Systematics of the Genus Ptychadena Boulenger

Home , Hildebrandtia (frog), Hildebrandtia (plant), Phrynobatrachus, Ptychadena, Ptychadena bibroni, Ptychadena guibei

University of Texas at El Paso DigitalCommons@UTEP

Open Access Theses & Dissertations

2010-01-01 Systematics of the genus Ptychadena Boulenger, 1917 (Anura: Ptychadenidae) from Democratic Republic of the Congo Katrina Marie Weber University of Texas at El Paso, [email protected]

Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Biodiversity Commons, Biology Commons, Evolution Commons, and the Genetics Commons

Recommended Citation Weber, Katrina Marie, "Systematics of the genus Ptychadena Boulenger, 1917 (Anura: Ptychadenidae) from Democratic Republic of the Congo" (2010). Open Access Theses & Dissertations. 2612. https://digitalcommons.utep.edu/open_etd/2612

This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. SYSTEMATICS OF THE GENUS PTYCHADENA BOULENGER, 1917 (ANURA: PTYCHADENIDAE) FROM DEMOCRATIC REPUBLIC OF THE CONGO

KATRINA M. WEBER

Department of Biological Sciences

APPROVED:

Eli Greenbaum, Ph.D., Chair

Max Shpak, Ph.D.

Jasper Konter, Ph.D.

Patricia D. Witherspoon, Ph.D. Dean of the Graduate School

Katrina M. Weber

2010

Dedication

This thesis is dedicated to my mother and father, my continual support system, who showed me how to learn for the sake of learning. I have become the person I am today because of you.

Also to Shawn T. Dash, I may not have always been appreciative of your assistance but this never would have gotten done without your help. SYSTEMATICS OF THE GENUS PTYCHADENA BOULENGER, 1917 (ANURA: PTYCHADENIDAE) FROM DEMOCRATIC REPUBLIC OF THE CONGO

KATRINA M. WEBER, B.Sc.

THESIS

Presented to the Faculty of the Graduate School of The University of Texas at El Paso

in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Department of Biological Sciences THE UNIVERSITY OF TEXAS AT EL PASO December 2010 Acknowledgements I would like to start by acknowledging my thesis committee, without which this thesis would never have been finished: Dr. Eli Greenbaum, who accepted me as a graduate student and introduced me to a new side of biology, Dr. Max Shpak for helping me with statistical analyses and providing new ideas, and Dr. Jasper Konter, who filled in the many gaps in my geological knowledge.

I would also like to thank all the other faculty members that have helped me along the way: Dr. Elizabeth Walsh, who has been a caring mentor since my time as an undergraduate and is always willing to provide help and advice and Dr. Vanessa Lougheed, who patiently explained statistics when I knew next to nothing.

Omar Hernandez, Dr. Carolina Lema, and Ana Betancourt in the UTEP DNA Analysis Core Facility (NIH grant #5G12RR008124) completed hundreds of DNA sequencing requests for me. Also, the UTEP Graduate School gave me two Graduate Student Research Grants, which paid for all the supplies needed for DNA sequencing. Field work conducted by Dr. Eli Greenbaum for this study was supported by National Geographic Research and Exploration Grant #8556–08. Garin Cael provided invaluable research assistance at the Royal Museum for Central Africa in Tervuren, Belgium.

Cesar Villanueva taught me all the lab procedures I know and was always the one to troubleshoot lab problems. The other members of the Greenbaum lab: Chris Anderson and Federico Valdez provided support and advice throughout my project. To my friends and fellow graduate students:

Jennifer Martinez, Kevin Floyd, and Shawn Dash, thank you for endless lunch discussions, project advice, and general encouragement.

v Abstract

This thesis increases the scope of previous phylogenetic analyses that revealed high levels of genetic differentiation within the anuran genus Ptychadena. Herein, I increase sampling of Central African populations of Ptychadena by over six times to examine their relationships to other African populations and to search for cryptic species. This study represents the most comprehensive molecular phylogeny of Ptychadena to date. A total of three mitochondrial (12S, 16S, and cyt b) and two nuclear (RAG1 and rhodopsin) genes were sequenced for 67 specimens of Ptychadena. Maximum parsimony, maximum likelihood, and Bayesian analyses were conducted. These analyses revealed a great deal of genetic diversity in the genus Ptychadena, suggesting that many species have been separated into distinct genetic lineages that will be recognizable as distinct species as some point. There are five genetically distinct lineages of P. mascareniensis sensu lato, three of which occur in the DRC.

Multivariate analyses of morphometric and meristic data did not reveal any distinct morphological differences between any of the clades except P. mascareniensis sensu stricto in Madagascar. Based on habitat structuring, two of these distinct lineages, a savannah-dwelling lineage and a forest-dwelling lineage, should be recognized as distinct species under the names P. nilotica and P. marchei, respectively. The formation of the Albertine Rift combined with subsequent climatic shifts that increased habitat heterogeneity likely led to the establishment of these savannah and forest forms of P. mascareniensis.

vi Table of Contents

Acknowledgements...... v

Abstract ...... vi

Table of Contents...... vii

List of Tables ...... ix

List of Figures ...... x

Chapter 1: General Introduction ...... 1 1.1 Amphibian Declines ...... 1 1.2 Introduction to Democratic Republic of the Congo ...... 4 1.3 Historical Geology and Climate of Africa ...... 5 1.4 Introduction to Ptychadena...... 7 1.5 Research Objectives ...... 14

Chapter 2: Materials and Methods...... 15 2.1 Specimen Acquisition...... 15 2.2 Molecular analysis ...... 15 2.3 Species Concepts ...... 20 2.4 Morphological Analysis ...... 20

Chapter 3: Results of Molecular Study ...... 22 3.1 Phylogenetic Analyses ...... 22 3.2 Combined Dataset ...... 25 3.3 Genetic diversity in Ptychadena ...... 29 3.4 Genetic diversity in Ptychadena mascareniensis...... 30 3.5 Haplotype Networks ...... 34

Chapter 4: Results of Morphological Analyses ...... 40 4.1 Diagnostic characters in Ptychadena mascareniensis ...... 40 4.2 Multivariate Statistical Analyses ...... 46

Chapter 5: General Discussion and Conclusions ...... 57 5.1 Genetic diversity in Ptychadena ...... 57 5.2 Genetic diversity in Ptychadena mascareniensis...... 58 5.3 Taxonomic status of Ptychadena mascareniensis ...... 60

vii 5.4 Biogeography ...... 66 5.5 Conservation Implications ...... 72 5.6 Conclusions and future directions ...... 72

Literature Cited ...... 74

Appendices ...... 93 Appendix A: Locality Information for All Specimens ...... 93 Appendix B: Morphometric and Meristic Characters Used for Morphological Analyses ...... 99 Appendix C: Complete Measurement Data for All Ptychadena Specimens ...... 102

Curriculum Vita ...... 118

viii List of Tables

Table 1.4.1: Summary of the supraspecific taxonomic history of the family Ptychadenidae...... 11 Table 2.2.1: Primers used for sequencing mitochondrial and nuclear genes ...... 16 Table 3.2.1: Models of evolution selected by jModelTest ...... 27 Table 3.3.1: Uncorrected mean pairwise (p) divergence values between clades...... 30 Table 3.4.1: Uncorrected mean pairwise divergence values within and between clades of specimens identified as P. mascareniensis ...... 31 Table 3.4.2 Analysis of molecular variance results...... 33 Table 3.4.3 – Pairwise genetic differentiation statistics (FST) between clades of P. mascareniensis...... 33 Table 4.1.1 – Illustration of the overlapping of morphometric and meristic characters frequently used to diagnose species of Ptychadena...... 42 Table 4.2.1 – Principal components analysis comparing clades of Ptychadena mascareniensis using natural log-transformed morphometric data...... 48 Table 4.2.2 – Principal components analysis comparing clades of Ptychadena mascareniensis using morphometric data regressed against snout–vent length...... 49 Table 4.2.3 – Principal components analysis comparing clades of Ptychadena mascareniensis using standardized meristic data...... 50 Table 5.3.1 – All subspecies of P. mascareniensis that have been described and subsequently synonymized by one or more authors...... 62

ix List of Figures

Figure 1.4.1: Majority rule consensus tree (51%) from parsimony analysis of the previously recognized subfamily Ptychadeninae using morphological characters...... 13 Figure 3.1.1 – Maximum parsimony phylogram of all Ptychadena haplotype sequences for the 16S rRNA gene...... 23 Figure 3.1.2 – Maximum likelihood phylogram of all Ptychadena haplotype sequences for the 16S rRNA gene...... 24 Figure 3.1.3 – Bayesian analysis phylogram of all Ptychadena haplotype sequences of the 16S rRNA gene...... 26 Figure 3.2.1 – Maximum likelihood phylogram of all DRC Ptychadena sequenced for the combined dataset (12S, 16S, cyt b, RAG1, and rhodopsin)...... 28 Figure 3.4.1 – Map of haplotype distribution for clades of Ptychadena mascareniensis...... 32 Figure 3.5.1 – Haplotype network of Clade A of Ptychadena mascareniensis, reconstructed using parsimony probability implemented in TCS and map showing sampling sites...... 35 Figure 3.5.2 – Haplotype network of Clade C of Ptychadena mascareniensis, reconstructed using parsimony probability implemented in TCS and map showing sampling sites...... 36 Figure 3.5.3 – Haplotype network of Clade D of Ptychadena mascareniensis, reconstructed using parsimony probability implemented in TCS and map showing sampling sites...... 37 Figure 3.5.4 – Haplotype network of all sequences of Ptychadena mascareniensis...... 39 Figure 4.1.1 – Illustration of the three positions of the male gular slits within Ptychadena...... 41 Figure 4.1.2 – Plate of morphological variation in the genetically identified clade A ...... 43 Figure 4.1.3 – Plate of morphological variation in the genetically identified clade B ...... 44 Figure 4.2.1 – Scatter plot of the first and second principal component scores for the analysis using natural log-transformed morphometric data...... 51 Figure 4.2.2 – Scatter plot of the first and second principal component scores for the analysis using morphometric data regressed against snout–vent length...... 52 Figure 4.2.3 – Scatter plot of the first and second principal component scores for the analysis using standardized meristic data...... 53 Figure 4.2.4 – Scatter plot of canonical scores calculated from discriminant function analysis using 6 species groups...... 55 Figure 4.2.5 – Scatter plot of canonical scores calculated from discriminant function analysis using 4 species groups...... 56

x Chapter 1: General Introduction

1.1 AMPHIBIAN DECLINES

The Order Anura is composed of over 5,800 extant species of frogs and toads, making it the most speciose taxon of amphibians (AmphibiaWeb 2010). Unfortunately, rapid declines in populations of many species are leading to an increasing number of amphibian extinctions. Concern over rapid amphibian declines began in 1989 at the First World Congress of Herpetology, but declines are thought to have started as early as 1970. Subsequent studies have revealed the severity of amphibian declines around the world (Pounds et al. 1997; Lips 1998; Houlahan et al. 2000; Young et al. 2001; Whitfield et al. 2007; Rohr and Raffel 2010).

A lack of a comprehensive picture of the extent and severity of amphibian declines prompted the IUCN (World Conservation Union) Global Amphibian Assessment (GAA), which aimed to gather data on the status of all amphibian species (Stuart et al. 2004). The study showed that a staggering 32.5% of the planet‘s amphibian species are globally threatened—listed by the IUCN Red List Categories (IUCN 2010) as Vulnerable, Endangered, or Critically Endangered, and 39 are now extinct (IUCN 2010). In comparison, only 23% of mammals and 12% of birds are globally threatened (Stuart et al. 2004). The threat to amphibians is likely severely underestimated, however, since another 1,294 species (22.5%) are so poorly known (i.e., Data Deficient) that their status cannot be assessed (Stuart et al. 2004).

1 The causes for amphibian declines are known for only 52% of affected species (Stuart et al. 2004). Factors that are understood to cause amphibian declines include introduced species (Vredenburg

2004; Hayes et al. 2010a; Vogel and Pechmann 2010), overexploitation (Lannoo et al. 1994; Stuart et al. 2004), and habitat loss, fragmentation, and alteration because of human activities (Fisher and Schaffer 1996; Sodhi et al. 2008; Becker et al. 2010). For the remaining 48% of declining species, the causes remain unknown (Stuart et al. 2004). Suggested causes of these rapid enigmatic declines include climate change (Pounds et al. 1999; Pounds et al. 2006; Rohr and Raffel 2010), UV radiation (Blaustein et al. 2003; Bancroft et al. 2008; Searle et al. 2010), chemical contaminants (Hayes et al. 2002; Relyea and Diecks 2008; Hayes et al. 2010b), infectious diseases such as chytridiomycosis (Daszak 1998; Lips et al.

2008; Lötters et al. 2009; Crawford et al. 2010), or a combination of these factors (Wake and Vredenburg 2008; D‘Amen and Bombi 2009; Hayes et al. 2010a). Enigmatic species declines represent the greatest challenge to conservation, especially because they frequently occur in already well- protected areas (Stuart et al. 2004). Although many aspects of rapid amphibian declines are poorly understood, declines show important regional and taxonomic patterns. For example, Neotropical species are affected more severely than Afrotropical or Indomalayan species. Additionally, species from Australia and New Zealand suffer from significantly more declines than the average for amphibians as a whole (Stuart et al. 2004). Causes of decline vary by region as well. Overexploited species tend to be concentrated in East and Southeast

Asia, whereas reduced-habitat species occur more widely. The effect of chytrid infections also varies by region. For example, chytrid infections are widespread at study locations in Kenya (Kielgast et al. 2010) and Uganda (Goldberg et al. 2007). Chytrid also may be the cause of severe amphibian declines in

Africa, including the apparent extinction in the wild of the Kihansi Spray Toad (Nectophrynoides asperginss) in Tanzania (Channing et al. 2006). Similar analyses in the DRC, however, found evidence of chytrid infections in only two frogs, and declines caused by the fungus are not yet evident

(Greenbaum et al. 2008). This discrepancy within tropical Africa may be a result of long-term coevolution between some African amphibians and the chytrid fungus, which is thought to have evolved in Africa in the genus Xenopus and subsequently spread globally through extensive trade for biomedical

2 research (Weldon et al. 2004). Enigmatic-decline species are mostly restricted to South America, Mesoamerica, Puerto Rico, and Australia (Stuart et al. 2004). Taxonomically, four amphibian families contribute overwhelming numbers to the total of rapidly declining species: Bufonidae (true toads), Leptodactylidae (typical Neotropical frogs), Hylidae (treefrogs), and Ranidae (true frogs) (Stuart et al. 2004). Causes of declines also display taxonomic patterns. Overexploitation has affected Ranidae most severely because of frequent harvest for human consumption, whereas enigmatic declines have been most catastrophic in the Bufonidae (Stuart et al. 2004). Habitat loss is a significant cause of decline in all families (Stuart et al. 2004). The wide variation in declines between families is likely a result of the nonrandom geographic pattern of declines discussed above.

Because of their declining population numbers and sensitivity to environmental change, amphibians are frequently used as indicators of environmental quality in both aquatic and terrestrial ecosystems (Welsh and Ollivier 1998; Sheridan and Olson 2003). Highly permeable skin, a biphasic life cycle most often including an aquatic and terrestrial stage, and a rudimentary immune system are thought to make amphibians sensitive to environmental stress, including water quality and UV radiation (Gerlanc and Kaufman 2005; Taylor et al. 2005; Halliday 2008; Wake and Vredenburg 2008). Their value as biometrics is further enhanced because they are relatively easy and inexpensive to measure (Wake 1991; Waddle 2006; Welsh and Hodgson 2008). Kerby et al. (2010) suggested that amphibians are not as useful as environmental indicators as previously thought. They argue that although amphibians are estimated to be going extinct at 200 times the background rate (Roelants et al. 2007), it does not necessarily follow that amphibians are the most sensitive taxa to environmental perturbations

(Kerby et al. 2010). Nonetheless, amphibians are likely symbols of a major biodiversity crisis (Wake

1991). It has been suggested that global biodiversity is being threatened with a sixth mass extinction (Wilson 1988; Leakey and Lewin 1996; Wake and Vredenburg 2008; Ceballos et al. 2010), and this process may be affecting amphibians more severely than other vertebrate taxa, making the decline of amphibians an indicator of a major ecological crisis (Halliday 2008; Wake and Vredenburg 2008). Despite increases in amphibian declines and extinctions, the rate of discovery of new species is increasing. During the last decade, the number of amphibian species has shown an overall global

3 increase of 19.4%, reaching 6,752 recognized species in November, 2010 (AmphibiaWeb 2010). This acceleration in the rate of newly discovered species stems mainly from recent studies in tropical areas

(e.g., Meegaskumbura et al. 2002; Faivovich et al. 2005; Manamendra-Arachchi and Pethiyagoda 2005; Stuart et al. 2006; Fouquet et al. 2007; De la Riva 2007). Even higher numbers of undescribed amphibians are expected to exist in poorly studied tropical areas (Meegaskumbura et al. 2002; Fouquet et al. 2007). This rapid rate of species discovery illustrates the relative lack of knowledge regarding amphibian biodiversity. Central Africa is one area where biodiversity is poorly known because of inadequate sampling resulting from a lack of government infrastructure, prevalence of tropical diseases, and political instability, especially in the Democratic Republic of the Congo (DRC), which has suffered from frequent warfare since its independence in 1960.

1.2 INTRODUCTION TO DEMOCRATIC REPUBLIC OF THE CONGO

This study will focus on specimens of Ptychadena collected in DRC, Central Africa. The DRC is home to the second largest block of undisturbed rainforest in the world, with 45% of the country still covered by closed-canopy forest. The biodiversity of the country is exceptionally high, stemming from the wide variety of ecosystems in the country: tropical lowland forest and swamp forest in the Central Basin, tropical forest in the north and south, dry savannah and woodlands, and afro-montane rainforests along the eastern border (Bashige et al. 2004). The DRC is also one of 18 countries in the world that have been identified as ―megadiverse‖. Together, these 18 countries represent over 70% of the world‘s biodiversity, making them important targets for conservation. The DRC is one of only 3 ―megadiverse‖ countries in Africa, and the only one in tropical continental Africa (Mittermeier et al. 1997). Despite conservation efforts, however, at least 190 plant and animal species in the DRC are now considered threatened or endangered (IUCN 2008), because of continued civil war, poor economic conditions, poaching, collections for the pet trade, and accelerating deforestation (Koenig 2008, Laporte et al.

2007). The importance of this biodiversity has been recognized by the country‘s government, leading to the establishment of seven national parks, 30 hunting and wildlife preserves, three biosphere reserves and several forest reserves. Together, they protect 12% of the country (Bashige et al. 2004).

4 Historically, however, the DRC has not been extensively sampled for amphibians, especially over the past 40 years (Poynton 1998, IUCN et al. 2008). Central Africa is, however, experiencing severe amphibian declines due mainly to habitat destruction (Laporte et al. 2007; IUCN 2008). Anurans may also face future declines because of an infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis that has been detected in the DRC, although declines are not currently evident (Greenbaum et al. 2008). Therefore, it is crucial to determine accurate herpetological diversity of the DRC immediately, since organisms cannot be conserved if they have never been identified (Mace 2004).

1.3 HISTORICAL GEOLOGY AND CLIMATE OF AFRICA

The geologic and climatic history of the continent of Africa and specifically the DRC provides ample opportunity for speciation, resulting in an overall high biodiversity (Burgess et al. 2004). Africa is a remnant of Gondwanaland, which started breaking up about 290 million years ago (Ma) and some geologic formations in South Africa date back to 3.5 billion years ago (Ga). The continent was stable over the 80 million years between the late Cretaceous and the middle Tertiary, but tectonic activity began 30 Ma, resulting in many of the current mountain ranges. The continent gradually moved northward and rotated into southern Europe, causing major faults in the crust of eastern Africa. Since then, extension has resulted in the creation of the East African Rift System (Meijer and Wortel 1999).

The Albertine Rift is the western section of Africa‘s two rift valleys, which are gigantic tectonic valleys 20 to 40 miles wide, bounded by faulted rift flanks and formed by lateral stretching in the earth‘s crust. The rift valleys are relatively young geologically, as indicated by the abruptness of their escarpments as well as active volcanism and earthquakes in the region. It has been suggested that their formation began in the early Miocene, approximately 20 Ma (Ebinger 1989). Areas of uplift and volcanic activity usually provide enhanced opportunities for new species to evolve allopatrically

(Burgess et al. 2004). Over the past one to two million years, ice age cycles have caused dramatic shifts in the African climate associated with periods of higher or lower rainfall, which lasted thousands of years (Gasse et al.

5 1990; Burgess et al. 2004). Maximum ice cover during the last glacial maximum occurred 18–20 thousand years ago (Ka). At that time, ice covered about 30% of the Earth‘s land surface, as compared with 10% today. This ice cover resulted in a lowering of temperature by 10–20°C in temperate regions, and 2–5°C in the tropics. Additionally, a wide body of evidence suggests that land not covered by glaciers was substantially drier than present. This was likely caused by decreased evaporation from oceans because of lower sea level and temperature, as well as increased evaporation on land because of strengthened wind systems and a 10% increase in dry land surface. In Africa, desert sand dunes advanced to within 5–10° latitude of the equator, covering many areas that are now dominated by rainforest (Tallis 1991).

The Pleistocene forest refuge hypothesis, first proposed in 1846 by Edward Forbes, suggests that the climatic and vegetational changes described above caused fragmentation of previously continuous species ranges into isolated refuges. Originally, this hypothesis was applied only to species in temperate zones, but the Pleistocene also caused drastic changes in vegetation distributions in the tropics and subtropics (Mayr and O‘Hara 1986). Periods of reduced rainfall caused temporary fragmentation of previously continuous rainforests into forest islands isolated by wide stretches of savannah. This resulted in isolation of tropical lowland fauna in these forest refuges and subsequent allopatric speciation. These former refuges are now thought to possess high levels of species richness and endemism (Diamond and Hamilton 1980). The effect of glacial refugia can be observed in areas with high species richness along the margins of the Congo Basin in contrast to the lower diversity of the central Congo Basin, an area thought to have lost most of its forest during the peak of glacial drying (Maley 1997; Burgess et al.

2004; Marks 2010). As rainfall again increased, the forests expanded and savannahs were reduced, many times leading to the establishment of contact zones between previously isolated forest taxa (Mayr and O‘Hara 1986). Therefore, many studies now assert that isolation by vegetational barriers could have been as effective in the tropics as in temperate regions (Chapin 1932; Moreau 1954; Snow 1978).

Current and past climactic patterns may be useful in explaining the current biogeographic structuring of amphibian populations, including those in the genus Ptychadena.

6 1.4 INTRODUCTION TO PTYCHADENA

This research is focused on the anuran genus Ptychadena, classified in the family Ptychadenidae Dubois, 1987. Ptychadenidae is composed of 3 genera: Ptychadena Boulenger, 1917 (49 species),

Hildebrandtia Nieden, 1907 (3 species), and Lanzarana Clarke, 1982 (1 species) (Frost 2010). Species of Ptychadena are distributed across Sub-Saharan Africa and the islands of São Tomé, Madagascar, the Seychelles, and the Mascarenes (Meyer 2004). Four species of Ptychadena are classified as Threatened or Endangered, whereas a large number (11) are classified as Data Deficient (IUCN 2010), highlighting the need for a more detailed conservation assessment of this genus. Ptychadena includes several species with considerable morphological variation, resulting in poorly defined species limits and confused taxonomic states. A confused taxonomy, in turn, makes protection of the most threatened taxa in the genus impossible because data to be used for conservation measures cannot be collected if specimens cannot be identified as distinct taxa (Mace 2004; Poynton 1970).

1.4.2 Natural History of Ptychadena

Knowledge and documentation of the natural history of Ptychadena is not only beneficial for our biological understanding of each species, which can inform conservation policy, but may be useful in modifying current taxonomies and determining evolutionary patterns. Ptychadena are medium-sized frogs that are often abundant in wetland areas and irrigated agricultural landscapes of sub-Saharan

Africa, including several islands (Channing 2001). Ptychadena diversity is centered in the tropics of continental Africa, with the heaviest concentration of species in the region of the Zambezi-Congo divide, a continental divide between the Zambezi and Congo Rivers (Poynton 1970). In the southern third of Africa, Ptychadena diversity is generally concentrated in savannahs but there are several sylvicolous (woodland) species in the north. For most species, prey preference has not been recorded. Ptychadena mascareniensis (Duméril

& Bibron, 1841), however, is known to consume earthworms, snails, arthropods, and other frogs (Channing 2001). Ptychadena pumilio (Boulenger, 1920) is known to feed mainly on spiders and orthopterans (Rödel 2000). Tadpoles are likely efficient predators on a wide variety of insects, as

7 evidenced by bioaccumulation of pesticides in larval tissues (Channing 2001). In turn, species of Ptychadena are preyed upon by several species of snakes, including the boomslang (Dispholidus typus), as well as birds such as cattle egrets (Bubulcus ibis) and yellow billed kites (Milvus aegyptius). Interestingly, the large slit-faced bat (Nycteris grandis) feeds exclusively on P. anchietae (Bocage, 1967), even when other frog species are present in the same pond. The bat finds male frogs by listening for their advertisement calls, and is apparently tuned in only to the calls of P. anchietae. In addition to wild predators, some tribes in West Africa are also known to consume these frogs, specifically P. mascareniensis (Channing 2001). When faced with a threat, species may attempt to escape by either leaping into the water or away from the water to take cover in vegetation. It has been suggested that further investigation of this behavior could lead to its use as a character for subgeneric classification (Channing 2001), but this is unlikely. The majority of species, including P. mascareniensis and P. subpunctata (Bocage, 1866) have a tendency to leap into water. Only a few species have a tendency to leap away from water, including P. anchietae, P. oxyrhynchus (Smith, 1849), and P. porosissima (Steindachner, 1867) (Poynton & Broadley 1985; Channing 2001). If captured, several species including P. mascareniensis, exhibit a ―foam and moan‖ display by producing skin secretions and making a moaning call while in a rigid posture. This behavior is designed to distract the predator long enough for the frog to jump to safety. More commonly, individuals will bow the body, stiffen and straighten the hind legs and squeak when caught (Channing

2001).

1.4.3 Reproduction in Ptychadena

In most Ptychadena species for which reproductive biology is studied, eggs are deposited on the surface of stagnant waters in clutches of 500–1500 eggs, with clutch size being positively correlated with female snout–vent length (Rödel 2000). Ptychadena tellinii (Peracca, 1904) has been found in breeding aggregations at shallow, temporary pools in grass savannah, which is the most common breeding behavior displayed in the genus. Ptychadena aequiplicata (Werner, 1898) was found depositing clutches on the ground in vegetation at the edges of dried forest ponds to ensure that eggs

8 come in contact with open water only after the ponds reach maximum capacity. Larvae of this species can survive in eggs for up to two weeks (Rödel et al. 2002). Uniquely, P. broadleyi Stevens, 1972 breeds in seeps on granite outcrops, although the details of breeding are unknown (Channing 2001). Species have also been documented reproducing in ponds, marshes, along stream banks, in grass savannah and in woodland-savannah mosaics (Inger 1968). Females will frequently deposit their eggs at multiple locations, a practice known as risk spreading (Rödel 2000). The breeding season length and timing varies between species. For the majority of species, it is known to begin with the wet season, with little evidence of breeding during the dry season (Schmidt and Inger 1959; Rödel 2000). Individuals in breeding condition are most abundant from the end of the dry season until about two months before the end of the wet season. Vocalizing males of Ptychadena species are generally first heard near the end of the dry season (Inger 1968). Males are known to call while floating in the water, hiding in vegetation, or from exposed sites on bare soil (Rödel 2000). Many adults disappear after the rains stop, which likely reflects secretive behavior in response to dry conditions (Inger 1968). Most species spend the dry season under stones on river banks, in crevices, or in the mud of dry ponds. Large inner metatarsal tubercles may assist some species in burrowing during the dry season (Rödel 2000). Ptychadena has typical ovoid tadpoles, although a few traits vary between species, which may be useful for identification (Inger 1968). The length of development is largely unknown, but in P. bibroni

(Hallowell 1845), metamorphosed young have been collected one month after the beginning of the wet season (Rödel 2000). Metamorphosis likely occurs in many species when the frogs reach at least 10 mm snout–vent length (Inger 1968), although unfavorable conditions may induce earlier metamorphosis

(Rödel 2000). In captivity, Ptychadena species have reached a lifespan of almost nine years, but their life span in the wild is unknown (Channing 2001).

1.4.4 Taxonomic History of Ptychadena

The genus Ptychadena is classified in the family Ptychadenidae, which was originally recognized as the tribe Ptychadenini within the family Ranidae Rafinesque, 1814. The tribe was

9 subsequently elevated to subfamily rank (Ptychadeninae) by Dubois (1992) and then further elevated to family by Frost et al. (2006). Currently, Ptychadenidae is considered to be the sister group to the family

Ceratobatrachidae Boulenger, 1884 (Batrachylodes Boulenger, 1887; Ceratobatrachus Boulenger, 1884; Discodeles Boulenger, 1918; Ingerana Dubois, 1987 ‗‗1986‘‘; Palmatorappia Ahl, 1927 ‗‗1926‘‘; and Platymantis Günther, 1858), which was also previously considered part of Ranidae (Frost et al. 2006).

The sister relationships of Ptychadenidae to other families have been well substantiated (Bossuyt et al. 2006). A lack of taxonomic clarity has led to an extensive literature that highlights disagreement over nomenclature, classification, and phylogenetic relationships. Taxonomic inconsistencies are visible at the supraspecific level for the three genera in Ptychadenidae, as illustrated in Table 1.4.1. Disagreement is mainly the result of poorly defined species that are highly polymorphic with extensive geographic variation and ambiguous diagnostic characters (Schmidt and Inger 1959; Largen 1997; Meyer 2004).

Despite disagreement over nomenclature, Ptychadenidae has been shown to form a monophyletic clade based on several synapomorphies. The synapomorphies shared by the family include: loss of the palatines, reduced clavicles, a short tapering sternal style that forms a compact bony element, the fusion of the eighth presacral and sacral vertebrae, dorsal protuberance of the illium that is not differentiated from the dorsal prominence, pronounced skin folds, separation of the outer metatarsal tubercle from the inner, and condition of the inner and outer metatarsal tubercles (Clarke 1981, 1982; Meyer 2004).

Within the genus Ptychadena, few studies (Laurent 1997; Meyer 2004; Vences et al. 2004; Measey et al. 2007) have attempted to resolve phylogenetic relationships and the majority of the literature consists only of species descriptions and collection accounts (e.g., Laurent 1954; Schmidt and

Inger 1959; Perret 1991; Largen 1997). Moreover, many valid species are buried in synonymies of other taxa, and other species have been described under several unique names (Guibé and Lamotte 1954). Therefore, species diversity and phylogenetic relationships remain inadequately investigated and unresolved. Laurent (1997) attempted to use osteological features to construct a phylogeny, but could find no clear morphometric differences. His study did, however, identify the osteological synapomorphies and autapomorphies of the genus. Apomorphies of Ptychadena include shortening of

Table 1.4.1: Summary of the supraspecific taxonomic history of the family Ptychadenidae. Modified from Meyer (2004).

Authority Order Family Subfamily Tribe Genus Subgenus (Date)

Parker1 Anura Ranidae ------Rana Abrana (1930) Ptychadena

Laurent Anura Ranidae Raninae ---- Ptychadena - (1954)

Guibé & Anura Ranidae ------Rana Ptychadena Lamotte2 (1957)

Poynton Anura Ranidae Raninae ---- Hildebrandtia - (1964) Ptychadena -

Clarke Anura Ranidae Raninae ---- Hildebrandtia - (1981) Lanzarana - Ptychadena -

Dubois Anura Ranidae ------Rana Hildebrandtia (1981) -- Ptychadena

Dubois Anura Ranidae Raninae Ptychadenini Hildebrandtia Hildebrandtia (1986) Ptychadena Lanzarana

Dubois Anura Ranidae Ptychadeninae ---- Hildebrandtia Parkerana (1992) Lanzarana Ptychadena Ptychadena -

Frost Anura Ranidae ------Hildebrandtia - (2003) Lanzarana - Ptychadena -

Frost et Anura Ptychadenidae ------Hildebrandtia - al. (2006) Lanzarana - Ptychadena -

1 Parker (1930) implies Ptychadena and Abrana may be genera instead of subgenera 2 Guibé & Lamotte (1957) essentially treat Ptychadena as both a genus and a subgenus

11 clavicles that stay separated on the midline, anterior branch of the pterygoid curved toward the middle of the skull, and reduction of the superposition of the medial branch of the pterygoid and the wing of the parasphenoid. Autapomorphies include widely separated cervical condyles, a broadly forked base of the omosternum, terminal phalanges of fingers and toes curved dorsoventrally, as well as longer vocal slits, feet and snout–nostril distance. The phylogeny hypothesized using these osteological characters consisted of many polytomies and therefore provided little resolution of interspecific relationships (Laurent 1997). Meyer (2004) completed the first comprehensive algorithm-based analysis of the then- recognized subfamily Ptychadeninae, using morphological characters. His study included 42 of the 49 recognized species of Ptychadena, and provided the most comprehensive analysis of evolutionary relationships within the genus at that time. Characters were analyzed using parsimony, and the resulting consensus tree, although relatively weakly supported, provides one of the only comprehensive phylogenies available (Fig. 1.4.1). Unlike Laurent‘s (1997) tree, there is only one polytomy, consisting of the basal species P. tellinii and P. trinodis (Boettger, 1881). Additionally, the widespread species P. mascareniensis was analyzed as multiple samples from disparate geographic areas, and the branch lengths separating several distinct geographical groups indicated the presence of more than one sibling species. Meyer (2004) hypothesized that the weak support for relationships between species of

Ptychadena may result from a general lack of distinct morphological characters. It is also possible that patterns of glacial refugia leading to species separation and subsequent hybridization have led to an indistinct morphology for many species within the genus. Furthermore, convergence or parallelism may have given rise to high levels of homoplasy that obscures the true evolutionary relationships. The result is a genus of frogs with morphological characters that are difficult to define, highly subjective, and limited in number (Meyer 2004). Obscure relationships may also reflect a recent divergence that is not yet evident in morphological characters (Poynton 1970; Meyer 2004). These ambiguous relationships

Figure 1.4.1: Majority rule consensus tree (51%) from parsimony analysis of the previously recognized subfamily Ptychadeninae using morphological characters. Consensus tree is based on two equally most parsimonious trees. Ptychadena mascareniensis was analyzed as six geographic groups in which, S=South, N=North, Sey=Seychelles, M=Madagascar, E=East, W=West. Figure redrawn from Meyer (2004).

13 based on morphology alone suggest it is imperative to use molecular data to elucidate the true species diversity and phylogenetic relationships within the genus.

Two molecular analyses of Ptychadena have been completed (Vences et al. 2004; Measey et al. 2007), both as parts of studies on transoceanic dispersal in amphibians. Vences et al. (2004) focused on P. mascareniensis, which is the only amphibian species found on the continent of Africa as well as on

Madagascar, the Seychelles Islands, and the Mascarene Islands. Historically, any specimen with vocal sacs located above the forearm insertion was classified as P. mascareniensis (Lamotte 1967). Molecular analyses (Vences et al. 2004; Measey et al. 2007) have found five distinct clades (> 5% divergence) of P. mascareniensis. These results suggest that P. mascareniensis represents a complex of cryptic species that has been capable of transoceanic dispersal in the past, but no studies have resolved the taxonomy of the clade (Vences et al. 2004; Measey et al. 2007). Distinct lineages were also evident within other Ptychadena species groups, suggesting that there is a large amount of cryptic speciation within the genus

(Vences et al. 2004).

1.5 RESEARCH OBJECTIVES

My research focuses on two interdependent questions: 1) Does contemporary taxonomy represent true species richness of Ptychadena in the DRC? 2) Can proposed phylogenetic relationships resolve current biogeographic patterns?

The objectives of my research are: 1) To determine the extent of genetic variation occurring across the genus and identify

lineages representing cryptic species. 2) To construct a phylogenetic framework based on molecular data that facilitates a thorough analysis of morphological variation between and within species.

3) To determine historical geological and climatological influences that may have led to current patterns of speciation.

14 Chapter 2: Materials and Methods

2.1 SPECIMEN ACQUISITION

Most examined specimens were collected by Dr. Eli Greenbaum in the DRC over three summers (2007–2009) and one winter season (2010). Sampling was completed during both wet and dry seasons in a multitude of habitats. Tissue samples were preserved in 95% ethanol and whole specimens were preserved in 10% buffered formalin. A total of 65 specimens of Ptychadena, two specimens in the outgroup Hildebrandtia ornata, and one specimen in the outgroup Phrynobatrachus acutirostris were collected. An additional five tissue samples were obtained from the North Carolina Museum of Natural Sciences (NCSM) and three tissue samples from University of Texas at Arlington Department of

Biology (UTA), resulting in a total of 76 tissues. An additional 75 Ptychadena 16S sequences as well as one outgroup sequence (Hildebrandtia ornata) were obtained from GenBank and were included in phylogenetic analyses for a total of 151 sequences.

Locality information, GenBank accession numbers as far as available, and voucher specimen data are included in Appendix A. Museum abbreviations follow standard codes (Sabaj Pérez 2010). Outgroup selection was based on previous molecular studies including Ptychadena (Vences et al. 2004;

Frost et al. 2006; Measey et al. 2007). This combination of specimens provides the best assessment of Ptychadena biodiversity ever completed for the DRC.

2.2 MOLECULAR ANALYSIS

2.2.1 DNA Extraction

DNA was extracted from tissues using the Qiagen DNeasy Blood and Tissue Kit (Valencia, CA).

A small piece of tissue, about 25 mg, was cut off and soaked in deionized water in a refrigerator at 0.2°C for 2 hr to allow ethanol to diffuse out of the tissue sample. After removing the water, samples were dried in a refrigerator at 0.2°C for 2 hr to desiccate the samples. After drying, 180 µl of Buffer ATL and

20 µl of Proteinase K were added to each sample. Samples were then incubated at 55°C for 30 min, after which they were vortexed for 20 seconds and then incubated overnight. After incubation, 200 µl of Buffer AL was added to each sample. Samples were vortexed for 20 seconds and incubated at 70°C for

15 10 min. Following incubation, 200 µl of 100% ethanol was added, and each sample was vortexed for 20 seconds. The resulting solution was transferred into Qiagen spin columns, which were centrifuged at

8000 rpm for 1 min. Liquid remaining in the spin columns was discarded and 500 µl of Buffer AW1 added to each sample. Samples were then centrifuged again at 8000 rpm for 1 min. The remaining liquid was discarded and 500 µl of Buffer AW2 added. Samples were then centrifuged at 14,000 rpm for 3 min. Liquid was again discarded, 200 µl of Buffer AE was added and allowed to incubate at room temperature for 1 min, and samples were then centrifuged at 8000 rpm for 1 min. The resulting solution was saved for use in PCR. The previous step was then repeated, resulting in a total of 400 µl of DNA solution to be used in PCR.

2.2.2 DNA Amplification and Purification

DNA fragments were amplified for 3 mitochondrial (16S, 12S, and cyt b) and 2 nuclear (RAG1 and rhodopsin) genes using the primers in Table 2.2.1. All amplifications included a negative control to ensure no contaminated DNA was amplified. Amplification was completed using a denaturation temperature of 95°C, annealing at 50°C, and extension at 72°C for 32 cycles (mt genes) or 34 cycles (nuclear genes). Amplification products were checked for success using 1.5% agarose gel

Table 2.2.1: Primers used for sequencing mitochondrial and nuclear genes

Primer Sequences Fragment Primer Source Length 16SA - 5'-CGCCTGTTTATCAAAAACAT-3' 600 bp Palumbi et al. 1991 16SB - 5'-CCGGTCTGAACTCAGATCACGT-3' 12SA – 5‘-AAACTGGGATTAGATACCCCACTAT-3‘ 300 bp Kocher et al. 1989 12SB – 5‘-GAGGGTGACGGGCGGTGTGT-3‘ Cyt b-C - 5'-CTACTGGTTGTCCTCCGATTCATGT-3' 600 bp Bossuyt & Cyt b-CBJ10933 - 5'-TATGTTCTACCATGAGGACAAATATC-3' Milinkovitch 2000 RAG1MartF1 - 5'-AGCTGCAGYCARTAYCAYAARATGTA-3' 900 bp Chiari et al. 2004, RAG1AmpR1 - 5'-AACTCAGCTGCATTKCCAATRTCA-3' Pramuk et al. 2008 Rhod1A – 5‘-ACCATGAACGGAACAGAAGGYCC-3‘ 350 bp Bossuyt & Rhod1D – 5‘-GTAGCGAAGAARCCTTCAAMGTA-3‘ Milinkovitch 2000

16 electrophoresis. The DNA was then purified using standard protocols with Agencourt AMPure XP (Beckman Coulter, Brea, CA).

2.2.3 DNA Sequencing and Alignment

Samples were sequenced on a capillary sequencer (ABI 3130xl) at The University of Texas at El Paso DNA core facility following standard protocols. Chromatograph data were interpreted using the program SeqMan (Swindell and Plasterer 1997). Sequences were compared to others in the GenBank database using BLAST to ensure no contamination or improper sequencing had occurred. Sequences were aligned using the ClustalW algorithm in the program MegAlign (Clewley and Arnold 1997) and adjusted by eye in MacClade v4.08 (Maddison and Maddison 2000). In the alignment of the 16S fragments, homology of base pairs (bp) in three hypervariable regions was difficult to assess. These hypervariable regions were excluded from the analysis, resulting in exclusion of 95 bp. Separate analyses including all sites did not result in relevant modifications of tree topology or support values (results not shown). Protein-coding genes were checked for accuracy by translation into amino acids in MacClade v4.08 (Maddison and Maddison 2000). All sequence data will be deposited in GenBank prior to publication in a peer-reviewed journal.

2.2.4 Phylogenetic Analyses

Phylogenies were constructed using maximum parsimony (MP), maximum-likelihood (ML), and Bayesian inference (BI) methods separately for the 16S (with GenBank data) and combined datasets. The 16S dataset had a total of 151 sequences, each with 505 bp. To facilitate computation of parameters during analyses, the 16S dataset was reduced by eliminating identical haplotypes. The final, reduced 16S dataset contained 75 sequences and 505 bp. The combined dataset was a 2790 bp concatenated set of the DNA sequences from all fives genes (16S, 12S, cyt b, RAG1, rhodopsin) and included the 67 samples collected from the DRC. Before combining data, separate analyses were completed for each gene individually to ensure that they do not conflict at well-supported nodes. Parsimony analyses were conducted in PAUP* 4.0b (Swofford 2002) using a heuristic search algorithm with 25 random-addition replicates, accelerated character transformation and tree-bisection-

17 reconnection (TBR) branch swapping. Zero-length branches were collapsed to polytomies and gaps were treated as missing data. Node support was assessed in resulting topologies using nonparametric bootstrapping (1000 pseudoreplicates) (Felsenstein 1985). A majority rule consensus tree was constructed from the resulting trees. For BI and ML phylogenies, the best fitting model of sequence evolution was first determined using the Akaike information criterion (AIC) implemented in jModelTest v1.0 (Posada 2008). Each gene was analyzed separately, and for protein-coding genes each codon position was also analyzed separately (Brandley et al. 2005). The Bayesian analyses were performed in MrBayes v3.1 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) for the 16S and combined datasets. For the combined dataset, a total of 11 data partitions were created: 12S, 16S, and 1st, 2nd, and 3rd codon positions for the cyt b, RAG1, and rhodopsin genes. These partitions were unlinked and optimized separately. To ensure convergence of results on the same topology, the MCMC search was run twice with parallel runs for 10 million generations. Each parallel search used four Markov chains initiated from a random tree, with trees saved every 1000 generations. The output files from MrBayes were examined in Tracer v1.4 (Rambaut and Drummond 2007) to determine the number of generations to be excluded as burn-in and to ensure convergence of all parameters. Additionally, Are We There Yet? (Wilgenbusch et al. 2004) was used to ensure that the multiple runs converged and that sampling was sufficient. The sump and sumt commands were executed in MrBayes, summing over the multiple convergent runs. Maximum-likelihood analyses were conducted using RAxML v7.2.6 (Stamatakis 2006) for the

16S and combined datasets using 100 replicate best tree inferences. Clade support was assessed using the rapid bootstrap algorithm (Stamatakis et al. 2008) for 1000 replicates. The combined dataset was partitioned by gene and codon position. Maximum-likelihood analyses were also conducted using GARLI v0.96b8 (Zwickl 2006) for the 16S and combined datasets, with 1000 bootstrap replicates.

18 2.2.5 Population Level Genetic Analysis

To provide a rough indication of divergence between haplotypes, sequence divergences were estimated using uncorrected p-distances calculated in MEGA v4.0 (Tamura et al. 2007). Additionally, sequences were grouped based on clades identified in the 16S ML tree and p-distances were calculated within and between each clade. Genetically distinct clades were identified when their sequence divergence exceeded previous estimates of species level divergence (5%; Vences et al. 2005).

To further investigate haplotype diversity within each clade, haplotype networks were constructed using TCS 1.21 (Clement et al. 2000), which uses statistical parsimony to connect haplotypes based on a 95% confidence interval. Gaps were treated as missing data in the analysis.

Haplotypes separated from other haplotypes by > 8 mutational steps could not be connected to the network with 95% confidence. Clades of P. mascareniensis identified in phylogenetic analyses were > 8 mutational steps away from all other clades. Because of this, three separate haplotype networks were constructed to investigate genetic structuring within each clade of P. mascareniensis and to attempt to identify the origin of diversification for each clade. To identify the origin of P. mascareniensis as a species, a haplotype network was constructed using all available sequences for all clades. Highly divergent haplotypes in Madagascar could only be connected to the network when the connection limit was set at 20 mutational steps, which did not fall in the 95% confidence interval. The number of mutational steps between each haplotype and the ancestral haplotype was counted to calculate, on average, how derived populations are within and between clades. Ancestral haplotypes are identifiable by their internal position in the network, the number of lineages that arise from them, and their commonness (Castelloe and Templeton 1994). Tip clades that are connected to the network by only one connecting branch are considered to be derived (Rowe et al. 2004). To investigate gene flow between populations, a hierarchical analysis of molecular variance (AMOVA) was performed in ARLEQUIN v3.5 (Excoffier et al. 2005). This allowed further quantification of the amount of genetic variation between and within populations. Additionally, pairwise genetic divergences between genetic clades were estimated using the Fixation index (FST) (Excoffier at al. 1992). This includes information about mitochondrial haplotype frequency (Weir and Cockerham

19 1984) and genetic distance between clades. The significance of the FST values was assessed using 10,000 permutations for each pairwise comparison.

2.3 SPECIES CONCEPTS

In this thesis I employ the Evolutionary Species Concept (Simpson 1961; Wiley 1978) expanded to the General Lineage Concept of species (de Queiroz 1998; 1999). Species delimitation is guided by an estimate of phylogenetic relationships based on molecular evidence and identification of characters for species diagnoses. Distinct lineages are considered to be populations that are genetically and morphologically distinct and for which conspecificity can be rejected based on genetic and morphological data.

2.4 MORPHOLOGICAL ANALYSIS

Most specimens for my morphological analysis were collected in eastern DRC by Dr. Eli Greenbaum. To account for unsampled localities, specimens were obtained from the Field Museum of

Natural History (FMNH). Additional specimens were examined during a visit to the Musée Royal de l'Afrique Centrale (MRAC) in Tervuren, Belgium. Specimens were identified and compared to original taxonomic descriptions, but were also individually measured and analyzed to determine variation within and between species groups. Morphological analyses were conducted using conventional morphometric characters previously used in cladistic studies of Ptychadena (Meyer 2004; Bwong et al. 2009) and several novel characters. All measurements were taken using vernier calipers to the nearest 0.1 mm.

Limb measurements were taken on the right side of the body. All characters used are included in Appendix B.

2.4.2 Multivariate Statistics

Multivariate statistics were used to assess membership of clades based on morphological characters and to help identify characters that are useful in species delimitation. Measurements taken for

20 35 specimens identified as P. mascareniensis in genetic analyses, as well as possible synonyms, were used for analysis. To allow for a greater sample size and to eliminate the effects of sexual dimorphism, only females were included. Females were identified by relatively large body size and the lack of male secondary sexual characters typical in the genus (i.e., presence of vocal slits). Males will be analyzed separately in future work. Locality information for all specimens measured is included in Appendix A.

Principal component analysis (PCA), a multivariate ordination technique, was used to explain patterns of variation and covariation within the morphometric data. PCA was conducted using Minitab 17 and significance was assessed using  = 0.05. Three separate PCA analyses were performed. In the first, all morphometric data were natural log-transformed prior to analysis to achieve normalization. In the second analysis, measurements were regressed against snout–vent length in order to reduce the effects of size on the analysis. Regressed data were not natural log-transformed as they appeared to satisfy requirements of normality. The final analysis used meristic data that were standardized prior to analysis to determine if meristic characters between species groups identified by molecular analysis. All PCA analyses were performed using the covariance matrix, rather than a correlation matrix, in order to retain all information about variance and covariance. A separate analysis using a correlation matrix produced similar results (results not shown). Those components accounting for 85% of the cumulative variance were examined in detail, and plots of principal component (PC) scores were used to identify the relationships of species groups in morphospace.

Discriminate function analysis (DFA) was then used to test patterns of interspecific similarity identified through the PCA and also proposed in the literature. The morphometric variables were treated as independent variables and a multivariate equation (function) was defined to ensure groups were maximally discriminated. The model used for DFA was produced using a stepwise procedure. The probability to stay in the model was a = 1.00 and to be removed was a = 0.00. Support for the tested interspecific patterns was evaluated using the values of Wilks‘  for the discriminant functions and also the ability of the model to classify the species groups correctly. Canonical analysis was used to visualize how well variables discriminate between groups. DFA was conducted using STATISTICA v9.1 (StatSoft, Inc. 2010).

21 Chapter 3: Results of Molecular Study

3.1 PHYLOGENETIC ANALYSES

A 505 bp fragment of the 16S rRNA gene was obtained from 65 specimens collected in the DRC and combined with an additional 75 Ptychadena sequences obtained from GenBank. The outgroups Hildebrandtia ornata and Phrynobatrachus acutirostris were included to establish the monophyly of the genus and root the tree. Species, localities, museum numbers, and GenBank accession numbers for specimens used in the study are included in Appendix A. Several specimens had haplotypes that were identical to other individuals so they were excluded from the phylogenetic analyses. The final matrix contained 75 sequences.

The maximum parsimony (MP) analysis (with TBR branch swapping) of the 16S fragment resulted in 34 equally parsimonious trees (204 variable characters, 154 of these parsimony-informative; 727 steps; consistency index 0.0406; retention index 0.819). A 50% majority-rule consensus tree of these is shown in Fig. 3.1.1. Well-supported relationships were indicated by a bootstrap support (1000 replicates) value greater than 70% (Felsenstein 1985). For the 16S dataset, jModelTest selected the GTR + G model of evolution as the best fit for the data. This model was implemented in RAxML and GARLI for maximum likelihood (ML) analyses. The two programs provided nearly identical results and for this reason, only the RAxML tree is presented here (Fig. 3.1.2). One best-scoring tree was found by RAxML (–ln L = 4277.34). The ML analysis of the

16S dataset was largely in agreement with the MP analysis, with only slight differences in the arrangement of some terminal taxa. The outgroups seem to be in slightly different positions, but this may be a result of long-branch attraction, which is a drawback of MP analysis. In the MP tree, H. ornata appears to be the most basal taxon, with Ptychadena and Phrynobatrachus acutirostris forming a monophyletic clade. In the ML tree, Phrynobatrachus acutirostris is the most basal taxon and a monophyletic clade is formed by Ptychadena and H. ornata. Neither of these arrangements, however, was sufficiently supported.

22 Figure 3.1.1 – Maximum parsimony phylogram (majority rule consensus of 34 equally parsimonious trees) of all Ptychadena haplotype sequences for the 16S rRNA gene. Numbers above branches are bootstrap support values in percent (1000 replicates). Colored circles distinguish clades of Ptychadena mascareniensis identified as distinct lineages, with the location of each clade displayed to the right of the tree. Black circles denote distinct lineages of Ptychadena anchietae, Ptychadena bibroni, Ptychadena oxyrhynchus, and Ptychadena porosissima grouped for analysis of pairwise divergence between lineages.

23 Figure 3.1.2 – Maximum likelihood phylogram of all Ptychadena haplotype sequences for the 16S rRNA gene. Numbers above branches are bootstrap support values in percent for maximum likelihood (1000 replicates) from RAxML (left) and Garli (right). Colored circles distinguish clades of Ptychadena mascareniensis identified as distinct lineages, with the location of each clade displayed to the right of the tree. Black circles denote distinct lineages of Ptychadena anchietae, Ptychadena bibroni, Ptychadena oxyrhynchus, and Ptychadena porosissima grouped for analysis of pairwise divergence between lineages.

24 Bayesian inference (BI) of the 16S dataset was run with the GTR + G model of evolution, as selected by jModelTest. Analyses in Are We There Yet? (Wilgenbusch et al. 2004) and Tracer v1.4 indicated convergence after 250,000 generations, so the first 25% of trees were discarded as burn-in. For the results of the BI analysis, a posterior probability of 0.95 or higher was considered to indicate well- supported relationships (Felsenstein 1985). The 50% majority-rule consensus tree obtained using BI methods (10 million generations) is shown in Fig. 3.1.3. The relationships recovered using BI analysis were the same as those recovered using ML, but BI analysis provided higher support values for the majority of clades. Basal relationships are well supported using BI analysis but were not sufficiently supported in

ML or MP analysis. The two outgroups in the BI analysis, Hildebrandtia ornata and Phrynobatrachus acutirostris form a well-supported (1.00) monophyletic group basal to all species of Ptychadena. Relationships between species in the genus Ptychadena are also well supported. Ptychadena anchietae is well supported (100% MP, 95% ML, 1.00 BI) as a monophyletic group, although there are two distinct lineages of P. anchietae within the group. Ptychadena anchietae is the sister group to a well-supported clade of P. oxyrhynchus (98% MP, 93% ML, 1.00 BI). There are also three apparently distinct lineages of P. oxyrhynchus within one monophyletic clade. Ptychadena bibroni forms a paraphyletic clade with P. christyi, although this may be due to errors in identification of GenBank sequences, the identities of which cannot be independently verified. The monophyly of P. bibroni should be tested in future studies.

In general, support values are high for derived clades, but basal relationships are not well supported.

3.2 COMBINED DATASET

For the combined dataset, four additional genes (12S, cyt b, RAG1, and rhodopsin) were sequenced for the 67 specimens collected in the DRC. When combined with the 505 bp of 16S sequence data, a total of 2790 bp were used for analysis (436 bp 12S; 582 bp cyt b; 876 bp RAG1; 347 bp rhodopsin). No hypervariable regions of 12S needed to be deleted. For the rhodopsin gene, sequences were not successfully obtained for 30 specimens. Analyses eliminating samples with missing data did

25 Figure 3.1.3 – Bayesian analysis phylogram of all Ptychadena haplotype sequences of the 16S rRNA gene. Numbers above branches are posterior probabilities calculated in MrBayes (10 million generations). Colored circles distinguish clades of Ptychadena mascareniensis identified as distinct lineages, with the location of each clade displayed to the right of the tree. Black circles denote distinct lineages of Ptychadena anchietae, Ptychadena bibroni, Ptychadena oxyrhynchus, and Ptychadena porosissima grouped for analysis of pairwise divergence between lineages.

26 not significantly change the topology or support values of trees (results not shown). The dataset was partitioned by gene and codon position (for protein coding genes) and a separate model of evolution was inferred for each partition. The models selected by jModelTest are shown in Table 3.2.1. In some cases, jModelTest selected a model that could not be implemented in MrBayes. Because the bias caused by overparameterization has been shown to be less pronounced than that caused by underparameterization

(Lemmon and Moriarty 2004) an alternate model with more parameters was implemented in MrBayes (Table 3.2.1). Phylogenetic trees recovered using MP, ML, and BI analyses produced identical results. For this reason, only the tree obtained using ML criteria in RAxML is presented, but includes support values from all three analyses (Fig. 3.2.1). The MP analysis resulted in two equally parsimonious trees

(907 variable characters, 755 of these parsimony-informative; 2523 steps). One best-scoring tree was found in ML analysis (–ln L = 15242.43). Specimens identified as P. mascareniensis form a monophyletic clade that is well supported

(100% MP, 100% ML, 0.99 BI). As in the previous trees, P. mascareniensis is separated into multiple distinct clades. This analysis shows that there are three distinct clades of P. mascareniensis collected in eastern DRC. The three clades (Clade A, Clade B, and Clade D) are all equally well supported (100%

Table 3.2.1: Models of evolution selected by jModelTest and the corresponding model of evolution used for Bayesian analysis

Gene partition Model Selected (jModelTest) Model Implemented (MrBayes) 16S SYM + G GTR + G 12S SYM + G GTR + G cyt b Codon 1 F81 + G JC + G cyt b Codon 2 GTR + G GTR + G cyt b Codon 3 GTR + I + G GTR + I + G RAG1 Codon 1 GTR + I + G GTR + I + G RAG1 Codon 2 GTR + I + G GTR + I + G RAG1 Codon 3 GTR + I + G GTR + I + G rhodopsin Codon 1 GTR + I GTR + I rhodopsin Codon 2 HKY + G HKY + G rhodopsin Codon 3 K80 + G HKY + G

27 Figure 3.2.1 – Maximum likelihood phylogram of all DRC Ptychadena sequenced for the combined dataset (12S, 16S, cyt b, RAG1, and rhodopsin). Numbers above branches are bootstrap support values (1000 replicates) calculated for maximum parsimony (left) and maximum likelihood (right). Numbers below branches are Bayesian posterior probabilities calculated in MrBayes (10 million generations). Colored circles distinguish clades of Ptychadena mascareniensis identified as distinct lineages and correspond to those recovered in the 16S rRNA analyses. Black circles denote distinct lineages of Ptychadena oxyrhynchus grouped for analysis of pairwise divergence between 16S rRNA sequences.

28 MP and ML; 1.00 BI). Clades B and D are most closely related to each other (98% MP, 93% ML, 0.99 BI), which is consistent with the 16S analyses. A more basal clade containing all other species of

Ptychadena is also well supported (100% MP, 99% ML, 1.00 BI). An important structural modification was noticed in the 12S fragment. There is a 16 bp insertion in the 12S fragment that is present in all specimens from the five clades of P. mascareniensis, but not in any other species of Ptychadena. This insertion has been noted by previous authors (Richards and Moore 1998; Vences 1999) and is corroborated here.

3.3 GENETIC DIVERSITY IN PTYCHADENA

To analyze the extent of genetic diversity in Ptychadena, sequences were grouped based on their position in the phylogenetic trees. Lineages grouped for analysis are identified by black circles on the phylogenetic trees (Figs. 3.1.1–3.1.3). Uncorrected p-distances were calculated both within and between these groups. Pairwise divergence comparisons of 16S sequences revealed high genetic diversity within several species of Ptychadena. Within samples assigned to P. bibroni, two major clades were identified with marked (greater than 6%) pairwise sequences divergence. Samples assigned to P. oxyrhynchus also seem to be separated into two major clades, but exhibited maximum pairwise sequence divergence of only 3.2%. Samples assigned to P. anchietae are separated into two clades in phylogenetic analyses, but they are only separated by 2% sequence divergence. Additionally, P. aequiplicata represents two distinct clades that are separated by 6.5% sequence divergence. Because both sequences of P. aequiplicata were from GenBank, their taxonomic status cannot be addressed here. Pairwise divergence comparisons of species exhibiting high genetic diversity are shown in Table 3.3.1. The average divergence between groups (assigned based on phylogenetic analysis) was 10%, whereas the average divergence within groups was < 1%.

Table 3.3.1: Uncorrected mean pairwise (p) divergence values (proportion of sites differing between haplotypes) within and between clades identified from phylogenetic analyses. Values in bold type represent intra-clade divergences, not calculated (n/c) for haplotypes represented by a single sequence. Standard error for divergence estimates was 0.01.

Clade Name O1 O2 P. oxyrhynchus A O1 0.003 P. oxyrhynchus B O2 0.032 0.002 Clade Name A1 A2 P. anchietae A A1 0.002 P. anchietae B A2 0.021 0.004 Clade Name B1 B2 P. bibroni A B1 0 P. bibroni B B2 0.061 0.002 Clade Name P1 P2 P. porosissima A P1 n/c P. porosissima B P3 0.028 n/c Clade Name AE1 AE2 P. aequiplicata A AE1 n/c P. aequiplicata B AE3 0.065 n/c

3.4 GENETIC DIVERSITY IN PTYCHADENA MASCARENIENSIS

In all analyses, a monophyletic clade containing all specimens putatively identified as P. mascareniensis and P. newtoni was well supported (88% MP; 87% ML; 1.00 BI). Ptychadena mascareniensis is paraphyletic with respect to P. newtoni (Bocage, 1886), a species endemic to São Tomé island (Equatorial Guinea) that was removed from the synonymy of P. mascareniensis by Perret

(1976). Within the P. mascareniensis complex there are five apparent clades (Fig. 3.1.1–3.1.3). Clade E is confined to Madagascar, the Seychelles, and the Mascarenes and represents the topotypic P. mascareniensis (type locality Réunion Island). The remaining four clades (A–D) are distributed across the African continent. Clade A has representatives from both eastern and southern DRC, Kenya, Tanzania, Rwanda, Uganda, and Egypt. The majority of specimens in Clade A were collected from savannah or savannah-forest mosaics. Clade B is restricted to west Africa (Cameroon and Benin). Clade

30 C is mostly composed of specimens from the DRC, with one specimen from Kenya, one from Uganda, and one from the Central African Republic. Specimens in this clade were collected from lowland rainforest habitats. Clade D includes specimens found in West Africa (Ivory Coast and Guinea) and three specimens collected in the lowland rainforest of Bitale (near Kahuzi Biega National Park), DRC. Clade A is sister to P. newtoni, both of which form a sister group to the clade containing all other lineages of P. mascareniensis. These genetic clades are mapped in Fig. 3.4.1. The five putative clades were grouped to estimate pairwise divergences based on the 16S gene to determine if they represent distinct species. Based on a moderately long branch length, Clades A, D, and C were divided into two separate clades for this analysis. Only the five major clades were separated by a high pairwise divergence estimate (> 5%). Divergence between lineages in the same clades was not enough (1.9–2.9%) to warrant separation into more than five clades. These five clades are divergent enough (> 5%) to distinguish them as distinct species (sensu Vences 2005). Clade A is moderately divergent (4.4%) from its sister species, P. newtoni. Additionally, Clade C1 is 3.7% divergent from its sister group, Clade B in West Africa. Clade E in Madagascar is deeply divergent (4.4–7.3%) from all other clades. In contrast, only small differences were found within clades, even in the widespread clade with haplotype localities ranging from Kenya to Egypt (< 1%). Pairwise divergence estimates are shown in Table 3.4.1 and the five distinct clades are mapped in Fig. 3.4.1.

Table 3.4.1: Uncorrected mean pairwise divergence values within and between clades of specimens identified as P. mascareniensis. Values in bold type represent intra-clade divergences, which were not calculated (n/c) for haplotypes represented by a single sequence. Standard error for divergence estimates was 0.01. Country abbreviations are as follows: DRC - Democratic Republic of the Congo, KE – Kenya, UG – Uganda, CAR – Central African Republic.

Clade Name 1 2 3 4 5 6 7 8 9 Clade A1 – East Africa 1 0.002 Clade A2 – East Africa 2 0.019 0.014 Clade D1 – Far West Africa 3 0.045 0.048 0.000 Clade D2 – DRC 4 0.045 0.048 0.026 0.000 Clade B1 – West Africa 5 0.057 0.061 0.049 0.042 0.010 Clade C1 – CAR 6 0.036 0.041 0.046 0.039 0.037 n/c Clade C2 – DRC, KE, UG 7 0.038 0.043 0.037 0.037 0.036 0.019 0.004 Clade E – Madagascar 8 0.042 0.037 0.056 0.059 0.069 0.055 0.051 0.008 Ptychadena newtoni 9 0.044 0.054 0.073 0.067 0.064 0.051 0.054 0.059 0.001 31 Figure 3.4.1 – Map of haplotype distribution for clades of Ptychadena mascareniensis. Symbols next to clades are those used for sample sites in the map. Gray overlay on map shows the approximate distribution of P. mascareniensis, modified after the Global Amphibian Assessment records. Clade numbers correspond to those used in phylogenetic trees (Figs. 3.1.1–3.2.1).

32 An AMOVA was calculated to investigate the possibility of gene flow between clades of P. mascareniensis (Table 3.4.2). There was more variation within clades (79.70%) than between clades

(20.30%), which is expected in widely distributed taxa. Significant FST values (FST = 0.105–0.244, P < 0.05) indicates that clades are genetically structured. All clades were significantly different from each other with the exception of Clade B from West Africa, which was not significantly different from any other clades. This may be a result of the small sample size for this clade (n = 2). Overall, results suggest a lack of gene flow between genetically distinct clades. Pairwise FST values are shown in Table 3.4.3.

Table 3.4.2 Analysis of molecular variance results indicating percentage of variation among groups, and within groups, with groups representing genetic clades. df - degrees of freedom.

Source of variation df Sum of squares Variance components % of variation Among groups 4 6.494 0.10073 Va 20.30 Within groups 66 26.098 0.39542 Vb 79.70 70 32.592 0.49616

Fixation index FST 0.2 0303

Table 3.4.3 – Pairwise genetic differentiation statistics (FST) between the different clades based on the phylogenetic data. Country abbreviations are as follows: DRC - Democratic Republic of the Congo, KE – Kenya, UG – Uganda.

1 2 3 4 5 Clade A (East Africa) 1 - Clade C (DRC, KE, UG) 2 0.244* - Clade D (DRC and Far West Africa) 3 0.177* 0.207* - Clade B (West Africa) 4 0.172 0.211 0.069 - Clade E (Madagascar) 5 0.172* 0.198* 0.105* 0.081 - *P < 0.05

33 3.5 HAPLOTYPE NETWORKS

A detailed analysis of the haplotypes assigned to each clade of P. mascareniensis using the TCS program revealed a distinct geographical structuring of haplotypes (Figs. 3.5.1–3.5.4). Clade A is composed of specimens mainly collected in savannah habitats over a wide geographic range, including Kenya, Uganda, Tanzania, DRC, and Egypt (Fig. 3.5.1). The haplotypes show a wide geographic distribution, with the haplotype collected in Egypt identical to one collected in Kenya. The ancestral haplotype of Clade A is difficult to determine, because the most common haplotype (A2) is not the same as the most internal haplotype (A1). Specimens from both haplotypes, however, were collected in the Albertine Rift region of the DRC, indicating that Clade A originated in the Albertine Rift and then subsequently dispersed to other areas in Africa. Another indication of this is that haplotypes become increasingly derived farther from the Albertine Rift. Haplotypes A1 and A2 are only separated by one step, and occur in close proximity to each other. Conversely, haplotypes A3, A4, and A5 in the southern

DRC and A8, A9, and A10 ranging from Kenya up to Egypt are 3–5 steps away from the Albertine Rift haplotypes, as seen on the map of haplotypes in Fig. 3.5.1. The same pattern is observed in Clade C (Fig. 3.5.2) and Clade D (Fig. 3.5.3). The ancestral haplotype in Clade C (C1) is also centered near the

Albertine Rift, and the only haplotype separated by more than one step (C2) is the furthest away (Kenya). A low sample size in Clade D makes conclusions difficult, but the three haplotypes in the DRC (D1, D2, and D3) are likely ancestral to a considerably more derived haplotype found in the Ivory Coast and Guinea (Fig. 3.5.3). The haplotype network constructed using all available sequences of P. mascareniensis also suggests that the DRC is a speciation hotspot (Fig. 3.5.4). Because this haplotype network could only be constructed by adjusting the minimum numbers of connecting steps rather than a 95% confidence interval, the robustness of the analysis is reduced. It does, however, show a total of 26 haplotypes in the P. mascareniensis complex. There are no shared haplotypes between any of the clades, and they are divergent enough that no clade takes an entirely central position, making determination of the ancestral haplotype difficult. Based on the number of haplotypes arising from it, however, Clade C centered in the Albertine Rift region of the DRC appears to be the most likely ancestral clade. Clades C and D are only

34 Figure 3.5.1 – Haplotype network of Clade A of Ptychadena mascareniensis, reconstructed using parsimony probability implemented in TCS (bottom) and map showing sampling sites (top). Analysis was restricted Clade A specimens identified by phylogenetic analyses with an uncorrected pairwise genetic divergences >5% to all other clades. Each circle in the network corresponds to one observed haplotype (A1–A10); the size of the circles is proportional to the number of individuals in which a haplotype was found (in parentheses). Small black dots represent hypothetical haplotypes needed to connect the network but not observed among samples. The different colors and patterns of the haplotypes correspond to those used in the map.

35 Figure 3.5.2 – Haplotype network of Clade C of Ptychadena mascareniensis, reconstructed using parsimony probability implemented in TCS (bottom) and map showing sampling sites (top). Analysis was restricted Clade C specimens identified by phylogenetic analyses with an uncorrected pairwise genetic divergences >5% to all other clades. Each circle in the network corresponds to one observed haplotype (C1–C6); the size of the circles is proportional to the number of individuals in which a haplotype was found (in parentheses). Small black dots represent hypothetical haplotypes needed to connect the network but not observed among samples. The different colors and patterns of the haplotypes correspond to those used in the map.

36 Figure 3.5.3 – Haplotype network of Clade D of Ptychadena mascareniensis, reconstructed using parsimony probability implemented in TCS (bottom) and map showing sampling sites (top). Analysis was restricted Clade D specimens identified by phylogenetic analyses with an uncorrected pairwise genetic divergences >5% to all other clades. Each circle in the network corresponds to one observed haplotype (D1–D4); the size of the circles is proportional to the number of individuals in which a haplotype was found (in parentheses). Small black dots represent hypothetical haplotypes needed to connect the network but not observed among samples. The different colors and patterns of the haplotypes correspond to those used in the map.

37 separated by seven steps, which is supported by the sister relationship of these clades on the phylogenetic trees (Figs. 3.1.1–3.2.1). Clade B in West Africa is derived from Clade D, which has two haplotypes in West Africa. Clade A, which is the sister group to P. newtoni, is separated by 19 steps from Clade D, suggesting that it is much more derived the Clades C and D. The most derived clade is the Madagascar clade, which is derived from both Clades A and D. Specimens on Madagascar are thought to have rafted over from the African mainland and are highly derived because of their long history of isolation on the island, possibly since the Pleistocene. Additionally, this clade is likely to be significantly affected by genetic drift as a result of its likely small founder population (Vences et al. 2004).

38 Figure 3.5.4 – Haplotype network of all sequences of Ptychadena mascareniensis, reconstructed using parsimony probability as implemented in TCS (right). Each circle in the network corresponds to one observed haplotype and the size of the circles is proportional to the number of individuals in which a haplotype was found (in parentheses). Highlighted clades correspond to clades identified in phylogenetic analyses. Small black dots represent hypothetical haplotypes needed to connect the network but not observed among samples. The different colors and patterns of the haplotypes correspond to those used in the maps in Figs 3.5.1–3.5.3. 39 Chapter 4: Results of Morphological Analyses

A total of 24 female and 9 male frogs identified in genetic analyses as P. mascareniensis were examined in this study. An additional 42 female and 27 male frogs of other Ptychadena species were examined for comparison. Complete morphometric and meristic data for all specimens are included in Appendix C. Through these examinations it was determined that the main diagnostic character that distinguishes P. mascareniensis from other species of Ptychadena is that it is one of the only species to have males with vocal sac slits that end above the insertion of the arms (superior position, Fig. 4.1.1 C). The vast majority of Ptychadena species are characterized by the slits of the vocal sacs ending posterior to or below the insertion of the arms (inferior position, Fig. 4.1.1 A and B). Therefore, all specimens belonging to the P. mascareniensis species complex are distinguishable based on both genetics and morphology. Within the P. mascareniensis complex, however, there are very few diagnostic characters.

4.1 DIAGNOSTIC CHARACTERS IN PTYCHADENA MASCARENIENSIS

Species descriptions of P. mascareniensis subspecies usually rely heavily on a few morphological characters. These characters include general body size, webbing formula, the size of limbs in proportion to the body, and head size in proportion to the body (Werner 1908; Laurent 1954; Schmidt and Inger 1959; Lamotte 1967). Many of these characters were found to be overlapping in genetic clades of P. mascareniensis. Commonly used characters that were too variable to be diagnostic in this analysis are summarized in Table 4.1.1. Coloration, specifically the presence of a light colored dorsal stripe or barring on the posterior of the thighs, is also commonly used with species descriptions. Coloration was examined here within and between each genetic clade, but could not be quantified accurately to include in morphometric analysis, and there were no discernable meristic patterns in coloration evident between genetic clades. Coloration, including the presence of a dorsal band, barring on the arms and legs, and the presence of a light tibia line all seem to be plastic within genetic clades. To illustrate this, photographs of Ptychadena specimens belonging to each genetic clade are provided in Figs. 4.1.2–4.1.4.

Figure 4.1.1 – Illustration of the three positions of the male gular slits within Ptychadena. Gular slits terminate level with the insertion of the arm (A), below the insertion of the arm (B), or above the insertion of the arm (C). Ptychadena mascareniensis is one of the only Ptychadena species to exhibit gular slits that terminate above the insertion of the arm (C). Figure redrawn from Meyer (2004).

Table 4.1.1 – Illustration of the overlapping of morphometric and meristic characters frequently used to diagnose species. Clade C and D are considered together here because of the sample size of Clade D. Clades C and D were shown to be genetically distinct from Clade A in genetic analyses (Figs. 3.1.1–3.2.1) but show no discernable morphological variation.

Clade A (n=12) Clade C and D (n=11) Morphometrics Min. Max. Avg. +  Min. Max. Avg. +  Snout–vent length 46.5 61.3 53.2 + 4.66 49.1 63.8 57.01 + 4.87 Toe IV length 22.2 31.8 27.55 + 2.49 25.4 34.4 30.58 + 2.76 Snout–nostril distance 3.3 4.4 4.17 + 0.29 3.3 4.9 4.29 + 0.41 Phalanges free on toe IV 2.5 3 2.92 + 0.19 2 3 2.73 + 0.33 Ratio - toe IV to foot length 0.57 0.72 0.62 + 0.04 0.59 0.62 0.61 + 0.01 Ratio - tibia to foot length 0.64 0.81 0.70 + 0.05 0.67 0.72 0.69 + 0.01 Ratio - tympanum to eye diameter 0.78 0.96 0.87 + 0.06 0.86 0.93 0.89 + 0.03 Ratio - head width to head length 0.77 0.95 0.86 + 0.05 0.55 0.9 0.80 + 0.10 Ratio of snout–nostril to eye–nostril distance 0.79 0.98 0.88 + 0.08 0.75 0.93 0.85 + 0.06 Ratio of eye diameter to snout–nostril distance 1.05 1.55 1.23 + 0.15 1.07 1.45 1.22 + 0.10

Meristic Characters Present Absent Present Absent Light colored dorsal band 7 5 8 3 Barring on posterior of thighs 9 3 8 3 Light colored tibia line 7 5 8 3 Gular pattern 12 0 9 2

42 Figure 4.1.2 – Plate of morphological variation in a genetically identified clade. All specimens shown here belong to Clade A identified through genetic analysis. Many morphological features, including color pattern and dorsal stripes previously used for classification are highly variable within the clade. Photos are labeled with the haplotype number identified using haplotype networks and their locality information. All photos taken by Dr. Eli Greenbaum

43 Figure 4.1.3 – Plate of morphological variation in a genetically identified clade. All specimens shown here belong to the forest dwelling Clade C identified through genetic analysis. Many morphological features, including color pattern and dorsal stripes previously used for classification are highly variable within the clade. Photos are labeled with the haplotype number identified using haplotype networks and their locality information. All photos taken by Dr. Eli Greenbaum

44 Figure 4.1.4 – Plate of morphological variation in a genetically identified clade. All specimens shown here belong to Clade D identified through genetic analysis. Specimens shown here were collected at only two localities in the DRC, both of which were in lowland forest at similar elevations. Many morphological features, including color pattern and dorsal stripes previously used for classification are highly variable within the clade. Photos are labeled with the haplotype number identified using haplotype networks and their locality information. All photos taken by Dr. Eli

Greenbaum

45 4.2 MULTIVARIATE STATISTICAL ANALYSES

To quantitatively analyze morphological differences between clades, multivariate analyses were conducted using 19 measurements and 27 meristic characters (Appendix C). A total of 32 females of

Ptychadena mascareniensis and associated synonyms were measured for morphological analysis. Through genetic analyses, 12 frogs were identified as P. aff. mascareniensis Clade A, 10 as P. aff. mascareniensis Clade C, and 2 as P. aff. mascareniensis Clade D. In addition, two museum specimens identified as P. mascareniensis sensu stricto, six as P. hylaea, and two as P. mascareniensis venusta were included to determine if any of the genetic clades matched previously described species. Three separate analyses were completed: one with natural log-transformed morphometric data, one with morphometric data regressed against snout–vent length, and a third with standardized meristic data. A one-way ANOVA showed that in the natural log-transformed data, all variables used for analysis were significantly different (p < 0.01) between clades except for head width, snout–nostril distance, and toe I length. The significance in these variables is likely a result of size differences between species, because when measurements are regressed against snout–vent length only tibia length, eye–nostril distance, and the lengths of toes IV and V are significantly different (p < 0.01) between species.

For the analysis using natural log-transformed data, the first two principal component axes account for 87.7% of the total variance in these data (Figs. 4.2.1, Table 4.2.1). Plots of these two axes reveal only one distinct species group, P. mascareniensis ‗E‘, which represents P. mascareniensis sensu stricto from Madagascar (Fig. 4.2.1). The remaining samples do not form any distinct groups. The loadings of characters on the first three axes is shown in Table 4.2.1. One-way ANOVA showed significant differences between species only for scores on the first principal component axis (P < 0.001).

On the first principal components axis (PC1), which accounts for 75.4% of the cumulative variance, all 18 variables load strongly and positively. This axis can therefore be taken as a general descriptor of body size. Ptychadena mascareniensis ‗E‘ (Madagascar) exhibits the smallest scores for PC1 (F = 7.43, P < 0.001; Fig. 4.2.1), which is expected since it was the smallest of the species measured. The second principal component axis (PC2), which accounts for 12.2% of the total variance, loads most positively on internarial distance and most negatively on lengths of fingers II, III, and IV (Table 4.2.1). One-way

46 ANOVA showed that no significant differences exist between species for scores on the second principal component axis (P = 0.064).

For the analysis using morphometric data regressed against snout-vent length, similar groups were recovered (Fig. 4.2.2). In this analysis, the first two principal component axes explain 80.7% of the total variance (Fig. 4.2.2, Table 4.2.2). Plots of these two axes reveal the same distinct group of P. mascareniensis ‗E‘ revealed by the previous analysis, but no other groups are recovered. One-way ANOVA shows significant differences between scores on the first principal component (P < 0.005), but not on the second axis. All variables load positively on PC1, with foot length and tibia length being the strongest. Therefore, this axis can be taken as a descriptor of leg size in proportion to body size. PC2 accounts for 13.4% of the total variance. This axis loads most positively on foot length and most negatively on lengths of fingers II, III, and IV, but does not provide any additional resolution between groups.

With regard to meristic data, all characters that were identical to each species were eliminated from the analysis, leaving a total of 27 characters. Meristic data collected for Ptychadena provided similar results to morphometric data in that the only clade recovered by plots of principal component scores was P. mascareniensis ‗E‘ (Fig. 4.2.3). The first two principal component axes explain only 31% of the total variance (Table 4.2.3). To explain 85% of the variation, 11 axes would need to be included. One-way ANOVA showed that there were significant differences between species on only on the first axis (P < 0.05). PC1 loads most strongly and positively on characters describing webbing formula. The second axis (PC2) loads most positively on the presence of warts on the legs and lateral sides and most negatively on webbing formula. These analyses all suggest that morphometric and meristic characters can distinguish between specimens on Madagascar and those in Africa, but do not provide resolution between specimens on continental Africa.

Table 4.2.1 – Principal components analysis comparing clades of Ptychadena mascareniensis using natural log-transformed morphometric data. Eigenvalues, percent variance, cumulative variance, and loadings are shown for the first three principal components.

PC1 PC2 PC3 Eigenvalue 0.31743 0.05153 0.01038 Proportion 0.754 0.122 0.025 Cumulative 0.754 0.877 0.901 Loadings Snout–vent length 0.166 0.124 –0.147 Foot length 0.22 0.186 0.152 Tibia length 0.238 0.201 0.106 Head width 0.141 0.095 –0.514 Head length 0.192 0.093 –0.144 Snout–nostril 0.163 0.108 –0.28 distance Eye–nostril distance 0.173 0.224 –0.041 Tympanum 0.164 0.101 –0.256 diameter Eye diameter 0.125 0.216 –0.357 Internarial distance 0.195 0.253 –0.168 Finger I Length 0.25 –0.13 0.224 Finger II Length 0.297 –0.398 0.016 Finger III Length 0.322 –0.349 0.105 Finger IV Length 0.4 –0.524 –0.211 Toe I Length 0.213 0.229 0.295 Toe II Length 0.241 0.148 0.149 Toe III Length 0.23 0.113 0.065 Toe IV Length 0.214 0.144 0.153 Toe V Length 0.241 0.172 0.338

Table 4.2.2 – Principal components analysis comparing clades of Ptychadena mascareniensis using morphometric data regressed against snout–vent length. Eigenvalues, percent variance, cumulative variance, and loadings are shown for the first three principal components.

PC1 PC2 PC3 Eigenvalue 0.01 0.002 0.0008 Proportion 0.672 0.134 0.056 Cumulative 0.672 0.807 0.863 Loadings Foot length 0.669 0.299 –0.184 Tibia length 0.419 0.123 0.457 Head width 0.089 –0.027 –0.757 Head length 0.151 –0.095 –0.347 Snout–nostril distance 0.031 0.001 –0.09 Eye–nostril distance 0.037 0.038 –0.046 Tympanum diameter 0.022 –0.004 –0.075 Eye diameter 0.005 0.058 –0.097 Internarial distance 0.027 0.033 –0.009 Finger I Length 0.108 –0.171 0.001 Finger II Length 0.104 –0.391 –0.043 Finger III Length 0.16 –0.569 0.096 Finger IV Length 0.151 –0.603 0.023 Toe I Length 0.062 0.034 –0.079 Toe II Length 0.145 –0.027 0.031 Toe III Length 0.214 –0.076 –0.044 Toe IV Length 0.334 0.047 0.054 Toe V Length 0.299 0.022 0.12

49 Table 4.2.3 – Principal components analysis comparing clades of Ptychadena mascareniensis using standardized meristic data. Eigenvalues, percent variance, cumulative variance, and loadings for included variables are shown for the first three principal components.

PC1 PC2 PC3 Eigenvalue 4.6348 3.4217 2.9326

Proportion 17.2 12.7 10.9 Cumulative 17.2 29.8 40.7 Loadings

Gular pattern –0.105 0.087 –0.128 Dorsolateral folds –0.036 –0.134 –0.155 Outermost dorsal ridge whitish 0.061 –0.215 0.232 Warts on legs –0.101 0.022 0.236 Warts on lateral sides 0.196 0.064 0.234 Light tibial line 0.104 –0.237 0.242 Light bands on posterior of femur –0.272 –0.337 0.014 Dark bands on posterior of femur –0.283 –0.359 0.031 Dark mottling on posterior of femur 0.167 0.207 0.116 Whitish spots on lower lip –0.058 –0.001 –0.129 Whitish ring around tympanum 0.03 –0.034 0.06 Light colored vertebral band 0.025 –0.299 0.253 Outer tarsal fold of foot 0.046 0.129 –0.083 Perforations on toe II –0.097 0.41 –0.079 Perforations on toe III –0.097 0.41 –0.079 Number subarticular tubercles on –0.262 –0.044 –0.376 hand

Total number of dorsal ridges –0.219 –0.08 0.019 Number of short dorsolateral ridges –0.205 –0.067 –0.018 Number of tubercles on Toe IV –0.082 –0.073 –0.033 Number of subarticular tubercles –0.232 –0.106 –0.267 on foot Phalanges free on Toe 1 0.273 –0.044 –0.254 Phalanges free on Toe 2 internal 0.297 –0.02 0.093 Phalanges free on Toe 3 internal 0.168 –0.185 –0.332

Phalanges free on Toe 3 external 0.252 –0.167 –0.159 Phalanges free on Toe 4 internal 0.221 –0.028 0.175 Phalanges free on Toe 4 external 0.31 –0.166 –0.273

Phalanges free on Toe 5 0.313 –0.125 –0.3

P. hylaea

P. mascareniensis ‗A‘ P. mascareniensis ‗D‘ P. mascareniensis ‗C‘ PC2 P. mascareniensis ‗E‘ P. mascareniensis venusta

PC1

Figure 4.2.1 – Scatter plot of the first and second principal component scores for the analysis using natural log-transformed morphometric data. The second principal component (PC2) is plotted against the first principal component (PC1), which here is an indicator of general body size. Colors used in the graph correspond to colors used to distinguish clades in genetic analyses (Figs. 3.1.1–3.2.1)

P. hylaea P. mascareniensis ‗A‘ P. mascareniensis ‗D‘

PC2 P. mascareniensis ‗C‘ P. mascareniensis ‗E‘ P. mascareniensis venusta

PC1

Figure 4.2.2 – Scatter plot of the first and second principal component scores for the analysis using morphometric data regressed against snout–vent length. The second principal component (PC2) is plotted against the first principal component (PC1), which here is an indicator of leg size in relation to body size. Colors used in the graph correspond to colors used to distinguish clades in genetic analyses (Figs. 3.1.1–3.2.1)

P. hylaea

P. mascareniensis ‗A‘ P. mascareniensis ‗C‘ P. mascareniensis ‗D‘ PC2 P. mascareniensis ‗E‘ P. mascareniensis venusta

PC1

Figure 4.2.3 – Scatter plot of the first and second principal component scores for the analysis using standardize meristic data. The second principal component (PC2) is plotted against the first principal component (PC1), which here describes components of webbing formula. Colors used in the graph correspond to colors used to distinguish clades in genetic analyses (Figs. 3.1.1–3.2.1)

53 Discriminant function analysis (DFA) was conducted using the morphometric data collected for each specimen. The results of the DFA support the results of the above PCAs. Two separate analyses were completed. In the first analysis, all specimens were grouped by species identified in genetic analyses or identified from morphology (6 groups). In the second analysis, both Clade B and P. hylaea were grouped in with Clade C based on their similar habitat and potential conspecificity hypothesized by previous authors (Vences et al. 2004; Bwong et al. 2009). In the first analysis, eight of the 17 morphometric characters were entered into the stepwise discriminate function before its discriminating power was exhausted. The characters not included were: foot length, head width, head length, eye diameter, internarial distance, the length of fingers I and IV, and the length of toes II, III, and IV. The partial Wilks‘  indicates that eye–nostril distance contributes the most to the overall discrimination, explaining 67.5% of the total variance, followed by snout–vent length, which explains an additional 16.6%. Only 80% of specimens were assigned the correct group, with the remaining 20% being misclassified. The misclassified specimens all belonged to Clade A, Clade C, or P. hylaea. All specimens belonging to Clade D, Clade E, and P. mascareniensis venusta were correctly classified. A total of five functions were defined, but only the first two were significant

(2: P < 0.001). The actual discrimination functions were computed using a canonical analysis to see how the variables discriminate between the species groups. The canonical scores for the first two discriminant function roots are plotted in Fig. 4.2.4.

In the second analysis, 10 of the 17 morphometric characters could be entered into the stepwise discriminate function before its discriminating power was exhausted. The characters not included in this analysis were: head length, internarial distance, length of fingers I and IV, and length of toes III, IV, and

V. The partial Wilks‘  indicates that in this analysis, snout–vent length contributes the most to the overall discrimination, and explains 70% of the total variation. The second most discriminating variable is eye–nostril distance, which explains an additional 24.2% of the variance. There was a slight decrease in misclassified specimens compared to the previous DFA analysis, with 82% being classified correctly. Of those that were misclassified, three were from Clade A and three from Clade C. A total of three

54 functions were defined, but again only the first two were significant (2: P < 0.001). The canonical scores for the first two discriminant function roots are plotted in Fig. 4.2.5.

Neither analysis was able to discriminate between Clades A and C, even when additional samples were included in Clade C. This corroborates the results of the PCA, which did not elucidate any distinct groupings between Clade A, Clade C, or P. hylaea.

Figure 4.2.4 – Scatter plot of canonical scores calculated from discriminant function analysis using six species groups. Root 1 and Root 2, which were both significant are plotted against each other. Colors correspond to colors used to distinguish clades in genetic analyses (Figs. 3.1.1–3.2.1)

Figure 4.2.5 – Scatter plot of canonical scores calculated from discriminant function analysis using four species groups. Clade D and P. hylaea were both grouped into a classification with Clade C. Root 1 and Root 2, which were both significant, are plotted against each other. Colors correspond to colors used to distinguish clades in genetic analyses (Figs. 3.1.1–3.2.1)

56 Chapter 5: General Discussion and Conclusions

The Ptychadena species of the DRC are historically understudied and poorly known. This is the first study to combine molecular and morphological data to address the phylogeography and species boundaries of Ptychadena. The results presented herein suggest that contemporary taxonomy underrepresents the diversity of Ptychadena. In addition, it has been found that a major diversification event in Ptychadena may have had its origin in the DRC and can be linked to climatic and geological changes in the Miocene epoch.

5.1 GENETIC DIVERSITY IN PTYCHADENA

Previous studies have hypothesized that genetic diversity is high in the genus Ptychadena and that several complexes of cryptic species may exist (Schmidt and Inger 1959; Lamotte 1967; Vences et al. 2004; Measey et al. 2007). In this study I used molecular sequence data from mitochondrial (12S, 16S, and cyt b genes) and nuclear (RAG1 and rhodopsin) genes to analyze interspecific and intraspecific phylogenetic relationships of Ptychadena. This study corroborates the presence of high genetic diversity in Ptychadena. Vences et al. (2004) suggested that P. aequiplicata, P. bibroni and P. porosissima represent complexes of cryptic species because of at least 5% sequence divergence among lineages in the 16S rRNA gene. The same species were investigated here but, unlike the previous study (Vences et al. 2004), hypervariable regions were excluded in pairwise divergence analyses to ensure homology of the sequences being analyzed. With the exclusion of hypervariable regions, P. aequiplicata and P. porosissima are not as divergent as previously thought (3%) (Table 3.3.1). Additionally, no studies have investigated the morphology of these species carefully enough to find diagnostic characters between lineages. Therefore, further study is needed to assess the validity of these taxa. It is likely that they represent independent genetic lineages on unique evolutionary trajectories and if sampled at a later point in evolutionary time will be diagnosable as distinct species (de Queiroz 1998). Ptychadena bibroni, however, is composed of at least two distinct clades separated by 6% sequence divergence, which is comparable to the distinct but closely related lineage of P. christyi. The taxonomic status of P. bibroni therefore warrants investigation and possible division into separate species in future studies. Overall,

57 Ptychadena is characterized by high genetic diversity, much of which may be the result of lineages in the early stages of speciation (de Queiroz 1998).

5.2 GENETIC DIVERSITY IN PTYCHADENA MASCARENIENSIS

This study also reveals major genetic breaks within P. mascareniensis. The 16S rRNA sequence divergence levels between each clade of P. mascareniensis are comparable to or exceed levels between other valid species of anurans. For example, several frog species in Madagascar that are well diagnosed by morphological, ecological, and bioacoustic characters are separated by only 2% sequences divergence in the 16S rRNA gene (Vences et al. 2002; 2003; Vences and Glaw 2002; Veith et al. 2003a, b). However, Hudson and Tuerlli (2003) warned that species inference should not be based solely on monophyly and divergence in mitochondrial markers. Mitochondrial genes are inherited only through the maternal line and in general are not subject to recombination. That makes it possible for different discrete haplotypes to co-occur in the same species or population. Moreover, any hybridization event may lead to introgression of divergent haplotypes (Avise 2000). Species status can only be confidently inferred in cases where nuclear loci provide similar levels of divergence and monophyly. Separate analyses of only the nuclear genes (RAG1 and rhodopsin) corroborated the results of the mtDNA analysis, and provided high support for five distinct lineages of P. mascareniensis (results not shown) as did analyses of a combined data set including both mitochondrial and nuclear genes (Fig. 3.2.1).

Additionally, there are high levels of divergence (5%) between lineages of P. mascareniensis, which is generally not the case in known instances of intraspecific haplotype sharing in amphibians (Vences et al. 2004). Taken together, these results provide compelling evidence for cryptic speciation in P. mascareniensis. Cryptic species are defined as two or more species that have been classified as a single nominal species because they are morphologically indistinguishable, at least superficially (Bickford et al. 2007).

Cryptic species are common among many taxa and found in all parts of the globe (Pfenninger and Schwenk 2007; Bickford et al., 2007; Mayer et al., 2007; Murray et al., 2008). Stuart et al. (2006) has even suggested that cryptic lineages occurring in sympatry may be the rule rather than the exception (at

58 least in forests of Southeast Asia). Several studies (Stuart et al. 2006; Inger et al. 2009; McLeod 2010) also found that when cryptic lineages occur in sympatry, they frequently are not sister lineages of one another. This is true of P. aff. mascareniensis Clade A and Clade C, which co-occur in the DRC. Clade C is sister to Clade B, restricted to West Africa, whereas Clade A is sister to P. newtoni, an endemic to São Tomé Island off the coast of western Central Africa (Figs. 3.1.1–3.14).

The hypothesis of cryptic speciation is further supported by the lack of gene flow between populations. The FST values calculated using AMOVA (Table 3.4.3) are a measure of population differentiation. All clades identified in genetic analyses were significantly different from each other, with the exception of Clade B from West Africa. This indicates that there is no gene flow occurring between each genetic clade, eliminating the possibility of introgression. This indicates that each clade is evolving separately from other lineages, which under the General Lineages Concept of Species indicates the presence of distinct species regardless of whether or not they are phenetically distinguishable, diagnosable, intrinsically reproductively isolated, or ecologically divergent (de Queiroz 1998, 2007) There are several reasons why speciation is not necessarily always accompanied by recognizable phenotypic change. The two general and recurrent themes are: cryptic species are under selection that preserved morphological stasis and/or they are differentiated by non-visual mating signals. Extreme environmental conditions can impose stabilizing selection on morphological characters. This reduces or eliminates the morphological change that generally accompanies speciation (Bickford et al. 2007). In the case of non-visual mating signals, there may not be appreciable differences in the morphological machinery that produces the different acoustic or olfactory signals needed for conspecific recognition and reproduction. Acoustic mating signals have been shown to be important in discriminating between closely related cryptic anuran species (Narins 1983; Wilczynski et al. 1993; Glaw and Vences 2002). This is likely the case in P. mascareniensis. Several studies have published frequencies, sonograms, or descriptions of calls from specimens identified at P. mascareniensis (Schiøtz 1964; Amiet 1974;

Passmore 1977; Van Den Elzen and Kreulen 1979; Lambiris 1989; Akef and Schneider 1995), but a comprehensive study combining acoustics and genetics is required to elucidate differences between genetic clades. Attempts at recording mating calls for this study were unsuccessful as Ptychadena tend

59 to be wary and stop calling when disturbed or threatened (E. Greenbaum, personal observation). Choruses in flooded fields have been recorded, however, and will be analyzed in future studies to attempt to isolate and analyze Ptychadena calls.

5.3 TAXONOMIC STATUS OF PTYCHADENA MASCARENIENSIS

Traditionally, P. mascareniensis has been treated as a ―junk-drawer‖ species (Pokorny 1967). That is, a taxon with poorly defined species limits into which multiple taxa are placed. Originally described in 1841, P. mascareniensis is one of the only Ptychadena species to have vocal sac slits that end above the insertion of the arms (superior position, Fig. 4.1.1 C). The vast majority of Ptychadena species are characterized by the slits of the vocal sacs ending posterior to and below the insertion of the arms (inferior position, Fig. 4.1.1 A and B). Since erection of P. mascareniensis, any specimens whose vocal sacs are positioned superior to the arm insertion have been assigned to this taxon, regardless of other morphological characters or location. This has resulted in a taxonomic history filled with poor subspecific designations (Seetzen 1855; Rochebrune 1885; Werner 1908; Laurent 1950; 1954) and subsequent synonymies (Peters 1869; Boulenger 1879; Boulenger 1882; Noble 1924; Loveridge 1936; Guibé and Lamotte 1957). In the past, emphasis has been placed on the degree of webbing of the feet as a character useful for distinguishing species; however I have found this to be variable between specimens from the same genetic clade (Table 4.1.1). This character can also be confounded by a marked reduction of webbing in preserved specimens (Poynton 1970). Another character commonly used to discriminate between taxa is a distinction between a striped or mottled pattern on the posterior face of the thigh. This character is also not diagnostic because of variability within genetic clades (Table 4.1.1). Moreover, it is sometimes difficult to make a distinction between intergrades of the two patterns as intergrades. Overall, there is a great deal of phenotypic plasticity in this species complex, even within specimens from the same genetic clade that were collected from the same locality (Figs. 4.1.2–4.1.4). The molecular results presented here (Figs. 3.1.1–3.14) elucidate five genetically distinct lineages that were all previously classified as P. mascareniensis (A–E). The only valid taxon attributable

60 to the name P. mascareniensis is found on Madagascar, the Mascarenes, and the Seychelles (Clade E). The type locality for P. mascareniensis is Réunion (an isolated oceanic island 722 km east of

Madagascar). Specimens at the type locality were likely introduced from Madagascar and were recovered in phylogenetic analysis as conspecific with the populations on Madagascar (Vences et al. 2004). The four remaining clades (Clades A–D) are genetically distinct from the Malagasy clade and thus represent either one of several available synonyms of P. mascareniensis or undescribed taxa. Because of the relatively small sample sizes of Clades B (West Africa) and D (DRC and West Africa), only two distinct species will be recognized in this study. Clade A, found mainly in savannah habitat, is monophyletic and considered one distinct species (hereafter referred to as the savannah lineage), and

Clades B, C, and D combined form a monophyletic clade (hereafter referred to as the forest lineage) that is considered to be a second species restricted to lowland rainforests. Subsequent discussion will focus on the taxonomic status of these two clades.

Several synonyms are potentially available to assign to the clades identified in genetic analyses, because various authors (Seetzen 1855; Rochebrune 1885; Schmidt and Inger 1959; Lamotte 1967) have attempted to describe distinct species of Ptychadena with superior vocal slits (Table 5.3.1). In general, however, these species were synonymized by previous authors (see Frost 2010) because their diagnostic features are not distinct from topotypic P. mascareniensis. There are at least five possible synonyms that may correspond to the genetic clades (Table 5.31). Previous authors that have identified forest and savannah clades of P. mascareniensis (Lamotte 1967; Vences et al. 2004; Bwong et al. 2009) have suggested the name P. hylaea for the forest species and Rana (Ptychadena) nilotica for the savannah species. Ptychadena hylaea was originally described as a subspecies of P. mascareniensis (Schmidt and

Inger 1959), but was raised to species status by Lamotte (1967). This name, however, is a junior synonym of P. marchei, which was collected from the banks of the Casamance River in Senegal (Rochebrune 1885). The name P. mascareniensis venusta has also been proposed for populations in

West Africa (Vences et al. 2004; Bwong et al. 2009). To help provide taxonomic clarity, discussions of the taxonomic status of each clade identified in genetic analyses are provided here. Comparisons with other species are taken from original descriptions (P. mascareniensis, Duméril and Bibron 1841; Rana

61 nilotica, Seetzen 1855; Rana marchei, Rocheburne 1885; P. mascareniensis venusta, Werner 1908; and P. hylaea, Schmidt and Inger 1959).

Table 5.3.1 – All subspecies of P. mascareniensis that have been described and subsequently synonymized by one or more authors. Subspecies described from Madagascar are not discussed here. An additional subspecies, P. mascareniensis hylaea, is not included in the table because it has since been elevated to specific status (Lamotte 1967).

Synonym Author, Year Type Locality Authors of synonymy Peters, 1869; Boulenger, 1879; Boulenger, Rana nilotica Seetzen, 1855 Cairo, Egypt 1882; Guibé and Lamotte, 1957 Rana savignyi Jan, 1857 Not stated Boulenger, 1879; Boulenger, 1882 Boulenger, 1879; Boulenger, 1882; Guibé Rana idae Steindachner, 1864 Madagascar and Lamotte, 1957 Boulenger, 1879; Boulenger, 1882; Guibé Rana nigrescens Steindachner, 1864 Madagascar and Lamotte, 1957 KwaZulu-Natal Rana spinidactyla Cope, 1865 Province, South Günther, 1866; Loveridge, 1957 Africa Casamance Rana marchei Rochebrune, 1885 Noble, 1924 River, Senegal Loveridge, 1936; Guibé and Lamotte, Rana venusta Werner, 1908 Sudan 1957

5.3.2 Taxonomic Discussions

Ptychadena aff. mascareniensis savannah lineage

Specimens in this lineage are distinguishable from the majority of species of Ptychadena by vocal slits that end above the insertion of the arm. Very few morphometric differences exist between this clade and other clades or previously described species. Some very subtle differences do exist. For example, savannah lineage specimens differ from P. hylaea in that the internarial distance is smaller, rather than equal to, the eye–nostril distance, the ratio of the tympanum diameter to eye diameter is greater than 0.8 (avg. = 0.87), and the tympanum diameter is equal to or less than the eye–nostril distance. Specimens in this clade differ from P. mascareniensis venusta in that the distance from the

62 nostril to the tip of the snout is only 1.2 times the eye diameter (1.5–1.7 in P. venusta). Additionally, the first finger is shorter than the second finger, whereas they are equal in P. venusta. There may be a distinction between this species and the forest lineage based on a proportionally longer head in relation to head width (avg. ratio of head width to head length = 0.86; forest lineage = 0.8) although this difference is very subtle. Summaries of important measurement data are provided in Table 4.1.1 and the complete measurement dataset is included in Appendix C. The main distinguishing factor between this clade and the other clade identified from the DRC (forest lineage) is habitat. All specimens from the savannah lineage were collected in savannah, savannah-forest mosaic or deforested areas at elevations ranging from 560 m to 1969 m. Based on a review of the taxonomic history of P. mascareniensis, the oldest name that applies to a specimen collected in savannah is P. nilotica (Werner 1908). The original description for P. nilotica was not available for comparison, but the type specimen was collected in Cairo, Egypt (Werner 1908; Frost 2010), and based the recorded distribution of P. aff. mascareniensis in

Egypt was likely collected from the banks of the Nile River (Baha El Din 2006). The habitat surrounding the Nile River is Nile Delta flooded savannah (Burgess et al. 2004). Because there are no definitive diagnostic characters that distinguish between the savannah lineage and the forest lineage, habitat is currently the only way to distinguish the clades. There does not appear to be an overlap in habitat between the two lineages based on current sample sizes, and therefore provenance/habitat is currently a good estimation of clade membership. The name P. nilotica should be resurrected and applied to all savannah dwelling specimens previously assigned to P. mascareniensis. Type specimens for P. nilotica were ―presumably deposited in Universität Humboldt, Zoologisches Museum (ZMB)‖

(Frost 2010) but their presence there has not be verified and they may be lost. For this reason, before the name is formally resurrected it would be beneficial to designate a neotype. According to the International Code for Zoological Nomenclature, a neotype is a name-bearing type designated when no name-bearing type specimen is believed to exist, but is necessary to define the nominal taxon objectively. Because of the unstable taxonomic history of the P. mascareniensis species complex, a name-bearing type specimen and accompanying description would be beneficial to taxonomic stability.

63 For this reason, the name P. nilotica is tentatively assigned to the savannah lineage but will be officially designated in a later publication.

Ptychadena aff. mascareniensis forest lineage

The specimens assigned to the forest lineage based on genetic analyses also differ from the majority of species of Ptychadena in having vocal slits that end above the insertion of the arm. In the forest lineage, the internarial distance is smaller than the eye–nostril distance, whereas it is equal in P. hylaea. Additionally, the ratio of the tympanum diameter to the eye diameter is greater than 0.8 as described for P. hylaea (avg. = 0.90). Specimens in this clade differ from P. mascareniensis venusta by having a relatively shorter snout: the distance from the nostril to the tip of the snout is only 1.2 times the eye diameter (1.5–1.7 in P. venusta). Moreover, the first finger of forest specimens is shorter than the second finger (equal in P. venusta). There are no evident morphological characters that successfully distinguish between this clade and the savannah lineage except a slightly shorter head in relation to head width (described above), but the two lineages are distinguished by habitat. Specimens from the forest lineage were all collected from forest habitats, mostly low to mid-elevation rainforest (728–2088 m), which distinguishes them from the savannah lineage. The oldest name that applies to forest-dwelling forms of P. mascareniensis is Rana (Ptychadena) marchei (Rochebrune 1885). This specimen was collected from the Casamance River in southwestern Senegal. This area of Senegal is in the Guinean forest-savannah mosaic ecoregion (Burgess et al. 2004). The banks of the river, however, likely contained gallery forest in the late 19th century when this specimen was collected. The original description of R. marchei also matches specimens from this clade despite the vague characters used in the original description. The only quantitative character provided from the description of P. marchei is that the snout length is 2 ½ times the eye diameter. If snout length is interpreted as the distance from the anterior corner of the eye to the tip of the snout then it corresponds to the eye–nostril distance plus the snout–nostril distance measurements taken in this study. The forest lineage has a slightly shorter snout in relation to eye diameter (2 times). Snout length may have been measured differently in the original description, however, or else may be a mistake in the translation. Despite this discrepancy, it can be

64 assumed based on provenance that specimens from Clade C are conspecific with the forest dwelling Rana marchei. It has also been noted that this name likely also applies to P. hylaea because other specimens from Senegal look very similar to P. hylaea (A. Ohler, personal observation). Based on this evidence, it is suggested here that the name P. marchei be resurrected and applied to the forest lineage previously assigned to P. mascareniensis. The type specimens for P. marchei have also been lost (A.

Ohler, personal communication; Frost 2010), so the designation of a neotype will be required for this species as well.

5.3.3 Taxonomic conclusions

The original description of P. mascareniensis (Duméril and Bibron 1841) does not include any diagnostic characters that permit delimitation of species within the P. mascareniensis complex identified by this study. Many of the characters described, including webbing and color pattern are highly plastic within genetically distinct clades. There is also significant overlap of character states between genetic clades (Table 4.1.1), which renders characters such as snout–vent length, body ratios, and webbing formulas useless for species delimitation. Multivariate analyses of traditional morphometric characters provided no resolution between genetically distinct lineages, nor did it provide evidence of conspecificity with the previously described subspecies available for measurement (Figs 4.2.1–4.2.4). To maintain taxonomic stability, further studies of morphological, behavioral, and acoustic characters are needed before these clades should be taxonomically recognized. Based on distinct structuring of habitat between forest and savannah dwelling populations, and a conservation approach to clade monophyly and sequence divergence, this study concludes that the savannah lineage corresponds to P. nilotica and the forest lineage corresponds to P. marchei. The forest dwelling clades assigned to P. marchei may represent more than one distinct species, but an increased sample size is needed to provide a definitive conclusion. In conclusion, the widespread species P. mascareniensis is actually composed of three distinct species: P. mascareniensis in Madagascar, P. nilotica in savannah habitats of continental West, Central, and East Africa, and P. marchei in forest habitats of West and Central Africa.

65 5.4 BIOGEOGRAPHY

The question that remains is the cause of diversification in P. mascareniensis. Haplotype networks constructed using sequences from the five clades of P. mascareniensis indicate that its ancestor likely originated in the DRC (Figs. 3.5.1–3.5.4), and subsequently dispersed to other parts of Africa. For this reason, the phylogeographic discussion is focused on the DRC. Three of the five clades identified in phylogenetic analyses are found in the DRC (A, C, D).

Specimens identified in genetic analyses as P. aff. mascareniensis ‗A‘ are widely distributed throughout the Albertine Rift as well as southern DRC, Kenya, Uganda, and Egypt. A similar biogeographic pattern is found in many mountain flora. Most mountain ranges in Africa are very different from each other, like islands, but there is an anomaly in that the Kenyan mountains have a great affiliation with the distant mountains in the DRC. This anomaly may also apply to fauna such as P. mascareniensis, although the mechanisms underlying the affinity are poorly understood (Werdelin and

Sanders 2010). The majority of specimens from this clade were collected from savannah or savannah- forest mosaic habitats. For example, some savannah-clade specimens were collected from Fizi (EBG 2157) and Lwiro (EBG 1163; EBG 1384; EBG 1416) in the DRC, which are both classified as agriculture-savannah mosaics (E. Greenbaum, personal communication). The specimens from Kenya were collected from the Aberdares, Mt. Kenya, and the Taita Hills, and although precise localities are not available, it is likely that these specimens were also collected from savannah. Mount Kenya and the

Aberdares, for example, are surrounded by moist savannah and the Taita Hills are classified as dry savannah (Spawls et al. 2002). Based on these localities, it can be concluded that this species is mainly restricted to savannah or savannah-forest mosaic. Previous studies have also hypothesized the existence of a savannah dwelling race of P. mascareniensis (Lamotte 1967) but assumed it to be conspecific with P. mascareniensis sensu stricto, which this study reveals is incorrect. Specimens from Clade C of P. aff. mascareniensis were almost all collected from the Albertine Rift, with one specimen from the Kakamega forest in Kenya and one from Uganda. A number of specimens collected in the DRC were collected in the vicinity of Epulu, a humid lowland rainforest. All localities for these clades were lowland rainforest habitat, providing evidence for niche partitioning between Clade A and Clade C. The Kakamega forest is the easternmost remnant of a once continuous

66 Guineo-Congolian forest belt that would have connected DRC and Kenya (Lötters et al. 2007). Ptychadena aff. mascareniensis Clade C likely dispersed between DRC and Kenya until drying of the climate 10,000 years ago reduced the forests, creating discontinuous forest pockets (Lötters et al. 2007). This accounts for the disjunct distribution of this forest-dwelling clade, but not enough time has elapsed for divergence to be evident between the two populations.

The Ptychadena aff. mascareniensis Clade D is represented by only five specimens. Three were collected from Bitale and Mwenga in the DRC, one from Guinea, and one from the Ivory Coast (West Africa). The localities in the DRC are humid rainforest, but the habitats from West Africa are unknown because specific localities are not provided by previous authors (Vences et al. 2004; Measey et al. 2007).

This highly disjunct distribution may be due to a lack of sampling at localities that might connect the two ranges, or may be an artifact of a previous forest corridor connecting these two areas. The close relationship between specimens of P. aff. mascareniensis ‗C‘ in the DRC and in West Africa ( > 2% divergence) is mirrored in patterns of vegetation in the Miocene. A recurring aspect of East African paleofloras in the early and middle Miocene is the presence of taxa related to plants found today in forests of Central and West Africa. For example, fossil woods from the Ethiopian plateau represent the same taxa that are currently found in West Africa (Lemoigne et al. 1974; Lemoigne 1978). Moreover, a similar geographically disjunct distribution has been observed in avifauna (Diamond and Hamilton 1980) and reptiles (Eaton et al. 2009; Wagner et al. 2009).

5.4.2 Drivers of Speciation in Ptychadena mascareniensis

The phenomenon of glacial refugia has been proposed multiple times to explain patterns of divergence in forest dwelling species (Moreau 1954; Diamond and Hamilton 1980; Crowe and Crowe 1982; Mayr and O‘Hara 1986; Petit et al. 2003; Hewitt 2004). This approach may be overly simplified, particularly as forests are non-linear systems that are always dynamically evolving. It is clear, however, that many habitats respond to climatic oscillations and glacial refugia can provide an explanation of large-scale survival and present-day composition and distribution of forest species (Lovett et al. 2005).

67 This study hypothesized that climatic changes in the Pleistocene created forest refugia that may have driven speciation in Ptychadena.

An estimate of the time of divergence of Ptychadena species can help resolve their phylogenetic history. Considering standard calibrations of the amphibian molecular clock (Caccone et al. 1994; Veith et al. 2003), a pairwise divergence of 5% corresponds to a separation occurring 5–20 Ma. Therefore, the five clades of P. mascareniensis identified in molecular analyses (Figs. 3.1.1–3.2.1) have been on their own evolutionary track for at least 5–20 million years, which corresponds to the Miocene epoch (5.332– 23.03 Ma). Hypothesized drivers of speciation in the Pleistocene, therefore, occurred much too recently (10 Ka) to have played a role in the origin of the clades, although they may have impacted subsequent diversification of haplotypes within clades. Thus, the Pleistocene refuge hypothesis is rejected, as it has been in studies of several other clades of African fauna (Bowie et al. 2004; Fjeldså and Bowie 2008; Njabo et al. 2008; Marks 2010 for birds; Blackburn and Measey 2009 for frogs). More likely, the drivers of speciation were geological and climatic changes occurring during the Miocene epoch. The Miocene in Africa is characterized by great magmatic and tectonic activity (Guiraud et al. 2005). The northern Angola and Congo margins are thought to have been uplifted in the Burdigaian

(~18 Ma) and to a lesser extent in the Tortonian (11–7 Ma) (Lavier at al. 2000; 2001). The faulting of the East African Rift System (EARS), a major geologic event, may have begun as early as the Oligocene, but uplift and rift faulting occurred on a much larger scale beginning in the early to mid-

Miocene (20–14 Ma) (Ebinger et al. 2000). The Albertine Rift is the western branch of the EARS, which extends nearly 2000 km. During the formation of the Albertine Rift, some of the highest mountains and deepest valleys in Africa were formed, creating multiple physical barriers to dispersal of sensitive amphibians. The formation of the Albertine Rift had profound and far reaching effects on Africa. Today, the faults of the Albertine Rift form a complex network of fractures and troughs. The main trough was formed by conjoining of distinctive basins. Concurrent uplifting during the formation of the troughs built spectacular horsts. In addition to geological effects, the formation of the rift had effects on climate. The rift intensifies the contrast between humid Central Africa and dry East Africa. It does not constitute

68 a barrier per se, but a large transition and contact zone between habitats. Despite cyclic climate variations over the last two millions years, the Albertine Rift it thought to have been able to maintain diverse habitats, making it a refuge for species (Vande weghe 2004). The diversity of habitats in the Albertine Rift has resulted in exceedingly high biodiversity in this region. The Albertine Rift is hypothesized to be a hotspot for diversification in several taxa (mammals:

Rodgers et al. 1982; insects: Tattersfield 1996; mollusks: Brühl 1997). The Albertine Rift region encompasses much of the western Rift valley down to Tanzania and Zambia. Previous biodiversity studies (Plumptre et al. 2007 and references therein) have defined the region as beginning 30 km north of Lake Albert and extending to the southern tip of Lake Tanganyika. The ecoregion of the Albertine

Rift includes all natural habitats within 100 km east of the border of the DRC and follows the 900 m contour line in eastern DRC (Plumptre et al. 2007). The Albertine Rift is the most species rich region for vertebrates on the African continent (Brooks et al. 2001; Plumptre et al. 2007). The region is also an

Endemic Bird Area according to Bird Life International (Stattersfield et al. 1998), a ‗Global–200‘ priority ecoregion designated by the World Wildlife Foundation (Olson and Dinerstein 1998; Burgess et al. 2000) and it is part of the Eastern Afromontane Hotspot recognized by Conservation International

(Brooks et al. 2004). A literature review of the amphibians of the Albertine Rift (Plumptre et al. 2007) found records of 119 species of amphibians in the region, including 29 genera and 11 families, with 3 genera and 36 species being endemic to the region. These numbers account for 19% of the biodiversity of amphibians in Africa, although this may be an underrepresentation due to a general lack of sampling for amphibians in this region (Evans et al., in press; Roelke et al., in press). The formation of the rift corresponds with divergence estimates in Ptychadena, and matches the hypothesized location of ancestral lineages. Significant climatic events that occurred in the mid-Miocene may have also played a role in isolating species of Ptychadena. Low grass pollen percentages in the early Miocene indicate the presence of extensive forested regions, but grass pollen increased in the mid-Miocene and became abundant by the late Miocene. This can be interpreted as the onset of pronounced seasonality of rainfall, resulting in the emergence of large savannah areas in the late Miocene (Morley and Richards 1993).

69 Fossil plants have been used to demonstrate that the earliest grasslands arose during this time (~14 Ma) (Retallack 1992). The increase of savannahs may have been a result of the interception of a large amount of the moisture brought inland from the Atlantic or Indian Ocean by the highlands formed in the EARS, leaving the rift valleys in local rain shadows. Patches of wet rain forest are known to have survived in many areas (Yemane et al. 1987), but extensive savannahs would have prevented gene flow between isolated forest patches for forest dwelling species. The change from forest to savannah was likely not unidirectional, but reflected the combined effects of global climate change and regional physiographic development, as evidenced by the variety of environments that were present in the evolving rift (6–13 Ma). Lowland or submontane forest dominated at 12.6 Ma, seasonally arid woodland or wooded savannahs at about 10 Ma, and dry forest to woodland and upland forests at 6.8 Ma (Werdelin and Sanders 2010). This increase in habitat heterogeneity could have provided opportunities for niche partitioning and subsequent sympatric speciation, rather than allopatric speciation by geologic separation. This hypothesis is supported by evidence of habitat partitioning between P. mascareniensis ‗A‘ and P. aff. mascareniensis ‗B‘ in savannah and forest habitat, respectively. Ancestral lineages could have dispersed into unique habitats to reduce competition and subsequently evolved prezygotic barriers to hybridization. This biogeographic pattern has been well documented in songbirds on several continents (Richman 1996; Slikas et al. 1996; Voelker 1999, 2002; García-Moreno and Fjeldså 2000; Filardi and Moyle 2005; Cadena et al. 2007; Voelker et al. 2009;

Voelker et al. 2010). There is no evidence of geographic structuring based on elevation or montane isolation between the three lineages of P. aff. mascareniensis. Moreover, P. mascareniensis is not limited to the highlands and has not been found above 2000 meters (Rödel et al. 2008), which eliminates the possibility of montane speciation. In the absence of other geographic structuring, the hypothesis of prezygotic barriers evolving after dispersal into a new habitat seems to be the most parsimonious explanation for the genetic pattern of the P. mascareniensis complex in DRC.

The most likely prezygotic barrier to reproduction in P. aff. mascareniensis Clade A and P. aff. mascareniensis Clade C is a change in mating cues and behavior. Anuran mating calls have been found to be species-specific: the call of each species differs distinctly from every other species in at least one

70 of the attributes of their calls (Blair 1958). Conversely, studies have also found significant genetic differentiation between anuran species with no associated variation in mating call (Heyer and Reid

2003), although seems to be the exception rather than the rule. The mating call is the most common and easily recognized call and is often a useful taxonomic tool for separating morphologically similar species as a result of its fundamental importance in mate recognition (Passmore 1977). The call of P. mascareniensis in South Africa has been shown to be distinct from four other species of Ptychadena (Passmore 1977). Thus, it is likely that there will be distinct differences between the five clades of P. mascareniensis recovered here, which is especially important in regions like the DRC where multiple clades occur sympatrically. A distinct call is likely the only way that these species could remain distinct.

Chorus calls, terrestrial calls, and distress calls have also been shown to be surprisingly complex and may also be useful in distinguishing species (Passmore 1977; Padial et al. 2008). Previous studies have also shown that calling behavior and reproductive biology, both of which may differ among species, are sensitive to environmental conditions. For example, Rana sylvatica will chorus only when the air temperature is 8–10°C (Howard 1980). Moreover, development rates and survivorship of anuran eggs and larvae critically depend on temperature (Moore 1939). Other factors known to influence anuran reproductive activity include light intensity (Heinzmann 1970), wind (Henzi et al. 1995), humidity (Bellis 1962), and barometric pressure (Fitzgerald and Bider 1974; Obert 1976; Bauch and Grosse 1989). More recently, studies have shown that anurans can change the frequency of their calls immediately in response to high traffic noise near breeding sites, which in turn made females less selective in mate choice (Wollerman and Wiley 2002; Cunnington and Fahrig 2010). Based on this, it is likely that when Ptychadena expanded to savannah habitats, their calls changed to adapt to the new environmental conditions. The drier air may have required a change in call frequency or duration, since sound travels better through humid than dry air (Harris 1966). Additionally, the different precipitation regime may have forced a shift in the time of breeding. This alteration in calls would have gradually resulted in reproductive isolation between individuals in different habitats and prevented later hybridization if forest and savannah species were to re-integrate. A detailed study of breeding seasons and acoustics in clades of P. mascareniensis may help determine the factors maintaining reproductive

71 isolation. Based on available evidence, the hypotheses are both supported as they seem to be interlinked. The formation of the Albertine Rift changed the precipitation regime in eastern DRC, which in turn caused the expansion of grasslands into which P. aff. mascareniensis ‗A‘ likely dispersed, leading to subsequent speciation.

5.5 CONSERVATION IMPLICATIONS

Molecular tools were used to investigate the nominal taxon Ptychadena mascareniensis that is currently recognized as a single, widespread species and categorized as an IUCN species of Least

Concern. This taxon is categorized as ―known not to be threatened‖ on the basis of widespread distribution, local abundance, and tolerance to disturbances. But this taxon instead represents a complex of at least three cryptic species, which are genetically distinct but morphologically similar. Additionally, many of the habitats in which specimens were collected are highly threatened. Natural montane forest habitat in the Albertine Rift is highly threatened because of the high density of people (500–600 km-2) (Plumptre and Williamson 2001). The Kakamega forest in Kenya is also distinctly threatened due to human encroachment (Lötters et al. 2007). Despite this, the distribution of two lineages newly recognized here, P. nilotica and P. marchei, are extensive enough that the species are not currently threatened by extinction, although knowledge of population numbers would be useful in future studies.

5.6 CONCLUSIONS AND FUTURE DIRECTIONS

This study resulted in several main conclusions. First, there is a great deal of genetic diversity in the genus Ptychadena, suggesting that many species have been separated into distinct genetic lineages that will be recognizable as distinct species as some point. Second, there are five genetically distinct lineages of P. mascareniensis sensu lato, three of which occur in the DRC. Finally, the formation of the Albertine Rift combined with subsequent climatic shifts that increased habitat heterogeneity led to the establishment of a savannah and forest form of P. mascareniensis. These clades should be recognized under the oldest available synonym for each. The savannah clade should be designated as P. nilotica and the forest form as P. marchei. Additionally, P. hylaea should be synonymized with P. marchei because

72 the former name represents a junior synonym of the latter. The name P. mascareniensis should apply only to populations on Madagascar, the Seychelles, and the Mascarenes.

The recognition of several lineages of P. mascareniensis presents several questions to be investigated in future studies. Additional research efforts should focus on the precise barriers to reproduction that allowed establishment and maintenance of these genetic lineages, specifically using a combination of genetic, morphological, and acoustic techniques to delimit species groups. An increased sample size would also help to provide resolution between clades. For example, more specimens of P. aff. mascareniensis ‗C‘ are needed before robust taxonomic conclusions are made. Moreover, it is important to sample at more sites across Africa to get a better idea of the genetic diversity and variation across the entire continent.

73 Literature Cited

Akef, M. S. and Schneider, A. H. 1995. Calling behavior and mating call pattern in the Mascarene Frog,

Ptychadena mascareniensis, (Amphibia, Anura, Ranidae) in Egypt. Journal of African Zoology, 109: 225–229. Amiet, J.-L. 1974. Voix d'amphibiens camerounais IV. Raninae: genres Ptychadena, Hildebrandtia et

Dicroglossus. Annales de la Faculté des Sciences du Cameroun, 18 : 109–128. AmphibiaWeb. 2010. Information on amphibian biology and conservation. [web app]. Berkeley, California: AmphibiaWeb. Available: http://amphibiaweb.org/. (Accessed: Nov 10, 2009).

Avise, J.C. 2000. Phylogeography: the history and formation of species. Harvard University Press, Cambridge, MA, USA. Bancroft, B.A., N.J. Baker, and A.R. Blaustein. 2008. A meta-analysis of the effects of

ultraviolet B radiation and its synergistic interactions with pH, contaminants, and disease on amphibian survival. Conservation Biology, 22: 987–996. Bashige, E., A Motingea, M. Ntayingi, P. On‘okoko, and S. a Tshiluila. 2004. Nature and

Culture in the Democratic Republic of Congo. Royal Museum for Central Africa: Tervuren, Belgium. Bauch, S. and W-R. Grosse. 1989, Der nachweis einer Nacklaichzeil beim Laub frosch, Hyla a. arborea

(L.) (Amphibia: Anura: Hylidae). Hercynia, 26: 425–429. Becker, C.G., C.R. Fonseca, C.F.B. Haddad, and P.I. Prado. 2010. Habitat split as a cause of

local population declines on amphibians with aquatic larvae. Conservation Biology, 24: 287

294. Bellis, E.D. 1962. The influence of humidity on woodfrog activity. American Midland Naturalist, 63: 139–148. Bickford, D., D.J. Lohman, N.S. Sodhi, P.K.L. Ng, R. Meier, K. Winker, K.K. Ingrams, and I. Das.

2007. Cryptic species as a window on diversity and conservation. Trends in Ecology and Evolution, 22: 148–155.

74 Blackburn, D.C., and G.J. Measey. 2009. Dispersal to or from an African biodiversity hotspot? Molecular Ecology, 18: 1904–1915.

Blair, W.F. 1958. Call structure and species groups in U.S. treefrogs (Hyla). Southwestern Naturalist, 3: 77–89. Blaustein, A.R., J.M. Romansic, J.M. Kiesecker, and A.C. Hatch. 2003. Ultraviolet radiation,

toxic chemicals and amphibian population declines. Diversity and Distributions, 9: 123–140. Bossuyt, F.R., and M.C. Milinkovitch. 2000. Convergent adaptive radiations in Madagascan and Asian ranid frogs reveal covariation between larval and adult traits. PNAS, 97: 6585–6590. Bossuyt, F. R. M. Brown, D. M. Hillis, D. C. Cannatella, and M. C. Milinkovitch. 2006.

Phylogeny and biogeography of a cosmopolitan frog radiation: late Cretaceous diversification results in continent-scale endemism in the family Ranidae. Systematic Biology, 55: 579–594. Boulenger, G. A. 1879. Synonymie de Rana mascareniensis. Bulletin de la Société Zoologique de

France, 4: 92–94. Boulenger, G. A. 1882. Catalogue of the Batrachia Salientia s. Ecaudata in the Collection of the British Museum. Second Edition. London: Taylor and Francis.

Bowie, R.C.K., J. Fjeldså, S.J. Hackett, and T.M. Crowe. 2004. Molecular evolution in space and through time: mtDNA phylogeography of the Olive Sunbird (Nectarinia olivacea/obscura) throughout continental Africa. Molecular Phylogenetics and Evolution, 33: 56–74.

Brandley, M.C., A. Schmitz, and T.W. Reeder. 2005. Partitioned Bayesian analysis, partition choice, and the phylogenetic relationships of scincid lizards. Systematic Biology, 54: 373–390.

Brooks, T., A. Balmford, N. Burgess, J. Fjeldså, L.A. Hansen, J. Moore, C. Rahbek, and P. Williams.

2001. Towards a blueprint for conservation in Africa. BioScience, 51: 613–624. Brooks, T., M. Hoffman, N. Burgess, A. Plumptre, S. Williams, R.E. Gereau, R.A. Mittermeier, and S. Stuart. 2004. Eastern afromontane. In: Mittermeier, R.A., P. Robles-Gil, M. Hoffman, J.D. Pilgrim,

T.M. Brooks, C.G. Mittermeier, J.L. Lamoreux, and G. Fonseca. (Eds.), Hotspots revisited: Earth‘s biologically richest and most endangered ecoregions, second ed. Cemex, Mexico, pp. 241–242.

75 Brühl, C. 1997. Flightless insects: a test case for historical relationships of African mountains. Journal of Biogeography, 24: 233–250.

Burgess, N.D., H. de Klerk, J. Fjeldså, T. Crowe, and R. Carsten. 2000. A preliminary assessment of congruence between biodiversity patterns in Afrotropical forest birds and forest mammals. Ostrich, 71: 286–290.

Burgess, N., J. D‘Amico Hales, E. Underwood, E. Dinerstein, D. Olson, I. Itoua, J. Schipper, T. Ricketts, and K. Newman. 2004. Terrestrial Ecoregions of Africa and Madagascar: A Conservation Assessment. Island Press: Washington, D.C. 644 pp. Bwong, B.A., R. Chira, S. Schick, M. Veith, and S. Lötters. 2009. Diversity of ridged frogs

(Ptychadenidae: Ptychadena) in the easternmost remnant of the Guineo-Congolian rain forest: an analysis using morphology, bioacoustics and molecular genetics. Salamandra,45:129–146. Caccone, A., M.C. Milinkovitch, V. Sbordoni, and J.R. Powell. 1994. Molecular biogeography: using

the Corsica-Sardinia microplate disjunction to calibrate mitochondrial rDNA evolutionary rates in mountain newts (Euproctus). Journal of Evolutionary Biology, 7: 227–245. Cadena, C.D., J. Klicka, and R.E. Ricklefs. 2007. Evolutionary differentiation in the Neotropical motane

region: molecular phylogenetics and phylogeography of Buarremon brushfinches (Aves, Emberizidae). Molecular Phylogenetics and Evolution, 44: 993–1016. Castelloe, J. and A. R. Templeton. 1994. Root probabilities for intraspecific gene trees under neutral

coalescent theory. Molecular Phylogenetics and Evolution, 3:102–113. Ceballos, G., A. García, and P.R. Ehrlich. 2010. The sixth extinction crisis: loss of animal

populations and species. Journal of Cosmology, 8: 1821–1831.

Channing, A. 2001. Amphibians of Central and Southern Africa. Cornell University Press: New York, NY. Channing, A., S. Finlow-Bates, S.E. Haarklau, and P.G. Hawkes. 2006. The biology and recent history

of the critically endangered Kihansi Spray Toad Nectrophrynoides asperginis in Tanzania. Journal of East African Natural History, 95: 117–138.

76 Chapin. J.P. 1932. The birds of the Belgian Congo, vol. 1. Bulletin of the American Museum Natural History, 65: 1–756.

Clarke, B.T. 1981. Comparative osteology and evolutionary relationships in the African Raninae (Anura: Ranidae). Monitore Zoologico Italiano, (N.S.) Suppl. XV(14): 225–284. Clarke, B.T. 1982. A new genus of ranine frog (Anura: Ranidae) from Somalia. Bulletin of the

British Museum of Natural History (Zoology), 43: 179–183. Clement, M., D. Posada and K. A. Crandall 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology 9 (10): 1657–1660.\ Clewley, J. P. & C. Arnold. 1997. MEGALIGN. The multiple alignment module of

LASERGENE. Methods Mol Biol, 70: 119–129. Crawford, A.J., K.R. Lips, and E. Bermingham. 2010. Epidemic disease decimates amphibian abundance, species diversity, and evolutionary history in the highlands of central Panama.

PNAS, 107: 13777–13782. Crowe, T.M. and A.A. Crowe. 1982. Patterns of distribution, diversity and endemism in Afro-tropical birds. Journal of Zoology, 198: 417–442.

Cunnington, G.M. and L. Fahrig. 2010. Plasticity in the vocalizations of anurans in response to traffic noise. Acta Oecologica, 36: 463–470. D‘Amen, M. and P. Bombi. 2009. Global warming and biodiversity: evidence of climate-linked

amphibian declines in Italy. Biological Conservation, 142: 3060–3067. Daszak, P. 1998. A new fungal disease associated with amphibian population declines: recent

research put into perspective. British Herpetological Society Bulletin, 65: 38–41.

De la Riva, I. 2007. Bolivian frogs of the genus Phrynopus, with descriptions of twelve new species (Anura: Brachycephalidae). Herpetological Monographs, 21: 247–277. de Queiroz, K. 1998. The general lineage concept of species, species criteria, and the process of

speciation: a conceptual unification and terminological recommendations. Pages 57–75 in Endless forms: Species and speciation (D. J. Howard, and S. H. Berlocher, eds.). Oxford University Press, New York.

77 de Queiroz, K. 1999. The general lineage concept of species and the defining properties of the species category. Species, New Interdisciplinary Essays, 49–89. de Queiroz, K. 2007. Species concepts and species delimitation. Systematic Biology, 56: 879–886. Diamond, A.W. and A.C. Hamilton. 1980. The distribution of forest passerine birds and Quaternary climate change in tropical Africa. Journal of Zoology, 191: 379–402.

Dubois, A. 1992. Notes sur la classification des Ranidae (Amphibiens Anoures). Bull. Mens. Linn. Lyon, 61: 305–352. Duméril, A. M. C., and G. Bibron. 1841. Erpétologie Genérale ou Histoire Naturelle Complète des Reptiles. Volume 8. Paris: Librarie Enclyclopedique de Roret.

Eaton, M.J., A. Martin, J. Thorbjarnarson, and G. Amato. 2009. Species-level diversification of African dwarf crocodiles (Genus Osteolaemus): a geographic and phylogenetic perspective. Molecular Phylogenetics and Evolution, 50: 496–506.

Ebinger, C.J. 1989. Tectonic development of the western branch of the East African rift system. Geological Society of America Bulletin, 101: 885–903. Ebinger, C.J., T. Yemane, D.J. Harding, S. Tesfaye, S. Kelley, and D.C. Rex. 2000. Rift deflection,

migration and propagation: Linkage of the Ethiopian and Eastern rifts, Africa. Bulletin of the Geological Society of America, 112: 163–176. Excoffier, L., P.E. Smouse, and J.M. Quattro. 1992. Analysis of molecular variance inferred from metrix

distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics, 131: 479–491.

Excoffier, L., G. Laval, and S. Schneider. 2005. Arlequin ver. 3.0: An integrated software package for

population genetics data analysis. Evolutionary Bioinformatics Online, 1: 47–50. Faivovich, J., C.F.B. Haddad, P.C.A. Garcia, D.R. Frost, J.A. Campbell, and W.C. Wheeler. 2005. Systematic review of the frog family Hylidae, with special reference to Hylinae:

Phylogenetic analysis and taxonomic revision. Bulletin of the American Museum Natural History, 294: 1–240.

78 Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39: 783–791.

Filardi, C.E. and R.G. Moyle. 2005. Single origin of a pan-Pacific bird group and upstream colonization of Australasia. Nature, 438: 216–219. Fisher, R.N., and H.B. Shaffer. 1996. The decline of amphibians in California's Great Central

Valley. Conservation Biology, 10: 1387–1397. Fitzgerald, G.J., and J.R. Bider. 1974. Seasonal activity of the toad Bufo americanus in southern Quebec as revealed by a sand-transect technique. Canadian Journal of Zoology, 52: 1–5. Fjeldså, J., and R.C.K. Bowie. 2008. New perspectives on the origin and diversification of Africa‘s

forest avifauna. African Journal of Ecology, 46: 235–247. Fouquet, A., A. Gilles, M. Vences, C. Marty, M. Blanc, and N.J. Gemmell. 2007. Underestimation of species richness in Neotropical frogs revealed by mtDNA analyses. PLoS

ONE, 2: e1109. Frost, D.R., T. Grant, J. Faivovich, R.H. Bain, A. Haas, C.F.B. Hasddad, R. O. De Sa, A. Channing, M. Wilkinson, S.C. Donnellan, C. J. Raxworthy, J.A. Campbell, B. L. Blotto, P.

Moler, R. C. Drewes, R. A. Nussbaum, J. D. Lynch, D. M. Green, and W. C. Wheeler. 2006. The amphibian tree of life. Bulletin of the American Museum of Natural History. 297: 1–370. Frost, D. R. 2010. Amphibian species of the world: An online reference. Version 5.3 (12

February, 2009). Database accessible at http://research.amnh.org/herpetology/amphibia/ American Museum of Natural History, New York, USA.

García-Moreno, J. and J. Fjeldså. 2000. Chronology and mode of speciation in the Andean avifauna.

Proceedings of the 4th International Symposium: isolated vertebrate communities in the tropics. Bonn Zoological Monographs, 46: 25–46. Gasse, F., A. Téhet, A. Durand, E. Gibert, and J. Ch. Fontes. 1990. The arid-humid transition in

the Sahara and the Sahel during the last deglaciation. Nature, 346: 141–146. Gerlanc, N. M., and G. A. Kaufman. 2005. Habitat origin and changes in water chemistry influence development of Western Chorus Frogs. Journal of Herpetology, 39:254–265.

79 Glaw, F. and M. Vences. 2002. A new cryptic frog species of the Mantidactylus boulengeri group with a divergent vocal sac structure. Amphibia-Reptilia, 23: 293–304.

Goldberg, T.L., A.M. Readel, and M.H. Lee. 2007. Chytrid fungus in frogs from an equatorial African montane forest in Western Uganda. Journal of Wildlife Diseases, 43: 521–524. Greenbaum, E., C. Kusamba, M.M. Aristote, and K. Reed. 2008. Amphibian chytrid fungus

infections in Hyperolius (Anura: Hyperoliidae) from Eastern Democratic Republic of Congo. Herpetological Review. 39: 70–73. Guibé, J. and M. Lamotte. 1954. Etude comparée de Rana (Ptychadena) longirostris Peters et R. (Pt.) aequiplicata Werner. Bull. Mus. Nat. Hist Nat. Paris, 2: 318–321.

Guibé, J. and M. Lamotte. 1957. Révision systématique des Ptychadena (Batraciens Anoures Ranidés) d‘Afrique occidentale. Bull. Inst. Franç. Afr. Noire, XIX sér A : 937-1003. Guiraud, R., W. Bosworth, J. Thierry, and A. Delplanque. 2005. Phanerozoic geological evolution of

Northern and Central Africa: An overview. Journal of African Earth Sciences, 43: 83–143. Halliday, T.R. 2008. Why amphibians are important. International Zoo Yearbook, 42: 7–14. Harris, C.M. 1966. Absorption of sound in air versus humidity and temperature. Journal of the

Acoustical Society of America, 40: 148–159. Hayes, T.B., A. Collins, M. Lee, M. Mendoza, N. Noriega, A.A. Stuart, and A. Vonk. 2002. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low

ecologically relevant doses. PNAS, 99: 5476–5480. Hayes, T.B., P. Falso, S. Gallipeau, and M. Stice. 2010a. The cause of global amphibian

declines: a developmental endocrinologist‘s perspective. Journal of Experimental Biology,

213: 921–933. Hayes, T.B., V. Khoury, A. Narayan, M. Nazir, A. Park, T. Brown, L. Adame, E. Chana, D. Buchholz, T. Stueve, and S. Gallipeau. 2010b. Atrazine induces complete feminization and

chemical castration in male African clawed frogs (Xenopus laevis). PNAS, 107: 4612–4617. Heinzmann, U. 1970. Untersuchungen zur bio-akustik und ökologie der geburtshelferkröte, Alytes o.obsteiricans (Laur.). Oecologia, 5: 19–55.

80 Henzi, S.P., M.L. Dyson, S.B. Piper, N.B. Passmore, and P. Bishop. 1995. Chorus attendance by male and female painted reed frogs (Hyperolius marmoratus): environmental factors and selection

pressures. Functional Ecology, 9: 485–491. Heyer, W.R., and Y.R. Reid. 2003. Does advertisement call variation coincide with genetic variation in the genetically diverse frog taxon currently known as Leptodactylus fuscus (Amphibia:

Leptodactyidae)? Anais da Academia Brasileira de Ciências, 75: 39–54. Hewitt, G.M. 2004. Genetic consequences of climatic oscillations in the Quaternary. Philosophical Transactions of the Royal Society of London, 359: 183–195. Houlahan, J.E., C.S. Findlay, B.R. Schmidt, A.H. Meyer, Kuzmin. 2000. Quantitative evidence

for global amphibian population declines. Nature, 404: 752–755. Howard, R.D. 1980. Mating behavior and mating success in wood frogs, Rana sylvatica. Animal Behavior, 28: 705–716.

Hudson, R.R. and M. Turelli. 2003. Stochasticity overrules the ―three-times rules‖: genetic drift, genetic draft, and coalescence times for nuclear loci versus mitochondrial DNA. Evolution: International Journal of Organic Evolution, 57: 182–190.

Huelsenbeck, J.P. and F. Ronquist. 2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics, 17: 754–755. Inger, R.F. 1968. Amphibia. Exploration du Parc National de la Garamba. Fascicule 52. Institut

des Parcs Nationaux. Republique Democratique du Congo, Kinshasa. Inger, R., B. Stuart, and D. Iskandar. 2009. Systematics of a widespread Southeast Asian frog, Rana

cholconota (Amphibia: Anura: Ranidae). Zoological Journal of the Linnean Society, 155: 123–147.

IUCN 2010. IUCN Red List of Threatened Species. Version 2010.4. . Downloaded on 10 November 2010. IUCN, Conservation International, and NatureServe. 2008. An Analysis of Amphibians on the

2008 IUCN Red List . Downloaded on 10 March 2008.

81 Kerby, J.L., K.L. Richards-Hrdlicka, A. Storfer, and D.K. Skelly. 2010. An examination of amphibian sensitivity to environmental contaminants: are amphibians poor canaries? Ecology

Letters, 13: 60–67. Kielgast, J., D. Rödder, M. Veith, and S. Lötters. 2010. Widespread occurrence of the amphibian chytrid fungus in Kenya. Animal Conservation, 13: 36–43.

Koenig, R. 2008. Critical time for African rainforests. Science. 320: 1439–1441. Lambiris, A. J. L. 1989. The frogs of Zimbabwe. Museo Regionale di Scienze Naturali, Torino. Monografie, 10: 102–223. Lamotte, M. 1967. Le problème des Ptychadena (Fam. Ranidae) du groupe mascareniensis dans

l‘ouest Africain. Bulletin du Muséum National D’Histoire Naturelle, 2e Serie, 39 : 647–656. Lannoo, M. J., K. Lang, T. Waltz, and G. S. Phillips. 1994. An altered amphibian assemblage – Dickinson County, Iowa, 70 years after Blanchard, Frank Survey. American Midland

Naturalist, 131: 311–319. Laporte, N.T., J.A. Stabach, R. Grosch, T.S. Lin, and S.J. Goetz. 2007. Expansion of industrial logging in Central Africa. Science, 316: 1451.

Largen, M.J. 1997. Two new species of Ptychadena Boulenger 1917 (Amphibia: Anura: Ranidae) from Ethiopia, with observations of the other members of the genus recorded from this country and a tentative key for their identification. Tropical Zoology, 10: 223–246.

Laurent, R. F. 1950. Reptiles et batraciens de la région de Dundo (Angola du Nord Est).(Primière note). Publicações Culturais. Companhia de Diamantes de Angola. Lisboa ,6: (preprint) 7–17, (published

in series) 128–136.

Laurent, R.F. 1954. Etude de quelques espèces méconnues du genre Ptychadena. Annales Musée Royal de Congo Belge, Sciences Zoologiques, 34: 1–34. Laurent, R.F. 1997. Morphometric approach of the evolution of the tribe Ptychadenini.

Proceeding of the Third Herpetological Association of Africa Symposium 1997: 183–187.

82 Lavier, L.L., M.S. Steckler, and F. Brigaud. 2000. An improved method for reconstruction of the stratigraphy and bathymetry of continental margins: Application to the Cenozoic tectonic and

sedimentary history of the Congo margin. Bulletin of the American Association of Petroleum Geologists, 84: 923–939. Lavier, L.L., M.S. Steckler, and F. Brigaud. 2001. Climatic and tectonic control on the Cenozoic

evolution of the West African margin. Marine Geology, 178: 63–80. Leaky, R. E. and R. Lewin. 1996. The sixth extinction: biodiversity and its survival. Weidenfeld and Nicolson: London. Lemmon, A.R. and E.C. Moriarty. 2004. The importance of proper model assumption in Bayesian

phylogenetics. Systematic Biology, 53: 265–277. Lemoigne, Y., J. Beauchamp, and E. Samuel. 1974. Étude paléobotanique des dépots volcaniques d'age tertiaire des bordures est et Ouest du système des rifts éthiopiens. Geobios, 7 : 267–288.

Lemoigne, Y. 1978. Flores tertiaires de la haute vallée de l'Omo (Ethiopie). Palaeontographica Abteilung B Band B165, Lieferung 4–6, p. 89–157. Lips, K.R. 1998. Decline of a tropical montane amphibian fauna. Conservation Biology, 12:

106–117. Lips, K.R., J. Diffendorfer, J.R. Mendelson, and M.W. Sears. 2008. Riding the wave: reconciling the roles of disease and climate change in amphibian declines. PLoS Biol, 6: e72.

Lötters, S., J. Kielgast, J. Bielby, S. Schmidtlein, J. Bosch, M. Veith, S.F. Walker, M.C. Fisher, and D. Rödder. 2009. The link between rapid enigmatic amphibian decline and the globally

emerging chytrid fungus. EcoHealth, 6: 358–372.

Loveridge, A. 1936. African reptiles and amphibians in Field Museum of Natural History. Field Museum of Natural History Publication. Zoological Series, 22: 1–111. Mace, G.M. 2004. The role of taxonomy in species conservation. Philosophical Transactions:

Biological Sciences. 359: 711–719. Maddison, D. R. and W. P. Maddison, 2000. MacClade 4: Analysis of phylogeny and character evolution. Version 4.0. Sinauer Associates, Sunderland, Massachusetts.

83 Maley, J., 1997. Middle to Late Holocene changes in tropical Africa and other continents: paleomonsoon and sea surface temperature variations. In: Third Millenium BC Climate

Change and Old World Collapse. NATO ASI Series. Series 1, Vol. 49, pp. 611–640. Manamendra-Arachchi, K., and R. Pethiyagoda. 2005. The Sri Lankan shrub-frogs of the genus Philautus Gistel, 1848 (Ranidae: Rhacophorinae), with description of 27 new species. The

Raffles Bulletin of Zoology, 12: 163–303. Mayer, F., C. Dietz, and A. Kiefer. 2007. Molecular species identification boosts bat diversity. Frontiers in Zoology, 4: 4. Mayr, E., and R.J. O‘Hara. 1986. The biogeographic evidence supporting the Pleistocene forest

refuge hypothesis. Evolution, 40: 55–67. McLeod, D.S. 2010. Of least concern? Systematics of a cryptic species complex: Limnonectes kuhlii (Amphibia: Anura: Dicroglossidae). Molecular Phylogenetic and Evolution, 56: 991–1000.

Measey, G.J., M. Vences, R.C. Drewes, Y. Chiari, M. Melo, and B. Bourles. 2007. Freshwater paths across the ocean: molecular phylogeny of the frog Ptychadena newtoni gives insights into amphibian colonization of oceanic islands. Journal of Biogeography, 34: 7–20.

Meegaskumbura, M., F. Bossuyt, R. Pethiyagoda, K. Manamendra-Arachchi, M. Bahir, M.C. Milinkovitch, and C.J. Schneider. 2002. Sri Lanka: an amphibian hot spot. Science, 298: 379. Meijer, P. Th., and M. J. R. Wortel 1999. Cenozoic dynamics of the African plate with

emphasis on the Africa-Eurasia collision, J. Geophys. Res., 104: 7405–7418. Meyer, M.J. 2004. A morphological phylogeny of the African anuran subfamily Ptychadeninae

(Dubois 1992) (Anura: Ranidae). Master‘s Thesis, Illinois State University. 144 pp.

Mittermeier, R.A., P. R. Gil, and C.G. Mittermeier. eds. 1997. Megadiversity: Earth‘s biologically wealthiest nations. Quebecor Printing, Canada. 501 pp. Moore, J.A. 1939. Temperature tolerance and rates of development in the eggs of Amphibia. Ecology,

20: 459–478. Moreau, R.E. 1954. The distribution of African evergreen-forest birds. Proceedings of the Linnean Society of London, 165: 35–46.

84 Morley, R.J., and K. Richards. 1993. Gramineae cuticle: a key indicator of Late Cenozoic climatic change in the Niger Delta. Review of Palaeobotany and Palynology, 77: 119–127.

Murray, T., U. Fitzpatrick, and M. Brown. 2008. Cryptic species diversity in a widespread bumble bee complex revealed using mitochondria DNA RFLPs. Conservation Genetics, 9: 653–666. Narins, P.M. 1983. Divergence of acoustic communication systems of two sibling species of

eleutherodactylid frogs. Copeia, 4: 1089–1090. Njabo, K.Y., R.C.K. Bowie, and M.D. Sorenson. 2008. Phylogeny, biogeography and taxonomy of the African wattle-eyes (Aves: Passeriformes: Platysteiridae). Molecular Phylogenetics and Evolution, 48: 136–149.

Noble, G. K. 1924. Contributions of the herpetology of the Belgian Congo based on the collection of the American Museum Congo Expedition, 1909–1915. Bulletin of the American Museum of Natural History 49: 147–347.

Obert, H-J. 1976. Some effects of external factors upon the reproductive behavior of the grass frog Rana t. temporaria L. (Ranidae, Anura). Oecologia, 24: 43–55. Olson, D.M., and E. Dinerstein. 1998. The global 200: a representation approach to conserving the

earth‘s most biologically valuable ecoregions. Conservation Biology, 12: 502–515. Padial, J.M., J. Köhler, A. Muñoz, and I. de la Riva. 2008. Assessing the taxonomic status of tropical frogs through bioacoustics: Geographical variation in the advertisement calls in the

Eleutherodactylus discoidalis species group (Anura). Zoological Journal of the Linnean Society, 152: 353–365.

Passmore, N.I. 1977. Mating calls and other vocalizations of five species of Ptychadena. South African

Journal of Science, 73: 212–214. Perret, J. 1991. Description de Ptychadena ingeri n. sp. (Anura, Ranidae) du Zaїre. Archs Sci. Genève, 44: 265–281.

85 Peters, W.C.H. 1869. Säugethiere und Amphibien gesammelt von Baron C.C. von der Decken auf seinen Reisen im äquatorialen Ostafrika, Baron Carol Claus von der Deckens Reisen in Ost-Afrika in den

Jahren 1859–1865. Volume 3 (Wissenschaftliche Ergebnisse. Erste Abtheilung: Amphibien). C. F. Wintersche Verlashandlung, Leipzig und Heidelberg, 1–18. Petit, R.J., I. Aguinagade, J. de Beaulieu, C. Bittkau, S. Brewer, R. Cheddadi, R. Ennos, S. Fineschi, D.

Grivet, M. Lascoux, A. Mohanty, G. Müller-Starck, B. Demesure-Musch, A. Palmé, J.P. Martin, S. Rendell, G.G. Vendramin. 2003. Glacial refugia: hotspots but not melting pots of genetic diversity. Science, 300: 1563–1565. Pfenninger, M., and K. Schwenk. 2007. Cryptic animal species are homogeneously distributed among

taxa and biogeographical regions. BMC Evolutionary Biology, 7: 121. Plumptre, A.J. and E.A. Williamson. 2001. Conservation-oriented research in the Virunga Region. In: Robbins, M.M., P. Sicotte, K.J. Stewart. (Eds.), Mountain Gorillas: Three Decades of Research at

Karisoke. Cambridge University Press, Cambridge, pp. 361–389. Plumptre, A.J, T.R.B. Davenport, M. Behangana, R. Kityo, G. Eilu, P. Ssegawa, C. Ewango, D. Meirte, C. Kahindo, M. Herremans, J.K. Peterhans, J.D. Pilgrim, M. Wilson, M. Languy, and D. Moyer.

2007. The biodiversity of the Albertine Rift. Biological Conservation, 134: 178–194. Posada, D. 2008. jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution, 25: 1253–1256.

Pounds, J.A., M.P.L. Fogden, J.M. Savage, and G.C. Gorman. 1997. Tests of null models for amphibian declines on a tropical mountain. Conservation Biology, 11: 1307–1322.

Pounds, J.A., M.P.L. Fogden, and J.H. Campbell. 1999. Biological response to climate change

on a tropical mountain. Nature, 398: 611–615. Pounds, J.A., M.R. Bustamente, L.A. Colman, J.A. Consuegra, M.P.L. Fogden, P.N. Foster, E. La Marca, K.L. Masters, A. Merino-Viteri, R. Puschendorf, S.R. Ron, A. Sánchez-Azofeifa,

C.J. Still, and B.E. Young. 2006. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature, 439: 161–167. Poynton, J.C. 1964. The Amphibia of Southern Africa. Ann. Natal Mus., 17 : 1–334.

86 Poynton, J.C. 1970. Guide to the Ptychadena (Amphibia: Ranidae) of the southern third of Africa. Ann. Natal Mus. 20: 365–375.

Poynton, J.C. 1998. Introduction to Amphibia: pp. i–ix. In Schmidt, K.P., and G.K. Noble. 1998. Contributions to the Herpetology of the Belgian Congo. Facsimile Reprints in Herpetology, Society for the Study of Amphibians and Reptiles, St. Louis, Missouri. 780 pp.

Poynton, J.C. and D.G. Broadley. 1985. Amphibia Zambesiaca 2. Ranidae. Ann. Natal Mus. 27: 115–181. Rambaut, A. and A.J. Drummond. 2007. Tracer v1.4, Available from http://beast.bio.ed.ac.uk/Tracer

Relyea, R.A. and N. Diecks. 2008. An unforeseen chain of events: lethal effects of pesticides on frogs at sublethal concentrations. Ecological Applications, 18: 1728–1742. Retallack, G.J. 1992. Middle Miocene fossil plants from Fort Ternan (Kenya) and evolution of African

grasslands. Paleobiology, 18: 383–400. Richman, A.D. 1996. Ecological diversification and community structure in the Old World Leaf Warblers (Genus Phylloscopus): A phylogenetic perspective. Evolution, 50: 2461–2470.

Rochebrune, A. T. d. 1885. Vertebratorum novorum vel minus cognitorum orae Africae occidentalis incolarum. Diagnoses (1). Bulletin de la Société Philomathique de Paris. Series 7, 9: 86–99. Rödel, M. D. Kratz, & R. Ernst. 2002. The tadpole of Ptychadena aequiplicata (Werner, 1898)

with the description of a new reproductive mode for the genus (Amphibia, Anura, Ranidae). Alytes, 20: 1–12.

Rödel, M. O., M. Largen, L. Minter, K. Howell, R. Nussbaum, M. Vences, and S. Baha El Din. 2008.

Ptychadena mascareniensis. In: IUCN 2008. 2008 IUCN Red List of Threatened Species. Downloaded on 14 April 2009. Rodgers, W.A., C.F. Owen, and K.M. Homewood. 1982. Biogeography of East African forest

mammals. Journal of Biogeography, 9: 41–54.

87 Roelants, K., D.J. Gower, M. Wilkinson, S.P. Loader, S.D. Biju, K. Guillaume, L. Moriau, and F. Bossuyt. 2007. Global patterns of diversification in the history of modern amphibians.

PNAS, 104: 887–892. Rohr, J.R. and T.R. Raffel. 2010. Linking global climate and temperature variability to widespread amphibian declines putatively caused by disease. PNAS, 107: 8269–8274.

Ronquist, F. and J. P. Huelsenbeck. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574. Rowe, K.C., E.J. Heske, P.W. Brown, and K.N. Paige. 2004. Surviving the ice: Northern refugia and postglacial colonization. PNAS, 101: 10355–10359.

Sabaj Pérez, M.H. (editor). 2010. Standard symbolic codes for institutional resource collections in herpetology and ichthyology: an Online Reference. Version 1.5 (4 Oct 2010). Electronically accessible at http://www.asih.org/, American Society of Ichthyologists and

Herpetologists, Washington, DC. Schiøtz, A. 1964. The voices of some West African amphibians. Vedenskabelige Meddelelser fra Dansk Naturhistorisk Forening, 127: 35–83.

Schmidt, K.P. and R.F. Inger. 1959. Amphibians. Exploration du Parc National de Upemba, 56 : 3–264 + 9 plates. Searle, C.L., L.K. Belden, B.A. Bancroft, B.A. Han, L.M. Biga, and A.R. Blaustein. 2010.

Experimental examination of the effects of ultraviolet-B radiation in combination with other stressors on frog larvae. Oecologia, 162: 237–245.

Seetzen, U. J. 1855. Reisen durch Syrien, Palästina, Phönicien, die Transjordan-Länder, Arabia

Petraea und Unter-Aegytpten. Volume 3. Berlin: G. Reimer. Sheridan, C. D., and D. H. Olson. 2003. Amphibian assemblages in zero-order basins in the Oregon Coast Range. Canadian Journal of Forest Research, 33:1452–1477.

Simpson, G.G. 1961. Principles of animal taxonomy. New York: Columbia University Press. Slikas, B., F.H. Sheldon, and F.B. Gill. 1996. Phylogeny of titmice (Paridae): I. Estimate of relationships among subgenera based on DNA-DNA hybridization. Journal of Avian Biology, 27: 70–82.

88 Snow, D.W. 1978. Relationships between the European and African avifaunas. Bird Study, 25: 134–148.

Sodhi, N.S., D. Bickford, A.C. Diesmos, T.M. Lee, L.P. Koh, B.W. Brook, C.H. Sekercioglu, and C.J.A. Bradshaw. 2008. Measuring the meltdown: Drivers of global amphibian extinction and decline. PLoS Biol, 3: e1636.

Spawls, S., K. Howell, R. Drewes, and J. Ashe, J. 2002. A Field Guide to the Reptiles of East Africa: all the reptiles of Kenya, Tanzania, Uganda, Rwanda and Burundi. Academic Press, San Diego, California. 543 p. Stamatakis, A. 2006. RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses

with thousands of taxa and mixed models. Bioinformatics, 22: 2688–2690. Stamatakis, A., P. Hoover and J. Rougemont. 2008. A Rapid Bootstrap Algorithm for the RAxML Web-Servers, to be published.

StatSoft, Inc. 2010. STATISTICA (data analysis software system), version 9.1. www.statsoft.com. Stattersfield, A.J., Crosby, M.J., Long, A.J., Wege, D.C., 1998. Endemic Bird Areas of the World: priorities for biodiversity conservation. BirdLife International Conservation series No. 7. BirdLife

International, Cambridge. Stuart, S.N., J.S. Chanson, N.A. Cox, B.E. Young, A.S.L. Rodrigues, D.L. Fischman, and R.W. Wallers. 2004. Status and trends of amphibian declines and extinctions worldwide. Science,

306: 1783–1786. Stuart, B.L., R.F. Inger, and H.K. Voris. 2006. High level of cryptic species diversity revealed by

sympatric lineages of Southeast Asian forest frogs. Biology Letters, 2: 470–474.

Swindell, S. R., and T. N. Plasterer. 1997. SEQMAN: Contig assembly. Methods Mol. Biol., 70:75–89. Swofford, D.L. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods).

Version 4.0b. Sinauer Associates, Sunderland, Massachusetts. Tallis, J.H. 1991. Plant community history. London: Chapman and Hall.

89 Tamura K, J. Dudley, M. Nei and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24:1596–1599.

Tattersfield, P. 1996. Local patterns of land snail diversity in a Kenyan rain forest. Malacologia, 38: 161–180. Taylor, B., D. Skelly, L. K. Demarchis, M. D. Slade, D. Galusha, and P. M. Rabinowitz. 2005.

Proximity to pollution sources and risk of amphibian limb formation. Environmental Health Perspectives, 113:1497–1501. Van Den Elzen, P. and Kreulen, D.A. 1979. Notes on the vocalisations of some amphibians from the Serengeti National Park, Tanzania. Bonner Zoologische Beitrage, 30: 385–403.

Vande weghe, J.P. Forests of Central Africa: Nature and Man. Protea Book House, Belgium. Vences, M. 1999. Phylogenetic studies on ranoid frogs (Amphibia: Anura) with a discussion of the origin and evolution of the vertebrate clades of Madagascar. Dissertation zur Erlangung des

Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich Wilhelms-Universität Bonn. 294 pp. Vences, M., J. Kosuch, M.O. Rödel, S. Lötters, A. Channing, F. Glaw, and W. Böhme. 2004.

Phylogeography of Ptychadena mascareniensis suggests transoceanic dispersal in a widespread African-Malagasy frog lineage. Journal of Biogeography, 31: 593–601. Vences, M., M. Thomas, R.N. Bonett, and D.R. Vieites. 2005. Deciphering amphibian diversity

through DNA barcoding: chances and challenges. Philosophical Transactions of the Royal Society B. 360: 1859–1868.

Voelker, G. 1999. Dispersal, vicariance and clocks: historical biogeography and speciation in a

cosmopolitan passerine genus (Anthus: Motacillidae). Evolution, 53: 1536–1552. Voelker, G. 2002. Systematics and historical biogeography of wagtails (Aves: Motacilla): dispersal versus vicariance revisited. Condor, 104, 725–739.

Voelker, G., S. Rohwer, D.C. Outlaw, and R.C.K. Bowie. 2009. Repeated trans-Atlantic dispersal catalyzed a global songbird radiation. Global Ecology and Biogeography, 18: 41–49.

90 Voelker, G., R.K. Outlaw, and R.C.K. Bowie. 2010. Pliocene forest dynamics as a primary driver of African bird speciation. Global Ecology and Biogeography, 19: 111–121.

Vogel, L.S. and J.H.K. Pechmann. 2010. Response of Fowler‘s Toad (Anaxyrus fowleri) to competition and hydroperiod in the presence of the invasive Coastal Plain Toad (Incilius nebuifer). Journal of Herpetology, 44: 382–389.

Vredenburg, V.T. 2004. Reversing introduced species effects: experimental removal of introduced fish leads to rapid recovery of a declining frog. PNAS, 20: 7646–7650. Waddle, J.H. 2006. Use of amphibians as ecosystem indicator species. Ph.D. Dissertation, University of Florida. 110 pp.

Wake, D. B. 1991. Declining amphibian populations. Science, 253: 860. Wake, D.B. and V.T. Vredenburg. 2008. Are we in the midst of the sixth mass extinction? A view from the world of amphibians. PNAS, 105: 11466–11473.

Wagner, P., W. Böhme, O.S.G. Pauwels, and A. Schmitz. 2009. A review of the African red-flanked skinks of the Lygosoma fernandi (Burton, 1836) species group (Squamata: Scincidae) and the role of climate change in their speciation. Zootaxa, 2050: 1–30.

Weldon, C., L.H. du Preez, A.D. Hyatt, R. Muller, and R. Speare. 2004. Origin of the amphibian chytrid fungus. Emerging Infectious Diseases, 10: 2100–2105. Welsh, H.H. and G.R. Hodgson. 2008. Amphibians as metrics of critical biological thresholds in

forested headwater streams of the Pacific Northwest, U.S.A. Freshwater Biology, 53: 1470 1488.

Welsh, H. H. J., and L. M. Ollivier. 1998. Stream amphibians as indicators of ecosystem stress:

A case study from California's redwoods. Ecological Applications, 8:1118–1132. Werdelin, L. and W.J. Sanders. 2010. Cenozoic Mammals of Africa. University of California Press, Los Angeles.

91 Werner, F. 1908 "1907". Ergebnisse der mit Subvention aus der Erbschaft Treitl unternommenen zoologischen Forschungsreise Dr. Franz Werners nach dem agyptischen Sudan und Nord-Uganda.

XII. Die Reptilien und Amphibien. Sitzungsberichte der Kaiserlichen Akademie der Wissenschaften, Mathematisch-Naturwissenschaftliche Classe, 116: 1823–1926. Whitfield, S.M., K.E. Bell, T. Philippi, M. Sasa, F. Bolaños, G. Chaves, J.M. Savage, and M.A.

Donnelly. Amphibian and reptile declines over 35 years at La Selva, Costa Rica. PNAS, 104: 8352–8356. Wiley, E.O. 1978. The evolutionary species concept reconsidered. Systematic Zoology, 27: 17 26.

Wilgenbusch J.C., D.L. Warren, and D.L. Swofford. 2004. AWTY: A system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference. http://ceb.csit.fsu.edu/awty.

Wilson, E.O. 1988. The current state of biological diversity. In: Biodiversity, E.O. Wilson (ed). National Academy Press: Washington, D.C. Wollerman, L. and H.R. Wiley. 2002. Possibilities for error during communication by neotropical frogs

in a complex acoustic environmental. Behavioral Ecology and Sociobiology, 52: 465–473. Yemane, K., C. Robert, and R. Bonnefille. 1987. Pollen and clay mineral assemblages of a late Miocene lacustrine sequence from the northwestern Ethiopian highlands. Palaeogeography,

Palaeoclimatology, Palaeoecology, 60: 123–133. Young, B.E., K.R. Lips, J.K. Reaser, R. Ibáñez, A.W. Salas, J.R. Cedeño, L.A. Coloma, S. Ron,

E. La Marca, J.R. Meyer, A. Muñoz, F. Bolaños, G. Chaves, and D. Romo. 2001. Population

declines and priorities for amphibian conservation in Latin America. Conservation Biology, 15: 1213–1223. Zwickl, D. J. 2006. Genetic algorithm approaches for the phylogenetic analysis of large

biological sequence datasets under the maximum likelihood criterion. Ph.D. dissertation, The University of Texas at Austin.

92 Appendices

APPENDIX A: LOCALITY INFORMATION FOR ALL SPECIMENS

Specimens used for genetic analyses: Species Origin Locality Museum Accession Number Number Hildebrandtia ornata DRC Manono ELI 359 - Hildebrandtia ornata DRC Manono ELI 361 - Phrynobatrachus acutirostris DRC Irangi EBG 1314 - Ptychadena pumilio Guinea Niger River, Somoria EBG 784 - Ptychadena aff. bibroni DRC Ngalula EBG 2462 - Ptychadena aff. mascareniensis '1' DRC Kayumega stream, EBG 1127 - Mbayo Ptychadena aff. mascareniensis '1' DRC Lwiro EBG 1163 - Ptychadena aff. mascareniensis '1' DRC Lwiro EBG 1384 - Ptychadena aff. mascareniensis '1' DRC Lwiro EBG 1386 - Ptychadena aff. mascareniensis '1' DRC Lwiro EBG 1416 - Ptychadena aff. mascareniensis '1' DRC Mulongwe, Uvira EBG 1566 - Ptychadena aff. mascareniensis '1' DRC Mulongwe, Uvira EBG 1581 - Ptychadena aff. mascareniensis '1' DRC Mulongwe, Uvira EBG 1582 - Ptychadena aff. mascareniensis '1' DRC Kamanyola River, EBG 1931 - Luvungi Ptychadena aff. mascareniensis '1' DRC Kamanyola EBG 1977 - Ptychadena aff. mascareniensis '1' DRC Kamanyola EBG 1978 - Ptychadena aff. mascareniensis '1' DRC Luvuba River, Luvungi EBG 1983 - Ptychadena aff. mascareniensis '1' DRC Nundu, Sangya River EBG 2149 - Ptychadena aff. mascareniensis '1' DRC Fizi EBG 2157 - Ptychadena aff. mascareniensis '1' DRC Fizi EBG 2158 - Ptychadena aff. mascareniensis '1' DRC Nyakasaza pond, EBG 2833 - Tshibati Ptychadena aff. mascareniensis '1' DRC Pueto EBG 3004 - Ptychadena aff. mascareniensis '1' DRC Pueto ELI 007 - Ptychadena aff. mascareniensis '1' DRC Pueto ELI 009 - Ptychadena aff. mascareniensis '1' DRC Lukanga River ELI 052 - Ptychadena aff. mascareniensis '1' DRC Kabongo ELI 111 - Ptychadena aff. mascareniensis '1' DRC Kyolo ELI 254 - Ptychadena aff. mascareniensis '1' DRC Kyolo ELI 255 - Ptychadena aff. mascareniensis '1' Rwanda Mukungwe Waypoint UTA - A58247 Ptychadena aff. mascareniensis '1' Rwanda Karisoke Waypoint: UTA - Northern Province A58254 Ptychadena aff. mascareniensis '2' DRC Ndjuma, Virunga EBG 1768 - Ptychadena aff. mascareniensis '2' DRC Ndjuma, Virunga EBG 1769 - Ptychadena aff. mascareniensis '2' DRC Kamango EBG 1860 -

93 Ptychadena aff. mascareniensis '2' DRC Idohu EBG 2307 - Ptychadena aff. mascareniensis '2' DRC Mafifi EBG 2313 - Ptychadena aff. mascareniensis '2' DRC Bunia EBG 2331 - Ptychadena aff. mascareniensis '2' DRC Bunia EBG 2334 - Ptychadena aff. mascareniensis '2' DRC Aboro EBG 2353/UT-EP 20195 Ptychadena aff. mascareniensis '2' DRC Aboro EBG 2359 - Ptychadena aff. mascareniensis '2' DRC Aboro EBG 2382 - Ptychadena aff. mascareniensis '2' DRC Mt. Aboro EBG 2426 - Ptychadena aff. mascareniensis '2' DRC Ituri River EBG 2448 - Ptychadena aff. mascareniensis '2' DRC Ituri River EBG 2454 - Ptychadena aff. mascareniensis '2' DRC Mambasa EBG 2479 - Ptychadena aff. mascareniensis '2' DRC Epulu EBG 2510 - Ptychadena aff. mascareniensis '2' DRC Epulu EBG 2511 - Ptychadena aff. mascareniensis '2' DRC Epulu EBG 2545 - Ptychadena aff. mascareniensis '2' DRC Epulu EBG 2583 - Ptychadena aff. mascareniensis '3' DRC Bitale EBG 1493 - Ptychadena aff. mascareniensis '3' DRC Bitale EBG 1495 - Ptychadena aff. mascareniensis '3' DRC Mwenga EBG 1541 - Ptychadena anchietae DRC Lwiro EBG 1385 - Ptychadena anchietae DRC Ndjuma, Virunga EBG 1785 - Ptychadena bibroni DRC Nengode, Epulu EBG 2569 - Ptychadena bibroni DRC Bazinga EBG 2593 - Ptychadena bibroni Guinea Motel du Port, Conakry EBG 671 - Ptychadena bibroni Gabon Estuaire Province, Monts NCSM - de Cristal National Park, 76852 Kinguele Ptychadena bibroni Gabon Estuaire Province, Monts NCSM - de Cristal National Park, 76853 Kinguele Ptychadena bibroni Gabon Estuaire Province, Monts NCSM - de Cristal National Park, 76854 Kinguele Ptychadena bibroni Gabon Woleu-Ntem Province, NCSM - Monts de Cristal 76855 National Park, Tchimbele Ptychadena bibroni Gabon Ogooue-Ivingo Province, NCSM - Ivindo National Park, 76856 Ipassa Station Ptychadena c.f. tellinii DRC Mulihi Village, Mt Teye EBG 1902 - Ptychadena christyi DRC Ndjuma, Virunga EBG 1787 - Ptychadena grandisonae DRC Kakunko village, Kiboro ELI 224 - Mtns Ptychadena guibei DRC Manono ELI 362 - Ptychadena oxyrhynchus DRC Kalemie EBG 2873 - Ptychadena oxyrhynchus DRC Kalemie EBG 2874 -

94 Ptychadena oxyrhynchus DRC Kalemie EBG 2875 - Ptychadena oxyrhynchus Guinea No specific locality EBG 763 - Ptychadena oxyrhynchus DRC Kabongo ELI 109 - Ptychadena oxyrhynchus DRC Kabongo ELI 110 - Ptychadena oxyrhynchus DRC Kakunko village, Kiboro ELI 228 - Mtns Ptychadena porosissima DRC Kakunko village, Kiboro ELI 219 - Mtns Ptychadena porosissima Rwanda Musanza District, UTA - Mukungwe River A58822 Ptychadena sp. DRC Manono ELI 374 - Ptychadena sp. DRC Kamango EBG 1861 -

Specimens used from Vences et al. (2004) P. mascareniensis Réunion St. Etienne River ZSM AY517587 1007/2000 P. mascareniensis Réunion St. Etienne River ZSM AY517587 1006/2000 P. mascareniensis Mauritius Pereybere ZSM AY517589 984/2000 P. mascareniensis Mauritius Cascade Chamarel ZSM AY517589 973/2000 P. mascareniensis Seychelles Praslin ZFMK 62876 AY517589 P. mascareniensis Madagascar Antsiranana ZSM 504– AY517594 506/2000 P. mascareniensis Madagascar Sambava ZSM AY517594 562/2000 P. mascareniensis Madagascar Nosy Be UADBA- AY517592 FG/MV 2001.02 P. mascareniensis Madagascar Ambanja ZSM AY517591 421/2000 P. mascareniensis Madagascar Torotorofotsy Voucher not AY517590 collected P. mascareniensis Madagascar Fierenana ZSM AY517590 252/2002 P. mascareniensis Madagascar Ambohimanarivo UADBA-MV AY517590 2001.1111 P. mascareniensis Madagascar Ambohimanarivo UADBA-MV AY517590 2001.1109 P. mascareniensis Madagascar Moramanga ZFMK 66683 AY517590 P. mascareniensis Madagascar Andasibe ZFMK 52754 AY517590 P. mascareniensis Madagascar Antananarivo ZSM AY517593 432/2000 P. mascareniensis Madagascar Mantasoa UADBA- AY517593 FG/MV 2000.04 P. mascareniensis Madagascar Ambatolampy Voucher not AY517593 collected

95 P. mascareniensis Madagascar Ambatolampy Voucher not AY517593 collected P. mascareniensis Madagascar Ambatolampy Voucher not AY517593 collected P. mascareniensis Madagascar Tolagnaro Voucher not AY517587 collected P. mascareniensis Madagascar Nahampoana ZSM AY517587 190/2002 P. mascareniensis Madagascar Ankarafantsika ZSM AY517587 702/2001 P. mascareniensis Madagascar Andohariana UADBA-MV AY517587 (Andringitra) 2001.531 P. mascareniensis Madagascar Andohariana ZSM AY517588 (Andringitra) 717/2001 P. aff. mascareniensis 'A' Tanzania Kibebe Farm, Iringa AC 1728 AY517595 P. aff. mascareniensis 'A' Tanzania Ihafu, Usangu Swamp, N AC 1824 AY517595 Mbeya P. aff. mascareniensis 'A' Egypt probably Rashid Voucher not AY517596 collected P. aff. mascareniensis 'A' Egypt Rashid Voucher not AY517596 collected P. aff. mascareniensis 'A' Egypt Esna Voucher not AY517596 collected P. aff. mascareniensis 'A' Egypt Gabala, Fayoum ZFMK AY517596 77757–758 P. aff. mascareniensis 'A' Kenya Runda-Gigiri NMK/A/3842 AY517596 P. aff. mascareniensis 'A' Kenya Mount Elgon NMK/A3843/ AY517596 1 P. aff. mascareniensis 'A' Kenya Aberdares Salient NMK AY517596 A/3844/7 P. aff. mascareniensis 'A' Kenya Aberdares Salient NMK AY517596 A/3840/2 P. aff. mascareniensis 'A' Kenya Aberdares Salient NMK AY517596 A/3844/3 P. aff. mascareniensis 'A' Kenya Aberdares Salient NMK/A3841/ AY517596 2 P. aff. mascareniensis 'A' Kenya Aberdares Salient NMK/A3844/ AY517596 6 P. aff. mascareniensis 'A' Kenya Aberdares Salient NMK/A3841/ AY517596 1 P. aff. mascareniensis 'B' Cameroon unknown ZFMK 68826 AF215408 P. aff. mascareniensis 'B' Benin Lama forest ZFMK 77100 AY517597 P. aff. mascareniensis 'C' Ivory Coast Mont Sangbé National MOR S01.40 AY517598 Park P. aff. mascareniensis 'D' Kenya Kakamega Forest NMK AY517599 A/3840/5 P. aff. mascareniensis 'D' Kenya Kakamega Forest NMK AY517599 A/3840/1 P. aequiplicata Ivory Coast Taї National Park MOR T01.44 AY517618 P. aequiplicata Ivory Coast Taї National Park MOR T01.3 AY517617

96 P. aequiplicata Ivory Coast Taї National Park MOR T01.19 AY517616 P. aff. aequiplicata Ivory Coast Marahoué National Park, Voucher not AY517614 forest collected P. aff. aequiplicata Ivory Coast Mont Sangbé National Voucher not AY517615 Park collected P. aff. aequiplicata Ghana Wli Waterfalls MOR G56 AY517613 P. aff. aequiplicata Benin Lama forest ZFMK 77098 AY517613 P. aff. aequiplicata Benin Lama forest ZFMK 77104 AY517613 P. anchietae South Africa Mtunzini Voucher not AF215404 collected P. anchietae South Africa St. Lucia Voucher not AF215405 collected P. anchietae Kenya Marich Field Study ZFMK 70824 — Center P. anchietae Kenya Runda-Gigiri SL coll. AY517612 (unnumbered)

Specimens used from Measey et al. (2007) Ptychadena aff. Cameroon Dja Reserve CAS 199182 DQ525919 aequiplicata Ptychadena anchietae Kenya Kararacha Pond CAS 214837 DQ525920 Ptychadena anchietae Somalia Karin, Bari Region CAS 227562 DQ525921 Ptychadena anchietae Somalia Karin, Bari Region CAS 227507 DQ525922 Ptychadena anchietae South Africa Mtunzini No voucher AF215404 Ptychadena mahnerti Kenya Mt. Kenya SL 171 DQ525918 Ptychadena mascareniensis Madagascar Nahampoana ZSM AY517587 190/2002 Ptychadena aff. Uganda Lake Victoria MVZ 234085 DQ525923 mascareniensis 'A' Ptychadena aff. Kenya Makuru MVZ 223624 DQ525924 mascareniensis 'A' Ptychadena aff. Kenya Mt. Kenya MVZ 234087 DQ525925 mascareniensis 'A' Ptychadena aff. Kenya Mt. Kenya MVZ 234086 DQ525926 mascareniensis 'A' Ptychadena aff. Kenya Taita Hills CAS 191517 DQ525927 mascareniensis 'A' Ptychadena aff. Kenya Taita Hills CAS 191518 DQ525928 mascareniensis 'A' Ptychadena aff. Tanzania Kibebe Farm AC 2087 DQ525929 mascareniensis 'A' Ptychadena aff. Guinea No precise locality gu03.2 DQ525930 mascareniensis 'C' Ptychadena aff. Uganda Kampala MVZ 234084 DQ525931 mascareniensis 'D' Ptychadena aff. Central African Dzanga-Sangha Reserve MOR DS 52 DQ525932 mascareniensis 'E' Republic Ptychadena newtoni São Tomé São Tomé CAS 219249 DQ525933 Ptychadena newtoni São Tomé São Tomé CAS 219250 DQ525934

97 Ptychadena newtoni São Tomé São Tomé CAS 219251 DQ525935 Ptychadena newtoni São Tomé São Tomé CAS 219252 DQ525936 Ptychadena newtoni São Tomé São Tomé CAS 219253 DQ525937 Ptychadena newtoni São Tomé São Tomé CAS 219263 DQ525938 Ptychadena oxyrhynchus Malawai No precise locality 359 (6 DQ525939 specimens) Ptychadena oxyrhynchus South Africa Mtunzini No voucher – Ptychadena oxyrhynchus South Africa Kwambonambi No voucher AF215403 Ptychadena aff. porosissima Tanzania Tatanda AC 2034 DQ525940 'A' Ptychadena porosissima Tanzania Mumba AC 2122 DQ525941 Ptychadena c.f. pumilio Guinea Mont Béro MOR Gu 212 DQ525942 Ptychadena taenioscelis Kenya Kakamega Forest NMKA DQ525943 3955–1 Ptychadena aff. Tanzania Njombe AC 1970 DQ525945 uzungwensis Ptychadena sp. Tanzania Mikumi AC 1976 DQ525944

Specimens used from Vences (1999) Ptychadena oxyrhynchus Namibia Rundu - AF215409 Ptychadena subpunctata Namibia Rundu - AF215410 Ptychadena porosissima South Africa Kwambonambi - AF215411 Ptychadena mascareniensis Madagascar No precise locality ZFMK 52754 AF215406 Ptychadena mascareniensis Madagascar Moramanga ZFMK 66683 AF215407 Hildebrandtia ornata Ivory Coast Little Brak - AF215402

98 APPENDIX B: MORPHOMETRIC AND MERISTIC CHARACTERS USED FOR MORPHOLOGICAL ANALYSES

Morphometric characters: SVL Snout–vent length – total length of the animal from tip of snout to end of vent

FL Foot length –proximal edge of the heel to the tip of Toe IV when foot is bent at the ankle TL Tibia length – measured from top of knee to bottom of heel when leg is bent at the knee HW Head width – width of the head measured across the center of tympanum

HL Head length – measured from the angle of jaw (end of mandible) to the tip of the snout SN Snout–nostril distance – tip of snout to center of nostril, measured using microscope EN Eye–nostril distance – from anterior corner of eye to center of nostril

TD Tympanum diameter – measured horizontally at the widest point, surround line excluded ED Eye diameter – diameter of the eyeball only, measured horizontally ID Internarial distance – distance separating the nostrils, measured from center to center

TER Tympanum-eye ratio – diameter of tympanum/diameter of eye FL1 Length of finger 1 – finger lengths measured from the wrist FL2 Length of finger 2

FL3 Length of finger 3 FL4 Length of finger 4 FL5 Length of finger 5

TL1 Length of toe 1 – toe lengths measured from the base of digits TL3 Length of toe 2 TL3 Length of toe 3

TL4 Length of toe 4 TL5 Length of toe 5

99 Meristic Characters: (1) Gular pattern

(2) Ridges on legs (3) Dorsolateral folds (4) Dorsal ridges

(5) Outermost dorsal ridge whitish (6) Warts on legs (7) Warts on lateral sides (8) Light tibial line

(9) Light bands on posterior of femur (10) Dark bands on posterior of femur (11) Dark mottling on posterior of thigh

(12) Pale triangle on snout (13) Whitish spots on lower lip (14) Whitish ring around tympanum

(15) Light colored vertebral band (16) Palmar tubercle (17) Accessory palmar tubercles

(18) Thenar tubercles (19) Supernumerary tubercles

(20) Outer metatarsal tubercle

(21) Inner metatarsal tubercle (22) Inner tarsal fold of foot (23) Outer tarsal fold of foot

(24) Plantar tubercle of foot (25) Supernumerary tubercles of foot (26) Row of tubercles on metatarsus

100 (27) Perforations on toe II (28) Perforations on toe III

(29) Number subarticular tubercles on all fingers (30) Total number of dorsal ridges (31) Number of short ridges

(32) Number of tubercles on Toe IV (33) Number of subarticular tubercles on all toes (34) # phalanges free toe I (35) # phalanges free external toe II

(36) # phalanges free internal toe II (37) # phalanges free external toe III (38) # phalanges free internal toe III

(39) # phalanges free external toe IV (40) # phalanges free internal toe IV (41) # phalanges free toe V

101 APPENDIX C: COMPLETE MEASUREMENT DATA FOR ALL PTYCHADENA SPECIMENS

Character abbreviations follow Appendix B. An asterisk indicates a specimen used in morphological analyses.

Morphometric Characters

P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis Clade A* Clade A* Clade A* Clade A* Clade A* Clade A* EBG 1977 EBG 1127 EBG 1980 EBG 1978 EBG1983 EBG 1566 SVL 58.50 53.20 53.50 46.50 61.30 49.50 FL 47.20 48.50 44.20 37.50 53.20 43.40 TL 38.40 33.00 30.40 25.30 35.40 29.20 HW 17.40 16.10 18.00 14.60 19.40 16.50 HL 20.40 18.50 21.60 15.30 22.20 20.30 SN 4.20 4.30 4.10 3.30 4.30 4.30 SN/EN 1.29 1.21 1.32 1.55 1.42 1.09 EN 5.20 5.40 5.20 4.10 5.30 4.50 TD 4.40 4.20 4.20 4.20 5.30 4.40 ED 5.40 5.20 5.40 5.10 6.10 4.70 ID 4.50 4.40 4.40 3.30 4.20 3.50 FL1 7.20 8.20 5.50 6.20 10.20 7.30 FL2 6.70 7.40 6.20 5.30 11.10 8.60 FL3 9.10 9.80 8.50 7.40 14.60 11.50 FL4 7.10 6.40 6.20 5.20 12.40 9.30 TL1 4.00 4.00 6.00 6.00 6.00 6.00 TL3 8.00 8.00 8.00 8.00 8.00 8.00 TL3 2.00 2.00 2.00 2.00 2.00 2.00 TL4 3.00 3.00 3.00 3.00 3.00 3.00 TL5 7.00 8.00 9.00 9.00 9.00 9.00 T4L/FL 1.00 1.50 1.50 1.00 1.00 1.00 TL/FL 2.00 2.00 2.00 2.00 2.00 2.00 HW/HL 1.50 1.50 1.00 1.00 1.00 1.00 SN/EN 2.50 2.50 2.25 2.75 2.75 2.50 TD/ED 1.50 1.50 1.50 2.00 1.50 1.50

102

P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis Clade A* Clade A* Clade A* Clade A* Clade A* Clade A* ELI 254 EBG 2833 ELI 009 EBG 1386 EBG 2149 EBG 2834 SVL 50.10 53.40 57.40 50.30 47.40 57.30 FL 33.80 43.20 45.80 48.10 46.10 46.40 TL 26.50 31.40 30.80 33.40 29.40 32.20 HW 14.20 16.30 17.50 16.20 15.40 18.20 HL 18.40 17.40 21.40 19.40 18.40 20.30 SN 4.20 4.20 4.40 4.20 4.10 4.40 SN/EN 1.05 1.21 1.23 1.07 1.10 1.20 EN 4.30 4.30 5.20 4.40 4.30 4.90 TD 4.10 4.30 4.40 4.20 4.30 4.80 ED 4.40 5.10 5.40 4.50 4.50 5.30 ID 3.60 4.10 4.00 3.40 3.50 4.10 FL1 7.40 8.30 8.10 8.30 7.40 8.30 FL2 8.40 8.30 8.50 9.10 7.90 9.20 FL3 11.20 11.20 12.40 12.50 12.10 12.10 FL4 9.30 9.30 10.40 9.50 9.30 10.30 TL1 5.60 5.40 6.40 6.10 6.10 7.10 TL3 11.20 11.40 12.40 11.40 11.30 12.30 TL3 16.80 18.60 20.30 19.10 19.30 20.30 TL4 24.30 26.20 28.20 29.40 28.40 28.20 TL5 18.20 18.30 18.40 19.80 19.40 20.20 T4L/FL 0.72 0.61 0.62 0.61 0.62 0.61 TL/FL 0.78 0.73 0.67 0.69 0.64 0.69 HW/HL 0.77 0.94 0.82 0.84 0.84 0.90 SN/EN 0.98 0.98 0.85 0.95 0.95 0.90 TD/ED 0.93 0.84 0.81 0.93 0.96 0.91

103

P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis Clade C* Clade C* Clade C* Clade C* Clade C* Clade C* EBG 2313 EBG 2448 EBG2426 EBG 2307 EBG 2583 EBG 2512 SVL 59.40 60.30 63.80 61.50 59.40 61.20 FL 51.40 53.20 55.60 52.60 52.20 56.30 TL 35.30 36.20 39.10 37.30 36.40 38.60 HW 16.40 16.30 12.50 18.50 18.20 18.40 HL 19.20 22.20 22.70 22.50 22.50 21.20 SN 4.50 4.70 4.40 4.30 4.90 4.40 SN/EN 1.20 1.26 1.23 1.23 1.12 1.25 EN 5.30 5.40 5.20 5.30 5.30 5.30 TD 4.70 5.10 4.70 4.70 5.00 5.10 ED 5.40 5.90 5.40 5.30 5.50 5.50 ID 4.20 4.60 4.60 4.50 4.50 4.60 FL1 7.10 8.80 9.20 8.80 9.20 10.10 FL2 6.40 10.20 10.20 10.10 9.30 10.30 FL3 9.60 14.10 14.30 13.80 13.20 13.60 FL4 6.90 11.10 11.10 11.00 10.60 11.90 TL1 6.30 7.20 7.10 7.20 7.30 7.50 TL3 13.20 15.30 14.30 13.60 13.80 14.50 TL3 20.40 23.20 23.20 22.80 22.30 23.20 TL4 31.50 32.30 34.10 31.50 31.80 34.40 TL5 20.30 23.30 24.30 22.40 22.30 24.80 T4L/FL 0.61 0.61 0.61 0.60 0.61 0.61 TL/FL 0.69 0.68 0.70 0.71 0.70 0.69 HW/HL 0.85 0.73 0.55 0.82 0.81 0.87 SN/EN 0.85 0.87 0.85 0.81 0.92 0.83 TD/ED 0.87 0.86 0.87 0.89 0.91 0.93

104

P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis Clade C* Clade C* Clade C* Clade C* Clade D* Clade E* EBG 1770 EBG 1181 EBG 2333 EBG 1179 EBG 1493 CNHM 79902 SVL 53.40 55.20 51.50 49.10 52.30 46.20 FL 45.80 49.40 42.30 44.40 51.30 40.80 TL 31.80 33.80 30.40 31.30 34.20 25.40 HW 15.60 18.10 15.80 16.40 16.40 15.30 HL 19.60 20.20 19.30 19.00 20.70 16.80 SN 4.20 4.10 4.10 3.30 4.30 4.00 SN/EN 1.07 1.12 1.24 1.45 1.19 1.10 EN 4.50 5.30 4.50 4.40 5.20 4.20 TD 4.10 4.30 4.40 4.40 4.50 3.80 ED 4.50 4.60 5.10 4.80 5.10 4.40 ID 4.20 4.30 4.20 3.50 4.20 3.40 FL1 8.30 9.10 7.40 8.10 8.20 7.30 FL2 8.40 9.40 7.70 8.70 6.40 7.80 FL3 11.30 13.30 11.40 12.30 10.20 10.30 FL4 9.30 11.20 8.40 10.20 5.80 8.00 TL1 5.60 7.00 5.40 6.20 8.30 5.30 TL3 11.40 13.30 11.50 11.20 13.20 10.80 TL3 18.90 21.30 17.40 18.40 20.20 17.30 TL4 28.30 29.30 25.40 27.50 30.30 24.30 TL5 19.40 21.40 16.50 19.30 23.40 16.80 T4L/FL 0.62 0.59 0.60 0.62 0.59 0.60 TL/FL 0.69 0.68 0.72 0.70 0.67 0.62 HW/HL 0.80 0.90 0.82 0.86 0.79 0.91 SN/EN 0.93 0.77 0.91 0.75 0.83 0.95 TD/ED 0.91 0.93 0.86 0.92 0.88 0.86

105

P. mascareniensis R. mascareniensis R. mascareniensis P. hylaea* P. hylaea* P. hylaea* Clade E* venusta* venusta* CNHM 7 RGMAC RGMAC RGMAC RGMAC RGMAC 9881 58212 39493 7367- B-1895 7367-B-1909 116081093 SVL 42.20 57.40 52.40 63.40 54.40 56.50 FL 33.10 53.60 56.30 52.30 47.10 50.50 TL 22.20 40.90 38.40 37.40 37.30 34.30 HW 12.30 19.40 17.30 17.40 17.40 15.30 HL 15.30 23.30 22.20 21.30 20.30 20.10 SN 3.30 5.10 5.20 4.50 3.30 4.50 SN/EN 1.09 1.06 1.00 1.24 1.39 1.09 EN 3.50 6.30 5.40 5.50 5.30 4.60 TD 3.10 4.60 4.80 5.30 4.40 4.50 ED 3.60 5.40 5.20 5.60 4.60 4.90 ID 2.90 4.50 4.60 4.40 4.30 4.20 FL1 5.80 9.30 9.50 9.10 8.90 8.10 FL2 6.30 9.60 10.30 9.30 9.20 8.30 FL3 8.50 14.40 13.40 18.20 14.40 12.60 FL4 5.40 11.40 11.40 11.30 11.20 10.30 TL1 4.40 7.20 5.40 6.30 6.80 6.30 TL3 8.40 13.40 14.20 13.40 12.40 12.40 TL3 13.30 22.10 22.30 21.90 21.10 20.20 TL4 19.60 33.50 32.30 32.50 29.40 30.30 TL5 13.50 24.20 23.30 22.40 21.80 21.30 T4L/FL 0.59 0.63 0.57 0.62 0.62 0.60 TL/FL 0.67 0.76 0.68 0.72 0.79 0.68 HW/HL 0.80 0.83 0.78 0.82 0.86 0.76 SN/EN 0.94 0.81 0.96 0.82 0.62 0.98 TD/ED 0.86 0.85 0.92 0.95 0.96 0.92

106

P. hylaea* P. hylaea* P. hylaea* P. hylaea* P. hylaea* P. aff. mascareniensis RGMAC RGMAC RGMAC FMNH FMNH 12232 RGMAC 104970-975 104970-975 104970-975 12232(1) (2) 116157-166 SVL 61.60 66.80 61.40 56.10 58.40 50.60 FL 57.20 56.20 57.50 51.20 48.30 47.10 TL 40.40 41.10 39.50 36.10 33.20 32.20 HW 19.40 20.10 19.30 15.80 15.30 12.80 HL 24.50 25.30 23.30 21.40 19.10 19.10 SN 5.30 5.10 4.90 4.60 4.40 4.30 SN/EN 1.13 1.04 1.10 1.11 1.20 1.00 EN 5.50 5.30 5.40 5.30 5.10 4.40 TD 5.30 5.30 5.20 4.30 4.40 4.30 ED 6.00 5.30 5.40 5.10 5.30 4.30 ID 5.20 5.30 5.20 4.30 3.90 3.80 FL1 10.30 9.10 10.20 9.20 8.20 7.30 FL2 10.50 9.90 10.30 9.40 8.40 7.50 FL3 15.20 14.40 15.30 13.20 11.50 11.40 FL4 12.60 11.90 12.80 10.30 8.80 9.30 TL1 7.50 7.50 8.10 6.20 6.20 5.30 TL3 15.10 14.40 15.20 15.10 12.20 11.50 TL3 23.50 23.80 24.40 21.30 20.60 19.30 TL4 33.30 33.30 34.40 32.20 31.10 29.30 TL5 24.20 23.50 25.40 23.20 21.20 20.30 T4L/FL 0.58 0.59 0.60 0.63 0.64 0.62 TL/FL 0.71 0.73 0.69 0.71 0.69 0.68 HW/HL 0.79 0.79 0.83 0.74 0.80 0.67 SN/EN 0.96 0.96 0.91 0.87 0.86 0.98 TD/ED 0.88 1.00 0.96 0.84 0.83 1.00

107

P. aff. P. aff. P. aff. P. aff. P. aff. P. aff. mascareniensis mascareniensis mascareniensis mascareniensis mascareniensis mascareniensis RGMAC RGMAC RGMAC RGMAC RGMAC RGMAC 67447-465 61228 67646-650 4707-4708 4707-4708 814-816 SVL 50.50 57.30 54.40 49.20 52.30 65.40 FL 40.30 52.40 43.40 42.30 45.80 52.40 TL 22.60 37.30 32.30 29.30 34.20 39.30 HW 14.40 16.60 15.30 14.60 15.60 17.40 HL 18.30 20.60 20.20 18.30 18.40 24.40 SN 3.60 4.30 4.30 3.60 3.40 5.30 SN/EN 1.22 1.23 1.02 1.25 1.26 1.00 EN 4.30 5.30 4.20 5.30 4.40 6.30 TD 4.20 4.20 4.80 4.10 4.30 4.30 ED 4.40 5.30 4.40 4.50 4.30 5.30 ID 3.60 4.50 4.20 4.30 3.40 5.30 FL1 8.30 8.80 8.40 6.60 8.30 9.40 FL2 8.60 9.30 9.30 7.50 8.80 10.20 FL3 12.20 12.30 12.30 11.20 11.30 14.20 FL4 10.40 10.30 10.40 9.30 10.30 10.80 TL1 6.40 5.60 6.80 6.10 6.40 5.80 TL3 11.40 16.40 12.80 10.40 11.50 13.50 TL3 19.20 22.40 19.60 18.30 18.60 22.40 TL4 25.30 32.30 27.80 26.30 29.20 33.50 TL5 18.80 22.40 20.30 18.30 20.30 23.30 T4L/FL 0.63 0.62 0.64 0.62 0.64 0.64 TL/FL 0.56 0.71 0.74 0.69 0.75 0.75 HW/HL 0.79 0.81 0.76 0.80 0.85 0.71 SN/EN 0.84 0.81 1.02 0.68 0.77 0.84 TD/ED 0.95 0.79 1.09 0.91 1.00 0.81

108

P. aff. P. aff. P. aff. P. aff. P. aff. P. aff. mascareniensis mascareniensis mascareniensis mascareniensis mascareniensis mascareniensis RGMAC RGMAC RGMAC RGMAC RGMAC RGMAC 116892902 67466-484 1224-1225 734-737 103923924 4707-4708 SVL 62.30 45.50 39.20 55.10 58.20 49.30 FL 54.40 38.20 35.10 46.80 50.30 42.30 TL 36.80 26.50 24.40 29.90 36.20 29.90 HW 17.60 13.20 12.30 17.40 17.40 14.60 HL 21.80 15.80 16.20 20.30 21.10 18.30 SN 4.50 3.30 3.20 4.40 4.30 3.40 SN/EN 1.13 1.30 1.13 1.23 1.02 1.32 EN 5.30 3.40 4.10 4.40 4.40 4.60 TD 4.80 3.50 3.10 4.40 4.30 4.00 ED 5.10 4.30 3.60 5.40 4.40 4.50 ID 4.60 3.20 3.50 4.50 4.40 3.50 FL1 9.30 7.40 5.30 8.30 9.20 7.10 FL2 10.30 7.60 6.30 8.60 9.60 8.30 FL3 14.10 11.30 9.20 11.30 13.20 11.30 FL4 11.60 9.20 7.30 9.40 11.60 9.30 TL1 7.50 6.20 4.20 7.30 7.10 5.30 TL3 14.30 10.40 8.30 12.40 13.20 10.40 TL3 23.10 16.40 13.60 20.60 21.40 15.40 TL4 34.10 24.30 21.60 29.30 32.10 26.30 TL5 23.50 17.30 13.30 20.80 22.40 18.30 T4L/FL 0.63 0.64 0.62 0.63 0.64 0.62 TL/FL 0.68 0.69 0.70 0.64 0.72 0.71 HW/HL 0.81 0.84 0.76 0.86 0.82 0.80 SN/EN 0.85 0.97 0.78 1.00 0.98 0.74 TD/ED 0.94 0.81 0.86 0.81 0.98 0.89

109

P. aff. P. aff. P. aff. P. aff. bibroni P. aff. bibroni P. aff. bibroni mascareniensis mascareniensis mascareniensis RGMAC 2257- RGMAC RGMAC RGMAC RGMAC RGMAC 2258 41.885-898 116577-578 1728 34520 2380-82 SVL 55.30 55.30 55.20 55.40 57.40 56.50 FL 48.60 45.30 46.20 50.50 56.30 50.30 TL 34.30 33.40 33.30 35.40 43.50 35.40 HW 19.30 16.20 16.30 16.50 19.80 18.10 HL 20.50 18.90 19.40 19.90 21.40 19.50 SN 20.40 4.30 4.10 4.10 5.30 4.20 SN/EN 0.24 1.02 1.12 1.27 0.96 1.24 EN 4.20 4.80 5.20 5.10 5.30 5.00 TD 4.40 4.30 4.40 4.40 4.40 4.40 ED 4.80 4.40 4.60 5.20 5.10 5.20 ID 4.20 4.10 4.30 4.30 4.50 4.10 FL1 8.20 7.40 7.40 9.20 9.10 8.20 FL2 9.10 8.50 8.40 9.30 10.30 9.40 FL3 11.30 12.40 11.40 12.20 14.20 11.50 FL4 9.40 10.60 10.30 11.10 10.50 9.60 TL1 6.20 5.60 6.40 6.40 6.60 6.30 TL3 12.40 12.30 11.80 12.50 12.60 12.20 TL3 19.80 19.40 19.80 20.10 23.10 20.40 TL4 29.30 28.40 29.30 32.20 31.10 31.10 TL5 20.20 16.60 19.40 21.70 24.40 20.40 T4L/FL 0.60 0.63 0.63 0.64 0.55 0.62 TL/FL 0.71 0.74 0.72 0.70 0.77 0.70 HW/HL 0.94 0.86 0.84 0.83 0.93 0.93 SN/EN 4.86 0.90 0.79 0.80 1.00 0.84 TD/ED 0.92 0.98 0.96 0.85 0.86 0.85

110

P. aff. bibroni P. aff. bibroni P. obscura P. obscura P. chrysogaster P. chrysogaster RGMAC RGMAC RGMAC RGMAC EBG 2462 EBG 2569 68696-702 68732 109096 42016-017 SVL 57.80 55.40 43.30 44.60 49.40 48.20 FL 51.40 49.70 33.50 36.10 46.20 46.80 TL 48.90 36.20 26.40 28.50 36.30 32.80 HW 18.40 16.90 13.40 13.60 15.50 15.50 HL 21.30 18.30 14.50 16.90 18.10 17.30 SN 4.70 5.20 3.10 4.10 4.20 3.30 SN/EN 1.32 1.12 1.39 1.05 1.05 1.27 EN 5.30 6.40 4.10 4.40 4.10 4.10 TD 5.20 4.40 3.50 3.30 4.40 4.20 ED 6.20 5.80 4.30 4.30 4.40 4.20 ID 5.20 5.10 3.60 4.10 4.40 4.00 FL1 7.20 7.80 6.10 5.40 8.10 8.30 FL2 5.60 6.70 6.10 6.50 8.80 8.40 FL3 9.20 10.20 9.10 9.30 12.20 11.60 FL4 6.20 7.40 7.60 7.30 9.30 9.30 TL1 7.30 7.40 4.10 4.30 6.40 5.30 TL3 13.40 12.40 8.30 9.30 11.50 11.30 TL3 20.40 20.30 13.30 15.30 19.40 20.20 TL4 32.50 29.40 19.30 20.80 29.20 29.40 TL5 22.50 22.30 13.20 15.30 21.80 19.60 T4L/FL 0.63 0.59 0.58 0.58 0.63 0.63 TL/FL 0.95 0.73 0.79 0.79 0.79 0.70 HW/HL 0.86 0.92 0.92 0.80 0.86 0.90 SN/EN 0.89 0.81 0.76 0.93 1.02 0.80 TD/ED 0.84 0.76 0.81 0.77 1.00 1.00

111 Meristic Characters Numbers correspond to numbered characters in Appendix B. For characters 1–28, a 1 indicates absence and a 2 indicates presence of the character.

P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis Clade A* Clade A* Clade A* Clade A* Clade A* Clade A* EBG 1977 EBG 1127 EBG 1980 EBG 1978 EBG1983 EBG 1566 1 2 2 2 2 2 2 2 1 1 1 1 1 1 3 1 1 1 1 1 2 4 2 2 2 2 2 2 5 2 2 2 2 2 2 6 1 1 1 1 1 1 7 1 2 1 1 1 1 8 2 2 1 2 2 2 9 1 1 1 2 2 2 10 1 1 1 2 2 2 11 2 2 2 1 2 2 12 1 1 1 1 1 1 13 1 2 1 2 2 2 14 2 1 2 2 1 1 15 2 2 1 2 2 1 16 2 2 2 2 2 2 17 1 1 1 1 1 1 18 2 2 2 2 2 2 19 1 1 1 1 1 1 20 1 1 1 1 1 1 21 2 2 2 2 2 2 22 2 2 2 2 2 2 23 1 1 2 1 1 1 24 1 1 1 1 1 1 25 1 1 1 1 1 1 26 1 1 1 1 1 1 27 1 1 1 1 1 1 28 1 1 1 1 1 1 29 4 4 6 6 6 6 30 8 8 8 8 8 8 31 2 2 2 2 2 2 32 3 3 3 3 3 3 33 7 8 9 9 9 9 34 1 1.5 1.5 1 1 1 35 2 2 2 2 2 2 36 1.5 1.5 1 1 1 1 37 2.5 2.5 2.25 2.75 2.75 2.5 38 1.5 1.5 1.5 2 1.5 1.5 39 3 3 3 3 3 3 40 2.75 2.5 2.75 2.75 2.5 2.5 41 1 1.25 1 1 1 1

112

P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis Clade A* Clade A* Clade A* Clade A* Clade C* Clade C* ELI 009 EBG 1386 EBG 2149 EBG 2834 EBG 2313 EBG 2448 1 2 2 2 2 1 1 2 1 1 1 1 1 1 3 1 2 1 1 1 1 4 2 2 2 2 2 2 5 2 2 2 1 2 2 6 1 1 1 1 1 1 7 1 1 1 1 1 1 8 1 1 2 1 2 2 9 2 2 2 2 2 2 10 2 2 2 2 2 2 11 1 1 1 1 2 2 12 1 1 1 1 1 1 13 2 2 2 2 2 2 14 1 1 2 1 1 2 15 1 2 2 1 2 2 16 2 2 2 2 2 2 17 1 1 1 1 1 1 18 2 2 2 2 2 2 19 1 1 1 1 1 1 20 1 1 1 1 1 1 21 2 2 2 2 2 2 22 2 2 2 2 2 2 23 1 1 1 1 1 1 24 1 1 1 1 1 1 25 1 1 1 1 1 1 26 1 1 1 1 1 1 27 1 1 1 1 1 1 28 1 1 1 1 1 1 29 6 6 6 6 5 5 30 8 8 8 8 8 8 31 2 2 2 2 1 2 32 3 3 3 3 3 3 33 9 9 9 9 9 9 34 1 1.5 1.5 1 1 1 35 2 2 2 2 2 2 36 1 1.25 1 1 1 1 37 2.25 3 2.75 2.5 2.25 2.25 38 1 1.75 1.5 1.5 1.5 1 39 2.5 3 3 3 3 3 40 2 2.75 2.5 2.5 2 2 41 1 1 1 1 1 1

113

P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis Clade C* Clade C* Clade C* Clade C* Clade C* Clade C* EBG2426 EBG 2307 EBG 2583 EBG 2512 EBG 1770 EBG 1181 1 2 2 2 2 2 2 2 1 1 1 1 1 1 3 1 1 1 1 1 1 4 2 2 2 2 2 2 5 2 2 2 1 2 2 6 1 2 1 1 1 1 7 1 1 1 1 1 1 8 2 2 2 1 1 2 9 2 2 2 1 2 1 10 2 2 2 1 2 1 11 1 2 1 2 1 2 12 1 1 1 1 1 1 13 2 2 2 2 2 2 14 1 1 1 1 2 1 15 2 2 2 1 1 2 16 2 2 2 2 2 2 17 1 1 1 1 1 1 18 2 2 2 2 2 2 19 1 1 1 1 1 1 20 1 1 1 1 1 1 21 2 2 2 2 2 2 22 2 2 2 2 2 2 23 1 1 1 1 1 1 24 1 1 1 1 1 1 25 1 1 1 1 1 1 26 1 1 1 1 1 1 27 1 1 1 2 1 1 28 1 1 1 2 1 1 29 5 5 6 6 6 6 30 8 8 8 8 8 8 31 2 2 2 2 2 2 32 3 3 3 3 3 3 33 9 9 9 9 9 9 34 1.5 1 1.5 1 1 1 35 2 2 2 2 2 2 36 1 1 1 1 1 1 37 2.25 2 2.5 2.5 2.5 2.5 38 1.25 1 1.5 1 1.5 1.75 39 2.5 2.5 2.75 2.5 2.75 3 40 2 2 3 2 2.25 2.75 41 1 1 1 1 1 1

114

P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis P. mascareniensis R. mascareniensis Clade C* Clade C* Clade D* Clade E* Clade E* venusta* RGMAC EBG 2333 EBG 1179 EBG 1493 CNHM 79902 CNHM 79881 58212 1 2 2 1 2 2 2 2 1 1 1 1 1 1 3 1 1 1 1 1 1 4 2 2 2 2 2 2 5 2 1 2 2 2 1 6 1 1 1 1 1 1 7 1 1 1 1 1 1 8 2 2 1 1 2 2 9 1 2 2 2 1 1 10 1 2 1 1 1 1 11 2 2 2 2 2 2 12 1 1 1 1 1 1 13 2 2 1 2 2 2 14 1 1 1 1 1 1 15 2 2 1 1 1 1 16 2 2 2 2 2 2 17 1 1 1 1 1 1 18 2 2 2 2 2 2 19 1 1 1 1 1 1 20 1 1 1 1 1 1 21 2 2 2 2 2 2 22 2 2 2 2 2 2 23 1 1 1 1 1 1 24 1 1 1 1 1 1 25 1 1 1 1 1 1 26 1 1 1 1 1 1 27 1 1 1 1 1 1 28 1 1 1 1 1 1 29 6 6 5 6 6 5 30 8 8 8 8 7 6 31 2 2 2 2 1 0 32 3 3 2 3 3 3 33 9 9 8 9 9 9 34 1.5 1 1.5 1.5 2 2 35 2 2 2 2 2 2 36 1 1 1 1 1.5 1.5 37 2.5 2.5 2.5 2.5 3 2.5 38 1.5 1.5 1.5 1.5 2 2 39 3 2 3 3 3 3 40 2.25 2.5 2.75 2.5 3 3 41 1 1 1 1 1 1

115

R. mascareniensis venusta* P. hylaea* P. hylaea* P. hylaea* P. hylaea* P. hylaea* RGMAC RGMAC RGMAC RGMAC RGMAC RGMAC 39493 7367- B-1895 7367-B-1909 116081093 104970-975 104970-975 1 2 2 2 2 2 2 2 1 1 1 1 1 1 3 1 1 1 1 1 1 4 2 2 2 2 2 2 5 1 2 2 2 2 2 6 1 1 1 1 1 1 7 1 1 1 1 1 1 8 1 2 2 2 2 2 9 2 2 2 2 2 2 10 2 2 2 2 2 2 11 2 1 1 1 2 2 12 1 1 1 1 1 1 13 2 1 1 1 1 1 14 1 1 1 1 2 1 15 1 2 2 2 2 2 16 2 2 2 2 2 2 17 1 1 1 1 1 1 18 2 2 2 2 2 2 19 1 1 1 1 1 1 20 1 1 1 1 1 1 21 2 2 2 2 2 2 22 2 2 2 2 2 2 23 1 1 1 1 1 1 24 1 1 1 1 1 1 25 1 1 1 1 1 1 26 1 1 1 1 1 1 27 1 1 1 1 1 1 28 1 1 1 1 1 1 29 6 6 6 6 6 6 30 10 8 8 8 8 8 31 4 2 2 2 2 2 32 3 3 3 3 3 3 33 9 9 9 9 9 9 34 1.5 1.5 1 1.5 1 1 35 2 2 2 2 2 2 36 1 1 1.5 1.5 1.5 1 37 3 3 2 2.5 3 2.5 38 1.5 1 1 1.5 1.5 1.5 39 3 3 2.5 3 3 3 40 2.75 2.5 2 2.5 2.5 2 41 1 1 1 1 1 1

116

P. hylaea* P. hylaea* P. hylaea* RGMAC 104970-975 FMNH 12232(1) FMNH 12232 (2) 1 2 1 1 2 1 1 1 3 2 1 1 4 2 2 2 5 2 2 2 6 1 1 1 7 1 1 1 8 2 2 2 9 2 2 2 10 2 2 2 11 1 2 2 12 1 1 1 13 2 2 2 14 1 1 1 15 2 2 2 16 2 2 2 17 1 1 1 18 2 2 2 19 1 1 1 20 1 1 1 21 2 2 2 22 2 2 2 23 1 1 1 24 1 1 1 25 1 1 1 26 1 1 1 27 1 1 1 28 1 1 1 29 6 5 5 30 8 8 8 31 2 2 2 32 3 3 3 33 9 9 9 34 1.5 1.5 1.5 35 2 2 2 36 1 1.25 1.25 37 2.5 3 3 38 1.5 2 2 39 3 3 3 40 2.5 3 3 41 1 1 1.25

117 Curriculum Vita

Katrina Weber entered the University of Texas at El Paso in January 2005 and received her

Bachelor‘s of Science in Environmental Science in May 2008. During her time at UTEP, she spent a semester abroad studying marine biology at James Cook University in Townsville, Australia. She also participated in the International Polar Year: Research Opportunity in Antarctica for Minorities (IPY-

ROAM) which allowed her to conduct research on aquatic invertebrates in Antarctica. As an undergraduate, she worked as a research assistant in the Walsh Lab for Molecular Systematics studying predatory defenses of the rotifer Sinantherina socialis. She presented her undergraduate research at the

American Society of Limnology and Oceanography meeting (February 2007) and the Ecological Society of America Meeting (August 2007). In 2008, she was named a Top Ten Senior at UTEP. She has been a teaching assistant for a number of courses since August 2004, including Introductory Biology,

Organismal Biology, and Invertebrate Zoology. She began her graduate degree at UTEP in August 2008. During her graduate career, she received two grants from the UTEP Graduate School to support her research. She is a member of the American Society for Ichthyologists and Herpetologists, the Society for the Study of Amphibians and Reptiles, and the Chicago Herpetological Society. She has presented her graduate research at the Southwestern Association of Naturalists Meeting (April 2010) and the Joint Meeting of Ichthyologists and Herpetologists (July 2010). She currently plans to use her passion for learning to educate the next generation of college students.

Permanent address: 2111 N. Kansas

El Paso, TX, 79902

This thesis/dissertation was typed by Katrina M. Weber

118