Systematics of Leptopelis (Anura: Arthroleptidae) from the Itombwe

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2012-01-01 Systematics of Leptopelis (Anura: Arthroleptidae) from the Itombwe Plateau, Eastern Democratic Republic of the Congo Francisco Portillo University of Texas at El Paso, [email protected]

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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 LEPTOPELIS (ANURA: ARTHROLEPTIDAE) FROM

THE ITOMBWE PLATEAU, EASTERN DEMOCRATIC REPUBLIC OF THE

CONGO

FRANK PORTILLO

Department of Biological Sciences

APPROVED:

______Eli Greenbaum, Ph.D., Chair

______Jerry D. Johnson, Ph.D.

______Rip Langford, Ph.D.

______Benjamin C. Flores, Ph.D. Dean of the Graduate School

2012

SYSTEMATICS OF LEPTOPELIS (ANURA: ARTHROLEPTIDAE) FROM

THE ITOMBWE PLATEAU, EASTERN DEMOCRATIC REPUBLIC OF THE

CONGO

FRANK PORTILLO, B.S.

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 2012

ACKNOWLEDGMENTS

First I would like to thank my family for their love and support throughout my life. As a child, my parents taught me how to appreciate wildlife and my love for the natural world began.

I was always encouraged by my parents to learn and explore the outdoors without causing any disturbances to nature. My siblings have been great partners on many of my outdoor excursions and have always been a great support. My family continues to be a big inspiration to this day.

I would like to thank my graduate committee, Dr. Eli Greenbaum, Dr. Jerry Johnson and

Dr. Rip Langford. I would like to give a special thanks to my thesis advisor Dr. Eli Greenbaum who took me on as a student when I had very limited lab experience, and who provided superb guidance throughout my research. I would also like to thank all other faculty members that have helped along the way, including Dr. Elizabeth Walsh and Dr. Craig Tweedie for providing opinions. I also thank Dr. Vanessa Lougheed who carefully explained multivariate statistics.

I thank Jens V. Vindum, Dr. David Blackburn (California Academy of Sciences) and

Gregory Watkins-Colwell (Yale Peabody Museum) for access to specimens. DNA samples were sequenced by Omar Hernandez, Dr. Carolina Lema and Ana Betancourt at the UTEP DNA

Analysis Core Facility (funded by a UTEP Master’s Thesis Grant). Additional funding was provided by the UTEP Graduate School, Department of Biological Sciences and College of

Science.

I am also thankful to lab members Chris Anderson who provided support and advice throughout the research process, Nancy Conkey and Fernie Medina for continual lab assistance and support throughout this project, and Katrina Weber and Cesar Villanueva for teaching me lab procedures when I was new.

iv ABSTRACT

Leptopelis, a genus of Central African treefrogs, includes 51 species that live in tropical forests and savannas. Currently, only two species of Leptopelis are known from the poorly explored

Itombwe Plateau in eastern Democratic Republic of the Congo (DRC). Itombwe is renowned among conservationists for its rich and endemic amphibian fauna, including: Xenopus itombwensis, Chrysobatrachus cupreonitens, Laurentophryne parkeri, Hyperolius leleupi and at least three species of Arthroleptis. Evolutionary relationships of Itombwe Leptopelis were examined by sequencing two mitochondrial genes (16S: 557 bp [base pairs], cyt b: 620 bp) and one nuclear gene (RAG1: 761 bp). Results recovered strong support for several new lineages.

Morphological characters and male advertisement call data were used to examine species boundaries in distinct lineages identified from the molecular phylogeny. Results indicated that the poorly known species Leptopelis fiziensis, originally described as a subspecies of L. modestus, is a distinct taxon from Cameroonian (topotypic) L. modestus, and a distinct east

African population of L. modestus is described. Moreover a distinct L. karissimbensis population from Itombwe with low genetic divergence but distinct morphology and advertisement calls is described. The distinct calls between the recently diverged new species and the partially sympatric taxon L. karissimbensis are a likely consequence of reinforcement of species boundaries. This thesis represents the first phylogenetic analysis of Central African Leptopelis, and emphasizes the importance of the Itombwe Plateau as a conservation site.

v TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... iv

ABSTRACT ...... v

TABLE OF CONTENTS ...... vi

LIST OF TABLES ...... viii

LIST OF FIGURES...... Ix

CHAPTER 1: INTRODUCTION ...... 1

1.1 Background ...... 1

1.2 Natural history ...... 4

1.3 Taxonomy and systematics ...... 8

1.4 Study site ...... 11

1.5 Research objectives ...... 17

CHAPTER 2: MATERIALS AND METHODS ...... 19

2.1 Specimen acquisition ...... 19

2.2 DNA extraction ...... 19

2.3 DNA amplification, sequencing and alignment ...... 21

2.4 Phylogenetic analyses ...... 22

2.5 Population genetic analyses ...... 23

2.6 Morphological analyses of adult frogs ...... 24

2.7 Morphological analyses of tadpoles...... 25

2.8 Acoustic analyses ...... 26

CHAPTER 3: RESULTS ...... 28

3.1 Phylogenetic analyses ...... 28

3.2 Population genetic analyses ...... 30

3.3 Itombwe Leptopelis morphology, coloration and calls ...... 37

vi 3.4 Description of Leptopelis anebos...... 55

3.5 Description of Leptopelis versicolor ...... 62

CHAPTER 4: DISCUSSION ...... 73

4.1 Taxonomic status and biogeography of Itombwe Leptopelis ...... 73

4.2 Conservation ...... 80

4.3 Conclusions and future directions ...... 82

LITERATURE CITED ...... 83

APPENDIX I ...... 101

APPENDIX II ...... 105

VITA ...... 108

vii LIST OF TABLES

Table 2.3.1 Primers used for sequencing mitochondrial and nuclear genes ...... 22

Table 3.1.1 Model parameters specified by jModelTest and alternative models implemented in MrBayes...... 28

Table 3.2.1 Uncorrected p-distances within and among selected individuals from the

Leptopelis phylogeny (16S)...... 31

Table 3.2.2 Uncorrected p-distances within and among selected individuals from the

Leptopelis phylogeny (cyt b)...... 32

Table 3.2.3 Uncorrected p-distances within and among selected individuals from the

Leptopelis phylogeny (RAG1)...... 33

Table 3.2.4 Hierarchical analysis of molecular variance among lineages of

Leptopelis...... 35

Table 3.3.1 Morphometric data (in mm) for specimens of Leptopelis...... 38

Table 3.3.2 Hand (manus) and foot (pes) webbing formulas for Leptopelis...... 40

Table 3.3.3 P-values obtained from one way ANOVA and ANCOVA of mensural data for Itombwe Leptopelis...... 40

Table 3.3.4 Eigenvalues and PC loadings obtained from a PCA of raw morphometric data. 46

Table 3.3.5 Eigenvalues and PC loadings obtained from a PCA of raw morphometric data regressed against snout–vent length...... 47

Table 3.3.6 Call characteristics measured from recordings of Leptopelis...... 50

Table 3.5.1 Morphometric data of Leptopelis versicolor tadpoles...... 69

viii LIST OF FIGURES

Fig. 1.1 Phylogeny redrawn from the Vences et al. (2003a) molecular analysis of

African treefrogs...... 9

Fig. 1.4 Localities of Leptopelis fiziensis from Central Africa...... 18

Fig. 2.1 Map of the Itombwe Plateau in eastern Democratic Republic of the Congo, with sampling localities...... 20

Fig. 3.1.1 Bayesian phylogenetic tree (16S, cyt b, and RAG1 genes) of treefrogs in the genus Leptopelis from Itombwe...... 29

Fig. 3.2.1 Haplotype networks for L . cf. karissimbensis and L. karissimbensis...... 36

Fig. 3.3.1 Leptopelis cf. karissimbensis and L. karissimbensis in life...... 42

Fig. 3.3.2 Leptopelis cf. modestus in life...... 43

Fig. 3.3.3 Leptopelis fiziensis in life...... 44

Fig. 3.3.4 Scatter plot of the first and second principal component scores for the analysis with mensural data...... 48

Fig. 3.3.5 Scatter plot of the first and second principal component scores for the analysis with mensural data regressed against snout–vent length...... 49

Fig. 3.3.6 Waveform diagram and spectrogram of calls of Leptopelis cf. karissimbensis and L. karissimbensis...... 53

Fig. 3.3.7 Waveform diagram and spectrogram of calls of Leptopelis cf. modestus

and L. fiziensis...... 54

Fig. 3.4.1 Dorsal, ventral and lateral view of the head of the preserved male holotype of Leptopelis anebos...... 56

Fig. 3.5.1 Dorsal, ventral and lateral view of the head of the preserved male

ix holotype of Leptopelis versicolor...... 63

Fig. 3.5.2 Lateral, dorsal and ventral views of the preserved Leptopelis versicolor tadpole (UTEP 20587, Gosner stage 38)...... 70

Figure 3.5.3 Larval mouthparts of Leptopelis versicolor (UTEP 20587)...... 71

CHAPTER 1: INTRODUCTION

1.1 Background

The Order Anura is the most speciose taxon of amphibians, with more than 6,213 species

(AmphibiaWeb, 2012). The number of species continues to steadily increase. Anurans provide important ecological niches, because they are both predators and prey. Some scientists consider anurans to be good environmental indicators, because they are sensitive to environmental perturbations (Welsh and Oliver, 1998). Anurans are thought to be sensitive to environmental perturbations because of their biphasic life histories, highly specialized physiological adaptations and specific microhabitat requirements (Bury, 1988; Vitt et al., 1990; Wake, 1990; Olson, 1992;

Blaustein, 1994; Blaustein et al., 1994; Stebbins and Cohen, 1995). During their aquatic stage, many stream-dwelling amphibian larvae are highly specialized in the use of their habitats for both foraging and shelter. With such specialized adaptations, amphibian larvae are susceptible to small environmental changes. Furthermore, the semipermeable skin of amphibians makes them highly sensitive to water quality and UV radiation in all life stages (Gerlanc and Kaufman, 2005;

Taylor et al., 2005; Halliday, 2008). Because amphibians also spend a large portion of their lives on land, they are also susceptible to many disturbances in their terrestrial habitats (Waddle,

2006). Additionally, many stream-dwelling amphibians are highly philopatric and long-lived, and they exist in relatively stable populations. These attributes make them more tractable and reliable indicators of potential biotic diversity in stream ecosystems than anadromous fish or macroinvertebrates, and amphibian relative abundance can be a useful indicator of stream condition. Habitat alterations, such as conversion of forests and wetlands to farmlands, can also

negatively impact amphibians because of their high dependence on moisture (Halliday, 2008).

Collins and Crump (2009) criticized the metaphor “canary in the coalmine” when applied to amphibians, because amphibians do not meet specific criteria to be considered ideal ecological indicators. Criteria include well known: natural history, geographic distribution and taxonomy; the group should also be easily accessible cost effective to monitor. Thompson (2010) revisited the metaphor of “canary in the coalmine” and stated the metaphor was appropriate by saying, “in contrast to the arguments against the metaphor (canary in the coalmine) it may serve as a message to say that humanity must pay attention to amphibian declines as coal miners paid attention to a dying canary.”

Because of habitat perturbation and alteration, and amphibian sensitivity to environmental changes, amphibians have become the most threatened group of vertebrates

(Blaustein and Wake, 1995; Gallant, 2007). Although there are several factors contributing to population declines in amphibians, biologists around the world agree that habitat loss, alteration and fragmentation are the primary cause (Collins and Crump, 2008). The amphibian chytrid fungus (Batrachochytrium dendrobatidis) has also plagued amphibians worldwide. The chytrid fungus has been associated with amphibian population declines in East and South Africa

(Hopkins and Channing, 2003; Channing and Howell, 2006). Batrachochytrium dendrobadtidis

(Bd) has also been found in African frogs from Ghana, Kenya and South Africa (Speare and

Berger, 2002). Bell et al. (2012) found 20 species of amphibians from Gabon infected with Bd.

That study presented the first record of Bd in Gabon after two previous studies had failed to detect the fungus (Daversa et al., 2011; Gratwicke et al., 2011; Bell et al., 2012). Surveys from eastern DRC and Nigeria also detected the presence of Bd (Greenbaum et al., 2008; Imausen,

2009). Some research suggests the chytrid fungus most likely originated from Africa, with the

2 likely candidate for worldwide spread from the pipid Xenopus laevis. In the wild, X. laevis does not show any clinical signs of infection, nor has the species experienced any sudden die-offs.

Once the chytrid fungus reached the New World, other amphibians could have distributed the fungus between and within countries (Weldon et al., 2004). However, the African origin of Bd is disputed by James et al. (2009), because there is lower genetic diversity in African isolates of Bd compared to North American ones.

Mortality rate and time to death is affected by fungal dose, temperature, age of frogs, and host species (Berger et al., 1999, 2004; Ardipradja, 2001, Lamirande and Nichols, 2002;

Woodhams et al., 2003). Dead and dying frogs generally have disorders of the epidermis, and often exhibit behavioral changes such as lethargy and loss of righting reflex (Weldon et al.,

2004). Another important factor is the relative abundance and genetic diversity of Bd strains between continents. The results from Farrer et al. (2011) and other studies (Collins and Crump,

2009), support the novel pathogen hypothesis, which suggests that Bd is a recently emerged pathogen (Skerratt et al., 2007). Farrer et al. (2011) suggested anthropogenic mixing of allopatric lineages of Bd has led to the generation of a hypervirulent Bd strain, and the lineage is undergoing further diversification by either mitotic or sexual recombination.

The threat of amphibian declines is most likely underestimated because of a lack of knowledge regarding data-deficient species (about 23% of all amphibians) (Stuart et al., 2004).

Taxonomic knowledge of amphibians in the tropics is deficient and can be seen in the rate of species descriptions. In 1986, 4,015 amphibians had been described (Halliday, 2008). Currently,

(November, 2012) this value has increased to 7,067 described species, an increase of 75% since

1986. Anurans alone represent about 6,200 species, or about 88% of the total number of amphibian species (AmphibiaWeb, 2012). Determining which amphibians are in need of

3 conservation can help identify ecosystems that are unhealthy and affected by environmental change. Therefore, the study of amphibians, including anurans, can be helpful assets for ecosystem protection and conservation.

The Family Arthroleptidae (first named by Mivart, 1869) is a group of frogs composed of two subfamilies: Arthroleptinae Mivart, 1869 (89 species) and Leptopelinae Laurent, 1972 (51 species) (Frost, 2011). Arthroleptinae is composed of seven genera: Arthroleptis Smith 1849 (44 species), Astylosternus Werner, 1898 (11 species), Cardioglossa Boulenger, 1900 (16 species),

Leptodactylodon Andersson, 1903 (15 species), Nyctibates Boulenger, 1904 (monotypic),

Scotobleps Boulenger, 1900 (monotypic) and Trichobatrachus Boulenger, 1900 (monotypic)

(Frost, 2011). The subfamily Leptopelinae Laurent, 1972 is composed of a single genus,

Leptopelis Günther, 1859 (51 species) (Frost, 2011). The research herein is focused on

Leptopelis, specifically from the Itombwe Plateau in eastern Democratic Republic of the Congo

(DRC). Leptopelis is a genus of forest and savanna treefrogs found throughout sub-Saharan

Africa (Laurent, 1973). The majority of Leptopelis species have a conservative, similar treefrog- like morphology, which often impedes efforts to identify species. Developing adequate conservation plans cannot be done without sufficient taxonomic knowledge and species descriptions (Mace, 2004).

1.2 Natural history

Throughout sub-Saharan Africa, Leptopelis generally inhabit forests and savannas.

Leptopelis are medium to large treefrogs, with vertical pupils and vomerine teeth. Leptopelis also have broad heads, large toe discs and extensive webbing along the digits (Channing and

4 Howell, 2006). Although members of the genus are nearly unmistakable, they do show some diversity from smooth-skinned, fully webbed arboreal forms to toad-like, warty fossorial forms with no webbing and no toe discs. Since many of these species have a very similar morphology, there has been a tendency to classify them as subspecies of each other (Schiøtz, 1999).

Laurent (1973) suggested the genus Leptopelis originated from a terrestrial Astylosternus that adapted to an arboreal lifestyle. Leptopelis evolved several morphological characters associated with this arboreal lifestyle, differentiating them from terrestrial arthroleptids, including greater size, webbed feet, and adhesive, powerful discs (e.g., L. palmatus) (Laurent,

1973). Females of different species are very similar and are difficult to identify. Males have certain morphological characters associated with them and thus are easier to identify, including blue, white or gray gular sacs, pectoral glands and an N-shaped dorsal pattern (Channing and

Howell, 2006).

Leptopelis are mainly generalists, and prey on a variety of invertebrates and small vertebrates. However, some species have highly specialized diets. Leptopelis brevirostris, for example, has an extremely short snout and feeds exclusively on mollusks and other slugs (Perret,

1966). In turn, species of Leptopelis are preyed upon by water snakes, vine snakes, boomslangs

(Dispholidus typus) and even wandering spiders (Ctenidae) (Barej et al., 2009).

Species of Leptopelis use camouflage as a means to avoid predation, but when faced with a predatory attack, some species use a defense mechanism known in other amphibian taxa: the unken reflex. The unken reflex is a warning display commonly seen in anuran and salamander species with aposematic coloration, including the Asian frog genus Bombina. The back is usually arched, the limbs raised over the body with soles upward, and the head is pulled back, all

5 aiding the display of colorful ventral surfaces (Lillywhite, 2008). The unken reflex has been observed in L. kivuensis and L. karissimbensis. The latter species will also open its mouth, exposing a blue lining (Roelke et al., 2011).

1.2.1 Reproduction

Data are limited on the reproductive habits of most species, but in the species of

Leptopelis where the mode of reproduction is known, the pattern is consistent. Like most anurans, Leptopelis use vocalizations as a means to attract mates. In many species of Leptopelis, there is an absence of sexual dimorphism in color pattern, suggesting that visual cues are unimportant for the identification of potential mates. Aside from vocal cues, tactile cues may be the most important for mate identification. Differences in size and skin texture are used for sex identification. Amplexus is stimulated by the greater size and firmness of the females. When both the male and female are ready to mate, the male will grasp the female. In the case of

Leptopelis, like most neobatrachians (an advanced suborder of anurans), amplexus is axillary. In axillary amplexus, the males grasp the females under the arms, placing their vents closer together

(Duellman and Trueb, 1994). In some species of Leptopelis (e.g., L. bocagii), the male will secrete a sticky material from pectoral glands underneath his body. This sticky material is used to more efficiently hold on to the females while mating (Channing and Howell, 2006). Adhesive glands for reproduction are also found in microhylid species such as Kaloula conjuncta (Taylor,

1920), K. picta and K. rigida (Inger, 1954), Gastrophryne olivacea (Fitch, 1956), G. carolinensis

(Conaway and Metter, 1967) and the brevicipitid genus Breviceps (Poynton, 1964; Visser et al.,

1982). Because Breviceps is not closely related to either microhylids or Leptopelis, adhesive glands must have evolved independently in each of those taxa (Siegel et al., 2008). Leptopelis usually bury large, unpigmented, yolk-filled eggs in the soil near water or in a depression where

6 rainwater will accumulate. The larvae develop rather slowly. They are eel-shaped and able to maneuver overland to water with great agility, and can overcome formidable obstacles. There are some indications that L. brevirostris has direct development without an aquatic larval phase, which is a reproductive strategy seen in other arthroleptids (e.g., Arthroleptis) (Schiøtz, 1999).

1.2.2 Voice

Most species of Leptopelis calls are unmelodious clacks, buzzing, or a combination of both. In some forest species, the clacks have a strange acoustic quality, being composed of numerous harmonics in rapid succession. A similar advertisement call structure can be found in a group of possibly related species from the eastern savannas (Schiøtz, 1999). There is some evidence (Schiøtz, 1999) to suggest that the clacks serve to attract females, whereas buzzing or creaking may have territorial functions in males. It is unknown whether all species of Leptopelis emit both types of calls or whether both call types are used specifically and independently to attract mates and set territorial barriers (Schiøtz, 1999). There is a similar division of calls in certain Afrixalus and Hyperolius (Hyperoliidae) species (Backwell, 1988; Schiøtz, 1999). Grafe et al. (2000) studied the vocal repertoire of L. viridis and made distinctions between call types.

In addition to clicks that serve for advertisement calls, males of L. viridis also produced soft aggressive calls, trills and multinote calls. The context in which clicks and soft calls were produced suggests that they function as advertisement and aggressive calls, respectively. The males also seemed to increase the proportion of aggressive calls when the researcher replayed advertisement calls with an increase in sound pressure. In addition, at frequent advertisement call playback levels, males changed their behavior from stationary advertisement calling to an aggressive approach, silent retreat, or in one case, call suppression. This behavioral response suggests that L. viridis males probably use advertisement call intensity to mediate spacing

7 between individuals. This behavior has also been seen in other anuran species such as the

Neotropical frog Eleutherodactylus coqui (Stewart and Bishop, 1994), Pseudophilautus leucorhinus (Arak, 1983) and Diasporus diastema (Wilczynski and Brenowitz, 1988); sound pressure level was shown to mediate spacing in these species (Grafe et al., 2000).

1.3 Taxonomy and systematics

Leptopelis is the single genus included in the subfamily Leptopelinae, one of the two subfamilies included in the family Arthroleptidae. Laurent originally proposed Leptopelini as a tribe within the family Hyperoliidae (Laurent, 1972). Vences et al. (2003a) provided evidence that Leptopelis was nested within the arthroleptid frogs and did not form a monophyletic clade with the hyperoliids. Using the mitochondrial genes 12S, 16S and cytochrome b, Vences et al.

(2003a) constructed a molecular phylogeny of African treefrogs. To date, the Vences et al.

(2003a) study provided one of the most comprehensive molecular analyses conducted on African treefrogs. Characters from mitochondrial DNA sequence data were analyzed using maximum likelihood, maximum parsimony and neighbour-joining approaches (Fig. 1.1). The molecular analysis was one of the first to show that Hyperoliidae (with Leptopelis included) was not monophyletic (Vences et al., 2003a).

Frost et al. (2006) provided additional morphological and molecular evidence and placed

Leptopelinae as the sister taxon to Arthroleptinae, under the family Arthroleptidae. The Frost et al. (2006) study has been highly criticized because of the methodology used in the research.

Criticisms most noted for the study include: lack of inclusion of type species for genera, the exclusion of the commonly used nuclear gene RAG-1, and questionable analytical methods that assume all characters evolve at equal rates (Wiens, 2007).

Figure 1.1 Phylogeny redrawn from the Vences et al. (2003a) molecular analysis of African treefrogs. Maximum likelihood phylogeny is based on 471 bp of the mitochondrial gene 16S. Strongly supported clades are indicated by an asterisk.

9 Currently, Arthroleptidae is considered the sister taxon to the family Hyperoliidae (Frost et al., 2006). According to Laurent (1986), arthroleptid frogs differ from ranids, but are similar to hyperoliids in having a cartilaginous sternum. Molecular evidence supports the inclusion of

Leptopelis in Arthroleptidae (Vences et al., 2003a). Species of Leptopelis lack a number of significant apomorphies common to the hyperoliids (Drewes et al., 1984), notably the gular sacs found in males of the latter group. According to Vences et al. (2003a), the inclusion of

Leptopelis within Hyperoliidae was largely based on the presence of an intercalary element, which is now known to be of little phylogenetic value. The distant relationship of Leptopelis with respect to Hyperoliidae is also supported by several lines of evidence from chromosomes.

First, representatives of Leptopelis have a variable chromosome number, although one of the species used in one study, L. calcaratus, is similar to hyperoliids in its diploid complement of 2n

= 24. Second, all Leptopelis studied so far have at least one telocentric chromosome pair, a state that is unknown in hyperoliids. In the hyperoliid genus Kassina, the twelfth pair of chromosomes looks similar to the Leptopelis telocentric chromosome(s), but Kassina chromosomes are subtelocentric (Odierna et al., 2007). Third, the loci of nucleolus organizer regions (NORs) were located on the long arm of the fifth chromosome pair in L. calcaratus.

These same loci of NORs are located on the ninth pair in all examined hyperoliids (Odierna et al., 2007).

Systematic studies of the genus Leptopelis have been burdened by a dearth of specimen collecting in Central Africa and the morphological similarities of several species (Schiøtz, 1999;

Köhler, 2009). Recently, Roelke et al. (2011) examined the taxonomic status of two Leptopelis from the Albertine Rift; Leptopelis kivuensis was previously synonymized with L. karissimbensis because the two forms are almost indistinguishable, and they have overlapping populations (de

10 Witte, 1941). Laurent (1972) revisited the taxonomic changes of de Witte (1941) and removed

L. kivuensis from the synonymy of L. karissimbensis. Although Laurent’s decision to remove L. kivuensis from the synonymy of L. karissimbensis is widely accepted, it is likely that he continued to confuse the two species. Laurent (1973) listed L. kivuensis from the Itombwe

Plateau, but the only species noted from Itombwe by Roelke et al. (2011) was L. karissimbensis, which was found in forest edges and grassland habitats. The latter study found no morphometric differences between L. kivuensis and L. karissimbensis (Roelke et al., 2011). Many Leptopelis are cryptic species (morphologically similar but genetically distinct) and it is likely there are multiple species that remain unrecognized and undescribed. For example, Schiøtz (1999) argued this is the case for the Central African species L. modestus. Leptopelis mackayi, which was described from Kakamega Forest in western Kenya, is one of the species that was formerly considered to be a disjunct population of L. modestus (Köhler et al., 2006).

1.4 Study site

This study will focus on Leptopelis collected from the Itombwe Plateau, DRC. The

Itombwe Plateau is located on the northwestern side of Lake Tanganyika. Doumage (1998) defined the Itombwe Plateau as an area appoximately 15,000 km2 occurring between 2°40′ and

4°35′ south, and 27°55′ and 29°05′ east, with an elevation ranging from 900–3475 meters.

Geological processes, including formation of the Albertine Rift, may be the driving force for the pattern of high biodiversity in montane landscapes of eastern DRC (Burgess et al., 2004). The last major tectonic foldings and movement in Africa took place during the Ordovician, 480 million years ago (mya). About 250 mya, at the end of the Permian, the continents were stuck together in a giant land mass called Pangaea. Ocean levels were low, global climate was cool and dry, and the exposed landmass was at its maximum. During the Triassic (around 248 mya)

11 Pangaea moved northward and began to break up, leading to the separation of Laurasia from

Gondwanaland. Around 206 mya (Jurassic) Gondwanaland started to break apart. The separation of Africa and the Antarctic was followed by massive eruptions of basalts. As a result of these eruptions, the Table Mountains were formed in the Cape Region in South Africa.

During the Cretaceous (180 mya), north-south faults opened the South and North Atlantic. This process caused Gondwanaland to separate into multiple landmasses forming present-day South

America, Africa, Antarctica, Madagascar, Indonesia and Australia (Vande weghe, 2004).

Africa remained fairly stable over the 80 mya between the late Cretaceous and the middle

Tertiary (Meijer and Wortel, 1999). Unlike other continents, the surface of Africa was only slightly affected by the drift of plates because of its central position and relative immobility.

Africa became isolated from the other continental landmasses during the Cretaceous about 70–80 million years ago. During this isolation period, modern tropical forest plants appeared in Africa.

Mammals, birds and squamates diversified. Although Africa’s surface remained fairly stable for nearly 400 million years, by the end of the Cretaceous, tectonic activity in the earth’s crust caused lifting of the western margin of Central Africa. This caused cracks on the earth’s surface and disrupted the hydrographic network. It reactivated erosion, renewing soils and slowly exposed present-day features. During the Miocene, Africa’s long isolation period ended with the formation of the Arabo-African Rift and the separation of Arabia 20 mya. As a result, a land bridge between Asia and Africa was formed. Although Cameroon and the Gulf of Guinea experienced little tectonic activity, the Arabo-African Rift greatly affected all of East Africa.

This enormous complex of faults split the earth’s crust for about 6,000 kilometers, from present- day Palestine to Mozambique. The successive up-thrusting of the relief set off intense erosion that cleared off Cretaceous surfaces and transformed hydrologic networks. The western branch

12 of the rift formed on the western edge of the East African Plateau. The Albertine Rift is the western section of Africa’s two rift valleys, which are deep tectonic basins 32 to 64 kilometers wide. In the Albertine Rift, volcanic eruptions did not take place until about 12.6 mya with the subsequent formation of Lake Tanganyika. The formation of the Virunga volcanic chain would greatly affect the landscape. Virunga’s lavas created an enormous natural dam, giving way to the formation of Lake Kivu. Virungua’s lavas also obstructed the small lateral valleys of the rift and created a multitude of smaller lakes (Vande weghe, 2004).

Additionally, ice age cycles have cause dramatic shifts in Africa’s climate over the past two million years (Gasse et al., 1990; Burgess et al., 2004). Maximum ice cover in the northern hemisphere during the last ice age occurred 18–20,000 years ago (kya). Around 30% of Earth’s surface was covered in ice, compared to 10% today. The ice cover resulted from a lowering of temperature by 10–20°C in temperate regions and 2–5°C in the tropics. Furthermore, evidence suggests that land not covered by glaciers was substantially drier than it is presently. This was likely caused by decreased evaporation on land because of strengthened wind systems and a 10% increase in dry land surface (Tallis, 1991).

The Pleistocene forest refuge hypothesis, first proposed by Edward Forbes in 1846, suggests that the climatic and vegetation changes described above caused fragmentation of previously continuous species ranges into isolated refuges. Originally this hypothesis applied only to species living in temperate regions, but the Pleistocene also caused dramatic changes in vegetation distributions in the tropics (Mayr and O’Hara, 1986). Periods of drought and reduced rainfall caused temporary fragmentation of once continuous forests. These forests became isolated regions surrounded by stretches of savanna. These former forest islets are now thought to contain high levels of species richness and endemism (Diamond and Hamilton, 1980). As

13 rainfall increased, the forests expanded, with a subsequent decrease in areas occupied by savannas (Mayr and O’Hara, 1986). Current and past climatic patterns could prove useful in explaining biogeographic patterns of Central African amphibian species, including Leptopelis.

Today, the Albertine Rift encompasses much of the western rift valley down to southern

Tanzania and northern Zambia. The Albertine Rift extends from 30 km north of Lake Albert to the southern tip of Lake Tanganyika. The Albertine Rit includes all habitats within 100 km east of the border of DRC. Habitats range from rocky glaciers at the top of the Ruwenzori mountains

(5100 m), down through alpine moorland (3400–4500 m), giant Senecio and Lobelia vegetation

(3100–3600 m), giant heather (3000–3500 m), raised bogs (3000–4000 m), bamboo forest

(2500–3000 m), montane forest (1500–2500 m), lowland forest (600–1500 m), savanna woodland (600–2500 m) and savanna grassland (600–2500 m). The Albertine Rift is edged by some of the highest mountains in Africa, including the Virunga Mountains, Mitumba Mountains and Ruwenzori Range. It contains some of the deepest lakes in the world, including Tanganyika

(Plumptre et al., 2007). Lake Tanganyika, at 650 kilometers long, has been likened to an interior sea. Although all the forests are not yet completely explored, the Albertine Rift is the richest islet of montane forest in Africa (Vande weghe, 2004).

The Itombwe Plateau, located west of Lake Tanganyika in South Kivu Province, DRC is one of the most undisturbed areas of the Albertine Rift’s montane forests. The forests of

Itombwe include the most humid montane formations and are the richest biological forests located southwest of Bukavu (Vande weghe, 2004). Since the first exploration in the early

1900s, Itombwe has been recognized as one of the most biologically distinctive regions in Africa

(Omari et al., 1999). The diversity and extent of habitat types associated with this plateau are among the most significant in Africa (Doumenge, 1998). This is in part because the plateau lies

14 at the intersection of three major biogeographical regions, including the lowland forests of the

Congo basin, the montane forests of the Albertine Rift, and the grasslands of eastern and southern Africa (White, 1983). The Itombwe Plateau lies in what is considered to be the largest forest refugium in Central Africa; this refugium is thought to have persisted through the dry periods of the Pleistocene (Sayer et al., 1992). The importance of forest refugia for conservation is great because they harbor high levels of species richness and endemism (Lovett et al., 2005).

The Itombwe Plateau is well known among conservationists for its rich and unique fauna

(Doumenge et al., 1998), many of which are endemic, including primates, shrews (Nicoll and

Rathburn, 1990), birds (Prigogine, 1971, 1977, 1978, 1984, 1985), butterflies (Plumptre et al.,

2003) and amphibians (Laurent 1964). The recently described pipid anuran Xenopus itombwensis Evans et al., 2008, monotypic hyperoliid Chrysobatrachus cupreonitens Laurent,

1951, monotypic bufonid Laurentophryne parkeri (Laurent, 1950), arthroleptid Arthroleptis hematogaster (Laurent, 1954) and the lacertid Congolacerta asukului Greenbaum et al., 2011, are a few of the species endemic to the Itombwe Plateau. The avifauna of the Itombwe Plateau is the most species-rich for any single forest in Central Africa and is considered the most important forest in Africa for bird conservation (Prigogine, 1977, 1985, 1988; Collar and Stewart, 1988).

Most of the exploration and research conducted in the Itombwe Plateau was undertaken before independence and was capped by Alexander Prigogine’s monumental collections of the region’s avifauna made between 1950 and 1967, totaling 565 species (Omari et al., 1999).

Following independence, large areas of Itombwe became occupied by armed rebels. Despite the potential for many new species discoveries and its significance as one of the most important forest areas for conservation in Africa (Prigogine, 1985; Collar and Stuart, 1988; Stattersfield et al., 1998), no further fieldwork was conducted until the late 1980s (Omari et al., 1999).

15 Biological surveys were resumed in Itombwe in 1989 when researchers made a brief reconnaissance trip to the northern and southern limits, during which they added one new bird species to Prigogine’s list (Omari et al., 1999). Two years later, Hall and Wathaut (1992) made a trip to Itombwe to evaluate gorilla populations. They documented extensive habitat degradation, especially in northern areas of Itombwe, and found that several gorilla populations documented in 1959 (Emlen and Schaller, 1960) had been extirpated. Hall and Wathaut (1992) concluded that much of Itombwe’s fauna and undisturbed forests were at risk.

Currently, Central Africa is experiencing severe amphibian declines due to habitat destruction and alterations (IUCN, 2008). The Itombwe Plateau, when considered among other

Albertine Rift sites, has the most threatened species of amphibians (Laurent, 1964; 1983; Evans et al., 2008; Stuart et al., 2008; Roelke et al., 2011), and the highest number of endemic amphibians, rendering the plateau the most important site for amphibian conservation in continental Africa (Plumptre et al., 2003; Greenbaum and Kusamba, 2012). Leptopelis calcaratus, L. christyi, L. cynnamomeus, L. fiziensis, L. karissimbensis, L. kivuensis and L. modestus are known to occur in or near the Itombwe Plateau (Laurent, 1972, 1973; Schiøtz,

1999; Roelke et al., 2011). Some of these recognized species have disjunct populations that are likely to be distinct lineages (Schiøtz, 1999). Additionally, one of these seven species (L. fiziensis) has not been seen since its original discovery by Laurent in 1956 (Laurent, 1973).

There is a photograph of L. fiziensis from Schiøtz (1999) that I believe is questionable because of the location where the specimen was found at Gombe Stream, Tanzania. The holotype of L. fiziensis was described by Laurent (1973) from Mokanga, South Kivu, DRC (near the town of

Fizi), with additional specimens collected from the nearby town of Fizi. There is approximately

70–90 km of freshwater (Lake Tanganyika) between Laurent’s DRC specimens and Gombe

16 Stream in Tanzania (Fig. 1.4). Although it is possible that suitable habitat may have once been continuous around the northern edge of Lake Tangayika.

According to Doumenge (1998), the Itombwe Plateau is the second most, if not the most important, location of highland forest in continental Africa. As many new species of reptiles and amphibians await description (Greenbaum et al., 2011), it is likely that the Itombwe Plateau’s importance as a center of endemism and conservation concern will increase as biological exploration continues (Greenbaum et al., 2011). Therefore, it is crucial, now more than ever, to determine the true anuran diversity of Itombwe, since organisms cannot be conserved if they are not discovered and recognized as distinct taxa (Mace, 2004).

1.5 Research objectives

1) Does current taxonomy represent true species richness of Leptopelis in Itombwe, DRC?

2) Can proposed phylogenetic relationships resolve current biogeographical patterns?

The objectives of my research are:

1) To determine the extent of genetic variation occurring in Itombwe populations of the

genus Leptopelis, and identify and describe lineages representing cryptic species.

2) To propose a phylogeny illustrating evolutionary relationships of Leptopelis from

Itombwe with available comparative data from GenBank and tissue samples from other

sites in Africa.

3) To describe, when appropriate and data are available, calls, tadpoles, color in life,

distribution and conservation status for new and poorly known species.

Figure 1.4 Localities of Leptopelis fiziensis from the original description by Laurent (1973), a photograph in Schiøtz (1999), and recently collected specimens used in this thesis. Black square = Mokanga, type locality; grey = Fizi (locality for paratypes); gold = Gombe Stream National Park, Tanzania; pink = recently collected specimens from Kasanjala and Kanguli.

18 CHAPTER 2: MATERIALS AND METHODS

2.1 Specimen acquisition

Most specimens included in this thesis were collected by Dr. Eli Greenbaum from the

Itombwe Plateau in DRC from 2007–2012. Field seasons from 2007 through 2009 were conducted during the dry season (summer). Field seasons 2009/2010, 2010/2011 and 2011/2012 were conducted during the wet season (winter months).

To compensate for specimens not currently available for examination, preserved specimens were requested from the California Academy of Sciences (CAS) and Yale Peabody Museum

(YPM). Outgroup samples included Scotobleps gabonicus Arthroleptis variabilis, Leptopelis barbouri, and Leptopelis parbocagii. A total of 177 preserved Leptopelis were examined.

Locations of Leptopelis used in the genetic analyses are plotted in Fig. 2.1.

2.2 DNA extraction

The Qiagen DNeasy Blood and Tissue Kit (Valencia, CA, USA) was used to extract DNA from tissue samples. Small pieces of tissue, about 25 mg, were cut, soaked in deionized water, and placed in a refrigerator at 0.2°C for two hours (h), allowing the ethanol to diffuse from the tissue. The samples were then allowed to dry at 0.2ºC for 2 hours. Once the 2 h drying period was completed, 180 μl of Buffer ATL and 20 μl of Proteinase K were added to the samples.

Samples were then placed on a hot plate for 30 minutes (min) at 55°C. After the 30 min incubation period, samples were removed, vortexed for 20 seconds (s), and then left overnight on the hot plate at 55°C. The next morning 200 μl of Buffer AL was added to each sample.

Figure 2.1 Map of the Itombwe Plateau, showing sampling localities of Albertine Rift Leptopelis.

Samples were vortexted for 20 s and placed on the hot plate for 10 min at 70°C. Following the

10 min incubation, 200 μl of 100% ethanol was added and each sample was vortexed for 20 s.

20 The resulting solution was placed into spin columns and centrifuged at 8000 revolutions per min

(rpm) for 1 min. Liquid remaining in the spin columns was discarded and 500 μl of Buffer AW1 was added. Samples were again centrifuged at 8000 rpm for 1 min. Liquid remaining in the spin columns was discarded and 500 μl of Buffer AW2 was added. Samples were 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 one min, and samples were then centrifuged at 8000 rpm for

1 min. The remaining solution was saved for polymerase chain reaction (PCR). The previous step was repeated to obtain a total of 400 μl of genomic DNA for PCR.

2.3 DNA amplification, sequencing and alignment

Two mitochondrial (16S and cyt b) and one nuclear (RAG1) genes were used in this study, and primers for each gene are specified in Table 2.3.1. Amplifications included a negative control to ensure no contaminated DNA was amplified. Amplifications were completed using a denaturing temperature of 95°C, annealing at 50°C and extension at 72°C for 32 cycles

(mitochondrial DNA) and 34 cycles (nuclear DNA). The PCR products were visualized using

1.5% agarose gel and SYBRsafe gel stain (Invitrogen, Carlsbad, CA, USA). Products from PCR were purified with AMPure XP magnetic bead solution with the manufacturer’s protocol

(Beckman Coulter, Danvers, MA).

Sequencing of forward and reverse strands of PCR products were done on an ABI 3700xl capillary DNA sequencer at the UTEP DNA Analysis Core Facility. Chromatograph data were interpreted using the program SeqMan (Swindell and Plasterer, 1997). Sequences were aligned using the program MEGALIGN (DNASTAR, Madison, WI, USA) and adjusted by eye in

MacClade v4.08 (Maddison and Maddison, 2000). Hypervariable regions from the 16S gene

21 data set were removed from the final analysis, resulting in the exclusion of 13 bp. Protein-

coding genes were checked for accuracy by translation to amino acids in MacClade v4.08

(Maddison and Maddison, 2000).

Table 2.3.1 Primers used for sequencing mitochondrial and nuclear genes.

Primer Name Primer Sequence Fragment Primer Source length

16SA 5'-CGCCTGTTTATCAAAAACAT-3' 600 bp Palumbi et al. (1991)

16SB 5'-CCGGTCTGAACTCAGATCACGT-3'

Cyt b-CBJ10933 5'-TATGTTCTACCATGAGGACAAATATC-3' 600 bp Bossuyt and Milinkovitch (2000) Cyt b-C 5'-CTACTGGTTGTCCTCCGATTCATGT-3'

RAG1MartF1 5'-AGCTGCAGYCARTAYCAYAARATGTA-3' 900 bp Chiari et al. (2004); Pramuk et al. (2008) RAG1AmpR1 5'-AACTCAGCTGCATTKCCAATRTCA-3'

2.4 Phylogenetic analyses

In total, the analyses included 63individuals from the genus Leptopelis, with multiple

representatives of L. karissimbensis, L. cf. karissimbensis, L. cf. modestus, L. fiziensis and L.

kivuensis, L. cf. kivuensis, L. sp, L. calcaratus, L. cf. calcaratus and L. modestus. Outgroup samples

included Scotobleps gabonicus, Arthroleptis variabilis, Leptopelis barbouri and L. parbocagii.

Sequences from 14 of these individuals have been published previously (Vences et al., 2000, 2003;

van der Meijden et al., 2007; Roelke et al., 2011; Greenbaum et al., 2012); new sequences were

deposited in GenBank (Appendix I). Sequence data were analyzed using maximum parsimony

(MP) in PAUP* 4.0b (Swofford, 2002), maximum likelihood (ML) in RAxML v7.2.6

22 (Stamatakis, 2006) and Bayesian inference (BI) in MrBayes v3.1 (Ronquist and Huelsenbeck,

2003). The MP analyses were conducted with a heuristic search algorithm with 25 random- addition replicates, accelerated character transformation and tree bisection-reconnection branch swapping. Zero-length branches were collapsed to polytomies. Node support was assessed with

1,000 nonparametric bootstrap pseudoreplicates (Felsenstein, 1985). The ML analyses of single- gene and combined datasets were conducted using the GTRGAMMA model in RAxML v7.2.6

(Stamatakis, 2006). Datasets for ML and BI analyses were partitioned by codon position in single gene datasets, and by gene and codon position in combined-gene datasets (Brandley et al.,

2005). Maximum-likelihood analyses were initiated with random starting trees, and utilized the rapid hill-climbing algorithm (Stamatakis et al., 2007). Support values for clades inferred by ML analyses were assessed with the rapid bootstrap algorithm with 1,000 replicates (Stamatakis et al., 2008). The Akaike information criterion in jModelTest (Posada, 2008) was used to find the model of evolution that best fitted the data for subsequent BI analyses. Bayesian inference analyses were conducted in MrBayes v3.1 (Huelsenbeck and Ronquist, 2001; Ronquist and

Huelsenbeck, 2003) for single-gene and various combinations of datasets. Bayesian analyses were conducted with random starting trees, run for 20,000,000 generations and Markov chains were sampled every 1000 generations. Are we there yet? (AWTY) (Nylander et al., 2008) was used to verify that multiple runs converged. Once convergence was met, 25% of trees were discared as “burn in.” Phylogenies were visualized using FigTree v1.2.3 (Rambaut, 2010).

2.5 Population genetic analyses

Levels of divergence between haplotypes were inferred using uncorrected p-distances in

MEGA v4.0 (Tamura et al., 2007). NETWORK 4.600 (http://www.fluxus-engineering.com) was used to construct haplotype networks (Bandelt et al., 1999). Two separate networks were

23 constructed: one from the combined mitochondrial dataset (16S and cyt b) and the other from the nuclear (RAG1) dataset. ARLEQUIN v3.5 was used to conduct a hierarchical analysis of molecular varience (AMOVA) to assess the most probable geographic configuration (Excoffier et al., 2010). Populations were grouped according to the different geographic hierarchies that matched the lineages recovered in the phylogenetic analyses. Additionally, pairwise genetic divergences between genetic clades were estimated with the fixation index (FST) (Excoffier et al.,

1992). This included information about mitochondrial haplotype frequency and genetic distance between clades.

2.6 Morphological analyses of adult frogs

Specimens examined for this study (Appendix II) were collected from eastern DRC, photographed in life, sampled for tissues (95% ethanol), fixed in 10% buffered formalin, and transferred to 70% ethanol for long-term storage in the herpetology collection at the University of Texas at El Paso Biodiversity Collections (UTEP). Characters examined were chosen from previous taxonomic studies of Leptopelis by Köhler et al. (2006) and Lötters et al. (2005). All measurements were taken with digital calipers under a dissecting microscope and rounded to the nearest 0.1 mm. All body measurements were taken on the right side of the body.

Morphological data consisted of 12 mensural and 15 meristic characters: snout–vent length

(SVL); head width (HW)—measured at widest point of head; head length (HL)—measured at angle of jaw, from posterior edge of mandible to tip of snout; interorbital distance (IO); eye diameter (ED); tympanum diameter (TD); eye–nostril distance (EN); nostril–nostril distance

(NN); tibia length (TL); foot length (FL)— measured from proximal edge of inner metatarsal tubercle to tip of longest toe; hand length (HaL); horizontal length of subocular mark taken at

24 two different points (SbDL)—measured at contact with inferior edge of upper jaw, and

(SbDE)—measured at contact with eye. Meristic data included hand and foot webbing formulas, which were assessed by counting the number of phalanges (recognizable by subarticular tubercles) free of webbing (Glaw and Vences, 1994).

Statistical comparisons of selected measurements were conducted with paired t-tests. To eliminate the effect of size, analysis of covariance (ANCOVA) were conducted with snout–vent length as the covariate (Packard and Boardman, 1999). Principal components analyses (PCA) of mensural data were conducted in Minitab 15 (Minitab ® Statistical Software, State Collage, PA,

USA), and were used to identify patterns of variation in the data. All analyses used the covariance matrix. We conducted one PC analysis on raw data from the 12 mensural characters, and a second analysis with residuals obtained from the ANCOVA, which eliminates the effects of size on the 12 mensural characters.

2.7 Morphological analyses of tadpoles

Two tadpoles of L. cf. modestus (EBG 1669a, b; identification confirmed with DNA sequence data) in Gosner developmental stages 38–39 were fixed and stored in 10% formalin after collection from a stream. One adult male L. cf. modestus was collected near the location where the larvae were present. Measurements and stage development for the tadpole description follow

Grosjean (2001) and are abbreviated as follows: (GS)—Gosner Stage; (SS)—distance from tip of snout to opening of spiracle; (SU)—distance from tip of snout to insertion of upper tail fin;

(SV)—snout–vent length; (VMTH)—distance vent–maximum height of tail; (VT)—vent–tip of tail length; (TL)—total length; (UF)—maximum height of the upper tail fin; (LF)—maximum height of lower tail fin; (HT)—maximum tail height; (BH)—maximum body height; (BW)—

25 maximum body width; (PP)—distance between pupils; (NN)—internarial distance; (ED)—eye diameter; (OWD)—oral disc width.

2.8 Acoustic analyses

Advertisement calls of 22 Leptopelis were recorded from 13 males in plastic containers within two hours of capture (Tumungu and Kalundu), one individual a day after capture

(Kitopo), seven individuals calling on vegetation from Kasanjala, and from eight individuals calling on vegetation (Kahuzi-Biega and Kizuka) with a Zoom H4 Handy Recorder (B&H Photo,

New York, NY, USA). Temperature was measured immediately after each recording to the nearest 1.0°C with a Sunto Core Multifunction Watch (REI, Sumner, WA, USA) in 2008 and a

Fisher Scientific Traceable Digital Hygrometer/Thermometer (Fisher Scientific, Houston, TX,

USA) in subsequent years; temperatures were recorded approximately 1 m above ground (the watch was suspended by a strap to eliminate bias from body heat), where the majority of frogs were perched on vegetation and calling. The most clear and complete single advertisement call from each recording was analyzed using Canary© and Raven Pro 1.4© (Charif et al., 1996,

2010) software. Oscillograms (waveforms), audiospectrograms (sonograms), and results of the

Fast Fourier Transformation (FFT; frequency spectrum) were examined for spectral and temporal characters following the methodology and terminology of Diesmos et al. (2002) and

Brown et al. (2002). Each call series was characterized by the following parameters: (1) call duration (seconds, s); (2) number of pulses per call (N); (3) dominant frequency component

(kHz); (4) low frequency component (kHz); (5) high frequency component (kHz); (6) mean intercall interval (s); and (7) intercall interval range (s). Call data for L. karissimbensis used by

Roelke et al. (2011) were re-analyzed to ensure their accuracy, and to examine call components not examined by those authors. When detailed call data were available for individuals of

26 proximate Leptopelis species in the Albertine Rift (i.e., Roelke et al., 2011), I compared them to my data with the student’s t-test.

27 CHAPTER 3: RESULTS

3.1 Phylogenetic analyses

Two mitochondrial DNA genes (16S = 557 bp; cyt b = 620 bp) and one nuclear gene

(RAG1 = 761 bp) were analyzed for 60 samples of Leptopelis. The total alignment for the dataset included 1938 bp. Nucleotide substitution models selected by jModelTest and alternative models implemented in MrBayes are presented in Table 3.1.1. Topology for the MP, ML and BI

Table 3.1.1 Model parameters specified by jModelTest and alternative models implemented in MrBayes.

Gene and codon Model selected by jModelTest Model implemented in MrBayes position 16S SYM+G GTR+G cyt b codon 1 TIM2ef+I+G K80+I+G codon 2 GTR+G GTR+G codon 3 GTR+G GTR+G RAG1 codon 1 TIM2+G GTR+G codon 2 TIM2+G GTR+G codon 3 HKY+I HKY+I

analyses were identical, with similar strong support values. The MP analysis resulted in 270,313 equally parsimonious trees (length = 1558, CI = 0.641, RI = 0.845); the ML analysis likelihood score was –9201.078390.

Strongly supported clades (> 95% bootstrap) from the analyses (Fig. 3.1.1) included the following monophyletic clades: L. cf. calcaratus from Bizombo and Kalundu, S. Kivu, DRC; L.

Figure 3.1.1 Bayesian phylogenetic tree (16S, cyt b, and RAG1 genes) of treefrogs in the genus Leptopelis. Set of numbers listed at node bases are maximum-parsimony bootstrap support values, maximum-likelihood bootstrap support values and Bayesian inference posterior probability values

29 karissimbensis from Mugaba, Mikenge and Shalashala, S. Kivu, DRC and Mt. Tshiaberimu, N. Kivu,

DRC; L. cf. karissimbensis from Tumungu and Bilimba (Itombwe), S. Kivu, DRC; L. kivuensis from the vicinity of Mugaba, S. Kivu, DRC; L. cf. kivuensis from Mt. Teye in Virunga National Park, N. Kivu,

DRC; L. fiziensis from Kanguli (Itombwe) and Kasanjala (near Fizi), S. Kivu, DRC; L. cf. modestus populations from Kalundu, Kitopo,Mwana River, Kiandjo and Baraka, S. Kivu, DRC; and L. sp. from multiple localities in eastern DRC. Leptopelis karissimbensis was recovered as a well-supported clade that included samples of L. cf. karissimbensis. Within this clade, Leptopelis cf. karissimbensis was recovered as a monophyletic group with strong support (MP bootstrap = 95%, ML bootstrap = 92%, BI posterior probability = 0.99). A well-supported clade of Leptopelis fiziensis was recovered as the sister taxon to the L. cf. karissimbensis/L. karissimbensis clade with moderate support (MP = 76%, ML =

80%, BI = 0.96). Leptopelis kivuensis was recovered as a well-supported clade with two genetically distinct populations (MP = 100%, ML = 97%, BI = 0.99). Leptopelis cf. modestus was recovered as a monophyletic clade with strong support (MP = 100%, ML = 98%, BI = 0.99), and it was recovered as the sister taxon to L. kivuensis with strong support (MP = 98%, ML = 97%, BI = 0.98).

3.2 Population genetic analyses

In order to examine genetic diversity among populations of Itombwe Leptopelis,

specimens were assigned to groups based on well-supported clades indicated in Fig. 3.1.1.

Uncorrected p-distances (Tables 3.2.1, 3.2.2 and 3.2.3) within and among clades of L.

karissimbensis, L. cf. karissimbensis, L. cf. modestus and L. fiziensis are provided for the 16S,

cyt b and RAG1 datasets. Sequence divergences within L. cf. karissimbensis were low (16S:

30 Table 3.2.1 Uncorrected p-distances (16S) within and among selected individuals from the Leptopelis phylogeny.

1 2 3 4 5 6 7 8 9 10 11 12

1 L. cf. kivuensis UTEP 20152

2 L. cf. kivuensis UTEP 20153 0.000

3 L. kivuensis UTEP 20145 0.015 0.018

4 L. karissimbensis UTEP 20126 0.039 0.039 0.041

5 L. karissimbensis UTEP 20112 0.039 0.039 0.041 0.001

6 L. karissimbensis UTEP 20124 0.039 0.039 0.041 0.002 0.002

7 L. cf. karissimbensis UTEP 20561 0.048 0.048 0.050 0.009 0.009 0.009

8 L. cf. karissimbensis UTEP 20560 0.052 0.052 0.054 0.011 0.011 0.010 0.002

9 L. fiziensis UTEP 20466 0.050 0.050 0.056 0.025 0.025 0.025 0.037 0.041

10 L. fiziensis UTEP 20454 0.048 0.048 0.055 0.025 0.025 0.025 0.030 0.030 0.002

11 L. modestus CAS 207807 0.052 0.052 0.054 0.039 0.039 0.039 0.048 0.048 0.052 0.048

12 L. cf. modestus UTEP 20565 0.029 0.029 0.031 0.039 0.039 0.039 0.048 0.052 0.039 0.054 0.043

13 L. cf. modestus UTEP 20586 0.022 0.022 0.025 0.033 0.033 0.033 0.041 0.045 0.045 0.048 0.037 0.006

31 Table 3.2.2 Uncorrected p-distances (cyt b) within and among selected individuals from the Leptopelis phylogeny.

1 2 3 4 5 6 7 8 9 10 11 12

1 L. cf. kivuensis UTEP 20152

2 L. cf. kivuensis UTEP 20153 0.002

3 L. kivuensis UTEP 20145 0.027 0.029

4 L. karissimbensis UTEP 20126 0.115 0.114 0.117

5 L. karissimbensis UTEP 20112 0.114 0.114 0.116 0.003

6 L. karissimbensis UTEP 20124 0.114 0.114 0.116 0.002 0.000

7 L. cf. karissimbensis UTEP 20561 0.116 0.113 0.113 0.005 0.007 0.005

8 L. cf. karissimbensis UTEP 20560 0.116 0.113 0.113 0.007 0.009 0.007 0.01

9 L. fiziensis UTEP 20466 0.109 0.109 0.107 0.038 0.036 0.036 0.034 0.038

10 L. fiziensis UTEP 20454 0.109 0.109 0.107 0.038 0.036 0.036 0.036 0.036 0.002

11 L. modestus CAS 207807 0.178 0.178 0.172 0.173 0.172 0.175 0.173 0.173 0.172 0.172

12 L. cf. modestus UTEP 20565 0.043 0.043 0.049 0.116 0.114 0.114 0.114 0.114 0.107 0.107 0.163

13 L. cf. modestus UTEP 20586 0.043 0.043 0.049 0.116 0.114 0.114 0.114 0.114 0.109 0.109 0.163 0.001

32 Table 3.2.3 Uncorrected p-distances (RAG1) within and among selected individuals from the Leptopelis phylogeny.

1 2 3 4 5 6 7 8 9 10 11 12

1 L. cf. kivuensis UTEP 20152

2 L. cf. kivuensis UTEP 20153 0.000

3 L. kivuensis UTEP 20145 0.002 0.002

4 L. karissimbensis UTEP 20126 0.000 0.000 0.000

5 L. karissimbensis UTEP 20112 0.000 0.000 0.000 0.000

6 L. karissimbensis UTEP 20124 0.001 0.000 0.000 0.000 0.000

7 L. cf. karissimbensis UTEP 20561 0.002 0.001 0.001 0.001 0.001 0.001

8 L. cf. karissimbensis UTEP 20560 0.001 0.001 0.001 0.001 0.001 0.001 0.000

9 L. fiziensis UTEP 20466 0.001 0.001 0.001 0.000 0.000 0.000 0.002 0.002

10 L. fiziensis UTEP 20454 0.002 0.002 0.002 0.001 0.001 0.001 0.001 0.001 0.000

11 L. modestus CAS 207807 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002

12 L. cf. modestus UTEP 20565 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.001 0.003

13 L. cf. modestus UTEP 20586 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.001 0.002 0.000

33 0.1%; cyt b: 0.1–0.2%; RAG1: 0%). The L. karissimbensis clade also had low intraclade sequence divergences (16S: < 0.1–0.2%; cyt b: < 0.1–0.3%; RAG1: 0%). Within the L. cf. modestus clade, sequence divergences were low (16S: < 0.1–0.3%; cyt b: < 0.1%; RAG1: 0%).

Intraclade genetic divergence was low to moderate within the L. fiziensis clade (16S: < 0.1–

0.2%; cyt b: < 0.1–0.2%; RAG1: 0%). Genetic divergence between L. karissimbensis and L. cf. karissimbensis was low (16S: 0.9–1.1%; cyt b: 0.5–0.9%; RAG1: 0.1–0.2%). By comparison, sequence divergences between L. karissimbensis and L. fiziensis were slightly higher (16S: 1.7–

2.2%; cyt b: 3.6–3.8%; RAG1: 0.1%). Sequence divergences between L. fiziensis and L. cf. karissimbensis were low to moderate (16S: 1.8–3.7%; cyt b: 2.8–3.6%; RAG1: 0.1–0.2%).

Sequence divergences between L. cf. modestus and its sister taxon (L. kivuensis) were moderate

(16S: 2.2–2.9%; cyt b: 3.8–4.3%; RAG1: 0.2%). The L. modestus and L. calcaratus clades had relatively high intraclade divergences and this is likely because the L. modestus and L. calcaratus clades represent species complexes.

In the AMOVA (Table 3.2.4), the greatest genetic variance among groups was found with seven groups ([karissimbensis] [cf. karissimbensis] [kivuensis] [cf. kivuensis] [fiziensis]

[modestus] [L. cf. modestus]; FCT = 0.828, P < 0.05). Mitochondrial and nuclear haplotype networks constructed for L. cf. karissimbensis and L. karissimbensis (Fig. 3.2.1) identified geographic variation within and among clades. Results from the mitochondrial network indicated L. cf. karissimbensis is separated by two mutations from L. karissimbensis. Based on the RAG1 dataset, L. cf. karissimbensis is separated only by one mutation from L. karissimbensis populations.

Table 3.2.4 Hierarchical analysis of molecular variance among examined lineages of Leptopelis (*P < 0.05; **P < 0.01; ***P < 0.001)

Groups FST FSC FCT % among % within populations populations All populations 0.828 82.91 17.1

2 Groups [karissimbensis, cf. karissimbensis, 0.862*** 0.734*** 0.481 38.13 13.76 fiziensis, modestus] [cf. modestus, kivuensis, cf. kivuensis] 2 Groups [karissimbensis, cf. karissimbensis, 0.847*** 0.809*** 0.199 64.82 15.24 fiziensis, modestus, kivuensis, cf. kivuensis] [cf. modestus] 2 Groups [karissimbensis, cf. karissimbensis, 0.863*** 0.823*** 0.228 63.57 13.61 fiziensis, cf. modestus, kivuensis, cf. kivuensis] [modestus] 3 Groups [karissimbensis, cf. karissimbensis, 0.862*** 0.689*** 0.556** 30.53 13.78 fiziensis] [cf. modestus, kivuensis, cf. kivuensis] [modestus] 3 Groups [karissimbensis, cf. karissimbensis, 0.854*** 0.710*** 0.495** 35.84 14.59 fiziensis, modestus] [cf. modestus] [kivuensis, cf. kivuensis] 3 Groups [karissimbensis, cf. karissimbensis] 0.845*** 0.722*** 0.442* 40.26 15.54 [fiziensis, modestus] [cf. modestus, kivuensis, cf. kivuensis] 3 Groups [karissimbensis, cf. karissimbensis, 0.831*** 0.820*** 0.060 77.15 16.84 kivuensis, cf. kivuensis] [fiziensis, modestus] [cf. modestus] 4 Groups [karissimbensis, cf. karissimbensis] 0.838*** 0.672*** 0.506* 33.21 16.16 [fiziensis, modestus] [cf. modestus] [kivuensis, cf. kivuensis] 4 Groups [karissimbensis, cf. karissimbensis, 0.853*** 0.623*** 0.612** 24.13 14.6 fiziensis] [modestus] [cf. modestus] [ kivuensis, cf kivuensis] 4 Groups [karissimbensis, cf. karissimbensis, 0.839*** 0.809*** 0.128 70.59 16.58 kivuensis, cf. kivuensis] [fiziensis] [modestus] [cf. modestus] 5 Groups [karissimbensis, cf. karissimbensis] 0.839*** 0.460** 0.702** 13.69 16.04 [kivuensis, cf. kivuensis] [fiziensis] [modestus] [cf. modestus] 5 Groups [karissimbensis, fiziensis] [cf. 0.837*** 0.671** 0.505* 33.23 16.25 karissimbensis] [cf. modestus] [kivuensis, cf. kivuensis] [modestus] 6 Groups [karissimbensis] [fiziensis] [cf. 0.830*** 0.683** 0.463 36.69 16.95 karissimbensis] [cf. modestus] [kivuensis, cf. kivuensis] [modestus]

7 Groups [karissimbensis] [fiziensis] [L. cf. 0.851*** 0.420** 0.828* 79.35 11.51 karissimbensis] [cf. modestus] [kivuensis] [cf. kivuensis] [modestus ]

Figure 3.2.1 Haplotype networks for L. cf. karissimbensis and L. karissimbensis calculated from mitochondrial (A) (16S and cyt b) and nuclear (B) (RAG1) datasets in the program NETWORK. Colors used in the networks correspond to colors used in (Figs 2.1 and 3.1.1).

3.3 Itombwe Leptopelis morphology, coloration and calls

Morphology.—Mensural data for adult specimens of L. cf. karissimbensis, L. karissimbensis, L. cf. modestus and L. fiziensis are shown in Table 3.3.1. There are no significant size differences between females of: L. karissimbensis and L. cf. modestus (t-test, P = 0.76). Statistical t-tests could not be conducted with female L. fiziensis or L. cf. karissimbensis because there was only one female available for each taxon. Among male Leptopelis, there were significant size differences between L. fiziensis and L. cf. modestus (t-test, P = 0.01), L. karissimbensis and L. cf. modestus (t-test, P = 0.03), and L. karissimbensis and L. fiziensis (t-test, P = 0.001). There was no significant difference in size between male L. cf. karissimbensis and L. karissimbensis (t-test,

P = 0.56) or between male L. cf. karissimbensis and L. cf. modestus (P = 0.43). The females of of two species are significantly larger than conspecific males (t-test, L. karissimbensis: P =

0.001; L. cf. modestus: P = 0.013). Webbing formulas for each species are similar, with only minor differences between species (Table 3.3.2). Univariate statistics (ANOVA and ANCOVA) indicated several mensural characters that were significantly different among Itombwe

Leptopelis (Table 3.3.3).

Coloration.—Adult male L. cf. karissimbensis have gray vocal sacs; blue coloration on the throat was observed in L. karissimbensis and L. cf. modestus. However the blue vocal sacs of L. cf. modestus were grayish blue when compared to noticeably darker blue vocal sacs of L. karissimbensis. Dorsal coloration for adult L. cf. karissimbensis ranged from gray to a light

Table 3.3.1 Morphometric data (in mm) for specimens of L. cf. karissimbensis, L. karissimbensis, L. cf. modestus and L. fiziensis of both sexes. For abbreviations, see Materials and Methods.

L. cf. SVL HW HL IO ED TD EN NN TL FL HaL SbDL SbDE karissimbensis Males (N = 4) mean 35.6 14.5 13.4 7.6 3.9 2.1 2.8 3.6 19.2 18.5 13.1 2.2 1.1

SD ±3.5 ±1.5 ±1.4 ±0.7 ±0.5 ±0.5 ±0.4 ±0.3 ±2.0 ±1.8 ±1.7 ±0.3 ±0.3

range 31.9–38.9 12.8–16.4 12.1–14.8 6.8–8.1 3.7–4.5 1.8–2.3 2.5–3.1 3.3–4.3 17.4–20.6 16.9–19.4 11.7–13.4 1.9–2.5 0.8–1.9

Females (N = 1) 48.7 19.8 17.8 10.1 4.8 2.8 3.6 5.1 24.6 24.3 16.7 2.6 1.1

Leptopelis karissimbensis Males (N = 19) mean 36.9 14.3 13.7 7.4 4.1 1.9 2.9 3.7 18.3 17.7 12.4 2.1 1.7

SD ±1.4 ±1.3 ±1.3 ±0.6 ±0.5 ±0.4 ±0.4 ±0.7 ±1.7 ±1.6 ±1.1 ±0.4 ±0.4

range 32.9–42.6 13.1–16.1 12.9–15.7 6.3–8.1 3.4–4.6 1.6–2.3 2.5–3.3 2.8–4.3 16.8–19.2 16.1–18.8 10.2–13.1 1.5–2.5 1.3–2.7

Females (N =5) mean 44.8 20.0 18.1 9.7 5.4 2.6 3.9 4.6 23.4 22.7 15.7 2.5 1.7

SD ±3.4 ±2.7 ±2.3 ±1.0 ±0.6 ±0.5 ±0.4 ±0.4 ±3.1 ±3.1 ±1.8 ±0.5 ±0.4

range 44.7–54.6 16.7–23.2 16.1–21.9 8.3–10.5 4.8–5.8 2.4–3.1 3.4–4.4 4.2–5.0 19.7–26.8 19.1–25.9 15.0–17.8 2.0–3.4 1.2–2.6

Table 3.3.1 continued

L. cf. modestus SVL HW HL IO ED TD EN NN TL FL HaL SbDL SbDE

Males (N = 17) mean 34.9 14.1 13.1 7.5 4.3 2.3 2.8 3.7 18.7 17.1 12.2 1.9 1.6 SD ±3.8 ±1.6 ±1.6 ±0.8 ±0.6 ±0.5 ±0.5 ±0.4 ±2.1 ±1.9 ±1.2 ±9.1 ±9.1 0.8– range 34.7–37.8 14.1–15.0 12.7–14.1 6.9–8.1 3.8–4.6 1.9–2.6 2.7–2.9 3.2–3.9 17.2–20.5 16.8–18.4 11.8–13.1 1.3–2.6 2.2 Females (N = 4) mean 49.2 19.9 17.9 10.1 5.0 2.7 4.0 4.6 25.4 24.7 16.5 2.6 2.0 SD ±5.2 ±3.3 ±2.8 ±1.3 ±0.6 ±0.3 ±0.4 ±0.5 ±3.8 ±3.6 ±1.9 ±0.4 ±0.4 1.6– range 45.1–55.6 18.6–21.8 16.3–19.0 9.2–10.6 4.4–5.5 2.4–3.1 3.8–4.4 4.3–4.8 23.7–27.3 23.3–27.2 13.9–18.5 2.1–3.8 2.3 Leptopelis fiziensis Males (N = 11) mean 31.7 12.7 12.3 7.1 3.7 2.0 2.8 3.6 17.0 15.1 10.5 1.5 1.3 SD ±3.4 ±1.4 ±1.4 ±0.7 ±0.5 ±0.4 ±0.3 ±0.3 ±1.7 ±1.7 ±1.2 ±0.3 ±0.4 1.0– range 29.7–34.8 11.9–13.7 11.5–13.6 6.5–7.7 3.3–4.1 1.8–2.1 2.7–3.1 3.3–3.9 16.4–17.5 14.2–15.8 10.1–11.3 1.2–1.9 1.7

Females (N =1) 34.9 13.6 13.1 7.4 4.1 2.3 2.8 3.5 16.7 15.1 10.8 1.6 1.3

Table 3.3.2 Hand (manus) and foot (pes) webbing formulas for L. cf. karissimbensis, L. karissimbensis, L. cf. modestus and L. fiziensis.

Manus 1 2i 2e 3i 3e 4 L. cf. karissimbensis 1.75 1.75 1.25–1.50 1.75–2.00 1.75–2.00 1.50–1.75 L. karissimbensis 1.75 1.75 1.00–1.5.0 2.00–2.25 2.00 1.50 L. cf. modestus 1.50–1.75 1.50–2.00 1.00–1.50 2.00–2.50 1.75–2.00 1.25–1.50 L.fiziensis 1.75 1.75 1.25–1.50 1.75–2.00 1.75 1.00–1.25

Pes 1 2i 2e 3i 3e 4i 4e 5 L. cf. karissimbensis 0.75–1.00 1.00–1.25 0.75–1.00 1.75–2.00 0.75 2.00 1.75–2.00 0.75–1.00 L. karissimbensis 0.75–1.00 1.00 0.50 2.00 0.50 2.00–2.25 2.00 0.50–0.75 L. cf. modestus 0.50–0.75 0.75–1.00 0.25–0.50 1.50–1.75 0.50–0.75 1.75–2.00 1.50–2.00 0.5–1.00 L.fiziensis 1.00 1.00–1.25 0.25–0.50 1.75–2.00 0.50–0.75 2.00 1.75 0.75–1.00

Table 3.3.3 P–values obtained from one way ANOVA and ANCOVA of mensural data for Leptopelis cf. karissimbensis, L. karissimbensis, L. cf. modestus and L. fiziensis.

Characters P-value P-value ANOVA ANCOVA SVL 0.006 – HW 0.001 0.000 HL 0.008 0.005 ID 0.005 0.000 ED 0.001 0.003 TD 0.060 0.045 EN 0.057 0.062 NN 0.005 0.016 TL 0.000 0.008 FL 0.000 0.009 HaL 0.003 0.001 SbDL 0.012 0.080 SbDE 0.086 0.995

40 grayish-brown, with slightly distinct brown or gray triangular patterns between the eyes and the sacrum; brown or gray crossbar-like bands on the limbs; and white, brown or gray spots on the lateral sides of the body. A few individuals did not show any band pattern on the limbs. All individuals had a round or square-like white, subocular mark, although not as distinct or large as it was in L. karissimbensis. Dorsal coloration for adult L. karissimbensis ranged from gray to a dark reddish-brown (most individuals were brown or dark brown), with well-defined, dark brown to black, triangular patterns between the eyes and the sacrum; dark brown, crossbar-like bands on the limbs; and irregular white and brown lines and/or spots on the flanks. Some L. karissimbensis had green and yellow spots/blotches on the flanks and dorsum (photograph D in

Fig. 3.3.1). All L. karissimbensis had two round or square-like, white, subocular marks. Dorsal coloration for adult L. cf. modestus ranged from a light cream to a dark reddish brown, with dark brown, well defined, rectangular patterns between the eyes and sacrum; dark brown, rectangular and reticulate banding on the dorsum; dark brown, well-defined, rectangular bars on the limbs

(some individuals did not have a distinct dorsal pattern, instead having brown spots or irregular, faded blotches); and white or cream-colored irregular blotches or spots along the flanks (Fig.

3.3.2). Some of the L. cf. modestus specimens had dark brown or black tubercles on their dorsums. Most individuals had white triangular, subocular marks that were noticeably larger at their contact with the eye (SbDE) when compared to L. cf. karissimbensis (Tables 3.3.1 and

3.3.3). Dorsal coloration for adult L. fiziensis ranged from a light cream or gray to light brown

(Fig. 3.3.3). Some individuals had brown reticulate patterns on the dorsum; other L. fiziensis lacked a dorsal pattern, instead having brown or black tubercles; a few specimens had fluorescent lime-green blotches or spots on their dorsums; brown rectangular bars were present on some L. fiziensis and absent from others (the brown limb bands were never as thick or

A B

C D

Figure 3.3.1 Leptopelis cf. karissimbensis adult males (UTEP 20561, 34.4 mm snout–vent length [SVL]) (A) and (UTEP 20559, 38.8 mm SVL) (B) in life; Leptopelis karissimbensis adult males (UTEP 20126, 38.4 mm SVL) (C) and (UTEP 20112, 42.6 mm SVL) (D) in life.

A B

C D

Figure 3.3.2 Leptopelis cf. modestus adult males (UTEP 20583, 37.6 mm snout–vent length [SVL]) (A), (UTEP 20578, 35.4 mm SVL) (B), (UTEP 20575, 34.6 mm SVL) (C) and (UTEP 20566, 34.3 mm SVL) (D) in life.

A B

C D

Figure 3.3.3 Leptopelis fiziensis adult males (UTEP 20459, 34.8 mm snout–vent length [SVL]) (A), (UTEP 20462, 29.7 mm SVL) (B), (UTEP 20458, 31.3 mm SVL) (C) and (UTEP 20454, 30.6 mm SVL) (D) in life.

conspicuous on L. fiziensis compared to the other Itombwe Leptopelis taxa); and white, cream and/or gray spots and blotches were present on the flanks of most individuals. All L. fiziensis had small, white triangular subocular marks.

Multivariate statistics.—A total of 46 male Leptopelis (four L. cf. karissimbensis, 19 L. karissimbensis, 17 L. cf. modestus and 11 L. fiziensis) were used for the statistical analyses. Two analyses were completed: one with mensural data (only males) (Table 3.3.4 and Fig. 3.3.4) and one with raw mensural data regressed against snout–vent length (females included) (Table 3.3.5 and Fig. 3.3.5). For the PC analysis with mensural data, the first principal component axis accounted for 89.9 % of the total variance in the data (Table 3.3.4 and Fig. 3.3.4). The PCA with raw mensural data indicated morphological differences between several Leptopelis. The first PC axis loaded positively for SVL; a positive value on the first PC axis indicated larger SVL (Fig.

3.3.4). The second PC axis was positively correlated with SVL and negatively correlated with

TL and FL; negative values on the second axis indicated animals with longer tibias and feet. The third PC axis (not shown) was positively correlated with HaL, indicating that animals with positive values on this axis have longer hands. For the analysis with raw morphometric data regressed against SVL, the first two principal component axes explained 62.3 % of the total variance (Table 3.3.5 and Fig. 3.3.5). The first PC axis loaded positively for RESI8 (SVL/TL) and RESI9 (SVL/FL); specimens with larger values on the first axis were associated with longer legs, relative to body size (Fig. 3.3.5). The second PC axis loaded negatively for RESI9

(SVL/FL) and RESI10 (SVL/HaL); negative values on this axis were correlated with larger feet and hands relative to body size. The third PC axis (not shown in Fig. 3.3.5) was negatively correlated with RESI8 (SVL/TL) and positively correlated with RESI10 (SVL/HL). Smaller values on the third axis were correlated with larger TL and shorter snouts relative to body size.

Table 3.3.4 Principal components analysis comparing populations of Albertine Rift Leptopelis with raw mensural data. Eigenvalues, percent variance, cumulative variance, and loadings are shown for the first three principal components.

Variable PC1 PC2 PC3

SVL 0.733 0.511 0.081

HW 0.284 −0.072 0.294

HL 0.246 0.071 0.322

ID1 0.116 0.019 0.128

ED 0.061 −0.072 0.194

TD 0.022 −0.095 0.106

EN 0.02 0.002 0.013

NN 0.052 0.004 0.062

TL 0.252 −0.730 0.341

FL 0.396 −0.396 −0.378

HaL 0.277 −0.116 −0.678

SbDL 0.050 0.071 −0.102

SbDE 0.021 0.082 0.069

Eigenvalue 11.516 0.860 0.417

Proportion 0.837 0.062 0.030

Cumulative 0.837 0.899 0.929

Table 3.3.5 Principal components analysis comparing populations of Albertine Leptopelis with raw mensural data regressed against snout–vent length. Eigenvalues, percent variance, cumulative variance, and loadings are shown for the first three principal components.

Variable PC1 PC2 PC3

RESI1 (SVL/HW) 0.223 0.218 0.709

RESI2 (SVL/HL) 0.285 0.261 0.414

RESI3 (SVL/IO) −0.015 0.105 0.152

RESI4 (SVL/ED) 0.150 0.184 0.141

RESI5 (SVL/TD) 0.135 0.109 0.048

RESI6 (SVL/EN) 0.107 0.010 0.011

RESI7 (SVL/NN) 0.039 0.047 0.126

RESI8 (SVL/TL) 0.693 0.429 −0.473

RESI9 (SVL/FL) 0.539 −0.483 0.034

RESI10 (SVL/HaL) 0.295 −0.659 0.112

RESI11 (SVL/SbDL) −0.009 −0.126 0.160

RESI12 (SVL/SbDE) −0.054 0.061 −0.038

Eigenvalue 1.264 0.420 0.325

Proportion 0.467 0.156 0.120

Cumulative 0.467 0.623 0.743

Figure 3.3.4 Scatter plot of the first and second principal component scores for the analysis with raw mensural data (males only). Comparisons were done among the putative species identified in the molecular phylogeny. The letter P indicates the paratype of L. fiziensis.

Figure 3.3.5 Scatter plot of the first and second principal component scores for the analysis with raw mensural data regressed against snout–vent length. Comparisons were done among the putative species identified in the molecular phylogeny. The letters P and H indicate the paratype and the holotype (L. fiziensis) respectively; the letter L indicates the lectotype for L. modestus.

Acoustic analyses.— Calls of L. cf. karissimbensis were recorded for three males (Table 3.3.6 and Fig. 3.3.6). The advertisement call of L. cf. karissimbensis is a single pulsed clack with relatively short intercall intervals. None of the L. cf. karissimbensis emitted a second pulse. Call duration (L. karissimbensis, P = 0.02; L. cf. modestus P = 0.02; L. fiziensis, P = 0.03), peak frequency (L. karissimbensis, P < 0.005; L. cf. modestus, P = 0.05) and mean intercall interval

Table 3.3.6 Call characteristics measured from recordings of L. cf. karissimbensis, L. karissimbensis, L. cf. modestus and L. fiziensis.

Taxon Museum Date Time Temperature Mass Call Number Dominant Low High frequency Mean intercall Intercall interval No. (°C) (g) Duration (s) of Pulses frequency frequency component(kHz) interval (s) range (s) component component (kHz) (kHz) L. cf. UTEP 4 Jan 2011 19:36 17.8 2.9 0.19 1 1.91 1.87 2.43 3.84 3.21–4.50 karissimbensis 20562 L. cf. UTEP 2 Jan 2011 20:40 17.6 3.5 0.12 1 1.87 1.68 2.06 2.90 2.74–3.01 karissimbensis 20561 L. cf. UTEP 3 Jan 2011 20:48 15.7 5.1 0.32 1 1.90 1.68 2.43 3.72 3.12–4.08 karissimbensis 20559 Mean 3.8 0.21 1.89 1.74 2.30 3.48 L. karissimbensis UTEP 26 May 19:37 15.6 3.9 0.95 1–2 1.47 1.12 1.50 10.54 8.72–12.38 20121 2009 L. karissimbensis UTEP 6 Jun 2008 19:40 14 4.0 0.34 1 1.49 1.12 1.50 13.86 12.48–15.25 20117 L. karissimbensis UTEP 6 Jun 2008 20:02 14 3.7 0.36 1 1.49 1.12 1.50 9.94 8.38–11.50 20118 L. karissimbensis UTEP 5 Jun 2008 20:02 13 4.4 0.34 1 1.12 1.10 1.50 18.76 15.25–22.22 20113 L. karissimbensis UTEP 6 Jun 2008 20:20 13 4.3 0.46 1 1.50 1.12 1.50 9.83 8.21–11.56 20119 L. karissimbensis UTEP 5 Jun 2008 20:42 13 2.9 0.93 1–2 1.50 1.12 1.50 13.86 12.48–15.28 20116 L. karissimbensis UTEP 5 Jun 2008 21:28 13 7.1 0.20 1 1.12 1.10 1.51 11.44 9.23–14.41 20114 L. karissimbensis UTEP 5 Jun 2008 21:35 13 4.6 0.89 1–2 1.25 1.10 1.50 17.43 9.71–30.12 20115 Mean 4.3 0.55 1.36 1.11 1.50 17.57

Table 3.3.6 continued

Dominant High Low frequency Mean Intercall Temperature Call Duration Number of frequency frequency Taxon ID Date Time Mass (g) component intercall interval (°C) (s) Pulses component component (kHz) interval (s) range (s) (kHz) (kHz) 26 Jun L. cf. modestus UTEP 20578 18:15 22 3.2 0.85 3–5 1.68 1.61 1.75 6.77 3.76–5.64 08 22 Dec L. cf. modestus UTEP 20567 18:40 18.5 4.1 0.61 3 1.68 1.50 1.69 11.20 7.45–23.95 10 22 Dec L. cf. modestus UTEP 20564 19:20 19.6 4.0 0.42 3–4 1.50 1.31 1.69 10.73 8.39–17.81 10 22 Dec L. cf. modestus UTEP 20570 19:30 19.2 2.8 0.51 3 1.68 1.50 1.69 9.20 7.26–11.91 10 Mean 3.5 0.59 1.68 1.48 1.70 9.47 13 Jan L. fiziensis UTEP 20464 19:25 21.1 2.1 0.52 2 2.06 2.06 2.23 3.19 2.45–4.39 2012 13 Jan L. fiziensis UTEP 20463 19:37 21 2.3 0.47 2–3 1.89 1.75 2.07 3.26 2.61–3.91 2012 13 Jan L. fiziensis UTEP 20459 19:46 20.9 2.4 0.59 2 1.89 1.75 2.06 3.53 2.75–4.32 2012 13 Jan L. fiziensis UTEP 20462 19:54 20.9 2.6 0.57 2 1.89 1.75 2.06 3.13 2.54–3.97 2012 13 Jan L. fiziensis UTEP 20458 20:08 20.9 3.1 0.55 2–3 1.89 1.75 2.06 3.19 2.70–3.44 2012 13 Jan L. fiziensis UTEP 20461 20:20 20.8 2.5 0.41 2 1.89 1.75 2.06 3.24 2.92–3.56 2012 13 Jan L. fiziensis UTEP 20460 20:30 20.8 3.0 0.63 2 2.06 1.89 2.23 3.63 3.61–4.41 2012 Mean 2.5 0.53 1.93 1.81 2.11 3.31

(L. karissimbensis, P = 0.03; L. cf. modestus, P = 0.03) were significantly different between L. cf. karissimbensis and other species. Peak frequency (P = 0.49) and mean intercall interval (P =

0.62) were not statistically significant between L. cf. karissimbensis and L. fiziensis. Calls of L. karissimbensis were recorded from eight males (Table 3.3.6, Fig. 3.3.6). A complete advertisement call of L. karissimbensis typically lasts almost a full second. Calls from L. karissimbensis had relatively long intercall intervals. The call typically consisted of a single pulsed clack, although some males occasionally emitted a second pulse (Table 3.3.6, Fig. 3.3.6).

Calls of L. cf. modestus were recorded from four males (Table 3.3.6, Fig. 3.3.7). The advertisement call of L. cf. modestus is a single pulsed, clack, repeated in rapid succession. The number of pulses per call ranged from three to five pulses. Call duration (L. cf. karissimbensis,

P = 0.02) peak frequency (L. karissimbensis, P < 0.005; L. cf. karissimbensis, P = 0.05; L. fiziensis, P = 0.04) and mean intercall interval (L. cf. karissimbensis, P = 0.03; L. fiziensis, P =

0.03) were significantly different between L. cf. modestus and other species. Call duration (P =

0.73) and mean intercall interval (P = 0.15) were not significantly different between L. cf. modestus and L. karissimbensis; call duration (P = 0.56) was not significantly different between

L. cf. modestus and L. fiziensis. Calls of L. fiziensis were recorded from seven males (Table

3.3.6, Fig. 3.3.7). Leptopelis fiziensis typically emits a double-pulsed clack with an occasional third pulse. Peak frequency (P < 0.005) and mean intercall interval (P = 0.03) were significantly different between L. fiziensis and L. karissimbensis (all other comparisons are shown above).

Call duration (P = 0.83) was not significantly different between L. fiziensis and L. karissimbensis.

A o

Br o te u

Figure 3.3.6 Waveform diagram and spectrogram of calls of L. cf. karissimbensis (A) (UTEP 20559) and (B) L. karissimbensis (UTEP 20116). MU and kU on the y-axis on r audiospectrograms represent Raven Pro 1.4© amplitude units. t te h x e 53

t r A o u

rB o te u x

Figure 3.3.7 Waveform diagram and spectrogram of calls of L. cf. modestus (A) (UTEP 20578) and (B) Leptopelis fiziensis (UTEP 20458). MU and kU on the y-axis on audiospectrograms r represent Raven Pro 1.4© amplitude units. t te h x e 54

t r 3.4 Description of Leptopelis anebos

Leptopelis anebos sp. nov. Young Itombwe Forest Treefrog (Figs. 3.3.1 and 3.4.1; Tables 3.3.1, 3.3.2 and 3.3.6)

Holotype.—UTEP 20561 (field no. ELI 765), adult male, 2 January 2011, DRC, South Kivu

Province, Tumungu, 3.56455°S, 28.66619°E (datum = WGS84), 1897 m, collected by E.

Greenbaum, C. Kusamba, W. M. Moninga and M. M. Aristote (Figs. 3.3.1 and 3.4.1).

Paratypes.—UTEP 20559–60, 20562 (field nos. ELI 736, 782, 800), three adult males collected by the same collectors in the same locality on 3 January 2011, 4 January 2011, and 8

January 2011, respectively; UTEP 20563 (field no. CFS 909) adult female, October 2010, South

Kivu Province, Bilimba, 3.4610°S, 28.7829°E (datum = WGS84), 2227 m, collected by M. M.

Aristote.

Diagnosis.—A medium-sized, arboreal Leptopelis with (1) adult male SVL 32–39 mm; (2) head wider than long; (3) eye relatively large with horizontal eye diameter almost twice distance from nostril to anterior corner of eye; (4) dorsal snout shape rounded; (5) tympanum distinct, its horizontal diameter slightly more than half eye diameter; (6) dorsal skin finely granular, with small scattered tubercles; (7) feet one half webbed, hands one third webbed; (8) well-developed subarticular tubercles and terminal discs present on all toes and fingers; (9) pectoral glands distinct in males; (10) lack of heel spurs; (11) male color in life dorsally gray or light creamy brown, with light gray/brown markings; white blotch present below eye, laterally cream with no pattern or flecking; ventrally creamy white, some specimens with a few scattered brown spots;

Figure 3.4.1 Dorsal (A), ventral (B) and lateral view of the head (C) of the preserved male holotype of Leptopelis anebos (UTEP 20561, 34.4 mm SVL).

56 iris brown with fine black reticulation; (12) advertisement call is a single clack/pulsed note, with a dominant frequency between 1.87–1.91 kHz.

Comparisons.—A medium-sized Leptopelis that is distinguished from most other congeners by a head wider than long; eye relatively large with horizontal eye diameter almost twice distance from nostril to anterior corner of eye; dorsal snout shape rounded; tympanum distinct with horizontal diameter slightly more than half eye diameter; feet one half webbed and hands one third webbed; well-developed subarticular tubercles and terminal discs present on all toes and fingers; pectoral glands distinct in males; lack of heel spurs; and male dorsal color in life gray or light creamy brown. Detailed comparisons between L. anebos and conspecifics in the AR are provided below.

Leptopelis anebos is most similar to L. karissimbensis (Table 3.3.1, Fig. 3.3.1). The webbing formulas for both species are similar with minor differences. The webbing formulas for

L. anebos and L. karissimbensis are shown in Table 3.3.2. The new species can be distinguished from L. karissimbensis by having more webbing on the inner third digit of the hand (1.75–2.00 vs. 2.00–2.25 in L. karissimbensis); dorsal coloration (gray or creamy brown vs. gray to reddish brown, but most specimens are brown to dark brown in L. karissimbensis); vocal sac color, which is grayish brown in L. anebos (blue in L. karissimbensis), significantly longer tibias (P =

0.001) and feet (P = 0.003), larger hands (P = 0.005), a higher dominant frequency component in the advertisement call (Table 3.3.6 and Fig. 3.3.6), and by occurring in mid- to high elevation montane rainforests and savannas (high elevation forest edges, meadows or swamps in L. karissimbensis). The new species can be distinguished from L. modestus by dorsal and vocal sac coloration; the former is gray or light creamy brown dorsally with a grayish-brown vocal sac, whereas the latter species is brown dorsally with a green or blue vocal sac. Geographically, L.

57 anebos is restricted to the Itombwe Plateau, whereas L. modestus is most likely restricted to the

Cameroonian highlands (Schiøtz, 1999, but see Laurent, 1973). The new species is most easily distinguished from L. fiziensis by advertisement call; L. anebos emits one clack/pulse (L. fiziensis emits 2–3 clacks/pulses). Morphologically, L. anebos can be differentiated from L. fiziensis by having less webbing on the fourth digit of the hand (1.50–1.75 vs. 1.00–1.25 in L. fiziensis) and second digit of the foot (0.75–1.00 vs. 0.25–0.50 in L. fiziensis), and dorsal coloration (gray or light creamy brown vs. brown, tan or dark grayish brown in L. fiziensis). Leptopelis anebos inhabits mid- to high elevation montane rainforests and savannas (low- to mid-elevation transitional forest in L. fiziensis) (Greenbaum et al., 2012). Leptopelis anebos is most easily distinguished from L. kivuensis by male advertisement call; L. anebos emits only one clack/pulse and has a mean dominant frequency of 1.89 kHz, whereas L. kivuensis emits up to 12 clacks/pulses per call and has a mean dominant frequency component of 1.48 kHz.

Morphologically, L. anebos and L. kivuensis differ in dorsal coloration (gray or light creamy brown vs. tan, reddish brown or lime green in L. kivuensis); by having more webbing on the inner third digit of the hand (1.75 vs. 2.00–2.25 in L. kivuensis) and interior of the fourth digit of the foot (2.00 vs. 2.25–2.50 in L. kivuensis), and by the presence of distinct subocular marks

(most L. kivuensis specimens lacked distinct subocular marks) (Roelke et al., 2011). The new species can be distinguished from L. mackayi by having complete toe webbing (L. mackayi lacks webbing on the first and exterior second digit of the foot), and male advertisement call (one clack/pulse vs. 12 clacks/pulses in L. mackayi) (Köhler et al., 2006). The new species can be distinguished from L. christyi by its dorsal pattern and coloration (gray or brown crossbars vs. a dark brown triangle and green or cream lateral band in L. christyi), by having more webbing on the interior of the fourth digit of the foot (2.00 vs. 2.25–2.50 in L. christyi), and presence of a

58 subocular mark (absent in L. christyi) (Schiøtz, 1975). The new species can be distinguished from L. calcaratus morphologically; males of the former species have a shorter snout (mean 13.4 mm vs. 14.5 mm in L. calcaratus), a smaller, less distinct tympanum (mean = 2.1 mm vs. 3.0 mm in L. calcaratus), lack of heel spurs (present in L. calcaratus), and advertisement call (one clack/pulse per call vs. multiple clacks/pulses per call in L. calcaratus) (Perret, 1966; Schiøtz,

1999).

Description of the holotype.—Adult male (Figs. 3.3.1 and 3.4.1); body slender, head is noticeably wider than body; snout is short and rounded in lateral view; tongue longer than wide; nostrils visible from dorsal view; canthus rostralis slightly curved; eye relatively large, about 1.5 times distance from eye to nostril; skin finely granular with a few larger tubercles present on dorsal surface; skin on ventral surfaces granular; pectoral glands present; white spots/blotches present on lateral surface of skin; hind limbs relatively long with tibia length reaching a little more than half SVL; hand webbing formula 1(1.75) 2i(1.75), 2e(1.50), 3i(1.75), 3e(2.00), 4(1.25); relative length of fingers: I < II < IV < III; outer metatarsal tubercle absent, inner metatarsal tubercle small, ovoid; palmar surfaces have tubercles; well-developed subarticular tubercles under all fingers; tips of all fingers with discs, each about 1.5 times width of phalanges; foot webbing formula 1(0.75), 2i(1.00), 2e(0.75), 3i(1.75), 3e(0.75), 4i(2.00), 4e(1.75), 5(1.00); relative length of toes, I < II < III < V < IV; outer metatarsal tubercle absent; inner metatarsal tubercles well developed, round subarticular tubercles under all toes; tips of toes end in round discs, each about

1.5 times wider than width of phalanges.

Coloration of holotype in preservative.—Dorsal surface is brown with no brown or gray bands on the dorsum; three slightly visible brownish bands are present on the forearms, and four slightly visible gray bands on the hind limbs; small white blotches present on the lateral surface;

59 small diffused, brown or black spots present on dorsal tubercles; small, cream subocular blotch is rectangular in appearance, which is slightly wider at its contact with the upper lip than at its contact with the bottom of the eye; brownish triangular pattern in the sacral region is slightly visible; tympanum is the same color as the dorsal surface; tip of the snout is dark brown; ventral surface is cream with no flecks or spots; vocal sac is brownish gray.

Coloration of holotype in life.—Dorsal coloration is grayish-cream with black or brown round tubercles; cream flecks scattered on the dorsum; a very diffuse and indistinct brownish reticulate pattern behind the eyes; canthal region is light brown/gray; cream subocular blotch, rectangular in appearance; triangular pattern in sacral region slightly visible; tympanum is the same color as the dorsal surface; ventral surface is white with grayish-blue flanks; vocal sac is gray.

Variation.—Morphological variation among the five specimens of L. anebos is shown in Table

3.3.1. Tympanum is nearly round in most specimens of L. anebos, but is slightly oval in UTEP

20561 and 20563. Dark brown to black dorsal spots and grayish brown bands may be more or less distinct in different individuals. White spots that are present on the holotype are not present in UTEP 20562. All L. anebos exhibit slightly distinct brown blotches on their dorsal surfaces with the exception of UTEP 20560, which exhibits dark brown and distinct bands on the dorsal surface of the limbs. A dark brown triangular pattern between the eyes is distinct in UTEP

20560.

Distribution and natural history.—Currently, L. anebos is only known from montane forests of the Itombwe Plateau in the vicinity of Tumungu, and a stream running through savanna (near montane forest) in the nearby village of Bilimba at elevations between 1897–2227 m (Fig. 2.1).

The type locality at Tumungu is a mosaic of montane grassland and forest, which was illustrated by Greenbaum and Kusamba (2012:fig.2). Adult male frogs at Tumungu were collected in

60 montane forest 1–2 m above the ground from plants and trees near streams. Other anurans collected from montane forest at the type locality included Amietophrynus cf. camerunensis

(Bufonidae), Hyperolius leucotaenius (Hyperoliidae), Phrynobatrachus acutirostris and

Phrynobatrachus sp. (Phrynobatrachidae), and Xenopus itombwensis (Pipidae). The adult female was collected from Bilimba at the edge of a stream in savanna, approximately 500 meters away from montane forest.

Etymology.—The specific epithet is chosen from the Greek word anebos meaning “young or immature; not yet into adulthood.” The name refers to the young age of the new species, a contention that is based on the relatively small branch lengths distinguishing it from L. karissimbensis.

3.5 Description of Leptopelis versicolor

Leptopelis versicolor sp. nov. Multicolor Forest Treefrog (Figs. 3.3.2 and 3.5.1; Tables Tables 3.3.1, 3.3.2 and 3.3.6)

Holotype.—UTEP 20566 (field no. ELI 586), adult male, 20 December 2010, DRC, South

Kivu Province, Kalundu, 3.15552°S, 28.42108°E (datum = WGS84), 1482 m, collected by E.

Greenbaum, C. Kusamba, M. M. Aristote, W. M. Moninga and F. I. Alonda (Figs. 3.3.2 and

3.5.1).

Paratypes.—UTEP 20564–65, 20567–69, 205670–72 (field nos. ELI 604, 624–26, 650–52,

657), 12 adult males, collected by the same collectors at the same locality from 21–23 December

2010; UTEP 20573 (field no. ELI 629), adult male, 22 December 2010, DRC, South Kivu

Province, bridge at Mwana River, 3.15724°S, 28.42340°E (datum = WGS84), 1443 m, collected by C. Kusamba, M. M. Aristote and F. I. Alonda; UTEP 20574 (field no. ELI 630), adult male,

22 December 2010, DRC, South Kivu Province, bridge at Mwana River, 3.15779°S, 28.43412°E

(datum = WGS84), 1451 m, collected by C. Kusamba, M. M. Aristote and F. I. Alonda; UTEP

20580 (field no. EBG 1643), adult male, 23 June 2008, DRC, South Kivu Province, vicinity of

Miki, 3.3726°S, 28.6779°E (datum = WGS84), 1965 m, collected by C. Kusamba, M. Zigabe and M. Luhumyo; UTEP 20581–89 (field nos. EBG 1661–66, 1668–69a, b), three adult males, three adult females and two tadpoles, 25 June 2008, DRC, South Kivu Province, Kiandjo,

3.3726°S, 28.6432°E (datum = WGS84), 1816 m, collected by C. Kusamba, M. Zigabe and M.

Luhumyo; UTEP 20576-79 (field nos. EBG 1675–76, 1681, 1683), 3 adult males and 1 adult

Figure 3.5.1 Dorsal (A), ventral (B) and lateral view of the head (C) of the preserved male holotype of Leptopelis versicolor (UTEP 20566, 34.3 mm SVL).

63 female, 26 June 2008, DRC, South Kivu Province, stream at Afeci Mountain near Kitopo,

3.4039°S, 28.5866°E (datum = WGS84), 1837 m, collected by W. M. Moninga, F. Akuku and A.

M’Mema (Fig. 3.3.2).

Other specimens examined.— UTEP 20575 (field no. ELI 671), adult male, 27 December 2010,

DRC, South Kivu Province, Baraka, 4.10832°S, 29.09705°E (datum = WGS84), 777 m, collected by M. M. Aristote and W. M. Moninga.

Diagnosis.—A medium-sized, arboreal Leptopelis with (1) adult male SVL 35–38 mm; adult female SVL 45–56 mm; (2) head wider than long; (3) eye relatively large, with horizontal eye diameter almost twice distance from nostril to anterior corner of eye; (4) dorsal snout shape rounded; (5) tympanum distinct, its horizontal diameter slightly more than half eye diameter; (6) dorsal skin finely granular, with small, scattered, usually black, tubercles; (7) feet one half webbed, hands one third webbed; (8) well-developed subarticular tubercles and terminal discs present on all toes and fingers; (9) pectoral glands distinct in males; (10) lack of heel spurs; (11) color in life dorsally brown, bronze, dark brown or gray with distinct dark brown bands; white rectangular blotch below eye; laterally cream with white spots; ventrally creamy white, some with a few scattered brown spots; iris reddish brown superiorly, fading into a golden color inferiorly, with fine black reticulation.

Comparisons.—Univariate statistics identified differences in several morphological characters between the new species and Cameroonian (topotypic) L. modestus. The ANOVA analyses identified significant differences between the new species and Cameroonian L. modestus for: IO

(P = 0.005), ED (P = 0.011), TD (P = 0.013) and EN (P = 0.001); ANCOVA analyses identified

64 significant differences between the new species and Cameroonian L. modestus for: HL (P =

0.001), IO (P = 0.016), ED (P = 0.001), TD (P = 0.002) and EN (P = 0.001).

The new species can be distinguished from Cameroonian L. modestus by its smaller size of EN in males (2.7–2.9 mm vs 3.3–3.8 mm in L. modestus, P = 0.04), wider head (13.3–16.1 mm vs 13.1–13.7 mm in L. modestus, P = 0.05), having more webbing on the inner third digit of the foot (0.50–0.75 vs. 1.00 in L. modestus); dorsal coloration (bronze, brown, reddish brown or gray vs. grayish brown in L. modestus); vocal sac coloration (grayish blue vs. green or blue in L. modestus), and by advertisement call with (tonal clacks/pulses repeted in rapid succession vs. unmelodious clack in Cameroonian L. modestus) (Schiøtz, 1999; Amiet, 2012). The new species is most easily distinguished from L. fiziensis by advertisement call and morphology.

Morphologically, L. versicolor can be differentiated from L. fiziensis by having a larger size in males (35–38 mm vs. 29–35 mm in L. fiziensis, P = 0.01), having more webbing on the interior of the third digit of the hand (2.00–2.50 vs. 1.75–2.00 in L. fiziensis), by having less webbing on the first digit of the foot (0.50–0.75 vs. 1.00 in L. fiziensis), dorsal coloration (bronze, brown or gray vs. brown, tan or dark grayish brown in L. fiziensis) and male advertisement call (3–5 clack/pulse and a mean dominat frequency of 1.68 kHz vs. 2–3 clacks/pulses per call and a mean dominant frequency component of 1.93 kHz in L. fiziensis) (Greenbaum et al., 2012). Leptopelis versicolor can be distinguished from L. kivuensis by the presence of dark tubercles (no dark tubercles on L. kivuensis); by having more webbing on the interior of the fourth digit of the foot

(1.75–2.00 vs. 2.25–2.50 in L. kivuensis); male advertisement call (L. versicolor emits only 3–5 clack/pulse and has a mean dominat frequency of 1.68 kHz, whereas L. kivuensis emits up to 12 clacks/pulses per call and has a mean dominant frequency component of 1.48 kHz); and by the presence of distinct subocular marks (most L. kivuensis specimens lacked distinct subocular

65 marks) (Roelke et al., 2011). The new species can be distinguished from L. mackayi by having complete toe webbing (L. mackayi lacks webbing on the first and exterior second digit of the foot), and male advertisement call (3–5 clacks/pulse vs. 12 clacks/pulse in L. mackayi) (Köhler et al., 2006). The new species can be distinguished from L. karissimbensis by dorsal and vocal sac coloration, the former is dorsally bronze, reddish brown or gray, with black tubercles and has a grayish blue vocal sac, whereas the latter species is grayish brown to reddish brown (most specimens are brown or dark brown) dorsally, lacks black tubercles and has a blue colored vocal sac. Geographically, L. versicolor is found in mid-elevation transitional rainforest, whereas L. karissimbensis is mainly found in high elevation forest edges, meadows or swamps.

Morphologically, L. versicolor tadpoles posses papillae in two rows laterally, whereas L. karissimbensis tadpoles posses papillae in two rows posteriorly (Roelke et al., 2009). The new species can be distinguished from L. christyi by its dorsal pattern and coloration (bronze, brown or gray with black tubercles and brown crossbars vs. a dark brown triangle and green or cream lateral line without tubercles in L. christyi), by having more webbing on the interior of the fourth digit of the foot (1.75–2.00 vs. 2.25–2.50 in L. christyi) and presence of a subocular mark (absent in L. christyi) (Schiøtz, 1975). The new species can be easily distinguished from L. calcaratus morphologically; males of the former species have a shorter snout (mean 13.1 mm vs. 14.5 mm in L. calcaratus), a smaller, less distinct tympanum (mean = 2.1 mm vs. 3.0 mm in L. calcaratus) and lack of heel spurs (present in L. calcaratus) (Perret, 1966; Schiøtz, 1999).

Description of the holotype.— Adult male; body slender, head is noticeably wider than body; snout is short and rounded in lateral view; tongue longer than wide; nostrils visible from dorsal view; cathnus rostralis slightly curved; eye relatively large, about 1.5 times distance from eye to nostril; skin finely granular with a few scattered, large, black tubercles present on dorsal surface;

66 skin on ventral surfaces granular; pectoral glands present; white spots/blotches present on lateral surface of skin; hind limbs relatively long with tibia length reaching a little more than half SVL; hand webbing formula 1(1.50) 2i(1.75), 2e(1.00), 3i(2.00), 3e(1.25), 4(1.50); relative length of fingers: I < II < IV < III; outer metatarsal tubercle absent, inner small, ovoid; palmar surfaces have tubercles; well-developed subarticular tubercles under all fingers; tips of all fingers with discs, each about 1.5 times width of phalanges; foot webbing formula 1(0.75), 2i(1.25), 2e(0.50),

3i(2.00), 3e(0.75), 4i(2.00), 4e(1.75), 5(0.50); relative length of toes, I < II < III < V < IV; outer metatarsal tubercle absent; inner metatarsal tubercle well developed, round subarticular tubercles under all toes; tips of toes have round discs, each about 1.5 times wider than width of phalanges.

Coloration of holotype in preservative.— Dorsal surface is brown with distinct dark brown bands on the dorsum; three distinct brown bands are present on the forearms, and four distinct brown bands on the hind limbs; small white blotches present on the lateral surface; small diffused, black spots present on dorsal tubercles; small, cream subocular blotch is rectangular in appearance, which is slightly wider at its contact with the upper lip than at its contact with the bottom of the eye; distinct, dark brown triangular pattern in the sacral region; tympanum is the same color as the dorsal surface; tip of the snout is dark brown; ventral surface is cream with small flecks or spots; vocal sac is brownish gray.

Coloration of holotype in life.— Dorsal coloration is reddish-brown with dark brown bands on the dorsum; black round tubercles present on the dorsum; a dark, distinct brown reticulate pattern behind the eyes; canthal region is dark brown; cream subocular blotch, rectangular in appearance; triangular pattern in sacral region distinct; tympanum is the same color as the dorsal surface; ventral surface is white with grayish-blue flanks; vocal sac is grayish blue.

Variation.—The tympanum is nearly round in most specimens of L. versicolor, but is slightly oval in ELI 671. The dorsal bands are diffuse and not distinct in UTEP 20575 and UTEP 20583.

Dorsal patterns may include a dark brown blotch covering most of the dorsum, thick, dark-brown crossbands, irregular tannish brown patches, or a complete absence of dorsal pattern; UTEP

20575 lacks black tubercles on the dorsum (C in Fig. 3.3.2). In the same specimen, dorsal coloration is grayish tan with slightly darker grayish brown bands on the dorsum and limbs.

Specimens UTEP 20572 and UTEP 20571 have small green or blue blotches on their dorsums.

Specimens UTEP 20583 and UTEP 20578 have indistinct dorsal bands and dorsal patterns (A and B in Fig. 3.3.2); UTEP 20583 has very large and numerous black tubercles. Cream spots on the lateral surface that are present on the holotype are present in all specimens sampled; specimens UTEP 20575 and UTEP 20583 have faded, less distinct white spots/blotches on the lateral surface Specimens UTEP 20581–83 have small, speckled white spots on the lateral surface, rather than the large, distinct white spots on the holotype.

Description of tadpole.— The following description is based on two larvae in Gosner stages 38 and 39 (UTEP 20587–88). Tadpole mensural data can be seen in Table 3.5.1. One of the two tadpoles had a damaged tails, and tail measurements were excluded. In dorsal view, body is elliptical with widest point at spiracle (Fig. 3.5.2); snout is in a trunk-like form; eyes are moderate in size (about 0.90 times SV), slightly bulging and separated by about 1.8 time the internarial distance, directed more laterally than dorsolaterally; nostrils are circular, small, not rimmed, positioned dorsolaterally, visible in lateral view, and are closer to anterior margin of snout than eye; in profile body appears depressed (BW/BH ~ 1.25) and is flattened dorsally; snout rounded but slightly truncate; spiracle sinistral, triangular, short tube attached to body wall, slightly closer to eye than to vent, positioned ventrolaterally and oriented posteriorly and slightly

Table 3.5.1 Morphometric data (mm) of Leptopelis versicolor tadpoles. See text for abbreviations.

UTEP 20587 UTEP 20588

SS 7.82 8.01 SU 11.60 12.12 SV 17.42 18.11 VMHT 14.41 — VT 27.28 — TL 43.08 — UF 2.51 — LF 1.75 — HT 7.79 — BH 6.43 6.78 BW 7.81 7.78 PP 3.89 4.20 NN 2.13 2.38 ED 1.39 1.42 ODW 3.19 3.40

dorsally. Spiracal opening is rounded, set approximately at horizontal plane of hind limbs and horizontal plane of ventral edge of caudal myotomes.

Tail is large, robust with good musculature; about equal in size for anterior two-thirds of tail, decreasing after that point. Tail fins are short in vertical directions with dorsal tail fin not extending onto body. There is no visible lateral line or synonymous gland. Oral disc is positioned anteriorly with rounded papillae; it is nearly as wide as end of body, bordered on lower edge; upper edge of oral disc lacks papillae and consists of small ridge. Jaw sheaths appear strong with upper beak being curved and much longer than lower beak. Oral disc (Fig.

3.5.3) oriented anteriorly to slightly ventrally. Oral disc nearly as wide as proximal end of body surrounded on the lower edge by double row of short, rounded papillae. Upper edge of oral disc lacks papillae. Oral papillae are in a single row posteriorly, with two rows of papillae laterally.

Figure 3.5.2 Lateral (A), dorsal (B) and ventral (C) views of the preserved Leptopelis versicolor tadpole (UTEP 20587, Gosner stage 38).

Figure 3.5.3 Larval mouth of Leptopelis versicolor (UTEP 20587, Gosner stage 38, tail length = 43.08 mm, scale bar represents 1 mm).

Jaw sheaths are keratinized along their inner margins, with fine denticulation. Labial tooth row formula is 4 (2–4) / 3 (Fig. 3.5.3).

In preservative, the back and flanks are dark reddish brown, getting lighter towards the venter. A dark brown ring encircles the opening of the spiracle. The caudal musculature is dark reddish-brown to brown and slightly mottled or reticulated. The upper tail fin is lighter reddish brown than caudal musculature and reticulated lightly. The lower tail fin is nearly transparent near the border with caudal musculature, and towards the distal edges. The venter is creamy, reddish-brown at the proximal end, lightening to nearly white or translucent at the vent and near the insertion of hind limbs. The coiled intestines are visible.

Distribution and natural history.— Currently, L. versicolor is known from the Itombwe

Plateau in eastern DRC (Kitopo, Mwana, Kiandjo, Kalundu and one specimen found near Lake

Tanganyika at Baraka [Fig. 2.1]). Most L. versicolor specimens were collected from transitional forests of the Congo Basin and montane rainforests of the Itombwe Plateau, inhabiting altitudes of 1451–1837 m (Kalundu and Kitopo). However, one specimen was collected from a non- forested habitat near lake Tanganyika at Baraka (777 m), which was a mosaic of reeds, grasses and bushes with a few small trees. With the notable exception of the latter specimen, all known specimens of L. versicolor were collected near streams at the edge of forests, or in forests on bushes or trees about 1–3 meters above the ground. The two tadpoles were collected in a stream running from a pristine forest to a pond where some Phlyctimantis verrucosus (Hyperoliidae) were also found. One of the adult specimens (UTEP 20585) was carrying eggs, which were deposited in a plastic container within a few hours of capture. Other anurans collected at the type locality included Leptopelis christyi, Leptopelis cf. calcaratus (Arthroleptidae), several

Hyperolius species, Afrixalus laevis, Afrixalus osorioi (Hyperoliidae), Xenopus pygmaeus

(Pipidae), Phrynobatrachus cf. dendrobates and Phrynobatrachus sp. (Phrynobatrachidae).

Additional fieldwork is needed to acquire a better understanding of the distribution patterns of L. versicolor, and to explain whether the aberrant record from Baraka is an outlier, or the species regularly occurs in non-forested habitats.

Etymology.—The specific epithet is chosen from the Latin word versicolor meaning “many colors or forms.” The name refers to the diversity of colors and patterns of the new species.

CHAPTER 4: DISCUSSION

4.1 Taxonomic status and biogeography of Itombwe Leptopelis

To date, this study represents the most comprehensive phylogenetic analysis of any group of Leptopelis. Six major lineages were recovered (L. anebos, L. fiziensis, L. karissimbensis, L. kivuensis, L. cf. kivuensis and L. versicolor). Leptopelis anebos is the 52nd described species of the genus and is nested within a clade with populations of L. karissimbensis. Because of the minor molecular differences and polytomy recovered between these two closely related species

(Fig. 2.1), the decision to recognize the latter taxon as a new species may seem controversial.

Genetic divergences between L. anebos and L. karissimbensis were relatively low (16S: 0.9–

1.1%; cyt b: 0.5–0.9%; RAG1: 0.1%), but comparable genetic divergences have been found in valid taxa of the Neotropical bufonid genus Osornophryne (as low as 0.12%; Páez-Moscoso and

Guayasamin, 2012), dendrobatids in the genus Oophaga (as low as 0.8%; Brusa et al., in press) and mantellids in the genus Mantidactylus (as low as 1.0%; Vences et al., 2005). Based on distinctive morphology, Kuramoto et al. (2011) described Odorrana ishikawae, despite low levels of 16S divergence (1.44–2.16%).

The concept and recognition of species has long been controversial, and in some cases, vague and subjective. Some suggest drawing the species boundary relatively early in the process of divergence, whereas others recognize the boundary later, perhaps when the separate lineages develop an intrinsic reproductive barrier. This is likely the reason why so many incompatible species concepts exist (de Queiroz, 1998, 2005, 2007). Herein, the General Lineage Concept

(GLC) is utilized, which recognizes species when they are separately evolving lineages. Lineage

73 discovery via the GLC is neither straightforward nor precise in the earliest stages of speciation, and it is expected that some uncertainty will be present in delimiting young species (de Queiroz,

1998; Hey et al., 2003). Fouquet et al. (2007) suggested a minimum of 3% of 16S genetic divergence (uncorrected p distances) to use as a species boundary for Neotropical anurans.

Herein, a similar position as Padial et al. (2009) was taken, by rejecting species boundaries/thresholds based solely on genetic sequence divergence. Genetic divergences can serve as reference points to determine the presence of new species rather than fixed thresholds

(Páez-Moscoso and Guayasamin, 2012). The formation of a monophyletic clade can take a relatively long time for species examined with nuclear gene data (Hudson and Coyne, 2002), suggesting that many valid species can exist without having yet achieved monophyly (Shaffer and Thomson, 2007). The use of single lines of evidence (e.g., genetics) or the application of species thresholds to delimit species may impair both the opportunity for discovery and understanding of evolutionary processes. An approach that combines as many lines of evidence

(e.g., morphology, acoustic data, molecular data, ecology, behavior, reproductive biology) as possible provides more accurate feedback for species discoveries (Padial et al., 2009).

Other criteria for species boundaries remain important, including morphological differences and prezygotic (e.g., advertisement call) reproductive barriers that are acquired in distinctive lineages as they diverge from each other (de Queiroz, 2007). Because L. anebos and

L. karissimbensis occur in partial sympatry, it is likely that character displacement of courtship behaviors, including advertisement vocalizations, took place. Differences in premating isolation mechanisms in areas of sympatry reinforce prezygotic barriers, which ultimately reduce hybridization. Male anuran advertisement calls serve to attract conspecific females and act as premating isolation barriers where similar species coexist (Blair, 1974; Gerhardt, 1994).

Reinforcement of advertisement calls in zones of sympatry is well documented for several anuran genera, including Anaxyrus (Leary, 2001), Gastrophryne (Loftus-Hills and Littlejohn,

1992) and Litoria (Littlejohn, 1965). In the case of L. anebos and L. karissimbensis, the divergence in call characters likely reinforced species boundaries so rapidly that genes, including mitochondrial genes, have yet to coalesce. Phrynobatrachus kakamikro and Litoria myola were described on the basis of advertisement call data and morphology; both of these species exhibited low sequence divergence (1.7% and 0.1% respectively) from their respective sister taxa (Hoskin,

2007; Schick et al., 2010). Hoskin (2007) noted that the extremely low genetic divergence between Litoria myola and southern populations of L. genimaculata might be due to rapid speciation by reinforcement of a small population in the contact zone.

Advertisement calls are thought to be important species boundary characters (Boul et al.,

2007), and have frequently been used as fundamental characters in anuran systematics (Platz and

Forester 1988; Platz, 1989; Gamble et al., 2008; Twomey and Brown, 2008). The distinct calls of

L. anebos and L. karissimbensis are taxonomically relevant characters, which justifies full- species status for the former taxon. Other anurans that have been described on the basis of advertisement call data in the presence of gene trees that were not reciprocally monophyletic include dendrobatids in the genus Ameerega (Brown and Twomey, 2009). Brown and Twomey

(2009) noted that they were hesitant to describe a species in the presence of genetic polytomies, but the distinct calls of Ameerega yoshina bolstered its unique taxonomic position. These authors stated that advertisement calls are thought to serve as important intraspecific signals and the polytomy may be explained by incomplete lineage sorting, a likely explanation for the unresolved clade with L. anebos and L. karissimbensis. These findings demonstrate an interesting contrast to studies of closely related species that do not occur in sympatry. For

75 example, Heyer et al. (2005) presented an example with the Neotropical frog genus

Leptodactylus, in which some closely related allopatric species had no adult morphological or male advertisement call differences, but they were distinguished by large mitochondrial DNA sequence divergences (9% or greater), larval morphology and ecology, juvenile color pattern and habitat. This scenario likely happened because the different species did not meet in nature

(Leptodaculys knudseni and Middle American “pentadactylus” were collected from Ecuador and

Panama respectively), and there was no impetus for reinforcement to prevent hybridization.

Although L. anebos and L. karissimbensis occur in partial sympatry, the two species appear to occupy slightly different elevations and habitats. All of the L. karissimbensis used in this study were collected in forest edges, meadows or swamps above 2100 m, with most specimens found between 2400–2700 m (Fig. 2.1). Leptopelis anebos specimens were found in montane forests and a stream in a savanna below 2250 m, with most specimens found below

1900 m (Fig. 2.1). Because only five specimens of L. anebos were collected, more surveys are needed for more conclusive biogeographic patterns, and to improve knowledge of its morphological variation. Unfortunately, since the five known specimens of L. anebos were collected, internecine warfare between several armed groups has engulfed the Itombwe Plateau, rendering further fieldwork impossible for the foreseeable future.

Leptopelis versicolor is the 53rd described species of the genus. Leptopelis versicolor is superficially similar to Cameroonian (topotypic) L. modestus, but results presented herein suggest the former species is a distinct taxonomic lineage. Werner (1898) described L. modestus from Cameroon, andthe type locality was later restricted to mid-elevations of southern Cameroon

(Buéa, Cameroon) by lectotype designation (Perret, 1967). Schiøtz (1967) remarked that

Cameroonian L. modestus were distinguishable from similar taxa by the presence of a green

76 vocal sac in males, which contrasts with L. versicolor, which possess a blueish gray vocal sac.

Schiøtz (1967) noted that topotypic material from Buéa differed significantly from his

Cameroonian L. modestus. Along with the Cameroonian populations, the name L. modestus was attributed to several other populations, including those from eastern DRC, Nigeria and

Kakamega forest in Kenya. Laurent (1973) remarked that he originally regarded Leptopelis fiziensis as a distinct species, but he decided to describe the new taxon as a subspecies of L. modestus after his “biometric” (i.e., morphological) analyses suggested a close relationship to the latter species. Schiøtz (1975) later elevated L. fiziensis to full species status. Schiøtz (1999) explained that he regarded L. fiziensis as a full species because of the unsettled taxonomy of L. modestus, but that the two taxa were morphologically similar. Schiøtz (1999) argued that the name L. modestus possibly refers to several species, although he continued to refer to Nigerian and Kakamega populations as L. modestus (Schiøtz, 1999; Köhler et al., 2006). Based on morphology, male advertisement call analyses and mitochondrial sequence data (16S gene),

Köhler et al. (2006) recognized a population of L. modestus from forested areas of western

Kenya as a new species, L. mackayi. Although the latter authors provided a morphological diagnosis of L. mackayi from L. fiziensis, no specific hypothesis about the relationship of their new species to other Leptopelis species was provided. Evidence from molecular, morphological and advertisement call analyses presented herein; demonstrate that L. versicolor and L. fiziensis are distinct species from the Cameroonian L. modestus and the Kenyan L. mackayi.

Several advertisement call characters appear to distinguish L. versicolor from other AR

Leptopelis. Leptopelis anebos and L. karissimbensis emit one pulse, with the latter species occasionally emitting a second pulse (Roelke et al., 2011; Portillo and Greenbaum, submitted).

Unlike L. anebos and L. karissimbensis, L. versicolor emits three to five pulses per call. Other

Leptopelis from the AR emit multiple pulses per call, including L. fiziensis and L. kivuensis, with the former species emitting two to three pulses and the latter emitting up to 12 pulses per call

(Roelke et al., 2011; Greenbaum et al., 2012). Leptopelis mackayi emits 6–12 pulses per call, which is also much higher than L. versicolor (Köhler et al., 2006). Dominant frequency component, intercall interval and call duration were also useful characters to distinguish L. versicolor from other AR Leptopelis (Table 3.3.6). Schiøtz (1967, 1999) described the call of L. modestus as a deep, unmelodious, drawn-out clack, occasionally repeated twice, which differs from the call of the new species.

Evidence from analyses of sequences, morphology and acoustic call data support the continued recognition of L. fiziensis as a distinct species. Morphometric results and color pattern description are mostly consistent with data provided by Laurent (1973) in the original species description, but the upper size limit of male specimens collected from the DRC (34.8 mm SVL,

Table 3.3.1) is slightly larger (ca. 33 mm SVL; Laurent, 1973: fig. 39). In contrast, the three male specimens described by Schiøtz (1975, 1999) from Gombe Stream National Park, Tanzania are larger (34–38 mm SVL), have “tiny light specks” on the dorsum, a grayish venter, reddish femur and golden iris. Channing and Howell (2006) provided a similar color pattern description to that of Schiøtz (1975, 1999), but they noted females reach 38 mm and males only 35 mm.

Several male advertisement call characters seem to be unique to L. fiziensis. The number of pulses (2–3) distinguishes L. fiziensis from other AR Leptopelis. Moreover, the mean high frequency (2.11 kHz) renders L. fiziensis one of the highest call frequencies of any Leptopelis examined from the AR (Table 3.3.6; Roelke et al., 2011). This finding is consistent with the relatively small body size of male L. fiziensis and the supposition of an inverse relationship between call frequency and male body size (Duellman and Trueb, 1994; Wells, 2007). In stark

78 contrast to the findings herein, Channing and Howell (2006) described the call of L. fiziensis as

“a deep unmelodic clack” that did not seem to be recorded or analyzed.

The Kasanjala specimens of L. fiziensis were found on shrubs and trees (ca. 1–3 meters above the ground) near a stream in transitional forest, which occurs in small natural swathes (i.e.,

“forest strips” sensu Laurent, 1973) in the forest/savanna mosaic in the region around Fizi.

Hyperolius langi (Hyperoliidae) and an unidentified species of Amietia (Pyxicephalidae) were also present at the Kasanjala forest locality. The Milembwe road specimen (UTEP 20454) was calling from a leaf of a tree (ca. 1.5 m above ground) near a stream at the edge of montane forest.

Schiøtz (1975, 1999) and IUCN (2012) noted the Gombe Stream males were collected on branches in dense low bush at the edge of a clearing in dry forest. IUCN (2012) described the record from southwestern Tanzania (i.e., Senga, 1650 m) as riverine forest within pristine miombo (Brachystegia spp.) woodland.

Given the differences in mophometric data, color pattern, male advertisement call and habitat between specimens from the DRC and those from Tanzania (Schiøtz, 1975, 1999;

Channing and Howell, 2006; IUCN, 2012), I suspect that the Tanzanian records are not conspecific with Leptopelis fiziensis, and further study is needed to clarify their taxonomic status. Because of nearly complete destruction of lowland forests in western Burundi (E.

Greenbaum, pers. obs.), unspecific records of L. fiziensis from the country are likely to be confused with other species. The findings herein are consistent with Plumptre et al. (2003), who considered Leptopelis fiziensis to be one of only 37 AR endemic amphibian species. Additional work is necessary to improve knowledge of the distribution of L. fiziensis in eastern DRC, but given the limited, currently known geographic distribution of the species and widespread

79 deforestation in the region around Fizi and Itombwe (Greenbaum and Kusamba, 2012), the current IUCN Red List status as data-deficient (IUCN, 2012) should be reconsidered.

Results from my morphometric and phylogenetic analyses indicated taxonomic issues that require further investigation. The Bioko Island specimens of L. modestus appear to be a unique lineage, which is likely endemic. There also appears to be at least three distinct lineages within the L. calcaratus complex (Fig. 3.1.1). Leptopelis calcaratus meridionalis and the eastern

DRC population of L. cf. calcaratus are likely not conspecific with Cameroonian (topotypic) L. calcaratus, and additional work should help clarify their taxomic status. Furthermore, there seems to be two lineages of L. kivuensis indicated in the phylogenetic analyses (Fig. 3.1.1).

Roelke et al. (2011) described the range of L. kivuensis extending from Kahuzi-Biega National

Park (DRC), east to the type locality at Gisenyi (Rwanda), and north through western Uganda to the Ruwenzori Mountains (DRC and Uganda). Results from the phylogenetic analyses (Fig.

3.1.1) suggest that there are at least two distinct evolutionary lineages of L. kivuensis within the range noted by Roelke et al. (2011), neither of which may be conspecific with topotypic material.

Moreover, specimens collected from Burundi and Rwanda appear distinct from eastern DRC populations (FP and E. Greenbaum unplubl. data). Further assessment of specimens from

Rwanda and Burundi may help clarify the unsettled taxonomy within the L. kivuensis complex.

4.2 Conservation

The recognition of Leptopelis anebos and L. versicolor stresses the importance of the

Itombwe Plateau as a center of endemism and biodiversity. The Itombwe Plateau, among other

AR sites, has the most threatened species of amphibians (Laurent, 1964; 1983; Evans et al.,

2008; Stuart et al., 2008; Roelke et al., 2011), and the highest number of endemic amphibians in the AR, rendering the plateau one of the most important sites for amphibian conservation in continental Africa (Plumptre et al., 2007; Greenbaum et al., 2012; Greenbaum and Kusamba,

2012). The recently described pipid anuran Xenopus itombwensis Evans et al., 2008, monotypic hyperoliid Chrysobatrachus cupreonitens Laurent, 1951, monotypic bufonid Laurentophryne parkeri (Laurent, 1950), arthroleptid Arthroleptis hematogaster (Laurent, 1954) and the lacertid

Congolacerta asukului Greenbaum et al., 2011, are a few of the species endemic to the Itombwe

Plateau. Leptopelis fiziensis has also been added to the list of amphibians inhabiting the

Itombwe Plateau (Greenbaum et al., 2012).

According to Sayer et al. (1992), the Itombwe Plateau is a relict of the ancient relief that predates the mountains of the AR, which began forming around 25–30 million years ago (Vande weghe, 2004). This concept also supports the theory that the Itombwe Plateau is in an area of a large forest refuguim, which likely harbored forest-endemic species throughout the Quaternary because of its ecological stability (Doumenge, 1998; Sayer et al., 1992; Greenbaum and

Kusamba, 2012). Lovett et al. (2005) noted that ecologically stable forests are characterized by the presence of aggregates of relict lineages, newly evolved species and ecological equilibration, where the forests are uniformly diverse over ecological gradients, thus optimizing diversity at a sustainable level. The importance of forest refugia for conservation is great because they harbor high levels of species richness and endemism (Lovett et al., 2005). It is likely that many new, young, and cryptic species/lineages await description in forest refugia. Deforestation, illegal mining and other forms of human perturbation continue to threaten the biodiversity of the

Itombwe Plateau. Further biological exploration needs to continue to justify more aggressive conservation measures in the future.

4.3 Conclusions and future directions

The results of this study suggest that the diversity of Leptopelis has been severely underestimated. At least five distinct lineages were recovered from the Itombwe Plateau.

Previously, only three taxonomic groups were assigned to Itombwe Leptopelis. Diversity of

Itombwe Leptopelis seems to be correlated with the multifarious habitats present in the plateau.

Each distinct clade should be recognized under the oldest available synonym, if present. The population inhabiting upper montane forest edges and swamps of 2200 m and above, from Mt.

Tshiaberimu (Virunga National Park) to the highlands of southwestern Uganda, Nyungwe

National Park of Rwanda, and Kahuzi-Biega National Park and the Itombwe Plateau of eastern

DRC, should be designated as L. karissimbensis; the taxon inhabiting montane forests of the

Itombwe Plateau below 2200 m should be designated as L. anebos. The name L. fiziensis should apply to populations inhabiting transitional forests of southern Itombwe to forest/savanna mosaic in regions surrounding Fizi. The population inhabiting transitional forests in western Itombwe

(1400–1935 m), to Baraka (777 m) should be designated as L. versicolor. The name L. modestus should apply only to populations inhabiting the mid-elevation forests ofCameroon, because L. fiziensis, L. mackayi and L. versicolor are now considered to be distinct species.

The recognition of several lineages of Itombwe Leptopelis presents several querries to be investigated in future studies. Additional research efforts should focus on the biogeographic patterns and reproductive isolation barriers that allowed the establishment of young, distinct evolutionary lineages. These queries should be investigated with a combination of genetic, morphological, and acoustic components to determine species boundaries. An increased sample size would help resolve geographic patterns and provide better resolution between clades. For example, further investigation of populations from Rwanda, Burundi and Tanzania are needed

82 for more robust taxonomic conclusions on the L. kivuensis complex. Furthermore, it is imperative to sample more African sites to get a better understanding of African biodiversity.

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100

APPENDIX I

Voucher numbers, localities and Genbank accession numbers for genetic samples. DRC = Democratic Republic of the Congo, EQ = Equatorial Guinea, KBNP = Kahuzi-Biega National Park, VNP = Virunga National Park. Specimens with asterisks are tadpoles.

Species Collection No. Field No. Locality 16S cyt b RAG1

Scotobleps gabonicus YPM 6991 — EQ: Provencia Centro Sur: Monte KC152645 KC152646 KC152647 Alen National Park Arthroleptis variabilis ZFMK 68794 — Cameroon: Nlonako AF124107 AY341737 AY571642 Leptopelis barbouri CAS 168472 RCD-10139 Tanzania: Tanga Region: Muheza JX996027 KC006028 KC005992 District Leptopelis parbocagii UTEP 20552 ELI 247 DRC: Katanga: Kyolo JX996026 KC006027 KC005991 Leptopelis kivuensis UTEP 20145 EBG 1509 DRC: South Kivu: KBNP, Mugaba HQ130766 KC006029 KC005993 Leptopelis kivuensis UTEP 20148 EBG 1514 DRC: South Kivu: KBNP, Mugaba HQ130769 KC006030 KC005994 Leptopelis kivuensis UTEP 20146 EBG 1510 DRC: South Kivu: KBNP, Mugaba HQ130767 KC006031 KC005995 Leptopelis kivuensis UTEP 20143 EBG 1475 DRC: South Kivu: KBNP, Mugaba HQ130765 KC006032 KC005996 Leptopelis kivuensis UTEP 20137 EBG 1452 DRC: South Kivu: KBNP, Mugaba HQ130764 KC006033 KC005997 Leptopelis cf. kivuensis UTEP 20152 EBG 1917 DRC: North Kivu: VNP, Mt. Teye HQ130774 KC006034 KC005998 Leptopelis cf. kivuensis UTEP 20153 EBG 1918 DRC: North Kivu: VNP, Mt. Teye HQ130775 KC006035 KC005999 Leptopelis karissimbensis UTEP 20128 EBG 1282 DRC: South Kivu: KBNP, Chinya HQ130778 KC006036 KC006000 Leptopelis karissimbensis UTEP 20126 EBG 1233 DRC: South Kivu: KBNP, Mugaba HQ130776 KC006037 KC006001 Leptopelis karissimbensis UTEP 20120 EBG 1638 DRC: South Kivu: Itombwe HQ130783 KC006038 KC006002 Plateau, Miki Leptopelis karissimbensis UTEP 20124 EBG 1448 DRC: South Kivu: KBNP, Mugaba HQ130762 KC006039 KC006003 Leptopelis karissimbensis UTEP 20114 EBG 1477 DRC: South Kivu: KBNP, Mugaba HQ130782 KC006040 KC006004 Leptopelis karissimbensis UTEP 20112 EBG 1742 DRC: North Kivue: VNP, Mt. HQ130772 KC006041 KC006005 Tshiaberamu Leptopelis karissimbensis UTEP 20125 EBG 1450 DRC: South Kivu: Mugaba JX996029 KC006042 KC006006 Leptopelis karissimbensis No voucher EBG 2137 DRC: South Kivu: Shalashala, JX996029 KC006043 KC006007 Itombwe Plateau

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Leptopelis karissimbensis UTEP 20553 EBG 2141 DRC: South Kivu: Itombwe JX996029 KC006044 KC006008 Plateau, Shalashala, Leptopelis karissimbensis UTEP 20554 ELI 700 DRC: South Kivu: Itombwe JX996030 KC006045 KC006009 Plateau, Mikenge Leptopelis karissimbensis UTEP 20555 ELI 701 DRC: South Kivu: Itombwe JX996031 KC006046 KC006010 Plateau, Mikenge Leptopelis karissimbensis UTEP 20556 ELI 703 DRC: South Kivu: Itombwe JX996032 KC006047 KC006011 Plateau, Mikenge Leptopelis karissimbensis UTEP 20557 CFS 902 DRC: South Kivu: Itombwe JX996038 KC006053 KC006017 Plateau, Miki Leptopelis karissimbensis UTEP 20558 CFS 904 DRC: South Kivu: Itombwe JX996039 KC006054 KC006018 Plateau, Miki Leptopelis anebos UTEP 20559 ELI 782 DRC: South Kivu: Itombwe JX996033 KC006048 KC006012 Plateau, Tumungu Leptopelis anebos UTEP 20560 ELI 736 DRC: South Kivu: Itombwe JX996034 KC006049 KC006013 Plateau, Tumungu Leptopelis anebos UTEP 20561 ELI 765 DRC: South Kivu: Itombwe JX996035 KC006050 KC006014 Plateau, Tumungu Leptopelis anebos UTEP 20562 ELI 800 DRC: South Kivu: Itombwe JX996036 KC006051 KC006015 Plateau, Tumungu Leptopelis anebos UTEP 20563 CFS 909 DRC: South Kivu: Itombwe JX996037 KC006052 KC006016 Plateau, Bilimba Leptopelis fiziensis UTEP 20454 ELI 682 DRC: South Kivu: Itombwe JX298074 KC006055 KC006019 Plateau, rd. near Kanguli Leptopelis fiziensis UTEP 20455 ELI 1474 DRC: South Kivu: Kasanjala JX298067 KC006056 KC006020 Leptopelis fiziensis UTEP 20457 ELI 1476 DRC: South Kivu: Kasanjala JX298072 KC006057 KC006021 Leptopelis fiziensis UTEP 20458 ELI 1477 DRC: South Kivu: Kasanjala JX298070 KC006058 KC006022 Leptopelis fiziensis UTEP 20459 ELI 1478 DRC: South Kivu: Kasanjala JX298068 KC006059 KC006023 Leptopelis fiziensis UTEP 20460 ELI 1479 DRC: South Kivu: Kasanjala JX298071 KC006060 KC006024 Leptopelis fiziensis UTEP 20462 ELI 1481 DRC: South Kivu: Kasanjala JX298073 KC006061 KC006025 Leptopelis fiziensis UTEP 20464 ELI 1483 DRC: South Kivu: Kasanjala JX298069 KC006062 KC006026 Leptopelis versicolor UTEP 20564 ELI 625 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis versicolor UTEP 20565 ELI 626 DRC: South Kivu: Itombwe — — — Plateau, Kalundu

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Leptopelis versicolor UTEP 20566 ELI 586 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis versicolor UTEP 20567 ELI 651 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis versicolor UTEP 20568 ELI 604 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis versicolor UTEP 20569 ELI 650 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis versicolor UTEP 20570 ELI 652 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis versicolor UTEP 20573 ELI 629 DRC: South Kivu: Itombwe — — — Plateau, Mwana Leptopelis versicolor UTEP 20574 ELI 630 DRC: South Kivu: Itombwe — — — Plateau, Mwana Leptopelis versicolor UTEP 20571 ELI 624 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis versicolor UTEP 20572 ELI 657 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis versicolor UTEP 20575 ELI 671 DRC: South Kivu: Baraka — — —

Leptopelis versicolor UTEP 20581 EBG 1665 DRC: South Kivu: Itombwe — — — Plateau, Kiandjo Leptopelis versicolor UTEP 20582 EBG 1662 DRC: South Kivu: Itombwe — — — Plateau, Kiandjo Leptopelis versicolor UTEP 20576 EBG 1681 DRC: South Kivu: Itombwe — — — Plateau, Kitopo Leptopelis versicolor UTEP 20577 EBG 1683 DRC: South Kivu: Itombwe — — — Plateau, Kitopo Leptopelis versicolor UTEP 20583 EBG 1661 DRC: South Kivu: Itombwe — — — Plateau, Kiandjo Leptopelis versicolor UTEP 20578 EBG 1675 DRC: South Kivu: Itombwe — — — Plateau, Kitopo Leptopelis versicolor UTEP 20580 EBG 1643 DRC: South Kivu: Itombwe — — — Plateau, vicinity of Miki Leptopelis versicolor UTEP 20584 EBG 1664 DRC: South Kivu: Itombwe — — — Plateau, Kiandjo

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Leptopelis versicolor UTEP 20585 EBG 1666 DRC: South Kivu: Itombwe — — — Plateau, Kiandjo Leptopelis versicolor UTEP 20586 EBG 1663 DRC: South Kivu: Itombwe — — — Plateau, Kiandjo Leptopelis versicolor UTEP 20589 EBG 1668 DRC: South Kivu: Itombwe — — — Plateau, Kiandjo Leptopelis versicolor UTEP 20587 EBG 1669a* DRC: South Kivu: Itombwe — — — Plateau, Kiandjo Leptopelis versicolor UTEP 20588 EBG 1669b* DRC: South Kivu: Itombwe — — — Plateau, Kiandjo Leptopelis versicolor UTEP 20579 EBG 1676 DRC: South Kivu: Itombwe — — — Plateau, Kitopo/Mwana Leptopelis calcaratus CAS 207299 RCD-13105 EQ: Bioko Island: Moka Malabo — — — Leptopelis calcaratus — DCB 34392 Cameroon: Southwest: Nsoung — — — Leptopelis calcaratus — ELI 482 DRC: South Kivu: Bizombo — — — Leptopelis calcaratus — ELI 478 DRC: South Kivu: Bizombo — — — Leptopelis calcaratus — ELI 646 DRC: South Kivu: Itombwe — — — Plateau, Kalundu/Mwana River Leptopelis calcaratus — ELI 627 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis calcaratus — ELI 628 DRC: South Kivu: Itombwe — — — Plateau, Kalundu Leptopelis sp. — ELI 396 DRC: South Kivu: Nundu — — — Leptopelis sp. — ELI 364 DRC: Katanga: Manono — — — Leptopelis sp. — ELI 196 DRC: Katanga: Mitwaba — — — Leptopelis sp. — ELI 180 DRC: Katanga: Mitwaba — — — Leptopelis sp. — ELI 136 DRC: Katanga: Kabongo — — — Leptopelis sp. — ELI 324 DRC: Katanga: Manono — — —

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APPENDIX II

Specimens Examined

Leptopelis anebos— Democratic Republic of the Congo: South Kivu Province: Itombwe Plateau,

Tumungu, 03.53541°S, 28.67411°E, 1835 m (UTEP 20560), 03.56455°S, 28.6661°E, 1897 m

(UTEP 20561), 03.5697°S, 28.66113°E, 1890 m (UTEP 20559), 03.55370°S, 28.67422°E, 1869 m (UTEP 20562); Itombwe Plateau, Bilimba, 03.4610°S, 28.7829°E, 2227 m (UTEP 20563).

Leptopelis calcaratus— Cameroon: East Province: Dja Reserve, 03.1126°N, 12.4842°E, 665 m

(CAS 199300–01); Equatorial Guinea: Bioko Island: Rio Lomo, 03.1929°N, 08.4016°E, no elevation data, (CAS 207296–98).

Leptopelis cf. calcaratus— Democratic Republic of the Congo: South Kivu Province: Bizombo,

03.0288°S, 28.2824°E, 1132 m (ELI 478, 482); Itombwe Plateau, Mwana River, 03.15552°S,

28.42108°E, 1482 m (ELI 627–28, 645–46).

Leptopelis fiziensis— Democratic Republic of the Congo: South Kivu Province:

Itombwe Plateau Kanguli, 03.8814°S, 28.8571°E, 1950 m (UTEP 20454); vicinity of Fizi,

Kasanjala, 04.3738°S, 28.9328°E, 1305 m (UTEP 20455–66).

Leptopelis karissimbensis— Democratic Republic of the Congo: South Kivu Province: Mugaba,

Kahuzi-Biega National Park, 02.27288°S, 28.66208°E, 2289 m (UTEP 20113–19); Mugaba

River, 02.26719°S, 28.66217°E, 2262 m (UTEP 20123–25); Mugaba, 02.2750°S, 28.6631°E,

2298 m (UTEP 20126–27); Mugaba, 02.2671°S, 28.6455°E, 2267 m, (UTEP 20128); Magunda village, Itombwe Plateau, 03.36255°S, 28.87713°E, 2526 m (UTEP 20129–32); forest near Miki,

Itombwe Plateau, 03.37261°S, 28.67794°E, 1965 m (UTEP 20111, 20120); Kisakala village,

Itombwe Plateau, 03.33744°S, 28.78844°E, 2277 m (UTEP 20133); Kizuka village, Itombwe

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Plateau, 03.03705°S, 28.46189°E, 2450 m (UTEP 20121–22); North Kivu Province: Virunga

National Park, Mt. Tshiaberimu, 00.13061°S, 29.43094°E, 2760 m (UTEP 20112).

Leptopelis kivuensis— Democratic Republic of the Congo: South Kivu Province: Kahuzi-Biega

National Park: vicinity of Tshivanga, 02.199°S, 28.459°E, approximately 2200 m (UTEP

20135); Mbayo, 02.2784°S, 28.7718°E, 2160 m (UTEP 20136); Chinya River near Mugaba

(Kahuzi-Biega National Park), 02.27301°S, 28.65862°E, 2290 m (UTEP 20137); Mugaba,

02.27288°S, 28.66208°E, 2289 m (UTEP 20138–42, 20144); Mugaba, 02.2713°S, 28.6666°E,

2268 m (UTEP 20145); Mugaba, 02.2713°S, 28.6610°E, 2275 m (UTEP 20146; Mugaba,

02.2714°S, 28.6643°E, 2275 m (UTEP 20147); Mugaba, 02.2713°S, 28.6666°E, 2277 m (UTEP

20148); Mugaba, 02.2713°S, 28.6664°E, 2285 m, (UTEP 20149–51 ); Uganda: Kabale District:

Bwindi Impenetrable National Park, Mubwindi Swamp, northwestern end, 01.0685°S,

29.7483°E, elevation not recorded, CAS 201703).

Leptopelis cf. kivuensis— Democratic Republic of the Congo: North Kivu Province: Virunga

National Park, Mt. Teye, 00.5672°N, 29.9127°E, 1702 m (UTEP 20152–53).

Leptopelis modestus— Cameroon: Nsoung, 04.9847°N, 09.8113°E, 1400 m (MCZ A-138022–

23).

Leptopelis parbocagii.—Democratic Republic of the Congo: Katanga: Kyolo, 08.0139°S,

27.1129°E, 560 m (UTEP 20552).

Leptopelis sp.— Democratic Republic of the Congo: Katanga: Kabongo, 08.7331°S, 28.2012°E,

1014 m (ELI 136); Mitwaba, 08.6267°S, 27.3392°E, 1568 m (ELI 180); Monano, 07.2777°S,

27.3898°E, 627 m (ELI 324, 364); South Kivu Province: Nundu, 02.7048°S, 28.9463°E, 1346 m

(ELI 396).

Leptopelis versicolor— Democratic Republic of the Congo: South Kivu Province:

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Itombwe Plateau, Kiandjo, 03.3726°S 28.6432°E, 1816 m (UTEP 20581–89); Kitopo (Itombwe

Plateau), 03.4039°S 28.5866°E, 1837 m (UTEP 20576-79); Itombwe Plateau, Kalundu,

03.1555°S 28.4210°E, 1482 m (UTEP 20564–69); Itombwe Plateau, Mwana River, 03.1572°S

28.4234°E, 1443 m (UTEP 20573–74), 03.1555°S 28.4210°E, 1482 m (UTEP 20570–72);

Baraka (vicinity of Lake Tanganyika) 04.1083°S 29.0970°E, 777 m (UTEP 20575).

Scotobleps gabonicus.—Equatorial Guinea: Provencia Centro Sur: Monte Alen National Park,

Rio Bong, ca. 5 km W of Asoc., 910 m (YPM 6991).

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VITA

Frank Portillo began his college career in 2006, completing a B.S. in biology at the

University of Texas at El Paso (UTEP) in December 2010. During his time at UTEP, he participated in several field biology courses working and with species from the Chihuahuan

Desert. During the spring of 2010, Frank became a volunteer for the El Paso Zoo. Assignments included care and maintenance of many species of animals in the Education Department. Frank began his graduate degree at UTEP in January 2011. During his graduate stint, he received a grant from the UTEP graduate school to support his research. Frank has also been a teaching assistant for Introductory Biology (BIOL 1107) and Human Anotomy and Physiology II (BIOL

2113). He is a member of the Society for the Study of Amphibians and Reptiles and the Chicago

Herpetological Society. He presented his graduate research at the World Congress of

Herpetology meeting in Vancouver, Canada in August 2012. He plans to pursue a doctoral degree in evolutionary biology at UTEP and would like to take a curatorial position at a museum or a research position at an academic institution, where he can continue making contributions to herpetology.

Permanent address: 3132 Edgerock Dr.

El Paso, TX, 79902

This thesis/dissertation was typed by Frank Portillo

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