), northeastern South Africa (P. semivariegatus 2) and central-northern South Africa, (P. semivariegatus 1), each well supported for both BI and ML (> 70 %, >0.95 pp). A total of five nodes lack support for one of two phylogenetic methods (BI, ML) and therefore sister relationships between some of the species are unresolved. The placement of P. punctatus was also unresolved (Fig. 2.2).

Genetic species delimitation

Both coalescent methods bGMYC and mPTP retrieved similar results for putative taxonomic units (Fig. 2.1, Appendix B2). There was support for seven of the described species between these two methods: P. macrops, P. hoplogaster, P. ruandae, P. heterodermus, P. ornatus, P. thomensis, and P. punctatus. Philothamnus semivariegatus comprised at least four subclades and two were identified in P. carinatus. All were sufficiently divergent to indicate the presence of cryptic species. Furthermore, the low genetic divergence between the currently recognised species P. dorsalis and P. girardi indicate that they are conspecific. The mPTP analysis suggests that the subspecies of both P. natalensis (P. n. natalensis and P. n. occidentalis) and P. nitidus (P. n. nitidus and P. n. loveridgei) constitute separate species. Similarly, mPTP analysis indicates that P. angolensis may also comprise two cryptic species, although this was not supported by bGMYC, which also considered the two subspecies of P. nitidus to be a single species. Bayesian GMYC further delimits P. n. natalensis as specifically distinct from P. natalensis occidentalis, but also indicates two additional candidate species within the latter. Despite the discrepancy between mPTP and bGMYC for species boundaries

15 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae) within the P. natalensis complex, neither of the two coalescent methods indicate that P. natalensis is a single species.

16 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

Table 2.1. Results of Shimodaira-Hasegawa test of paraphyly for Philothamnus relative to Hapsidophrys. Left: results without optimization of parameters. Right: results with optimization of parameters. Tree In L Diff In L p - value In L Diff In L p - value Unconstrained -85585.33 0.000 0.500 -80029.60 0.000 0.497 Constrained -86265.57 680.242 0.000 -80951.41 921.810 0.000

Sequence divergence for the intra- versus interspecific threshold amongst Philothamnus ranged from 3.1–3.8 % for 16S (Fig. 2.3). Divergence values within the interspecific range between eight of the described species (P. angolensis, P. nitidus, P. macrops, P. hoplogaster, P. ruandae, P. heterodermus, P. ornatus, and P. punctatus) accord to coalescent species delimitations (Appendix B2). In contrast to the findings of the two coalescent methods, P. thomensis was not specifically distinct from P. dorsalis/girardi. Interspecific sequence divergence were found between the four lineages of P. semivariegatus as well as between the two P. carinatus lineages, which were in further agreement with both coalescent methods. P. angolensis and P. nitidus were identified as two valid species, which correspond with results from bGMYC analysis. The two P. natalensis subspecies were indistinct within the intraspecific sequence divergence range.

1200 Intraspecific 1. P. carinatus 1, P. carinatus 2 Interspecific 1000 2. P. angolensis, P. nitidus, P. punctatus, P. semivariegatus 4 3. P. semivariegatus 2, P. semivariegatus 3 800 4. P. n. natalensis, P. n. occidentalis 5. P. dorsalis, P. girardi, P. thomensis 6. P. n. nitidus, P. n.loveridgei 600 3 400 2 5 6 1

Frequency pairwise differences pairwise Frequency 200 4

0.5- 1.0 0.5- 1.5- 2.0 1.5- 3.0 2.5- 4.0 3.5- 5.0 4.5- 6.0 5.5- 7.0 6.5- 8.0 7.5- 9.0 8.5-

9.5- 10.0 9.5-

10.5- 11.0 10.5- 12.0 11.5- 13.0 12.5- 14.0 13.5- 16.0 15.5- 17.0 16.5- 18.0 17.5- 19.0 18.5- 20.0 19.5- 14.49- 15.0 14.49- Sequence divergence for 16S (%)

17 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

Discussion

The phylogeny suggests there are several previously unrecognised lineages of Philothamnus, which represent up to seven candidate species, two of which are presently considered subspecies. The detection of cryptic diversity within the genus has resulted from more inclusive taxon and intraspecific sampling compared to previous investigations. In addition, Philothamnus was recovered as a monophyletic genus (Jesus et al., 2009; Tonini et al., 2015), and is not paraphyletic with respect to Hapsidophrys as previous suggested (Pyron et al., 2013; Zheng & Wiens, 2016). In agreement with previous phylogenetic investigations of the genus (Jesus et al., 2009; Pyron et al., 2013; Tonini et al., 2015; Zheng & Wiens, 2016), P. thomensis + P. girardi, P. heterodermus + sensu lato P. carinatus and P. angolensis + P. nitidus + sensu lato P. semivariegatus were consistently recovered, despite lack of resolution at basal nodes, whilst the placement of P. natalensis remains unresolved, as does that of the newly sequenced P. punctatus.

Given that coalescent species delimitation methods applied during this study account for phylogenetic uncertainty, 18 species are provisionally identified of which seven are new candidate species. A conservative approach has been adopted in which candidate species are recognised only when supported by at least two of the species delimitation methods employed (except for P. natalensis which requires special consideration: see discussion below). Of the 12 species and four subspecies included in the phylogeny, nine correspond to currently described species differentiated by diagnostic morphological characters and need not be revised. They are: P. angolensis, P. heterodermus, P. hoplogaster, P. macrops, P. nitidus, P. ornatus, P. punctatus, P. ruandae and P. thomensis. Taxonomic revision is required for the other taxa studied, including seven new candidate species: three additional candidate species for P. semivariegatus, one additional species for P. carinatus, and the subspecies P. n. natalensis and P. n. occidentalis should be elevated to full species. In contrast, there was little genetic support for the specific distinction of P. girardi from and P. dorsalis with which it should be synonymised.

There were some conflicting results between the two coalescent-based species delimitation methods for a minority of the candidate species. The methods used rely on different criteria to delineate species (Kapli et al., 2016; Reid & Carstens, 2012). The generalized mixed Yule-coalescent model (GMYC) approach relies on the expectation that intraspecific coalescent branching proceeds discernibly quicker than speciation, which is modelled as a Yule process. Therefore, species can be identified in a gene tree as clusters of terminals separated by longer internal branches. (Fontaneto et al., 2015). The Poisson Tree Process model (PTP: Zhang et al., 2013) also uses coalescent theory to distinguish

18 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae) between population-level and species-level processes, but in contrast to the GMYC approach, it assumes that intraspecific and interspecific substitutions follow two distinct Poison processes, and that intraspecific substitutions are discernibly fewer than interspecific substitutions because they have less time to accumulate. In turn, distance-based methods such as the barcoding approach do not take evolutionary relationships into account. Moreover, sequence divergence thresholds are dependent on the genetic variation incorporated for the given taxon and markers. Therefore intra- and interspecific thresholds will shift given the taxa and datasets under investigation; i.e. the method relies on an observable ‘barcoding-gap’ (Meyer & Paulay, 2005; Wiemers & Fiedler, 2007; Meier et al., 2008).

Of note, the bGMYC method suggested there are two candidate species within the subspecies P. n. occidentalis, although this was not recognized based on the other two methods. There are potentially ecological differences between these lineages, where one is restricted to the sub-escarpment savanna bioregion and the other associated with Albany thicket vegetation. However, shallow branch lengths and low sequence divergence between these two P. n. occidentalis lineages point to population level structure, rather than species boundaries. Despite the discrepancy between the two coalescent methods for species boundaries within P. natalensis, i.e. mPTP indicating two candidate species and bGMYC three candidate species, neither of the two methods suggests that only one species occurs. In contrast, the barcoding approach does suggests that P. natalensis is a single species with only 1.59- 2.45% sequence divergence which falls within the intra specific range. However, the sequence divergence threshold between the P. natalensis subspecies were likely influenced by the uneven sampling between the two subspecies (P. n. natalensis n = 2; P. n. occidentalis n = 15, i.e. number of individuals as well as geographical coverage). Similarly, uneven sampling across distinct lineages may influence coalescent methods (Bagley et al., 2015). However, mPTP incorporates different levels of intraspecific genetic diversity deriving from difference in either evolutionary history or sampling of each species. As such, in the particular case of P. natalensis, the mPTP results should be more reliable than bGMYC and distance-based methods regarding biased intraspecific sampling (Blair & Bryson, 2017). Notwithstanding, both bGMYC and mPTP indicate P. n. natalensis to be specifically distrinct from P. n. occidentalis.

In general, each type of species delimitation method has caveats and pitfalls, and care should be taken when interpreting the results (Carstens et al., 2013; Fontaneto et al., 2015; Flot, 2015). The use of multiple delimitation approaches together with other lines of evidence (e.g. morphology, ecology, behaviour and geography) allows for more defensible conclusions than would any one method (Carstens et al., 2013; Fontaneto et al., 2015; Puillandre et al., 2012; Pante et al., 2015; Padial et al.,

19 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

2010; Schlick-Steiner et al., 2010). Accordingly, I propose that the two subspecies of P. natalensis be elevated to full species.

This action is supported by both morphological and genetic evidence. Broadley (1966) based his subspecies primarily on the presence of subcaudal keeling in P. n. natalensis and its absence in P. n. occidentalis, as well as difference in the number of upper labials (P. n. natalensis usually 9, with the 5-6th entering the orbit; P. n. occidentalis usually 8, with the 4-5th entering the orbit). Differences in colouration patterns also appear to be consistent between the two candidate species (Broadley, 1966; 1983; Alexander, 1990). The distributions of the two candidate species are almost allopatric, with P. n. natalensis occurring on the coastal plain from central Mozambique to Maputoland, and P. n. occidentalis above the escarpment in Limpopo and Mpumalanga provinces, and along a narrow coastal strip through the eastern and southern Cape to the Brede River. A small region of sympatry occurs between Maputoland and Durban.

The phylogenetic tree and species delimitation methods convincingly show a number of previously unrecognised candidate species are present. The results clearly show that taxonomic revision is required for P. carinatus sensu lato and P. semivariegatus sensu lato, as well as P. dorsalis/girardi. The type locality for P. carinatus was restricted to Mapanja in south-western Cameroon (Wallach, 2014), but samples from Cameroon were not included in phylogenetic and species delimitation analyses for P. carinatus sensu lato. However, due to proximity it is likely that Cameroon individuals will be genetically similar to those from Gabon and the Republic of Congo, i.e. ‘P. carinatus 2’, and this populations is thus referable to Philothamnus carinatus (Anderson, 1901). Loveridge (1958) lists no other names in the synonymy of P. heterodermus carinatus. Although Wallach et al. (2014) and Uetz & Hošek (2017) both list Philothamnus nigrofasciatus (Buchholz & Peters 1875) in the synonymy of P. carinatus, this name is referred to P. nitidus by Bauer et al. (1995). Moreover, the type locality for P. nigrofasciatus is “Cameruns”, and is thus not available for individuals of “P. carinatus 1” for which a new name will be needed.

Two of the four candidate species for P. semivariegatus sensu lato (‘P. semivariegatus 1’ and ‘P. semivariegatus 4’) are geographically nearly equidistant from the type locality, which is Springbok in south-western South Africa (Wallach, 2014). Therefore, the inclusion of genetic material from the type locality of P. semivariegatus is needed for the taxonomic revision of P. semivariegatus sensu lato. Among names currently in synonymy Philothamnus smithii (Bocage, 1882b) may be available for ‘P. semivariegatus 4’ should ‘P. semivariegatus 1’ retain the original name.

20 Chapter Two: Species boundaries in African Green and Bush snakes, Philothamnus (Serpentes: Colubridae)

Genetic samples of P. dorsalis from Soyo, extreme northwest Angola used in the analysis are almost topotypic material, the type locality being “Molembo, Afrique occidentale,” [= Molembo, West Africa]. Emended to Molembo de la Côte de Loango au nord du Zaire fide Bocage (1882b) (= Malembo, Cabinda Prov., ext. NW Angola, 05°20’S, 12°11’E, elevation 110 m) (Wallach et al., 2014). The taxa P. dorsalis (Bocage, 1866) and P. girardi (Bocage, 1893) were shown to be conspecific during this study, and as the former is the oldest available name it therefore has taxonomic priority, for which Philothamnus girardi Bocage, 1893 is here considered a junior subjective synonym.

Overall, these proposed changes would increase the number of species in the genus to 26 species. The inclusion of the eight missing species in future phylogenetic and species delimitation investigations may reveal additional hidden diversity within the species-rich Philothamnus.

Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia

Introduction

Wide-ranging species often occupy ecologically and climatically heterogeneous habitats that have the potential to impose divergent selection pressures on different populations (Rundle & Nosil, 2005; Gavrilets & Losos, 2009; Rundell & Price, 2009). If assumed that gene expression is tailored at the population level to suit local conditions (Aubret et al., 2004), environmental differentiation can reduce homogenising gene flow and similarly correlate to neutral genetic population differentiation (Shafer & Wolf, 2013). Therefore, ecological variation within widespread species becomes important for directing and corroborating taxonomic hypotheses under the General Lineage Concept. The latter encompasses several species concepts, such as the Evolutionary Species Concept (Queiroz, 1998; Fujita et al., 2012), thus supporting more holistic criteria of species recognition. Under the Evolutionary Species Concept (Simpson, 1951; Wiley, 1980), molecular phylogenetic approaches are ideal in identifying species level genetic divergence among populations of widely distributed species, which are potentially linked to ecological and morphological differences.

The Afrotropical region accommodates several wide-ranging species in the Colubridae, a snake family that has received very little attention with regards to molecular phylogenetic systematics in relation to African landscape dynamics at broad geographical scales. Wide-ranging African representatives of the sub-family Colubrinae include 18 species in the genera Crotaphopeltis, Dasypeltis, Dipsadoboa, Dispholidus, Hapsidophrys, Meizodon, Natriciteres, Philothamnus, Rhamnophis, Teloscopus, Thrasops and Toxicodryas (Wallach, 2014; Uetz & Hošek, 2017). Representatives of these genera were included in phylogenetic studies addressing higher taxonomic classifications (e.g. Lawson et al., 2005; Vidal et al., 2007; Pyron et al., 2011; 2013; 2014; Chen et al., 2013; Zheng & Wiens 2016). The genera Dasypeltis, Dispholidus, Philothamnus, Rhamnophis and Thelotornis received focused molecular phylogenetic assessments with broad taxon sampling (Eimermacher, 2012; Trape et al., 2012; Chapter Two). For Dasypeltis, several new taxa are recognised in West Africa, all supported by genetic and morphological analysis (Trape et al., 2012). Genetic lineages for Dasypeltis spp., D. typus, as well as P. semivariegatus, appeared to be associated with vegetation types in different biomes across their distributions, whilst physical barriers also affect D. typus (Eimermacher, 2012). Phylogenetic relationships and species status within Thelotornis spp. and the widespread Rhamnophis aethiopissa remain unresolved, and require improved geographical

22 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia. coverage for resolution (Eimermacher, 2012). These findings imply that wide-ranging African colubrids may comprise cryptic lineages and therefore more comprehensive molecular phylogenetic investigations are required to better understand their taxonomy and evolutionary history.

Crotaphopeltis is a wide-ranging African colubrid genus that requires a focussed molecular phylogenetic investigation. The genus occurs throughout sub-Saharan Africa and is currently comprised of six species, of which five are relatively range restricted: C. barotseensis in south east Africa, C. braestrupi in southern Somalia and southeast Kenya, C. degeni in central Africa, C. hippocrepis in West Africa, and C. tornieri in Tanzania and northern Malawi. Crotaphopeltis hotamboeia is comparatively widespread and occurs in sympatry with all five of its congeners in different climatic regimes and associated habitats of the sub-Saharan region (Wallach, 2014; Broadley, 1962). Crotaphopeltis is poorly represented in previous phylogenetic analyses, with only two of the six species, i.e. C. tornieri and C. hotamboeia having been previously included (e.g. Gravlund 2002; Lawson et al., 2005; Pyron et al., 2013; Tonini et al., 2016; Zheng & Wiens 2016). Gravlund (2002), in particular, focused on the intraspecific relationships within C. tornieri, with C. hotamboeia included as the presumed sister taxon of C. tornieri. Crotaphopeltis tornieri was found to be paraphyletic with respect to C. hotamboeia, questioning the species status of the two taxa. From Gravlund’s (2002) findings, it is evident that the wide-ranging C. hotamboeia may comprise cryptic species, and this requires further investigation in a phylogenetic framework.

Previous taxonomic classifications for C. hotamboeia were based on morphological and geographic distributions. Additionally, taxonomic classifications of C. hotamboeia included C. tornieri as a synonym (Barbour & Amaral 1927), of which the latter was subsequently treated as a subspecies of C. hotamboeia (Barbour & Loveridge, 1928). Broadley (1968) showed that two distinct forms, i.e. C. hotamboeia as the typical form and C. tornieri as the montane forest form, are altitudinally segregated and therefore should be considered as distinct species. Additional subspecies for C. hotamboeia also include Crotaphopeltis h. ruziziensis (Laurent 1956) from the Ruzizi-Kivu basin in Congo and C. h. kagleri (Uthmöller, 1939) from Kilimanjaro. The taxonomic status of the former has not been re- assessed since its description, but Crotaphopeltis hotamboeia kagleri, described from two specimens, was later recognised as a valid taxon but based on skull morphology was transferred to the sister genus Dipsadoboa (Laurent, 1951; Rasmussen, 1979, Rasmussen, 1993).

Supralabial colour variation in C. hotamboeia has been investigated (Mertens, 1955; Laurent, 1960; Leach, 1819), with the resurrection from synonymy of C. h. bicolor Leach, 1891. However, supralabial colour appears to be only loosely correlated with geographical range, and Broadley (1962)

23 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia. noted the upper labials are red in Gauteng, Limpopo, Mpumalanga, eastern North-West, Kwazulu- Natal and the Eastern Cape provinces of South Africa, yellow in the Western Cape Province, and white to brown or black in other parts of the continent (for example in Zimbabwe and east Africa). It was proposed that the name typical C. h. hotamboeia be restricted to the red-lipped form, whilst C. h. bicolor be used for other populations (Mertens, 1955; Laurent, 1960). However, the pattern is not concordant with meristic characters and taxonomic recognition of such an arrangement has not received acceptance (Broadley 1990, Wallach, 2014).

For this study the phylogenetic relationships between four out of the six Crotaphopeltis species were investigated, and genetic divergence within the widespread C. hotamboeia was assessed to evaluate whether the documented morphological and geographical variation within the species (Mertens, 1955; Laurent, 1960; Leach, 1819; Broadley, 1962) was of species level genetic divergence. It is hypothesised that because C. hotamboeia is widespread across heterogeneous habitat there will be strong genetic structure and possibly cryptic diversity. This hypothesis was tested by a molecular phylogenetic approach, using multiple nuclear and mitochondrial markers, and including geographical sampling across the entire distribution range of C. hotamboeia.

24 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

Materials & Methods

Sample collection

A primary focus was to place C. hotamboeia in a phylogenetic framework, with geographical sampling from 12 countries, covering the known distribution (n = 69). Additional taxa included were two samples of C. barotseensis, one of C. degeni, and 15 C. tornieri (Fig.3.1; Appendix B3). Crotaphopeltis braestrupi and C. hippocrepis were not available for inclusion in the phylogenetic analysis. Crotaphopeltis tornieri sequences were downloaded from GenBank and were only available for one marker (cyt–b). Boiga forsteni, Dasypeltis fasciata, Toxicodryas pulverulenta, Dipsadoboa unilcolor, and Telescopus semiannulatus, were included as outgroup taxa, of which the first three were downloaded from GenBank and the latter two were sequenced during this study (Appendix B3).

Figure 3.1. Sampling localities throughout sub-Saharan Africa for four species of Crotaphopeltis investigated during this study. Samples of C. hotamboeia are shown as circles.

25 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

DNA extraction, amplification & sequencing

Phylogenetic analyses

Sequences were aligned in Geneious v. 4. 8. 5 (Kearse et al., 2012) and screened for base ambiguities and stop codons (in coding genes). A total of 59 individuals were sequenced for 16S (486 bp), 77 individuals and for cyt–b (583 bp), 71 individuals for ND4 (650 bp) and tRNA (70bp), 45 individuals for c–mos (545 bp) and 25 individuals for RAG1 (961 bp) (Appendix B3). The ribosomal 16S was independently aligned in Block Mapping and Gathering with Entropy alignments Mobyle portal (BMGE: http://mobyle.pasteur.fr/cgi-bin/portal.py) to remove ambiguously aligned regions (Criscuolo & Gribaldo, 2010). PartitionFinder was used to identify the best fit dataset partitioning scheme and accompanying substitutional models (Lanfear et al., 2014). A user defined search was conducted for pre-defined partitions into firstly genes, and secondly 16S and by codon position for the four remaining genes with pos 1 + pos2, pos 3 and the tRNA that follows ND4. As a third option, a greedy search was conducted on 14 data blocks that included 16S separately, trRNA separately, and all codon positions for the remaining four genes separately. Settings applied for both AIC and BIC criteria were: branch lengths linked and unlinked, searching for all models. The AIC selection criteria were recovered as the best, with branch lengths linked for the codon (pos 1 + pos2 and pos 3) partitioning scheme.

26 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

Phylogenetic trees were constructed in both Maximum Likelihood (ML) and Bayesian Inference (BI) frameworks (RAxML-VI-HPC and v. 3.2 MrBayes; Stamatakis 2006; Ronquist et al., 2012) for three datasets: cyt–b only (given that only this marker was available for C. tornieri), mitochondrial, and mitochondrial and nuclear genomes combined. Both analyses were run at the CIPRES Science Gateway v. 3.3 (Miller et al., 2010). For Bayesian Inference, four Markov chains were run in parallel, with each chain starting from random trees, running for 10 million generations, sampling each tree every 1,000 generations. The analyses were run according to the substitution models and partitioning scheme inferred by PartitionFinder: 16s rRNA (GTR + I + G), cyt–b pos1 + pos2(HKY + I + G), cyt– b pos 3 (GTR + G), ND4 pos1 + pos2(TVM + I + G), ND4 pos 3 (TrN+ I + G), ND4 tRNA (HKY + G), c–mos pos1 + pos2 (K81uf + I), c–mos pos 3 (TrN + I + G), RAG1 pos1 + pos2(HKY + I + G) and RAG1 pos 3 (HKY +G). Ten percent of the trees were discarded as burn-in, determined using Tracer v 1.6 (Rambaut et al., 2007) by examining the number of generations at which the effective sample size exceeded 200 for all parameters. A 50% majority-rule consensus tree was generated from the trees retained and nodes with posterior probabilities (pp) ≥ 0.95 were considered well supported. A partitioned Maximum Likelihood analysis was run with the same partition as Bayesian Inference, with the GTR + G model of evolution as implemented in RAxML and automatic halting bootstrapping. Bootstrap values ≥ 70 % were considered well-supported (Wilcox et al., 2002).

Genetic species delimitations

To investigate putative taxonomic units within Crotaphopeltis three species delimitation methods were used of which two were coalescent-based species delimitation methods: 1) Bayesian General Mixed Yule-Coalescent Model bGMYC), 2) a multi-rate Poisson Tree Processes (mPTP), and 3) a distance-based barcoding approach. Bayesian General Mixed Yule-Coalescent Model (bGMYC) was implemented in R using the package bGMYC v. 3.0.1(Reid & Carstens, 2012; R Core Team 2013). The traditional GMYC employs speciation and neutral coalescent models and maximizes likelihood score by identifying the transition point between intraspecific (coalescent) and interspecific (speciational/divergence) branching processes on an ultrametric tree derived from a single locus (Yule, 1925; Hudson, 1990; Pons et al., 2006). The bGMYC overcomes two limitations of GMYC, in that the Bayesian implementation provide flexible prior distributions and accounts for error in phylogenetic estimation and uncertainty in model parameters by integrating over uncertainty in tree topology and branch lengths in parameters of the model via MCMC. Bayesian GMYC was conducted with the single tree method for 10, 000 generations, discarding the first 1000 trees as burn-in and sampling every 100 generations. The best ML tree from RAxML was converted to an ultrametric tree using package APE in R (Paradis et al., 2004). In addition to bGMYC, a multi-rate Poisson Tree

27 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

Processes (mPTP) was conducted using the web server http://mptp.h-its.org/#/tree. Compared to bGMYC the Poisson Tree Processes (PTP) models the branching processes based on the number of accumulated expected substitutions between subsequent speciation events, determining the transition point from within and between species by assuming a two parameter model one parameter for speciation and a second parameter for the coalescent process that best fits the data. In comparison, mPTP incorporates different levels of intraspecific genetic diversity deriving from differences in either the evolutionary history or sampling of each species. Additionally, the mPTP fits the branching events of each species to a distinct exponential distribution, instead of one exponential distribution across all species (Kapli et al., 2017). The total evidence (mtDNA + nuDNA) ML tree was used for the two coalescent species delimitation methods (bGMYC and mPTP).

Distance-based methods, such as, DNA-barcoding applications, considers a nucleotide distance threshold, below which specimens are considered conspecific and above, they are considered to belong to different species (Fontaneto et al., 2015). The basis for this method is that intraspecific and interspecific variations do not overlap, which is known as the “barcode gap”. The barcode gap presumably exists because individuals of a given species are genetically more similar than individuals from different species. A sequence divergence threshold is therefore placed at the barcode-gap that can be used to delineate species (Lefébure et al., 2006). SpeciesIdentifier v1.8 (Meier et al., 2006) was used to compare frequency distributions between intra- and interspecific sequence divergence. Sequence divergence thresholds are dependent on the genetic variation incorporated for the given taxon and markers, therefore intra- and interspecific thresholds will shift given the taxon and dataset under investigation (Meyer & Paulay, 2005; Wiemers & Fiedler, 2007; Meier et al., 2008). Thus, the barcoding analysis was carried out on the cyt–b dataset as this was the most complete dataset.

28 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

Results

Genetic species delimitation

Results for bGMYC and mPTP were discordant for the total evidence dataset (Fig. 3.2). Bayesian GMYC indicates that the south-western and north-eastern Tanzanian clades for C. tornieri represent two distinct species. Species status for C. hotamboeia, C. barotseensis and C. degeni was confirmed. The mPTP analyses retrieved similar results except that three species for C. tornieri were recovered, which included an additional species for north-eastern C. tornieri. Thus, according to the mPTP analyses the three candidate species for C. tornieri occur in three different geographic regions: 1) the Rungwe Mountains (south-western Tanzania), 2) Usambara Mountains (north-eastern Tanzania) and 3) Uzungwa and Uluguru Mountains (north-eastern Tanzania).

29 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

30 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

The barcoding approach for the cyt–b dataset was concordant with results of the bGMYC analysis. The cyt–b sequence divergence was within the intraspecific range amongst all C. hotamboeia clades. Furthermore, sequence divergence was within the interspecific range amongst C. hotamboeia, C. barotseensis and C. degeni, north-eastern C. tornieri and south-western C. tornieri (Fig. 3.3). The barcoding gap between intra– and interspecific cyt–b sequence divergence for Crotaphopeltis species was 12.84%. The two lineages for C. tornieri (south-western and north-eastern Tanzania) were distinct at species level ranging from 15.11% to 18.65% sequence divergence. Sequence divergence between the two north-eastern C. tonieri, i.e. between the Usambara mountains clade and the clade in Uzungwa and Uluguru mountains was within the intraspecific range, 6.67% to 10.96%.

250

200

Intraspecific 150 Interspecific

100

50 Frequency pairwise differences pairwise Frequency

0.5- 1.0 0.5- 2.0 1.5- 3.0 2.5- 4.0 3.5- 5.0 4.5- 6.0 5.5- 7.0 6.5- 8.0 7.5- 9.0 8.5-

9.5- 10.0 9.5-

16.5- 17.0 16.5- 10.5- 11.0 10.5- 12.0 11.5- 13.0 12.5- 14.0 13.5- 16.0 15.5- 18.0 17.5- 19.0 18.5- 20.0 19.5- 14.49- 15.0 14.49- Sequence divergence for cyt–b (%)

31 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

Phylogenetic analyses

A total of 3295 bp from four mitochondrial and two nuclear markers were available for analysis. All three datasets (cyt–b, total mitochondrial, mitochondrial, and nuclear combined) and both BI and ML analyses supported the monophyly of Crotaphopeltis with respect to the diverse colubrids employed as outgroup, and corresponding topologies were recovered (Fig. 3.2). Similarly, the monophyly for the widely sampled C. hotamboeia was well-supported by all datasets and both analyses (100% ML bootstrap and 1.00 Bayesian pp). Crotaphopeltis barotseensis is the sister lineage to other Crotaphopeltis species included in this study, with C. degeni, C. hotamboeia and C. tornieri forming a separate clade. Crotaphopeltis tornieri is polyphyletic with the north-eastern Tanzanian lineage sister to C. degeni and the placement of the south-western Tanzania lineage unresolved (Fig. 3.1 and Fig. 3.2). Within C. hotamboeia two major clades were recovered, one clade restricted to the south- western Kenya (clade 1, Fig. 3.2) and the second clade spans the rest of sub-Saharan Africa. This geographically widespread clade is sub-divided into four clades: the western region of Africa (clade 2a), central and east Africa (clade 2b), southern margin of Africa (clade 2c) and south-eastern Africa (clade 2d).

Discussion

Within Crotaphopeltis, C. hotamboeia is a single species comprising several clades that are geographically distinct, suggesting strong population level structure. The spatial distribution of clades is broadly congruent with the biogeographical regions proposed for reptiles by Linder et al. ( 2012). There are a total of five clades of which one is wide-ranging and corresponds to the Zambezian biogeographic region. Another two clades are geographically restricted in west and east Africa, and are respectively associated with the Congolian and Somalian biogeographic regions. Across southeast Africa, two clades correspond with the biogeographic break between the Zambezian and South African region. The genetic partitioning of C. hotamboeia in relation to the biogeographic regions for reptiles is roughly comparable to that shown by D. typus and P. semivariegatus (Eimermacher, 2012, Chapter Two). Each of these species has a wide-ranging clade in central Africa and share similar biogeographic breaks relative to the Great Escarpment in South Africa. Furthermore, C. hotamboeia and D. typus each have isolated east African clades that are small in geographical extent. Congruity in genetic partitioning across the landscape suggests that the distributions of these species have been influenced by either a common underlying history, or by the same ecological parameters (Linder et al., 2012).

32 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

Shared biogeographic breaks amongst these wide-ranging species also can be associated with the different Köppen-Geiger climate classes for the sub-Saharan region (Peel et al., 2007). The climatic classification of Peel et al. (2007) presents an empirical relationship between climate and natural vegetation. Accordingly, climatic regimes and associated vegetation differentiation appear to be of evolutionary importance for these widely distributed colubrids. Clades that are associated with the Zambezian biogeographic region are, for instance, associated with the subtropical climate and associated savanna biome, while the geographically restricted clades in east Africa are associated with the warm semi-arid climates in the region. The biogeographic breaks in southern Africa, in turn, can be associated with the gradual shift from subtropical climate to temperate climate in South Africa. These similarities in genetic delineation across the landscape are possibly influenced by similar ecological factors (Shafer & Wolf, 2013), that are associated with the diverse habitats across the sub- Saharan region.

The geographical variation in supralabial colour for C. hotamboeia more or less corresponds with the geographic extent of the genetic clades. Populations in Gauteng, Limpopo, Mpumalanga, eastern North-West, Kwazulu-Natal and the Eastern Cape provinces of South Africa in which the upper labials are red (Broadley 1962) correspond to clade 2d, which spans south-eastern Africa. Yellow upper labial colouration in the Western Cape Province (South Africa) corresponds to the southern clade 2c, whilst the white, and brown to black labials found in other parts of the continent (e.g. Zimbabwe and east Africa) correspond to the geographically widespread clade 2b in the central regions to east Africa. It is unclear what might be the basis for the congruence between supralabial colour and genetic partitioning. The specific supralabial colour may have become fixed within ‘populations’ as a result of various reasons, e.g. genetic drift or strong positive selection for this trait.

Crotaphopeltis hotamboeia displays lip-flaring behaviour, and in association with lip coloration may serve as an agonistic behaviour for the deterrence of visual predators, as in some other colubrids (Sousa do Amaral, 1999). Lip-flaring in snakes may also be a prey handling adaptation for feeding on sticky and slimy soft-bodied prey, such as gastropods (slugs and snails), in which lip-flaring behaviour prevents the labial scales from becoming fouled with slime (de Queiroz, 1997, Gans, 1975). Crotaphopeltis hotamboeia feeds predominantly on amphibians, which may fit the ‘sticky soft- bodied’ criteria, and lip-flaring behaviour in this species may be important for prey handling. However, agonistic lip-flaring in the absence amphibians and allied with bright labial coloration suggests that C. hotamboeia exhibits lip-flaring also for predator deterrence. Thus, agonistic lip- flaring is possibly an exaptation (sensu Gould & Vrba, 1982), and supralabial colour (irrespective of the specific colour) possibly intensifies this behavioural display.

33 Chapter Three: Molecular systematics within the genus Crotaphopeltis (Serpentes: Colubridae), with a special focus on the widespread C. hotamboeia.

All three species delimitation methods confirm the species status of the widely distributed C. hotamboeia, as well as for C. barotseensis and C. degeni. Overall, two candidate species were recognised for C. tornieri. The type locality for C. tornieri is the Usambara Mountains, Tanga Province, north-eastern Tanzania (Wallach, 2014) corresponds to the distribution of clade ‘C. tornieri 1’, to which the original name would be applicable. The species has a fragmented distribution in the Afromontane forests of Tanzania and northern Malawi. If similar species level divergence to that of Tanzanian populations occurs elsewhere, it is likely that wider geographical sampling will reveal additional lineages within this species.

Similar to previous findings (Gravlund, 2002), C. tornieri was shown not to be monophyletic. The paraphyly and species level distinction between the south-western and north-eastern Afromontane forests populations in Tanzania, suggests that lineage diversification was induced by a relatively old vicariance event (Gravlund, 2002). Coalescent species delimitation methods account for phylogenetic uncertainty, though the inclusion of C. braestrupi and C. hippocrepis, as well as improved geographical sampling for C. barotseensis, C. degeni, and C. tornieri, are still required to fully understand the phylogenetic relationships within Crotaphopeltis.

Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

Introduction

Epochal climatic oscillations were one of the most influential abiotic factors that contributed to the successive change of the African landscape. Habitat transformations included spatial shifts of major biomes and the emergence of novel habitats (e.g. Axelrod & Raven, 1978; Hewitt, 2004). For example, the landscape of the African continent has transformed from a pan-African forest since the Oligocene (Bobe, 2006; Couvreur et al., 2008; Kissling et al., 2012) to primarily savanna at present (Edwards et al., 2010). During the Early and Middle Miocene a heterogeneous landscape developed, which was not dominated by either forest or savanna, as a result of the global decline in temperature and greater rainfall seasonality (Shipman et al., 1981; Cerling et al., 1991; Retallack et al., 1990; Zachos et al., 2001; Couvreur et al., 2008; Bobe, 2006). These conditions were followed by the appearance of grass-dominated ecosystems (e.g. savanna) in many regions of the continent during the Plio/Pleistocene, with wet and dry alternation of African climate, punctuated by periods of increased aridity (Cerling, 1992; Bobe & Behrensmeyer, 2004; Bobe, 2006; Wynn, 2004). The progression therefore, has been from a continent dominated by closed forest habitats, toward open habitat (e.g. savanna). Additional factors that influenced the formation of open habitat were the interaction between geological processes, edaphic and phytogeographical dynamics, and factors such as herbivory, fire, and in modern times, human cultural practices (Huntley, 1982). Topography, affected moisture transport such as rainfall patterns, as well as hydrological modifications that could have induced strong shifts in vegetation (Sepulchre et al., 2006).

The impact of these progressions on the evolution of African faunal groups is relatively well documented for mammals and birds (e.g. Pomeroy & Ssekabiira, 1990; Fjeldsa & Lovett, 1997; Barnett et al., 2006; Brown et al., 2007; Dehghani et al., 2008; Miller et al., 2011; Ishida et al., 2011; Lorenzen et al., 2012; Levinsky et al., 2013; Smitz et al., 2013). A notable trend of increased diversity of large herbivorous mammals is linked to the growing dominance of open habitat formation during the Late Miocene to Early Pleistocene (Kappelman et al., 1997; Bobe & Behrensmeyer, 2004; Bobe, 2006; Edwards et al., 2010). Similar parallels can be drawn for African reptiles. The African palaeoenvironment induced lineage diversification for several reptile taxa for example, African cobras (Wüster et al., 2007), forest vipers (Menegon et al., 2014), semiaquatic varanids (Dowell et al., 2016), skinks (Medina et al., 2016) and chameleons (Tolley et al., 2008; Ceccarelli et al., 2014) since the Eocene, during which African reptile diversity increased significantly, especially throughout

35 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

the Miocene period. Such common patterns for diverse reptile taxa suggest that there could be some general processes (for example the concomitant contraction and expansion of the forest and savanna biomes during the Miocene period) that played a key role in African reptile evolution.

African reptile genera that emerged prior to the Miocene period and have species distributed in both forest and savanna habitat should show a genetic signature of the possible evolutionary impact of these biome developments. The snake family, Colubridae, appear to have diverged from their sister clade, (Elapidae+Lamprophiidae), during the Eocene suggesting that genera within Colubridae are younger than 50 Mya (Zheng & Wiens, 2016). The African colubrid snake genera, Crotaphopeltis and Philothamnus are widespread throughout sub-Saharan Africa and their species are mainly associated with either forest or savanna. Species within these two genera are therefore good candidates for additional comparisons of African reptile evolution with respect to historical major biome developments. Accordingly, divergence of Crotaphopeltis and Philothamnus from their forest sister groups (Dipsadoboa and Hapsidophrys) and radiation into open habitat could have been initiated during the Oligocene/Miocene transition, when habitats became more heterogeneous. Species in both genera possibly radiated from closed habitat to open habitat, as the landscape transformed from predominant forest habitats to primarily savanna habitat. Therefore, multiple shifts of species from closed to open habitat probably occurred. Subsequent punctuated aridification of the African landscape and grass-dominated ecosystems during the Plio/Pleistocene period most likely induced intraspecific radiation of species within these two genera.

Here, the temporal and spatial evolutionary patterns of Crotaphopeltis and Philothamnus will be examined. Synchronised timing of lineage diversification in response to habitat contraction or expansion events, as well as radiation patterns of species into open habitat will be investigated and compared between the two genera. In particular, I hypothesised that 1) closed habitat is the ancestral habitat state for the two genera, and that species within these genera recently radiated into open habitat, and 2) species diversification events correspond to periods of biome contraction/expansion events. Timing of lineage diversification estimation and ancestral state reconstruction methods will be employed to investigate and infer the biogeographic history of Crotaphopeltis and Philothamnus with respect to the evolution of the African forest and savanna biomes.

36 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

Material and Methods

Species background

The putative species identified within Philothamnus (Chapter Two) and Crotaphopeltis (Chapter Three) were used for analysis of lineage diversification estimation and ancestral habitat reconstruction in this chapter. Results of phylogenetic and species delimitation analyses within Philothamnus (Chapter Two) indicated several previously unrecognised lineages in Philothamnus. Philothamnus semivariegatus was found to be paraphyletic, comprising four cryptic lineages, P. carinatus comprised two cryptic lineages, the two subspecies of P. natalensis were identified as candidate species, P. dorsalis and P. giraradi were conspecific, and the species status of P. nitidus was confirmed, with the two recognised subspecies (P. n. nitidus and P. n. loveridgei) monophyletic, but with only intra-specific levels of divergence.

Philothamnus species are arboreal in nature and are widely distributed across the Afrotropical realm. Currently recognised species and putative species show variation with regards to the level of habitat specialisation and can be divided into habitat classes in which they typically occur. Philothamnus species and putative species included in this Chapter were: P. carinatus 1, P. carinatus 2, P. heterodermus, P. n. natalensis, P. nitidus, P. ruandae and P. thomensis that have their ranges predominantly in the forests across Sub-Saharan Africa, and the Sao Tomé, Principé and Annobón islands in the Gulf of Guinea. Philothamnus ornatus and P. semivariegatus 1 are associated with savanna in southern Africa. The remaining species and putative species: P. semivariegatus 2, P. semivariegatus 3, P. semivariegatus 4, P. angolensis, P. dorsalis/girardi, P. hoplogaster, P. n. occidentalis, P. macrops and P. punctatus can be considered habitat generalists, occupying both closed (forest) and open (primarily savanna) habitat types (Broadley, 1983; Loveridge, 1958; Broadley, 1983; Hughes, 1985; Wallach 2014; Trape & Baldé 2014).

In comparison, phylogenetic and species delimitation analyses of the terrestrial Crotaphopeltis (Chapter Three) indicated that Crotaphopeltis tornieri is not monophyletic with respect to C. degeni and C. hotamboeia, of which the latter was found to be a valid, widespread taxon without cryptic diversity (Chapter Three). Crotaphopeltis species and putative species included in this Chapter were: C. barotseensis that occupies moist grasslands in south east Africa, C. tornieri 1 and C. tornieri 2 in Afromontane forest in Tanzania and northern Malawi, C. degeni in grassland, savanna and shrublands in central Africa, and the widespread sub- Saharan C. hotamboeia, which occurs in all habitats except tropical rainforest and arid regions of the southwest (Broadley, 1983; Rasmussen, 1993; Rasmussen

37 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

& Hughes, 1997; Rasmussen et al., 2000; Wallach 2014).

Lineage divergence estimates

Previously constructed phylogenies for Crotaphopeltis and Philothamnus (Chapter Two and Three), were not dated due to a lack of internal calibration points. The subfamily, Colubrinae is paraphyletic, with Crotaphopeltis and Philothamnus forming part of the subfamily “Colubrinae 2”, sensu Zheng & Wiens (2016). Therefore, to estimate timing of lineage diversification within the two genera simultaneously, additional colubrid taxa from “Colubrinae 1” were included for a temporal framework. Additional taxa were also chosen so that calibration points could be placed and included closely related forest relatives for each of the genera (Dipsadoboa unicolor and Hapsidophrys spp. for Crotaphopeltis and Philothamnus respectively). Sequences for 37 additional colubrid taxa were retrieved from GenBank for cyt–b, ND4 and c–mos gene fragments (Appendix B4). Target taxa for Crotaphopeltis and Philothamnus were represented by species and putative species within the respective genera (identified in Chapter Two and Three). Representative species from closely related Colubridae subfamilies (Grayiinae, Calamariinae and Sibynophiinae) were used as outgroup taxa (Pyron et al., 2014; Zheng & Wiens, 2016).

Lineage diversification was estimated in Bayesian Evolutionary Analysis Sampling Trees (BEAST, v. 2.3.0, Bouckaert et al., 2014), using the total evidence molecular dataset for both genera (cyt–b, ND4 and c–mos, Chapter Two and Three). This Bayesian framework uses a probabilistic model to define rates of molecular sequence evolution over time, on an unconstrained phylogeny, using the MCMC methods to derive clade ages. An uncorrelated lognormal molecular clock and the reversible- jump based substitution model of Bouckaert et al. (2013) were employed. For each gene partition, a gamma distribution of among-site rate heterogeneity with four rate categories was assumed. A Yule speciation process was imposed, and a total of three calibration points were used. Two of the three calibration points have been previously used (Nagy et al., 2003; Wüster et al., 2007; Pook et al., 2009; Tamar et al., 2016) and a third was recently identified by Head et al. (2016). Previously used calibration points (nodes) are not recognised by Head et al. (2016) and were included in this study given the sampled taxa for a temporal framework. The three calibration points were: C1) Hemorrhois divergence between the eastern (H. ravergieri and H. nummifer) and western (H. algirus and H. hippocrepis) subclades that occurred after the contact of Africa-Arabia with Eurasia, 16-18 Mya (Nagy et al., 2003), C2) Hierophis subclade divergence, including Eirenis 18 Mya according fossil data (Ivanov, 2002), and C3) a Pantherophis fossil with a minimum age of 11.93 Ma, calibrating the divergence between the extant genera Pantherophis and Pituophis (Head et al., 2016). A normal

38 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae) distribution prior with a mean of 18Myr and standard deviation of 2Myr was applied to the biogeographical calibration, C1 (95% confidence interval of 14.7-21.3 Mya), as well as a normal distribution prior for C2 with a mean of 18Myr and standard deviation of 1Myr (95% confidence interval of 16.4-19.6 Mya). A lognormal prior was applied to C3, using the fossil age, 11.93 Mya as zero-offset, the default lognormal mean of 1 and standard deviation of 1, to specify a mode slightly older than the fossil in order to model the likelihood of actual date of the node (Ho & Phillips, 2009). Ten independent MCMC chains were run for 100 million generations and sampled every 100,000 generations. The convergence of the three independent chains was verified by the effective samples size for each parameter in TRACER v. 1.6.0 (Rambaut & Drummond, 2007) after a 10% burn-in. Trees in the ten chains were combined using LOGCOMBINER v. 2.3.0 (Rambaut & Drummond, 2014a) and were assessed using TREEANNOTATER v. 1.8 (Rambaut & Drummond, 2014b). The final chronogram and node ages were visualised in FigTree v.1.3.1 (Rambaut 2010).

Ancestral habitat state reconstruction

Ancestral habitats were reconstructed for Crotaphopeltis and Philothamnus separately, using likelihood optimisation and the Markov k-state, one-parameter model (all states equally probable) in MESQUITE 3.03 (Maddison & Maddison, 2015). The ultrametric BEAST tree was pruned for Crotaphopeltis and Philothamnus separately and species from closely related sister genera for each genus were retained to allow for habitat state at the base of the clade to be estimated in the ancestral habitat reconstruction analyses. All genetic samples for Philothamnus and Crotaphopeltis were mapped onto the Terrestrial Ecoregions of the Afrotropical region (Olson et al., 2001) (Fig. 4.1), as well as biomes of South Africa (Mucina, Rutherford & Mucina, 2006). Literature sources were additionally used to confirm classification of species into biome classes, in which they typically occur (Loveridge, 1958; Broadley, 1983; Hughes, 1985; Rasmussen, 1993, 1997; 2000). Biome classifications were further categorised into closed (all forest classes) and open (fynbos, grasslands and savanna) habitat which was used as the character states for ancestral state reconstruction. Terminal taxa were coded accordingly and generalist taxa that occur in both closed and open habitat were coded to have a multistate. Species from sister genera that are closely related to Crotaphopeltis and Philothamnus were coded similarly, according to their distributions (Wallach, 2014), in biome classes (Olson et al., 2001). Species from sister genera that are closely related to Crotaphopeltis were coded as follows: Boiga forsteni, multistate, Telescopus semiannulatus, multistate; Dipsadoboa unicolor, closed habitat. Species from sister genera that are closely related to Philothamnus were coded as follows: Thrasops jacksonii, closed habitat; Thelotornis kirtlandii, closed habitat; Dispholidus typus, multistate and all three Hapsidophrys species, closed habitat.

39 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

40 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

Results

Phylogenetic results from the BEAST analysis was similar to phylogenetic findings in Chapters One and Two, except for the unresolved placement of P. semivariegatus 1 here, that showed a sister relationship to P. semivariegatus 2 + P. semivariegatus 3 + P. semivariegatus 4 + P. nitidus + P. angolensis in Chapter Two (Fig. 4.2; Appendix A4). Divergence times and associated credibility intervals indicate that both Crotaphopeltis and Philothamnus diverged from their forest relatives, D. unicolor and Hapsidophrys spp. during the Late Oligocene (24.56 Mya, 95% HPD: 28.99-20.67 and 24.59 Mya; 95% HPD: 28.79-20.42 respectively).

Ancestral habitat reconstruction unequivocally indicates a closed habitat ancestral state for Philothamnus (0.99; Fig. 4.2; Table 4.1). Lineage diversification estimation shows a major diversification event within this genus during Early Miocene (19.62 Mya, 95% HPD: 23.21-16.46; Fig. 4.2). This initial divergence within Philothamnus resulted in two major clades one that comprises species that have their distributions mainly in the northern regions of the Afrotropical realm, while the second clade comprises species that are distributed in the rest of sub-Saharan Africa (Clade 1 and 2, Fig. 4.2). Species from the northern Afrotropical realm (Clade 1) remained predominantly in closed habitat (P. carinatus 1, P. carinatus 2, P. heterodermus, P. ruandae), with the exception of P. hoplogaster and P. macrops. The two latter species diverged from their forest sister species during the Early/Mid-Miocene (17.9 Mya, 95% HPD: 21.20-14.68), occur in both closed and open habitat and are therefore considered the oldest habitat generalist species in the genus. The majority of species in the Clade 2 show several independent transitions into open habitat until the Pliocene. However, two species, P. nitidus and P. thomensis and one subspecies, P. n. natalensis within Clade 2 remained in closed habitat and diverged from their generalist sister taxa during the Late Miocene/Pliocene transition (7.75 Mya, 95% HPD: 9.79-5.82; 6.60 Mya, 95% HPD: 9.16-4.16; 6.58 Mya, 95% HPD: 8.92-4.57 respectively). Within the same clade, P. ornatus and P. semivariegatus 1 specialised into open habitat, since 17-13 Mya (95% HPD: 23.21- 14.21) (Early/Mid-Miocene).

In contrast, the ancestral habitat type for Crotaphopeltis was equivocal (Fig. 4.2; Table 4.1). Crotaphopeltis barotseensis, which is the oldest species in the genus, specialised into open habitat during the Oligo/Miocene transition period (20.59 Mya, 95% HPD: 24.44-16.83). The second diversification event within Crotaphopeltis was between C. degeni + C. tornieri 1 and C. hotamboeia (17.59 Mya, 95% HPD: 21.09-14.16). Crotaphopeltis degeni diverged from C. tornieri 1 during the Mid-Miocene. (12.34 Mya, 95% HPD: 15.89-8.77) and specialised into open habitat, while C. tornieri 1 radiated into Afromontane forest habitat. The widespread C. hotamboeia maintained an equal

41 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae) preference for both habitat types. Intraspecific diversification for C. hotamboeia commenced during the Late Miocene (6.45 Mya, 95% HPD: 7.88-5.04; Appendix A4). The sister relationship of C. tornieri 2 to C. hotamboeia is unsupported (similar to Chapter Three), but this taxon appears to have diverged from sister taxa during the Miocene period, as the inclusion within C. degeni + C. hotamboeia is supported.

42 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

Philothamnus 1 0.99 0.0074 2 0.99 0.0085 3 0.90 0.9500 4 0.99 0.0009 5 0.99 0.0003 6 0.99 0.0003 7 0.95 0.0490 8 0.48 0.524 9 0.48 0.5200 10 0.95 0.0460 11 0.90 0.0960 12 0.95 0.0510 13 0.96 0.0390 14 0.90 0.0960 15 0.97 0.0300 16 0.93 0.0690 17 0.50 0.0500 Crotaphopeltis 1 0.49 0.5000 2 0.49 0.5000 3 0.50 0.4900 4 0.49 0.5000

43 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

Discussion

Crotaphopeltis and Philothamnus show corresponding timing of lineage diversification in relation to climatic oscillations and associated major biome shifts. Both the genera diverged from their forest relatives during the Late Oligocene, a period that coincides with the fragmentation of the pan-African forest, due to global cooling and drier climate in equatorial Africa (Coetzee, 1993; Zachos et al., 2001; Couvreur et al., 2008). Within both genera more species became associated with open habitat probably due to the growing dominance of open habitat on the landscape.

Initial divergence within Philothamnus gave rise to two major clades during the Early Miocene. Within one of the major clades, a clade of four species (P. carinatus 1, P. carinatus 2, P. heterodermus and P. ruandae) remained predominantly in closed habitat within the northern Afrotropical realm. Lineage diversification amongst these forest species seems to follow the east African and west/central biogeographic break observed for rainforest tree families as well as animal taxa (e.g. White, 1979; Wasser & Lovett, 1993; Burgess & Clarke, 2000; Loader et al., 2007; Zimkus et al., 2016). Forest habitat is thought have extended in a central belt running coast to coast during Early Miocene with fragmentation during Early/Mid-Miocene transition and again during Late Miocene, after the initiation of geological activity in the western east African Rift System (Axelrod & Raven, 1978; Wasser & Lovett, 1993; Jacobs et al., 1999; Jacobs, 2004; Sepulchre et al., 2006; Couvreur et al., 2008). Accordingly, the diversification dates amongst the four northern Afrotropical Philothamnus forest species correspond to the series of connection-isolation events between east African and Guinea-Congolian forests. Forest fragmentation during the Early/Mid-Miocene transition was concomitant with the advent of savanna in east Africa (Bobe, 2006; Couvreur et al., 2008), and was primarily driven by the aridification of the Congo basin (Senut et al., 2009), the end of a warming trend (18-14 Ma) and onset of marked global cooling (Zachos et al., 2001). Therefore, the Early/Mid- Miocene savanna habitat in east Africa could have provided opportunities for species to radiate into open habitat, such as Philothamnus hoplogaster and P. macrops. These two species occur in both closed and open habitat, diverged from the northern Afrotropical forest species during the Early/Mid Miocene and are therefore the oldest habitat generalists in the genus. Philothamnus hoplogaster and P. macrops have eastern Africa distributions, where the former is more widespread from east Africa to South Africa, and the latter is restricted to Tanzania (Van Wallach, 2014). It is therefore suggested that Philothamnus hoplogaster and P. macrops radiated into open habitat with the concomitant advent of savanna and forest fragmentation ine east Africa during the Early/Mid-Miocene transition (Bobe, 2006; Couvreur et al., 2008).

44 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae)

The second major clade within Philothamnus comprises a number of species that radiated into open habitat. In fact, one P. semivariegatus lineage became an open habitat specialist in southern Africa during the Early/Mid-Miocene transition, a time period when sclerophyll vegetation dominated the landscape of southern Africa (Axelrod & Raven, 1978). Most species in this Philothamnus clade are habitat generalists with relatively wide distributions. Since the Mid-Miocene, south-western Africa became more arid with upwelling of cold waters associated with the Benguela current (Sieser, 1978; Udeze & Oboh-Ikuenobe, 2005; Senut et al., 2009). Thus, aridification of south-western Africa possibly contributed to a significantly more heterogeneous environment that could have facilitated a speciation pulse and radiation into novel habitats, especially into southern Africa. For example, P. n. natalensis, P. nitidus and P. thomensis remained in forest habitat, P. ornatus specialised into savanna habitat in southern Africa, while the majority of species became widespread habitat generalists.

Crotaphopeltis shows timing of lineage diversification that could be linked to the development of the savanna biome. Furthermore, lineage diversification within Crotaphopeltis corresponds with several divergence events within Philothamnus, although patterns between the genera are not identical. For example, open habitat in most parts of southern Africa during the Early Miocene (Axelrod & Raven, 1978), possibly provided opportunities for open habitat (moist grasslands) specialisation of the oldest species within Crotaphopeltis, C. barotseensis. Similarly, the Mid-Miocene aridification in east Africa could have caused allopatric speciation of the montane forest species, C. tornieri. This species occurs in fragmented populations in Tanzania and Malawi and comprises two cryptic lineages that are divergent at species level, in north-eastern and south-western Tanzania (Chapter Three). During the same time period (Mid-Miocene), the divergence of C. degeni and specialisation into open habitat could have been induced by the presence of savanna habitats specifically in Ethiopia, Kenya and Uganda (Bobe, 2006). The fragmented occurrence of this species and morphological differentiation between populations of C. degeni (Rasmussen, 1997), further supports specialisation into the patchy savanna environment of the Mid-Miocene east Africa.

There are two widespread species in each genus (C. hotamboeia and P. angolensis), that diverged from their sister taxa at different time periods. Crotaphopeltis hotamboeia diverged from its east African sister taxa, during the Mid-Miocene and underwent rapid intraspecific diversification during the Late Miocene. Radiation of this species coincides with significant aridification and open habitat formation, that is attributable to western east African Rift System and related uplift of the central Tanganyikan plateau. The subsequent Miocene/Pliocene transition further contributed to the expansion of open habitat due to an increase of C4 vegetation (Cerling et al., 1997; Wasser & Lovett, 1993; Sepulchre et al., 2006), and possibly facilitated further radiation of C. hotamboeia into novel

45 Chapter Four: Snakes on an African plain: the radiation of Crotaphopeltis and Philothamnus into open habitat (Serpentes: Colubridae) habitats. In comparison, P. angolensis diverged from its forest sister species P. nitidus during the Miocene/Pliocene boundary (ca. 6myr) with initial intraspecific divergence at ca 4 Mya. Both C. hotamboeia and P. angolensis show Pliocene/Pleistocene intraspecific diversification that broadly coincides with the substantial increase in C4 vegetation as well as punctuated aridity around 3.6 Ma, 2.5 Ma and again about 1.8 Ma (deMenocal, 1995; Wynn, 2004). Thus, the extensive open habitat since the Late Miocene could have enabled widespread radiation of these two species into open habitat, irrespective of different emergence times.

For Crotaphopeltis and Philothamnus species investigated during this study, lineage diversification and associated shifts into novel habitats correspond to the concomitant contraction and expansion of the forest and savanna biomes in the Afrotropical realm, since the Oligocene to the Late Miocene. Therefore the overarching hypothesis that the development of these major biomes as a driver for cladogenesis and species radiation patterns within Crotaphopeltis and Philothamnus is well supported. The Miocene epoch signifies a period of increased diversity within both Crotaphopeltis and Philothamnus, which is similar to findings of other African reptile taxa (Wüster et al., 2007; Tolley, Chase & Forest, 2008; Ceccarelli et al., 2014; Menegon et al., 2014; Dowell et al., 2016; Medina et al., 2016). Philothamnus shows notably more cladogenic events relative to Crotaphopeltis, irrespective of similar emergence times for the two genera. Given that Philothamnus has an ancestral preference for forest habitat, the fragmentation of this biome since the Oligocene must have had a greater evolutionary impact on the distribution of species in this genus, than it did on Crotaphopeltis that have an equal preference for both closed and open habitats. Furthermore, the evolution of habitat generalist species in Philothamnus since the Early Miocene, is in agreement with the hypothesis that the origin of generalist species is triggered by a physically and temporally variable environment (Futuyma & Moreno, 1988; Remold, 2012). Radiation of Philothamnus and Crotaphopeltis species into novel habitats are possibly facilitated by ecological opportunity (Losos & Mahler, 2010) associated with the development of major biomes since the Oligocene. Thus, investigation of ecomorphological and ecophysiological traits between species within each genus presents a future research avenue.

Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub- Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis

Introduction

Species are often closely tied to certain habitat types and can therefore be classified as either specialist or generalist (Julliard et al., 2006). Ecological theory states that specialists adapt to an increasingly narrow subset of possible environments whereas generalists have the ability to exploit a range of resources (the “jack-of-all-trades is master of none” hypothesis) enabling them to occupy broader niche breadths (Futuyma & Moreno, 1988; Julliard et al., 2006; Poisot et al., 2012). The propensity to evolve in either direction is influenced by the diversity of environments that compose a species habitat (Remold, 2012). As such, a spatially heterogeneous and/or temporally variable environment tends to drive species toward ecological generalisation, while a homogeneous or temporally stable environment can induce ecological specialisation (MacArthure, 1972; Roughgardern, 1972; Futuyma & Moreno, 1988; Kassen, 2002; Remold, 2012).

The African continent harbours many wide-ranging reptile taxa, and featured among these are colubrid snakes with sub-Saharan distributions (Van Wallach, 2014; Chapter Three). Underlying evolutionary drivers for their wide geographical distributions are, however, unexplored. Crotaphopeltis hotamboeia and Philothamnus angolensis are two colubrid snake species that are widely distributed across sub-Saharan Africa. Although both species are wide ranging, they have different geographical distribution patterns (Wallach 2014). The widespread geographical distribution of C. hotamboeia relative to its congeners is consistent with the ecological specialist- generalist hypothesis. Congeners of C. hotamboeia are habitat specialists, with restricted or fragmented geographical distributions in either savanna or Afromontane forest (Chapter Four). In comparison, C. hotamboeia has a relatively continuous sub-Saharan distribution covering six habitat classes of the sub-continent (coastal lowland forest, tropical lowland forest edges, fynbos, grasslands, savanna and succulent Karoo), but excluding tropical lowland rainforest and the hyper-arid regions of the southwest (Broadley, 1983; Rasmussen, 1993; 1997; 2000; Wallach 2014). Crotaphopeltis hotamboeia diverged from its sister species during the Early/Mid-Miocene period (ca. 17 Mya; Chapter Four), though intraspecific divergence commenced during the Miocene/Pliocene transition period (ca. 6 Mya; Chapter Four). The Early to Mid-Miocene period was characterised by periodic

47 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis climatic oscillations that gave rise to heterogeneous habitats (Sieser, 1978; Zachos et al., 2001; Udeze & Oboh-Ikuenobe, 2005; Senut et al., 2009), relative to the stable subtropical climatic conditions of the Late Miocene period that are associated with the establishment of the savanna biome (Zachos, 2001; Jacobs, 2004, Bobe, 2006). Consequently, the climatically variable and heterogeneous landscape during the Early/Mid-Miocene period could have triggered ecological generalisation of C. hotamboeia.

In comparison, P. angolensis has a wide-ranging but fragmented geographical distribution throughout eastern sub-Saharan Africa within two habitat classes, i.e. lowland tropical forest and savanna in the southern regions of Tanzania, southern Democratic Republic of Congo, northern and central Namibia, Zambia, Malawi, eastern and central Zimbabwe as well as northern KwaZulu-Natal, South Africa (Branch, 1998; Broadley, 1983). In comparison to the continuous geographical distribution of C. hotamboeia in multiple different habitat types, the fragmented geographical distribution of P. angolensis exclusive to lowland tropical forest and savanna suggests that P. angolensis is potentially an ecological specialist. Philothamnus angolensis diverged from its sister species during the Late Miocene (ca.8 Mya; Chapter Four) with intraspecific divergence commencing during the Early Pliocene (ca 4 Mya; Chapter Four). Therefore, the relatively stable environment of the established savanna biome since the Late Miocene could have been an evolutionary precursor for ecological specialisation of P. angolensis.

The climatic progressions during the Quaternary period were characterised by climatic oscillations, during which the wet and dry oscillations of the Plio/Pleistocene transition were accompanied by periods of increased aridity around 3.6 Ma, 2.5 Ma and 1.8 Ma (Bobe, 2006). These Quaternary climatic progressions could have affected subsequent geographical distributions of both these wide- ranging species. During the Pleistocene period, the Last Glacial Maximum (LGM, ca. 21,000 years BP) and Last Interglacial (LIG, 140, 000-120,000 years BP) periods (Deacon, 1983; Tyson, 1986) were two periods of climatic extremes of which the LIG was relatively warmer and wetter, and the LGM cooler and drier than present-day climatic conditions (Deacon, 1983; Tyson, 1986; Prömmel et al., 2013; Gavashelishvili & Tarkhnishvili, 2016). These climatic extremes probably caused the contraction and expansion in the geographical distribution of C. hotamboeia on the margins of hyper arid and tropical climate regions. Similarly, the contrasting climatic conditions would have caused the contraction of lowland forest and savanna. Thus, the fragmented geographical distribution of P. angolensis could result from this species tracking these habitats across sub-Saharan Africa. In this context, Gavashelishvili & Tarkhnishvili (2016) developed a model for the geographical distributions

48 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis of African biomes during the LGM using climate, terrain, hydrographic, fossil pollen, and plant macrofossil data, whereas Prömmel et al. ( 2013) studied the impact of tectonic and orbital forcing on African precipitation and vegetation during the LIG. Accordingly, these studies provide points of comparison for the climatic distribution of these two species with respect to African biome distribution during the LGM and LIG.

Given that climate has a major influence on vegetation type (e.g. Holdridge, 1947; Whittaker, 1975; Prentice et al., 1992; Peel et al., 2007; Prömmel et al., 2013; Gavashelishvili & Tarkhnishvili, 2016), it is possible that climatic shifts, especially those that influence vegetation composition, were an underlying evolutionary driver for the wide-ranging geographical distribution patterns of C. hotamboeia and P. angolensis. Accordingly, the Köppen-Geiger climatic classification (Peel et al., 2007), which is an empirical relationship between climate classes and natural vegetation, provides a simple framework to describe climate and biomes in an integrative way (e.g. Chen & Chen, 2013; Wang & Overland, 2004, Gnanadesikan & Stouffer, 2006; Kleidon et al., 2000). Therefore, this climatic classification can also provide a practical approach for investigating the evolution of a species climatic niche with respect to biome developments in a phylogenetic framework. The ‘Köppen-Geiger’ climatic classification recognises thirty climate types globally, of which three main climate classes, tropical climate (class A), arid climate (class B) and temperate climate (Class C) are present in Africa (Peel et al., 2007). The patchy sub-Saharan distribution of P. angolensis in lowland forest and savanna is linked to subtropical climate (Fig.5.2), which collectively constitutes tropical savanna climate, humid subtropical climate, humid subtropical climate/subtropical oceanic highland climate and warm semi-arid climate (Branch, 1998; Broadley, 1983; Peel et al., 2007). In comparison, the multiple habitat types in which C. hotamboeia occurs are linked to subtropical climates, tropical climate on forest edges, temperate climate as well as the semi-arid climates (Fig.5.2) of sub-Saharan Africa (Rasmussen, Chirio & Ineich, 2000; Branch, 1998; Broadley, 1983; Peel et al., 2007; Wagner et al., 2008).

On the basis of the historical environmental conditions for C. hotamboeia and P. angolensis, in combination with their current geographical distribution patterns within specific climatic and associated habitat types, I postulate that C. hotamboeia is a climatic generalist and P. angolensis is a climatic specialist. Furthermore, the present-day geographical distributions of these species, i.e. widespread continuous distribution in multiple climate classes (C. hotamboeia), and a fragmented distribution in subtropical climate (P. angolensis), suggests that each species maintained its generalist/specialist distribution with respect to climatic oscillations. Therefore, the impact of climatic

49 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis shifts on: 1) the evolution of the climatic niche and 2) the climatic distributional changes during opposing climatic conditions were investigated for each species. I hypothesised that: 1) C. hotamboeia is a climatic generalist, with a multistate for ancestral climate preference, and that P. angolensis is a climatic specialist with an ancestral preference for subtropical climate; and 2) that each species has maintained its generalist/specialist spatial distribution through time. To test these hypotheses, the regional ‘Köppen-Geiger’ climatic classifications of Peel et al. (2007) were used in analyses of ancestral state reconstruction to investigate the evolution of the climatic niches of C. hotamboeia and P. angolensis in a phylogenetic framework. Additionally, Species Distribution Models of C. hotamboeia and P. angolensis were constructed, focussing on the climatic extremes of the Pleistocene period, i.e. the Last Glacial and Interglacial Maxima. The distribution models were used to examine if species maintained their climatic generalist/specialist distribution characteristics during contrasting climatic conditions.

50 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis

Materials and Methods

Ancestral climatic state reconstruction

Ancestral climatic state reconstruction was conducted separately for .C. hotamboeia and P. angolensis, using likelihood optimization and the Markov k-state, one-parameter model (all states equally probable) in MESQUITE 3.03 (Maddison & Maddison, 2015). The total evidence RAxML phylogenies (5 genes: 16S, cyt–b, ND4, c–mos and RAG1) generated at the genus level (Chapters Two and Three) were pruned for relevant terminal taxa, and converted to ultrametric trees using the packages APE and PHYTOOLS in R (Paradis et al., 2004; Revell, 2012). The pruned phylogenies contained individuals representing intraspecific clades within each species (identified in Chapter Two and Three), from across the geographical distribution covering the different climate classes in which each species occur. In addition, sister taxa were retained in both phylogenies to allow for the climatic state at the base of the clade to be constructed. Sister species for C. hotamboeia included C. barotseensis, C. degeni and both lineages of C. tornieri, and for P. angolensis sister species in the P. angolensis subclade (P. semivariegatus, P. punctatus and P. nitidus) were included. The localities for all genetic samples of P. angolensis and C. hotamboeia were mapped onto the Köppen-Gieger climatic classifications of the sub-Saharan region, in order to code terminal taxa for the climatic classes in which they occur (Peel et al., 2007). Terminal tips that occur in more than one climatic class were coded to have a multistate of the Köppen-Geiger climatic classes.

Species distribution modelling

Species Distribution Models (SDMs) were constructed for P. angolensis and C. hotamboeia from occurrence records retrieved from: the Global Biodiversity Information Facility (GBIF: http://www.gbif.org), the global network of herpetological collections for natural history museums (herpNET: http://www.herpnet.org), the Southern African Reptiles database (SARCA: http://sarca.adu.org.za/index.php), the South African National Biodiversity Institute (SANBI DNA bank: http://www.sanbi.org/programmes/conservation/dna-bank), literature records, and newly collected genetic samples (Chapter Two and Three). Records were thinned at 100 km distance to account for a possible sampling bias from auto-correlated occurrence (Boria et al., 2014), which resulted in 31 and 131 unique locality records for P. angolensis and C. hotamboeia, respectively.

51 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis

Species Distribution Models were constructed using the maximum entropy algorithm implemented in Maxent (version 3.3.3k, Phillips et al., 2006; Philipps & Dudík, 2009). Maxent models were built using linear and quadratic features, data were randomly partitioned into 75% training and 25% test data via bootstrapping and we produced 100 replicate runs to evaluate model accuracy. To evaluate model accuracy the area under the curve (AUC) statistic and the True Skill Statistic (TSS; Allouche, Tsoar & Kadmon, 2006) as well as the table of variable contributions and the three jack-knife test (regularised training gain, test gain and AUC) were compared. As predictors, bioclimatic variables were downloaded from the WorldClim database (Hijmans et al., 2005) (http://www.worldclim.org/paleo-climate1) at 5 arc-min resolution (~ 10 km), which was the overall scale for the model, and palaeoclimatic data were retrieved for the CCSM4 Global Climate Model. To avoid model overfitting and data collinearity pairwise Pearson correlation coefficients between recent climatic variables were examined (ExDet tool v1.1; Mesgaran et al., 2014) and correlated variables with absolute values higher than 0.75 were omitted. Preliminary models were run in a stepwise procedure to identify the climatic variable combination that predict the climatic niche of each species most accurately. Analyses were initialised with a full predictor set, and after each step excluded variables that added no gain or led to a decline in the predictive accuracy of the model, until optimal accuracy was reached. This procedure led to the same four climatic variables for both species, temperature seasonality (Bio 4), mean temperature of wettest quarter (Bio 8), mean temperature of the warmest quarter (Bio 10), and annual precipitation (Bio 12). These final models were used to predict the climatic distributions of the species for the LIG and LGM.

A threshold-based (maxSSS: maximum test sensitivity plus specificity logistic threshold) prevalence estimation approach was used to transform Species Distribution modelling results into binary output (presence-absence) maps for each species separately. The maxSSS, has been proved valid to use with presence-only data when random occurrence points are used instead of true absences, where sensitivity is the proportion of correctly predicted presences among all the presences, and specificity is the proportion of correctly predicted absences among all absences (Liu et al., 2013; 2016). To estimate climate suitability through the two Pleistocene periods (LIG and LGM), the resulting binary maps for both time periods were superimposed using the raster overlay method. In raster overlay, each cell of each layer references the same geographical location, which makes it well suited to mathematically combine numerical values for multiple layers into a single layer, and assign a new value to each cell in the output layer. Accordingly, the presence-absence values (1 = presence, 0 = absent i.e. no data) were summed across identical geographically referenced grid cells of the LIG and LGM maps, to estimate climatic suitability during both periods. Suitability values per 5 arc-min

52 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis resolution (~ 10 km) were classified as 0 (no data/not present in either time period: not suitable ), 1 (present at one of the time periods: low suitability) and 2 (present at both time periods: high suitability). The summed raster layer was then used to map out the areas that were highly suitable across both time periods. All GIS raster calculations and processing were conducted in Quantum Geographic Information System processing tools (version 2.18, QGIS, 2015).

Results

Ancestral climatic state reconstruction

The ancestral climatic association for Crotaphopeltis hotamboeia was subtropical climate with a likelihood of 0.98 (node 5) for tropical savanna climate (Aw; Fig. 5.1a). The initial radiation within this species was associated with subtropical savanna climate (node 6). Subsequent diversification within this species was concurrently into the subtropical climate of the central sub-Saharan region (node 8) and a combination of climates at node 7 the humid subtropical/subtropical oceanic highland climate (Cwb), warm semi-arid climate (BSh) as well as the temperate oceanic climate (Cfb) of southeastern Africa. The most recent climatic shifts for this species (at tree terminals) includes the occupation of temperate oceanic climate (Cfb), temperate Mediterranean climate (Csb), cold semi- arid climate (BSk), warm desert climate (BWh), monsoon climate (Am) as well as equatorial climate (Af).

The likelihood of the ancestral climatic state (node 4; Fig. 5.1b) for Philothamnus angolensis was 0.58 for Aw (tropical savanna climate), 0.18 for Cwa (humid subtropical climate) and 0.16 for Cwb (humid subtropical/subtropical oceanic highland climate). These fine-scaled Köppen-Geiger climatic classifications combined make up the subtropical climate across sub-Saharan Africa. Therefore, the ancestral climatic state for Philothamnus angolensis could be considered subtropical climate with a combined likelihood for all subtropical climate classes at node 4 of 0.92. The climatic state at the node representing the recent divergence within P. angolensis was also primarily associated with subtropical climate (node: 5; Aw, 0.25; Cwa, 0.61). Only one terminal contains an equatorial climate state (Am), suggesting that the association of P. angolensis with equatorial climate is a recent occurrence, while remaining terminals indicate that this species primarily occupies subtropical climate regions.

53 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis

Figure 5.1. Chronograms and ancestral climatic state reconstruction for a) Crotaphopeltis hotamboeia and b) Philothamnus angolensis. Tree terminals are colour-coded according to the Köppen-Geiger climatic classifications. Black symbols (tree terminals and map) distinguish intraspecific clades within each species. Likelihood values for each reconstructed Köppen-Geiger climate class are represented by pie charts at each numbered node. The map presents the recent Köppen-Geiger climate classes: Af (Equatorial climate), Am (Monsoon climate), Aw (tropical savanna climate), BSh (Warm semi-arid climate), BSk (Cold semi-arid climate), BWh (warm desert climate), Cfb (temperate oceanic climate), Csb (temperate mediterranean climate), Cwa (humid subtropical climate), and Cwb (humid subtropical climate/subtropical oceanic highland climate), adapted from Peel et al. (2007). Time scale in millions of years (Ma).

54 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis

Species distribution modelling

The Species Distribution Models for current climatic conditions produced good resulst over the majority of the range (Fig. 5.2a & b). AUC values for the test data indicated good predictive accuracy of the models (mean AUC = 0.835, standard deviation = 0.0257; mean AUC = 0.879, standard deviation = 0.0487, for C. hotamboeia and P. angolensis respectively). Mean TSS values were 0.555 and 0.605 respectively. Over-prediction at the southern tip of the continent and under-prediction in southern Africa was marginal for P. angolensis. Similarly, marginal over-prediction for C. hotamboeia was within the central and southwestern regions of Africa (Fig. 5.2).

55 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis

The superimposed map for the two Pleistocene periods indicate that highly suitable climatic conditions for C. hotamboeia were largely continuous and more widespread relative to present-day occurrence (Fig. 5.3a). This species showed marginal climatic range expansion into the Horn of Africa, southeastern Africa and the southwestern region of the continent during the LIG (Fig. 5.3a, Appendix A5a). However, these areas were considered areas of low overall climatic suitability, as these areas were climatically unsuitable during the LGM (Fig. 5.3a, Appendix A5c). Similary, the central African region was an area of low climatic suitability and in some regions predicted to be completely unsuitable for of C. hotamboeia. A similar prediction of highly suitable climatic conditions over large continuous areas for C. hotamboeia suggests that the species probably occupied several different climatic classes during the climatic extremes of the Pleistocene period.

Highly suitable climatic regions throughout the LIG and LGM appear to be fragmented for P. angolensis (Fig. 5.3b), and associated with the extent of the savanna biome (Prömmel et al., 2013; Gavashelishvili & Tarkhnishvili 2016) during both these periods. Given that the savanna biome is governed by subtropical climate classes (Peel et al., 2007), the results suggest that P. angolensis remained within subtropical climate regions during both these climatic extremes. The LIG climatic

56 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis prediction showed a substantial fragmented climatic distribution that largely matches the present-day geographical and climatic distribution of this species (Fig. 5.2b; Appendix A5b), with an isolated area in the northern and southern regions of central Africa as well as within regions of southern Africa. The LGM prediction was wider and more continuous relative to the climatic distribution of the LIG, especially in the southeastern and central regions of Africa (Appendix A5b & d), which were considered areas of low climate suitability for P. angolensis.

Discussion

Ancestral climatic state reconstruction indicates that P. angolensis has a climatic niche that is primarily associated with savanna and lowland forest habitat, which has not changed since the Late Miocene (ca.8 Mya). In comparison, C. hotamboeia initially (ca. 7 Mya) occupied subtropical climate and moved into diverse climatic classes, with wetter climates such as the equatorial and monsoon climates, where this species is found at forest edges (e.g. Wagner et al., 2008), temperate climate that is associated with the fynbos and coastal habitats, as well as drier climates of the grasslands and the succulent Karoo. Although P. angolensis and C. hotamboeia are spatially widespread over sub- Saharan Africa, C. hotamboeia has a continuous climatic distribution, associated with multiple different climate classes, whereas P. angolensis has a fragmented climatic distribution that correspond to subtropical climate. This suggests that the former is a climate generalist and the latter a climate specialist. The absence of C. hotamboeia in habitats with extremes climates (associated with tropical lowland rainforest and hyper-arid habitats) indicate that this species may not be capable of occupying habitats that require specialised physiological adaptations, which is in accord with the ‘jack-of-all-trades is master of none’ hypothesis for generalist species (Futuyma & Moreno, 1988).

The palaeoclimatic distribution of C. hotamboeia appears to have been relatively stable throughout the Pleistocene period, which is likely attributable to the ability of this species to occupy a range of climatic classes and persist through fluctuating climatic conditions. The modelled distribution of C. hotamboeia during these two periods was similar to its present-day occurrence, in that the climatic regions that were associated with hyper-arid habitats were not suitable for C. hotamboeia. However, the LIG and LGM predictions of C. hotamboeia overlapped with geographical areas that were known to be associated with tropical rainforest (Prömmel et al,. 2013; Gavashelishvili & Tarkhnishvili 2016), which is probably an artefact of marginal over-prediction for present-day climate conditions. In view of this, the climatic distributions of generalist species are not easily modelled with accuracy compared to specialist species (e.g. Segurado & Araújo, 2004; M. McPherson & Jetz, 2007; Tsoar et

57 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis al., 2007). Habitat tolerance of generalist species may impede model algorithms from distinguishing between suitable and the overall environment. That is to say, abundant occurrence of species could influence the balance between observations of presence and absence which unless controlled for influence model accuracy through statistical artefacts (McPherson & Jetz, 2007). Although the models accurately model the current climatic distribution, predicted suitable areas for past conditions should be regarded as hypothetical, given that suitable climatic conditions in the past could have existed in areas that could have been physically inaccessible to the species (Araújo et al., 2005; Devitt et al., 2013).

In contrast, the projected palaeoclimatic distribution of P. angolensis corresponds with the geographical distribution of the savanna during the Pleistocene period (Prömmel et al., 2013; Gavashelishvili & Tarkhnishvili 2016). The climatic conditions of the LIG involved strong seasonal differences in precipitation over large parts of the continent that specifically caused prevailing tropical climate conditions over central Africa (Prömmel et al., 2013), that could have been instrumental in the climatic range contraction of P. angolensis in the Congo Basin proximity. Temperature declines and increased aridity of the LGM period was conducive for the expansion of savanna habitat. Correspondingly, the more continuous and widespread climatic distribution of P. angolensis during the LGM mirrors the geographical distribution of the savanna biome defined by Gavashelishvili & Tarkhnishvili (2016), and therefore suggests that the occurrence of this species is largely bound to the subtropical climate that is predominantly associated with the extent of the natural savanna.

Certainly, both a climatic generalist and subtropical climate specialist state could have allowed for the current wide-ranging geographical distributions of these species across the sub-continent assuming that physical barriers and dispersal capabilities were not limiting factors. Generalisation, by definition, enables species to have a wider niche breadth, which can equate to a wider geographical range relative to specialist species (Futuyma & Moreno, 1988; Julliard et al., 2006; Poisot et al., 2012; Slatyer et al., 2013). In contrast, P. angolensis is wide-ranging, with a discontinuous geographical distribution within subtropical climate patches across eastern sub-Saharan Africa. Contrasting climatic conditions would have caused the concomitant contraction and expansion of habitats that are associated with subtropical climate, such as lowland forest and savanna. For this reason, the fragmented occurrence of P. angolensis could be a result of this species tracking subtropical climate and associated habitats through time. However, because species ranges are not solely limited by the abiotic environment (Nogués-Bravo, 2009), further investigations for these species as well as congeners, should focus on biotic interactions such as interspecific competition, predation,

58 Chapter Five: The potential impact of historical climate oscillations on the evolution of the climatic niche and distribution patterns of two sub-Saharan colubrid snakes; Crotaphopeltis hotamboeia and Philothamnus angolensis physiological adaptions, amongst other things, as determinants of species’ ecological niches and related geographical distributions and patterns.

Climate fluctuation is the primary cause for historical changes in the natural composition of vegetation. However, fire cycles, herbivory and in modern times agriculture and urbanisation also contribute to landscape disturbance and fragmentation (Huntley, 1982; Sankaran et al., 2005; Lehmann et al., 2011; Murphy et al., 2014). Though it is expected that most species will be negatively affected by habitat fragmentation and unnatural disturbance, evidence suggests that specialist species such as P. angolensis, may be at a higher risk than generalist species assuming they can tolerate landscape degradation (Devictor et al., 2008). This is particularly relevant to C. hotamboeia that appears to be a climate and habitat generalist and is abundant around human settlements (Broadley, 1983). The presumed tolerance of C. hotamboeia for disturbed habitats probably contributes to its large continuous geographical distribution across sub-Saharan Africa.

Historical climatic conditions and associated habitats appear to have shaped different wide-ranging climatic and geographical distribution patterns for C. hotamboeia and P. angolensis across a similar geographical extent. Changes in the extent of the suitable climatic regions during the Pleistocene climatic extremes, as well as the present-day distribution furthermore indicate that both species have maintained their climatic generalist and specialist distribution characteristics through time. It remains to be tested if the same environmental factors also contributed to the wide-ranging geographical distributions of other African colubrid species.

Chapter Six: General conclusion and summary of dissertation

The research presented here demonstrates that development of habitat throughout the Late Oligocene influenced the radiation patterns of the colubrid snake genera, Crotaphopeltis and Philothamnus. Knowledge of the evolutionary relationships for the two genera were needed to construct their biogeographic histories reliably. Genealogical relationships amongst species within each of the two snake genera were examined in a phylogenetic framework, and species boundaries were identified under the Evolutionary Species Concept (Chapters Two and Three). Both genera were found to be monophyletic with respect to diverse African colubrids studied. Phylogenetic species delimitation methods proved to be useful as the majority of described species were recognised, and in cases where morphology appeared conservative, cryptic candidate species were identified. For example, the wide- ranging P. semivariegatus showed hidden diversity with four lineages divergent at the species level. Other wide-ranging species within the two genera, for example, P. angolensis and C. hotamboeia, showed only intraspecific divergence without evidence of cryptic species. Despite the differences in the level of regional diversification within wide-ranging species, the spatial distribution of clades is broadly congruent. Genetic partitioning within wide-ranging Crotaphopeltis and Philothamnus species appears to be influenced by climatic regimes and associated vegetation differentiation across the sub-Saharan region.

Taxonomic considerations

The findings of this dissertation reveal that several taxonomic conflicts need to be addressed. Candidate cryptic species identified under the Evolutionary Species Concept require further investigation for additional evidence (e.g. morphology, ecology, behaviour, and geography), in favour of the General Lineage Concept, to allow confident species recognition. The newly identified species level diversity revealed within Crotaphopeltis and Philothamnus provide a basis for future taxonomic revisions.

Philothamnus

Of the 12 species and four subspecies included in the Philothamnus phylogeny, nine species correspond to currently described species, i.e. P. angolensis, P. heterodermus, P. hoplogaster, P. macrops, P. nitidus, P. ornatus, P. punctatus, P. ruandae and P. thomensis, and are well differentiated by diagnostic morphological characters. For most, there is no reason on current knowledge to expect them to contain cryptic diversity. However, for two species with large and/or disjunct distributions,

60 Chapter Six: General conclusion and summary of dissertation only a few samples were available for analysis, e.g. P. heterodermus and P. ornatus. Additional sampling from new localities, e.g. Zimbabwe populations of P ornatus, may show greater genetic divergence and cryptic diversity. Although genetic divergence within the numerous samples of P. hoplogaster did not exceed intra-specific levels, samples from north east Angola and Tanzania showed the deepest divergence, and their relationship to the recently described P. hughesi from the northern Congo basin (Trape & Rouw-Esteve, 1990) should be investigated. Taxonomic adjustments or revisions are required for some species, and are summarised below.

Philothamnus dorsalis and P. girardi

These two species had levels of genetic divergence comparable to intra-specific variation in other species. Both species were described by Bocage, but both treated as subspecies of P. semivariegatus by Loveridge (1958). Hughes (1985) convincingly argued that this arrangement was untenable, and raised both to specific status, with P. girardi restricted to Annobón Island (= Pagalu Is.), Equatorial Guinea, Trape & Roux-Esteve (1990) described additional material from the Congo Republic, but it remains unknown from Gabon (Pauwels & Vande weghe 2008). Topotypic material of P. girardi from Annobón Island was sequenced, and was conspecific with P. dorsalis samples. It remains possible that the Congo specimens referred to P. girardi by Trape & Roux-Esteve (1995) represent a different, possibly new taxon, and further studies are required.

Philothamnus carinatus

Philothamnus carinatus was found to comprises two cryptic species. One is geographically placed in Gabon and the Republic of Congo region, while the other occurs further east in the Democratic Republic of Congo. Although genetic material from the type locality in south-western Cameroon was not available, it is likely to be genetically similar to material sequenced from western parts of the distribution. The only synonym if the species is Philothamnus nigrofasciatus Buchholz & Peters, 1875 whose type locality is also Cameroon, and would therefore not be available for the eastern DRC population. Resolution of the the status of the eastern DRC population reuires more inclusive geographical sampling, which will also contribute to a better understanding of the spatial boundaries between the two candidate species.

61 Chapter Six: General conclusion and summary of dissertation

Philothamnus semivariegatus

Genetic material from the type locality for P. semivariegatus is essential before taxonomic conclusions can be made, as two western special-level clades occur in the region of the type locality, “Busman Flat, South Africa”. Bocage’s (1882) Philothamnus smithii is the only name available for northern populations currently assigned to P. semivariegatus. They may be available for the western and central clade (P. semivariegatus 4) identified during this study, and which Trape & Baldé (2014) have recently revived for West African material. No West African P semivariegatus material was included in this study, this is required to confirm that clade 4 is conspecific with populations in West Africa.

Philothamnus natalensis

Although there was discrepancy between the two coalescent methods for species boundaries within P. natalensis (mPTP indicated two candidate species, bGMYC three candidate species), neither indicated they were conspecific. Based on conservative interpretation of the genetic species delimitation results the two subspecies of P. natalensis currently recognised are elevated to full species. This action is supported by both morphological and genetic evidence (see Chapter Two).

Phliothamnus nitidus

Genetic material for both subspecies of P. nitidus were available, including two samples of P. n. loveridgei from eastern DRC, close to the type locality (Itula, Kivu Province, DRC), and samples from the Republic of Congo, which are probably referable to typical P. nitidus (type locality: Lagos, Nigeria, fide Boulenger 1894). Although the samples are reciprocally monophyletic, they are also well separated geographically. Moreover, their intraspecific divergence level falls at the transition between intra- and interspecific sequence divergence, and acceptance of them either as subspecies or species depends on acceptance of the concept of subspecies in herpetology (Frost and Hillis 1990). Further studies on the species, including additional, more inclusive molecular analysis and modern morphological analysis, e.g. of skull morphology and dentition, are required. Conservatively the current taxonomy of Laurent (1960) and Hughes (1985) is maintained.

Future phylogenetic and species delimitation studies including material from the eight missing Philothamnus species may reveal additional hidden diversity within the species-rich genus. Formal

62 Chapter Six: General conclusion and summary of dissertation description of the candidate species revealed during these studies may raise the number of species within the genus from 20 to 26 species.

Crotaphopeltis

Within Crotaphopeltis, the widespread C. hotamboeia was found to comprise a monophyletic lineage with only intra-specific divergence between the widespread samples and no evidence of cryptic species. In contrast, C. tornieri comprises two cryptic lineages that are divergent at species level and require taxonomic revision. Coalescent species delimitation methods account for phylogenetic uncertainty, though the inclusion of C. braestrupi and C. hippocrepis, as well as improved geographical sampling for C. barotseensis, C. degeni and C. tornieri, is still required to fully understand the phylogenetic relationships within Crotaphopeltis.

Crotaphopeltis tornieri

The type locality for C. tornieri is the Usambara Mountains, Tanga Province, north-eastern Tanzania (Wallach, 2014). Therefore, in the clade in north-eastern Tanzania would retain the original species name. As no available names are currently included in the synonymy of C. tornieri the south-western Tanzania clade represents a new species. Furthermore, C. tornieri has a fragmented distribution range in the Afromontane forests of Tanzania as well as northern Malawi, and wider geographical sampling of these populations may reveal additional cryptic species.

Radiation patterns within Crotaphopeltis and Philothamnus

63 Chapter Six: General conclusion and summary of dissertation

The Miocene epoch in particular, signifies a period of increased diversity within both Crotaphopeltis and Philothamnus. The evolution of climatic and habitat generalists species within the two genera since the Early Miocene, is in agreement with the hypothesis that the origin of generalist species is triggered by a physically and temporally variable environment (Futuyma & Moreno, 1988; Remold, 2012), whereas a stable environment tends to drive species toward ecological specialisation (Chapters Four and Five). It specifically appears that the climatic oscillations during Early/Mid-Miocene facilitated the evolution of C. hotamboeia as a climatic generalist, while the Late-Miocene climatic conditions induced specialisation of P. angolensis in subtropical climates (Chapter Five). Changes in the extent of the climatic niche during the Pleistocene climatic extremes, as well as their present-day distributions furthermore indicate that both species have maintained their climatic niche through time.

Directions for future research

Radiation of Philothamnus, as well as Crotaphopeltis species into novel habitats are possibly adaptive due to ecological opportunity (e.g. Losos & Mahler, 2010) associated with the development of major biomes since the Oligocene. Thus, investigation of ecomorphological and ecophysiological traits between species within each genus presents a future research avenue. Of particular interest may be the cranial and feeding adaptations in Philothamnus and C. hotamboeia, as well the functional morphology related to climbing behaviour in diverse Philothamnus species.

Cranial and feeding adaptations

It has been demonstrated that the variation in body proportions and cranial feeding apparatus (bones and tooth morphology) for snake species can be associated with variation in habitat use, daily activity, prey preference and prey size (Mori & Vincent, 2008; Bonnet et al., 2001; Aubret et al., 2004; Vincent et al., 2006; Herrell et al., 2008; Vincent et al., 2009; Hampton et al., 2011; Liu et al., 2012; Henderson et al., 2013). Therefore, the relationship between feeding morphology and prey availability for Philothamnus species in differing habitats is a research interest that I would like to pursue. Additionally, I would like to explore the functional morphology and ecological significance of the lip-flaring behaviour in Crotaphopeltis hotamboeia. Cranial adaptations for lip-flaring can be related to features such as muscle attachment, arched mandibles, and different feeding behaviours (Kofron, 1982; Sazima, 1989; Rossman & Myer, 1990). Moreover, C. hotamboeia may possess carina-bearing teeth, i.e. teeth with sharp edges, and if present these structures may assist in venom delivery during lip-flaring displays, as seen for other colubrid snakes (Sousa do Amaral, 1999).

64 Chapter Six: General conclusion and summary of dissertation

Venom delivery in such cases may immobilize prey and thus decrease prey-handling time (Salomão & Laporta-Ferreira, 1994).

Functional morphology for climbing behaviour

Philothamnus species are predominantly arboreal and despite the fact that all species regularly climb, some are more proficient than others. Species in this genus show varying degrees of keeled scales on ventral surfaces, and in some species ventral keeling is present along the entire ventral area, while in others it is only present in a certain section of the ventrum or tail. Ventral keeling within Philothamnus presumably assists in climbing performance, and the resulting ventral ridge possibly allows the body surface to grip onto rough surfaces (Hughes, 1985). In contrast, the absence of keeling could reduce traction when fast, smooth moving is required through thick vegetation. Furthermore, fine-branching vegetation is possibly used for body support and therefore ventral keeling could become less important in such habitats. Open habitat specialists (e.g. P. semivariegatus) can ascend, for example, vertical rocky slabs and will need to compensate for the lack of vegetation support. Thus, the presence or increased ventral keeling could be due to the selection for this trait in open habitats with less complex vegetation. Thus, it appears that the historical biome developments have influenced the distribution of wide-ranging African colubrid species, and possibly contributed to their ecological diversification as well. It remains to be tested if the same environmental factors had a similar evolutionary impact on other wide-ranging African colubrid species.

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Appendix A: supplementary figures

Appendix A1. Maximum Likelihood topology for the total evidence DNA data (16S rRNA, cyt–b, ND4, c– mos and RAG1) for Philothamnus labelled with all samples from 14 countries. Support values for both posterior probability (> 0.95 pp) and bootstrap (> 70%) are indicated by half black circles (BI on the left, ML on the right) at the nodes.

75 Appendix A: supplementary figures

Appendix A2. Maximum likelihood topology for constrained relationship of Hapsidophrys as sister to (Philothamnus natalensis + P. hoplogaster).

Appendix A: supplementary figures

Appendix A3. Maximum Likelihood topology for the total evidence DNA data (16S rRNA, cyt–b, ND4, c– mos and RAG1) for Crotaphopeltis labelled with all samples from 13 countries. Support values for both posterior probability (> 0.95 pp) and bootstrap (> 70%) are indicated by half black circles (BI on the left, ML on the right) at the nodes.

Appendix A: supplementary figures

77 Appendix A: supplementary figures

Appendix B: supplementary tables

Appendix B1. Sample information for Philothamnus and outgroup taxa. Specimen ID numbers refer to field numbers for the DNA samples, and voucher indicates museum accession numbers (PEM = Port Elizabeth Museum, MVZ = Museum of Vertebrate Zoology at Berkeley, MTSN = Museo Tridentino di Scienze Naturali, USNM = United States National Museum). Locality information and GenBank accession numbers are given, where NA denotes missing information and TBA, GenBank accession numbers to be added. Genus species ID number Voucher Latitude Longitude Country 16S cyt–b ND4 c–mos RAG1 Dispholidus typus EBG2673 NA NA NA NA TBA TBA TBA TBA TBA Dispholidus typus JGL146 NA -6.057697 37.477316 Tanzania TBA NA TBA TBA TBA Hapsidophrys lineatus EBG1672 NA NA NA NA TBA TBA TBA TBA NA Hapsidophrys lineatus EBG2610 NA NA NA NA TBA TBA TBA TBA TBA Hapsidophrys lineatus J345 PEM R2307 4.90277777 -7,3161111 Ivory Coast AY611873 AY612055 NA NA NA Hapsidophrys principis 592 592 NA NA Príncipe island FJ913476 FJ913492 NA NA NA Hapsidophrys principis 606 606 NA NA Príncipe island FJ913481 FJ913494 NA NA NA Hapsidophrys principis 769 769 NA NA Príncipe island FJ913482 FJ913493 NA NA NA Hapsidophrys smaragdinus EBG2607 NA NA NA NA TBA TBA TBA TBA TBA Hapsidophrys smaragdinus J349 PEM R5383 -1.9152777 9.87138888 Gabon TBA TBA NA NA NA Hapsidophrys smaragdinus USNM57606 USNM57606 NA NA NA TBA NA TBA TBA NA Macroprotodon cucullatus MVZ186073 MVZ186073 36.3325 -5.7 Spain AY188065.1 AF471087.1 AY487064.1 AF471145.1 NA Philothamnus angolensis EBG3003 NA -6.984461 29.695299 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus angolensis EI_0166 NA -17.705757 35.356353 Mozambique TBA TBA TBA TBA TBA Philothamnus angolensis EI_0167 NA -17.706632 35.357115 Mozambique TBA TBA TBA TBA TBA Philothamnus angolensis EI_0168 NA -17.70362 35.359275 Mozambique TBA TBA TBA TBA TBA Philothamnus angolensis ELI239 NA -0.935398 27.818891 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus angolensis J382 PEM R13207 -16.937612 38.669111 Mozambique AY611886 AY612068 NA NA NA Philothamnus angolensis MO999 NA -15.833333 35.7 Malawi NA NA TBA NA TBA Philothamnus angolensis MOZ14-253 NA -15.366361 37.033311 Mozambique TBA TBA TBA TBA NA Philothamnus angolensis MTSN10482 MTSN10482 -3.05503 28.97772 Democratic Republic of Congo TBA TBA TBA TBA NA Philothamnus angolensis MTSN7239 MTSN7239 -9.13333 33.6667 Tanzania TBA NA TBA TBA NA Philothamnus angolensis MTSN7240 MTSN7240 -8.53178 33.6387 Tanzania NA NA TBA NA NA Philothamnus angolensis WC4145 NA -14.59 26.907584 Zambia NA TBA TBA NA TBA

79 Appendix B: supplementary tables

Appendix B1. (continued) Genus species ID number Voucher Latitude Longitude Locality 16S cyt–b ND4 c–mos RAG1 Philothamnus carinatus EBG1363 NA -1.515563 28.502912 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus carinatus EBG2613 NA 1.405462 28.563105 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus carinatus MBUR03196 NA -3.490917 12.136824 Republic of Congo TBA TBA TBA TBA TBA Philothamnus carinatus MBUR03243 NA -2.303232 12.809195 Republic of Congo TBA TBA TBA NA TBA Philothamnus carinatus MBUR03303 NA NA NA Republic of Congo TBA TBA TBA TBA TBA Philothamnus carinatus MBUR03314 NA NA NA Republic of Congo NA NA TBA TBA TBA Philothamnus carinatus MBUR03474 NA NA NA Republic of Congo TBA NA TBA TBA TBA Philothamnus carinatus MBUR03481 NA -2.300119 12.812609 Republic of Congo TBA TBA TBA TBA TBA Philothamnus carinatus MBUR03482 NA -2.300119 12.812609 Republic of Congo TBA TBA TBA TBA TBA Philothamnus carinatus MBUR03491 NA NA NA Republic of Congo TBA TBA TBA TBA TBA Philothamnus carinatus MBUR03542 NA -2.351316 12.778693 Republic of Congo TBA NA TBA TBA TBA Philothamnus carinatus J335 PEM R5441 -2.152983 9.58917 Gabon AY611870 AY612052 NA NA NA Philothamnus carinatus J354 PEM R5938 -2.169564 10.030479 Gabon FJ913483 FJ913498 NA NA NA Philothamnus dorsalis Nov-76 NA -6.139914 12.359009 Angola TBA NA TBA TBA TBA Philothamnus dorsalis 13-114 NA -6.139914 12.359009 Angola TBA NA TBA TBA TBA Philothamnus dorsalis 13-80 NA -6.139914 12.359009 Angola TBA NA TBA NA NA Philothamnus dorsalis MBUR02944 NA -4.383897 11.620395 Republic of Congo NA NA TBA TBA TBA Philothamnus dorsalis MBUR08097 NA -4.620168 12.167282 Republic of Congo TBA TBA TBA TBA TBA Philothamnus girardi 621 621 -1.439346 5.632839 Annobon island FJ913475 FJ913495 NA NA NA Philothamnus heterodermus J238 JCL762 NA NA No locality information AY611856 AY612038 NA NA NA Philothamnus heterodermus MBUR03181 NA NA NA Republic of Congo NA NA TBA TBA TBA Philothamnus heterodermus MBUR08066 NA -4.668881 11.897547 Republic of Congo TBA NA TBA TBA TBA Philothamnus heterodermus MBUR08206 NA -4.682329 11.907075 Republic of Congo TBA TBA TBA TBA TBA Philothamnus heterodermus MBUR08250 NA -4.668187 11.8966 Republic of Congo TBA TBA NA TBA NA Philothamnus hoplogaster EI_0063 NA -17.7850483 37.15739 Mozambique NA NA TBA NA NA

Appendix B: supplementary tables

Appendix B1. (continued) Genus species ID number Voucher Latitude Longitude Locality 16S cyt–b ND4 c–mos RAG1 Philothamnus hoplogaster EI_0064 NA -17.785118 37.157398 Mozambique TBA TBA TBA NA TBA Philothamnus hoplogaster EI_0069 NA -17.773618 37.178032 Mozambique TBA TBA TBA TBA TBA Philothamnus hoplogaster EI_0080 NA -17.767702 36.31951 Mozambique NA NA TBA NA TBA Philothamnus hoplogaster KTH436 NA -34.160852 24.655631 South Africa NA NA TBA NA NA Philothamnus hoplogaster MBUR01495 NA -25.488379 30.979838 South Africa TBA NA TBA TBA NA Philothamnus hoplogaster MOZ14-110 NA -16.281476 36.443768 Mozambique TBA NA TBA TBA NA Philothamnus hoplogaster MOZ14-298 NA -14.915912 38.316496 Mozambique TBA TBA TBA TBA NA Philothamnus hoplogaster MTSN8178 MTSN8178 -6.133333 37.533333 Tanzania NA TBA NA NA NA Philothamnus hoplogaster MTSN8204 MTSN8204 -6.029494 37.516237 Tanzania TBA TBA TBA TBA NA Philothamnus hoplogaster J389 PEM R 13214 -17.064444 38.743889 Mozambique TBA TBA NA NA NA Philothamnus hoplogaster PEMR17609 PEM R17609 -34.157206 24.710833 South Africa TBA NA NA NA NA Philothamnus hoplogaster SPN031 NA -18.643385 34.814202 Mozambique TBA TBA TBA TBA NA Philothamnus hoplogaster TB47 PEM R19906 -9.39706 20.43195 Angola TBA NA TBA TBA TBA Philothamnus hoplogaster TGET2-53 NA -27.8781 32.36307 South Africa TBA NA TBA TBA TBA Philothamnus hoplogaster WCDNA957 NA -32.037373 29.108333 South Africa TBA NA TBA TBA NA Philothamnus n. loveridgei ELI510 NA -3.30767 27.299394 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus n. loveridgei USNM57625 USNM57625 -1.901227 26.221795 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus n. nitidus MBUR03179 NA -2.303232 12.809195 Republic of Congo TBA NA TBA TBA TBA Philothamnus n. nitidus MBUR03543 NA 0.8667 14.8167 Republic of Congo TBA NA TBA TBA NA Philothamnus n. nitidus MBUR08282 NA 0.8667 14.8167 Republic of Congo TBA NA NA TBA NA Philothamnus n. nitidus J337 PEM R5397 -2.091046 9.961789 Gabon TBA TBA NA NA NA Philothamnus macrops 665 NA -16.301304 36.565883 Mozambique TBA TBA NA TBA NA Philothamnus macrops EI_0396 NA -10.967 40.358278 Mozambique TBA NA TBA NA NA Philothamnus macrops MTSN5227 MTSN5227 -8.32 35.93 Tanzania TBA NA TBA NA NA Philothamnus macrops MTSN5271 MTSN5271 -8.32 35.93 Tanzania TBA TBA NA NA NA

Appendix B: supplementary tables

Appendix B1. (continued) Genus species ID number Voucher Latitude Longitude Locality 16S cyt–b ND4 c–mos RAG1 Philothamnus macrops MTSN8200 MTSN8200 -5.985489 37.501176 Tanzania NA TBA TBA TBA NA Philothamnus n. natalensis JMOZ154 NA -26.008822 32.954468 Inhaca island TBA NA TBA NA NA Philothamnus n. natalensis Tyr01 NA -29.7092 31.0975 South Africa TBA TBA TBA TBA TBA Philothamnus n. occidentalis J383 PEM FN441 -33.886519 22.399253 South Africa AY611887 AY612069 NA NA NA Philothamnus n. occidentalis WC10-107 NA -31.28984 29.98994 South Africa TBA TBA TBA TBA NA Philothamnus n. occidentalis WC10-144 NA -31.28879 30.01221 South Africa TBA NA TBA TBA NA Philothamnus n. occidentalis WC3634 NA -33.498436 23.628839 South Africa TBA NA TBA TBA TBA Philothamnus n. occidentalis WC2771 NA -32.76522 26.618776 South Africa TBA NA TBA TBA TBA Philothamnus n. occidentalis WC3635 NA -33.498436 23.628839 South Africa TBA NA TBA NA TBA Philothamnus n. occidentalis WC3685 NA NA NA NA TBA NA TBA NA TBA Philothamnus n. occidentalis WCDNA0087 NA -34.003372 25.546479 South Africa TBA TBA TBA TBA NA Philothamnus n. occidentalis WCDNA633 NA -32.70232 28.35091 South Africa TBA TBA NA TBA NA Philothamnus n. occidentalis WCDNA721 NA -33.973014 25.257143 South Africa TBA TBA TBA TBA NA Philothamnus n. occidentalis WCDNA781 NA -31.65903 29.49207 South Africa TBA TBA TBA TBA NA Philothamnus n. occidentalis WCDNA816 NA -31.65949 29.29955 South Africa TBA TBA TBA TBA TBA Philothamnus n. occidentalis WCDNA879 NA -33.973014 25.257143 South Africa TBA TBA TBA TBA NA Philothamnus n. occidentalis WCDNA962 NA -32.031667 29.109722 South Africa TBA TBA TBA TBA TBA Philothamnus n. occidentalis WCDNA983 NA -34.009692 25.590385 South Africa TBA TBA TBA TBA TBA Philothamnus ornatus PEMR18950-73 PEM R18950-73 -12.139759 25.393734 Zambia TBA NA TBA TBA TBA Philothamnus ornatus WC12-A136 NA -14.606222 18.467225 Angola TBA TBA TBA TBA NA Philothamnus ornatus WC3925 NA -12.689351 18.360115 Angola TBA TBA TBA NA TBA Philothamnus ornatus WC4034 NA -12.689351 18.360115 Angola TBA NA NA NA TBA Philothamnus punctatus 759 NA -4.76750000 38.2736111 Tanzania TBA NA TBA TBA NA Philothamnus punctatus MTSN8190 MTSN8190 -6.133333 37.533333 Tanzania TBA TBA TBA NA NA Philothamnus punctatus MTSN8411 MTSN8411 -6.133333 37.533333 Tanzania NA TBA TBA NA NA

Appendix B: supplementary tables

Appendix B1. (continued) Genus species ID number Voucher Latitude Longitude Locality 16S cyt–b ND4 c–mos RAG1 Philothamnus punctatus T3148 NA NA NA NA TBA NA TBA TBA TBA Philothamnus ruandae EBG1689 NA -3.407026 27.413229 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus ruandae ELI4892 NA NA NA NA TBA TBA NA TBA TBA Philothamnus ruandae ELI780 NA -3.391543 27.767083 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus ruandae MTSN7373 MTSN7373 -2.527987 29.27855 Rwanda TBA TBA TBA TBA NA Philothamnus semivariegatus 603 NA -2.192632 33.963303 Tanzania TBA NA TBA TBA NA Philothamnus semivariegatus 748 NA -12.273517 25.38055 Zambia TBA NA TBA TBA NA Philothamnus semivariegatus Cape777 NA -33.768639 25.414139 South Africa TBA NA TBA TBA NA Philothamnus semivariegatus CMRK006 NA -18.920008 29.822128 Zimbabwe TBA NA TBA TBA NA Philothamnus semivariegatus EI_0068 NA -17.773577 37.177928 Mozambique TBA TBA TBA NA TBA Philothamnus semivariegatus EI_0075 NA -17.77248 37.18085 Mozambique TBA NA TBA TBA TBA Philothamnus semivariegatus ELI210 NA 0.935248 27.818471 Democratic Republic of Congo TBA NA TBA TBA TBA Philothamnus semivariegatus ELI214 NA -0.934293 27.818156 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus semivariegatus ELI215 NA -0.933876 27.818341 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus semivariegatus ELI261 NA -0.933524 27.818105 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus semivariegatus ELI262 NA -0.934422 27.818653 Democratic Republic of Congo TBA NA TBA TBA NA Philothamnus semivariegatus HB282 NA -22.681755 29.101272 South Africa TBA TBA TBA TBA TBA Philothamnus semivariegatus HB283 NA -20.472149 16.656589 Namibia TBA TBA NA TBA TBA Philothamnus semivariegatus J390 PEM R13189 -17.058518 38.699018 Mozambique AY611889 AY612071 NA NA NA Philothamnus semivariegatus KTH09-287 NA -15.02518 13.376214 Angola TBA TBA TBA TBA TBA Philothamnus semivariegatus MB20869 NA -29.039444 22.273889 South Africa TBA TBA NA TBA NA Philothamnus semivariegatus MBUR00278 NA -23.28389 28.816389 South Africa NA TBA NA TBA NA Philothamnus semivariegatus MBUR01494 NA NA NA South Africa TBA TBA TBA TBA TBA Philothamnus semivariegatus PEMR13977 PEMR13977 -9.07383 18.066705 Angola NA NA TBA TBA NA Philothamnus semivariegatus SPN091 NA -18.643425 34.814223 Mozambique TBA TBA TBA TBA NA

Appendix B: supplementary tables

Appendix B1. (continued) Genus species ID number Voucher Latitude Longitude Locality 16S cyt–b ND4 c–mos RAG1 Philothamnus semivariegatus VM1023 NA -14.43159 35.246838 Malawi TBA NA NA TBA NA Philothamnus semivariegatus WRB670 NA -25.288039 30.553937 South Africa TBA NA TBA TBA TBA Philothamnus semivariegatus Z37930 NA -17.410266 14.196233 Namibia TBA NA TBA TBA NA Philothamnus thomensis 567 567 0.248863 6.624783 São Tomé island FJ913477 FJ913490 NA NA NA Philothamnus thomensis 568 568 0.167081 6.533273 São Tomé island FJ913473 FJ913489 NA NA NA Philothamnus thomensis 570 570 0.260273 6.621059 São Tomé island FJ913478 FJ913491 NA NA NA Philothamnus thomensis 573 573 0.166337 6.526164 São Tomé island FJ913480 FJ913487 NA NA NA Philothamnus thomensis 631 631 0.250816 6.611107 São Tomé island FJ913474 FJ913486 NA NA NA Philothamnus thomensis 632 632 0.165959 6.533996 São Tomé island FJ913479 FJ913488 NA NA NA Thelotornis kirtlandii ELI373 NA NA NA NA TBA NA TBA TBA TBA Thrasops jacksonii ELI509 NA NA NA NA TBA NA TBA TBA NA

Total: 138 121 73 102 93 53

Appendix B: supplementary tables

Appendix B2. Species recognised for Philothamnus, identified through two coalescent species delimitation methods and a barcoding approach (method indicated in brackets). Check marks indicate that species status is supported by the particular method. Crosses indicate no support for species status. Species status was inferred if supported by at least two out of the three species delimitation methods. Asterisks indicate currently described species that are differentiated by diagnostic morphological characters and need not be revised. The remainder would be considered candidate species. Inferred species Species delimitation method Taxa Coalescent Coalescent Barcoding (distance (bGMYC) (mPTP) based) P. angolensis* Yes √ X √ P. angolensis 1 No X √ X P. angolensis 2 No X √ X P. carinatus 1 Yes √ √ √ P. carinatus 2 Yes √ √ √ P. dorsalis No X X X P. Girardi No X X X P. dorsalis/girardi Yes √ √ X P. dorsalis/girardi /thomensis No X X √ P. heterodermus* Yes √ √ √ P. hoplogaster* Yes √ √ √ P. macrops* Yes √ √ √ P. n. natalensis Yes √ √ X P. n. occidentalis Yes X √ X P. n. occidentalis 1 No √ X X P. n. occidentalis 2 No √ X X P. nitidus* Yes √ X √ P. nitidus nitidus No X √ X P. nitidus loverigei No X √ X P. ornatus* Yes √ √ √ P. punctatus* Yes √ √ √ P. ruande* Yes √ √ √ P. semivariegatus 1 Yes √ √ √ P. semivariegatus 2 Yes √ √ √ P. semivariegatus 3 Yes √ √ √ P. semivariegatus 4 Yes √ √ √ P. thomensis* Yes √ √ X

Appendix B: supplementary tables

Appendix B3. Sample information for Crotaphopeltis and outgroup taxa. Specimen ID numbers refer to field numbers for the DNA samples, and voucher indicates museum accession numbers (CAS = California Acedemy of Sciences, MTSN = Museo Tridentino di Scienze Naturali, PEM = Port Elizabeth Museum, YPM = Yale Peabody Museum of Natural History). Locality information and GenBank accession numbers are given, where NA denotes missing information. TBA, GenBank accession numbers to be added. Genus species ID number Voucher Latitude Longitude Country 16s cyt–b ND4 c–mos RAG1 Crotaphopeltis hotamboeia JM01442 NA -3.392835 38.400478 Kenya TBA TBA TBA TBA NA Crotaphopeltis hotamboeia JM02670 NA -3.195364 38.373642 Kenya TBA TBA TBA NA NA Crotaphopeltis hotamboeia JM02962 NA -3.357276 38.821557 Kenya TBA TBA TBA TBA NA Crotaphopeltis hotamboeia JM2532 NA -3.195364 38.373642 Kenya NA TBA TBA NA NA Crotaphopeltis hotamboeia CFS3911 NA -4.98203 12.91946 Democratic Republic of Congo TBA TBA TBA TBA NA Crotaphopeltis hotamboeia MBUR02932 NA -4.160064 11.685889 Republic of Congo TBA NA NA TBA NA Crotaphopeltis hotamboeia MBUR02978 NA -4.186228 11.666861 Republic of Congo TBA NA TBA TBA NA Crotaphopeltis hotamboeia MBUR08044 NA -4.548426 12.100251 Republic of Congo TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia MBUR08065 NA -4.619483 12.06317 Republic of Congo TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia MBUR08113 NA -4.630568 12.155163 Republic of Congo TBA NA TBA TBA TBA Crotaphopeltis hotamboeia MBUR08194 NA -4.630562 12.155169 Republic of Congo TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia MBUR08279 NA -4.586649 12.145568 Republic of Congo TBA NA TBA TBA TBA Crotaphopeltis hotamboeia WC12-A109 NA -14.671556 17.73525 Angola TBA TBA TBA TBA NA Crotaphopeltis hotamboeia WRB933 NA -6.141752 12.351946 Angola TBA TBA TBA TBA NA Crotaphopeltis hotamboeia WC3951 NA -12.736749 18.393102 Angola NA TBA TBA NA TBA Crotaphopeltis hotamboeia EBG2219 NA 0.568056 29.477839 Democratic Republic of Congo TBA TBA TBA TBA NA Crotaphopeltis hotamboeia ELI107 NA -7.343184 25.575727 Democratic republic of Congo TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia Eli3209 NA -3.65579 26.12786 Democratic republic of Congo TBA NA TBA TBA NA Crotaphopeltis hotamboeia Eli3245 NA -3.67679 26.11742 Democratic republic of Congo TBA NA TBA TBA NA Crotaphopeltis hotamboeia MBUR08458 NA 10.66238 34.39922 Ethiopia TBA TBA TBA NA TBA Crotaphopeltis hotamboeia MBUR08480 NA 10.664714 34.419664 Ethiopia TBA NA TBA NA TBA Crotaphopeltis hotamboeia MBUR08502 NA 10.69275 34.49775 Ethiopia TBA NA TBA NA TBA Crotaphopeltis hotamboeia MBUR08574 NA 10.6959722 34.457361 Ethiopia TBA NA TBA TBA TBA Crotaphopeltis hotamboeia PEM R18947 PEM R18947 -12.040805 25.333333 Zambia TBA NA TBA NA NA

Appendix B: supplementary tables

Appendix B3. (continued) Genus species ID number Voucher Latitude Longitude Country 16s cyt–b ND4 c–mos RAG1 Crotaphopeltis hotamboeia TMHC2013.07.252 NA 5.784454 36.565912 Ethiopia TBA TBA TBA NA NA Crotaphopeltis hotamboeia TMHC2015.07.637 NA 5.113302 40.654359 Ethiopia TBA TBA TBA TBA NA Crotaphopeltis hotamboeia VM1201 NA -14.436057 33.820121 Malawi TBA TBA NA NA TBA Crotaphopeltis hotamboeia WC-ANG0017 NA -7.751639 19.955389 Angola TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia WC12-A082 NA -13.596278 16.877083 Angola TBA TBA TBA TBA NA Crotaphopeltis hotamboeia WRB927 NA 8.871097 -12.040623 Sierra Leone TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia WRB928 NA 8.871097 -12.040623 Sierra Leone TBA TBA TBA NA NA Crotaphopeltis hotamboeia R631263 NA -9.100114 33.633282 Tanzania NA AF428021.1 NA NA NA Crotaphopeltis hotamboeia MBUR08428 NA 10.643687 34.411388 Ethiopia NA TBA TBA NA NA Crotaphopeltis hotamboeia ARD00023 NA -34.189724 18.366388 South Africa TBA TBA TBA NA NA Crotaphopeltis hotamboeia FFNR002 NA -32.682741 26.495723 South Africa TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia KTH09-320 NA -29.486639 30.017517 South Africa TBA TBA TBA TBA NA Crotaphopeltis hotamboeia KTH303 NA -34.068056 24.904444 South Africa TBA TBA TBA NA NA Crotaphopeltis hotamboeia MBUR02436 NA -34.1914 24.714 South Africa TBA TBA TBA TBA NA Crotaphopeltis hotamboeia SVN450 NA -31.719444 24.635278 South Africa TBA TBA TBA NA NA Crotaphopeltis hotamboeia WC10-122 NA -31.27613 30.02296 South Africa TBA TBA TBA TBA NA Crotaphopeltis hotamboeia WCDNA659 NA -32.281916 28.87195 South Africa TBA TBA TBA TBA NA Crotaphopeltis hotamboeia WCDNA702 NA -32.601653 26.921943 South Africa TBA TBA TBA NA NA Crotaphopeltis hotamboeia HB019 NA -33.606657 22.195209 South Africa NA TBA TBA NA NA Crotaphopeltis hotamboeia KTH132 NA -33.994444 24.703333 South Africa NA TBA TBA TBA NA Crotaphopeltis hotamboeia WCDNA416 NA -31.81806 29.30017 South Africa NA TBA TBA NA NA Crotaphopeltis hotamboeia WCDNA974 NA -32.44516 28.59825 South Africa NA TBA TBA TBA NA Crotaphopeltis hotamboeia BHMP023 NA -23.865002 31.700514 South Africa TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia DNA890 NA -15.555149 32.844436 Mozambique TBA TBA TBA TBA NA Crotaphopeltis hotamboeia EI_0114 NA -27.681285 28.7443 South Africa TBA TBA TBA NA TBA

Appendix B: supplementary tables

Appendix B3. (continued) Genus species ID number Voucher Latitude Longitude Country 16s cyt–b ND4 c–mos RAG1 Crotaphopeltis hotamboeia EI_0149 NA -24.837122 29.412195 South Africa TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia EI_0072 NA -17.77453 37.175708 Mozambique TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia EI_0096 NA -17.897675 35.83339 Mozambique TBA TBA TBA NA TBA Crotaphopeltis hotamboeia PEM R18440 PEM R18440 -29.656538 26.187499 South Africa TBA TBA TBA TBA TBA Crotaphopeltis hotamboeia MBUR01497 NA -23.974167 31.075001 South Africa TBA TBA TBA TBA NA Crotaphopeltis hotamboeia TGET1-38 NA -24.44571 30.85745 South Africa TBA NA NA NA NA Crotaphopeltis hotamboeia TGET1-67 NA -24.46119 30.8699 South Africa TBA TBA TBA TBA NA Crotaphopeltis hotamboeia TGET1-7 NA -25.38841 27.56052 South Africa TBA NA TBA NA NA Crotaphopeltis hotamboeia TGET2-27 NA -27.78382 32.42762 South Africa TBA NA TBA NA NA Crotaphopeltis hotamboeia TGET2-38 NA -27.70765 32.45638 South Africa TBA TBA TBA NA NA Crotaphopeltis hotamboeia TGET2-81 NA -26.10476 31.87118 Swaziland TBA TBA TBA TBA NA Crotaphopeltis hotamboeia R631265 NA -9.100114 33.633282 Tanzania NA AF428022.1 NA NA NA Crotaphopeltis hotamboeia R631266 NA -9.100114 33.633282 Tanzania NA AF428023.1 NA NA NA Crotaphopeltis hotamboeia CCH02 NA -25.47474 30.97008 South Africa NA TBA TBA NA NA Crotaphopeltis hotamboeia CCH03 NA -25.47474 30.97008 South Africa NA TBA TBA NA NA Crotaphopeltis hotamboeia CCH04 NA -25.47474 30.97008 South Africa NA TBA TBA TBA NA Crotaphopeltis hotamboeia EI_0193 NA -16.101247 33.884958 Mozambique NA TBA TBA TBA TBA Crotaphopeltis hotamboeia MBUR00822 NA -23.955 31.025833 South Africa NA TBA TBA NA NA Crotaphopeltis hotamboeia MTSN7478 MTSN7478 -9.840193 34.607639 Tanzania NA TBA TBA TBA NA Crotaphopeltis hotamboeia TGET2-12 NA -24.5002 31.03353 South Africa NA NA TBA NA NA Crotaphopeltis barotseensis WC3903 NA -12.689351 18.360115 Angola TBA TBA TBA TBA TBA Crotaphopeltis barotseensis WC3950 NA -12.736749 18.393102 Angola NA NA NA NA TBA Crotaphopeltis degeni TMHC2015.01.605 NA 8.245298 34.59175 Ethiopia NA TBA TBA NA NA Crotaphopeltis tornieri MTSN7300 MTSN7478 -9.335337 33.991886 Tanzania TBA TBA TBA NA NA Crotaphopeltis tornieri R631329 NA -4.722572 38.352506 Tanzania NA AF428028.1 NA NA NA

Appendix B: supplementary tables

Appendix B3. (continued) Genus species ID number Voucher Latitude Longitude Country 16s cyt–b ND4 c–mos RAG1 Crotaphopeltis tornieri R631257 NA -9.100114 33.633282 Tanzania NA AF428020.1 NA NA NA Crotaphopeltis tornieri R631258 NA -9.100114 33.633282 Tanzania NA AF428024.1 NA NA NA Crotaphopeltis tornieri R631259 NA -9.100114 33.633282 Tanzania NA AF428025.1 NA NA NA Crotaphopeltis tornieri R631338 NA -5.014767 38.625201 Tanzania NA AF428026.1 NA NA NA Crotaphopeltis tornieri R631339 NA -4.722572 38.352506 Tanzania NA AF428027.1 NA NA NA Crotaphopeltis tornieri R631120 NA -5.014767 38.625201 Tanzania NA AF428034.1 NA NA NA Crotaphopeltis tornieri R631127 NA -5.014767 38.625201 Tanzania NA AF428035.1 NA NA NA Crotaphopeltis tornieri R631130 NA -4.722572 38.352506 Tanzania NA AF428036.1 NA NA NA Crotaphopeltis tornieri R631165 NA -7.15715 37.653351 Tanzania NA AF428029.1 NA NA NA Crotaphopeltis tornieri R631166 NA -7.15715 37.653351 Tanzania NA AF428032.1 NA NA NA Crotaphopeltis tornieri R631243 NA -7.746296 36.698192 Tanzania NA AF428019.1 NA NA NA Crotaphopeltis tornieri R631244 NA -7.746296 36.698192 Tanzania NA AF428030.1 NA NA NA Crotaphopeltis tornieri R631245 NA -7.746296 36.698192 Tanzania NA AF428031.1 NA NA NA T. semiannulatus EI_0079 NA -17.765188 36.323413 Mozambique TBA TBA TBA TBA NA B. foresteni RAP0540 NA NA NA NA KC347348.1 KC347468.1 KC347506.1 KC347388.1 NA D. unicolor EI_0346 NA 0.520833 30.300806 Uganda TBA TBA TBA TBA TBA T. pulverulenta CAS220642 CAS220642 NA NA NA KX660187.1 KX660460.1 KX660586.1 NA NA D. fasciata YPM13202 YPM13202 NA NA NA KX660190.1 KX660463.1 KX660589.1 KX660328.1 NA

Total: 92 59 77 71 45 25

Appendix B: supplementary tables

Appendix B4. Sequence sources for lineage diversification estimation analyses. Taxa in bold were generated in this study (Chapter Two and Three). NA indicates missing data and TBA indicate sequence data to be deposited on GenBank. GenBank accession number Family (Subfamily) Taxon cmos cyt-b ND4 Colubridae (Colubrinae) Bamanophis dorri NA AY039173 AY188001 Colubridae (Colubrinae) Boiga dendrophila AF471128 AF471089 U49303 Colubridae (Colubrinae) Boiga forsteni KC347388.1 KC347468.1 KC347506.1 Colubridae (Calamariinae) Calamaria pavimentata KX694804.1 KX694890.1 NA Colubridae (Colubrinae) Coronella girondica AF471113 AF471088 AY487066 Colubridae (Colubrinae) Crotaphopeltis barotseensis WC3903 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis degeni TMHC2015.07.605 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia EI_0072 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia FFNR002 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia JM01442 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia MBUR8574 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia PEMR18440 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia TGET1-67 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia TMHC2015.07.637 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia WC12-A082 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia WC12-A109 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia WC-ANG001 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis hotamboeia WRB933 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis tornieri 1 R631120 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis tornieri 1 R631245 TBA TBA TBA Colubridae (Colubrinae) Crotaphopeltis tornieri 2 MTSN7300 TBA TBA TBA Colubridae (Colubrinae) Dipsadoboa unicolor TBA TBA TBA Colubridae (Colubrinae) Dispholidus typus TBA TBA TBA Colubridae (Colubrinae) Dispholidus typus TBA TBA TBA

Appendix B: supplementary tables

Appendix B4. (continued) GenBank accession number Family (Subfamily) Taxon cmos cyt-b ND4 Colubridae (Colubrinae) Dolichophis caspius AY376797 AY039173 AY487039.1 Colubridae (Colubrinae) Dolichophis jugularis AY376798 AY376740 AY487046 Colubridae (Colubrinae) Eirenis eiselti AY376805 AY376747 AY487069.1 Colubridae (Colubrinae) Eirenis levantinus AY376823 AY376765 AY487071.1 Colubridae (Colubrinae) Eirenis modestus AY486957 AY486933 AY487072.1 Colubridae (Colubrinae) Eirenis punctatolineatus AY376813 AY376755 AY487073.1 Colubridae (Colubrinae) Elaphe quatuorlineata AY486955 AY486931 AY487067 Colubridae (Grayiinae) Grayia tholloni DQ486175 DQ486351 DQ486326 Colubridae (Colubrinae) Hapsidophrys lineatus TBA TBA TBA Colubridae (Colubrinae) Hapsidophrys principis TBA TBA TBA Colubridae (Colubrinae) Hapsidophrys smaragdinus TBA TBA TBA Colubridae (Colubrinae) Hemerophis socotrae AY188003 AY188042 NA Colubridae (Colubrinae) Hemorrhois algirus AY486935 AY486911 AY487037 Colubridae (Colubrinae) Hemorrhois hippocrepis AY486940 AY486916 AY487045 Colubridae (Colubrinae) Hemorrhois nummifer AY376800 AY376742 AY487049 Colubridae (Colubrinae) Hemorrhois ravergieri AY486944 AY486920 AY487050 Colubridae (Colubrinae) Hierophis gemonensis AY376799 AY039183 LN552101.1 Colubridae (Colubrinae) Hierophis viridiflavus AY486949 AY486925 FJ430660.1 Colubridae (Colubrinae) Pantherophis bairdi DQ902061.1 GU073395.1 NA Colubridae (Colubrinae) Pantherophis obsoletus FJ627805.1 DQ538343.1 KM655218.1 Colubridae (Colubrinae) Pantherophis spiloides NA NA KM655241.1 Colubridae (Colubrinae) Philothamnus angolensis EBG3003 TBA TBA TBA Colubridae (Colubrinae) Philothamnus angolensis EI_0166 TBA TBA TBA Colubridae (Colubrinae) Philothamnus angolensis ELI239 TBA TBA TBA Colubridae (Colubrinae) Philothamnus angolensis MO999 TBA TBA TBA

Appendix B: supplementary tables

Appendix B4. (continued) GenBank accession number Family (Subfamily) Taxon cmos cyt-b ND4 Colubridae (Colubrinae) Philothamnus angolensis MOZ14-253 TBA TBA TBA Colubridae (Colubrinae) Philothamnus angolensis MTSN10482 TBA TBA TBA Colubridae (Colubrinae) Philothamnus angolensis MTSN7239 TBA TBA TBA Colubridae (Colubrinae) Philothamnus angolensis MTSN7240 TBA TBA TBA Colubridae (Colubrinae) Philothamnus angolensis WC4145 TBA TBA TBA Colubridae (Colubrinae) Philothamnus carinatus 2 MBUR03481 TBA TBA TBA Colubridae (Colubrinae) Philothamnus carinauts 1 EBG1363 TBA TBA TBA Colubridae (Colubrinae) Philothamnus dorsalis MBUR08097 TBA TBA TBA Colubridae (Colubrinae) Philothamnus girardi 621 TBA TBA TBA Colubridae (Colubrinae) Philothamnus heterodermus MBUR08206 TBA TBA TBA Colubridae (Colubrinae) Philothamnus macrops MTSN8200 TBA TBA TBA Colubridae (Colubrinae) Philothamnus natalensis natalensis Tyr01 TBA TBA TBA Colubridae (Colubrinae) Philothamnus natalensis occidentalis WCDNA0087 TBA TBA TBA Colubridae (Colubrinae) Philothamnus natalensis occidentalis WCDNA816 TBA TBA TBA Colubridae (Colubrinae) Philothamnus nitidus ELI510 TBA TBA TBA Colubridae (Colubrinae) Philothamnus nitidus MBUR03179 TBA TBA TBA Colubridae (Colubrinae) Philothamnus ornatus WC12-A136 TBA TBA TBA Colubridae (Colubrinae) Philothamnus punctatus 759 TBA TBA TBA Colubridae (Colubrinae) Philothamnus ruandae MTSN7373 TBA TBA TBA Colubridae (Colubrinae) Philothamnus semivariegatus 1 HB282 TBA TBA TBA Colubridae (Colubrinae) Philothamnus semivariegatus 2 MBUR01494 TBA TBA TBA Colubridae (Colubrinae) Philothamnus semivariegatus 3 SPN091 TBA TBA TBA Colubridae (Colubrinae) Philothamnus semivariegatus 4 EI_0138 TBA TBA TBA Colubridae (Colubrinae) Philothamnus thomensis 567 TBA TBA TBA Colubridae (Colubrinae) Philothamnushoplogaster MOZ14-298 TBA TBA TBA

Appendix B: supplementary tables

Appendix B4. (continued) GenBank accession number Family (Subfamily) Taxon cmos cyt-b ND4 Colubridae (Colubrinae) Pituophis catenifer FJ627790.1 NA JF308311.1 Colubridae (Colubrinae) Pituophis catenifer vertebralis FJ627789.1 FJ627819.1 AF141126.2 Colubridae (Colubrinae) Pituophis deppei KX694814.1 FJ627818.1 AF138766.1 Colubridae (Colubrinae) Pituophis lineaticollis FJ627804.1 NA AF138768.1 Colubridae (Colubrinae) Pituophis melanoleucus FJ627797.1 AF337100.1 EU272832.1 Colubridae (Colubrinae) Pituophis ruthveni DQ902092.1 NA KJ938643.1 Colubridae (Colubrinae) Platyceps (rubriceps)collaris AY486946 AY486922 AY487053.1 Colubridae (Colubrinae) Platyceps karelini AY486942 AY486918 AY487047.1 Colubridae (Colubrinae) Platyceps najadum AY486936 AY486912 AY487048.1 Colubridae (Colubrinae) Platyceps rhodorachis AY486945 AY486921 AY487051.1 Colubridae (Colubrinae) Platyceps rogersi AY188002 AY188041 AY487052.1 Colubridae (Colubrinae) Ptyas mucosus AF471151 AF471054 AY487063 Colubridae (Colubrinae) Scaphiophis albopunctatus DQ486169 DQ486345 DQ486321 Colubridae (Colubrinae) Sibynophis subpunctatus KC347411.1 KC347471.1 KC347516.1 Colubridae (Colubrinae) Spalerosophis diadema AF471155 AF471049 AY487059 Colubridae (Colubrinae) Telescopus semiannulatus TBA TBA TBA Colubridae (Colubrinae) Thelotornis kirtlandii TBA TBA TBA Colubridae (Colubrinae) Thrasops jacksonii TBA TBA TBA