Morphometric, Molecular Phylogenetic and Gene Expression Approaches towards the Understanding of the Adaptive Radiations of the East African

Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)

an der

Universität Konstanz, Mathematisch-naturwissenschaftliche Sektion Fachbereich Biologie vorgelegt von Céline Clabaut, M.Sc.

Konstanz, Dezember 2005

1 ACKNOWLEDGMENTS

I first want to thank Prof. Axel Meyer for welcoming me in his laboratory and giving me the opportunity of completing a doctoral degree in the University of Konstanz.

I also thank Dr. Walter Salzburger for getting me started on all the projects that could not have happened without his work. I am grateful for all the ideas he shared with me, and his availability to answer my questions.

Dr. Paul Bunje gave me the chance of experiencing a fruitful collaboration by improving the morphometric work, and I am grateful to him for this (as well as all the corrections he did of my English writing).

I am grateful to people belonging to the Meyer lab in general. I have never been denied or refused help, time, book or answers, each time I have been asking for them. I have to particularly acknowledge Arie and Dirk, since computer-related issues are the field where I have been more demanding, and Elke for being so efficient.

I am so thankful to the friends I made here in Konstanz. It took a while for me to meet them, but they really made life here more enjoyable. Some even provided support in addition to fun, and I am particularly grateful to them. I also want to thank my friends from France and my family. They have proven to be even more important with the distance than before (was it possible?). Thanks for listening, believing and being proud and being there. Finally, I want to mention here Arne, because he makes my life so beautiful.

2

“On dit aussi que les savants qui fréquentent les princes sont les pires des savants.” Amin Maalouf, Samarcande

3 TABLE OF CONTENTS

ACKNOWLEDGMENTS 2 TABLE OF CONTENTS 4

Chapter I: Introduction 6 THE CICHLIDS 7 Cichlids around the world 7 Cichlids of East Africa 9 ADAPTIVE RADIATION 11 Trait utility 11 Phenotype-Environment correlation 12 Common ancestry 12 Rapid speciation 13 RADIATION OF THE EAST AFRICAN CICHLIDS 14 Adaptive stages of the radiation 14 Non adaptive stage of the radiation through sexual selection 15

Chapter II: Geometric Morphometric Analyses Provide Evidence for the Adaptive Character of the Tanganyikan Radiations 16 ABSTRACT 17 INTRODUCTION 18 MATERIALS AND METHODS 23 Specimens 23 Data collection 23 Phylogenetic analyses 28 Statistical analysis 28 Disparity within and among lineages 30 RESULTS 31 Phylogenetic analysis 31 Description of the morphospace 33 Influence of phylogeny on body shape evolution 36 Influence of ecology on body shape evolution 37 Disparity within and among 42 DISCUSSION 46 The adaptive character of the radiations 47 Potential for diversification 50 Independent adaptive radiations in Lake Tanganyika 51 ACKNOWLEDGMENTS 52

4 Chapter III: Comparative Phylogenetic Analyses of the Adaptive Radiation of Lake Tanganyika Cichlid : Nuclear Sequences are Less Homoplasious but also Less Informative than Mitochondrial DNA 53 ABSTRACT 54 INTRODUCTION 55 MATERIAL AND METHODS 61 Molecular Methods 61 Phylogenetic analyses 62 Analysis of Character Evolution 64 Pairwise Genetic Distances Comparison 64 RESULTS 66 Results of the Phylogenetic Analysis 66 Mapping of Molecular Character Evolution 70 Pairwise Distances Comparisons 72 DISCUSSION 74 Missing Data and Support Values 74 The Phylogeny of the Lake Tanganyika Cichlid Assemblage 75 Contribution of Nuclear DNA 78 ACKNOWLEDGMENTS 82

Chapter IV: Characterization of genes involved in cichlid coloration using microarrays 83 ABSTRACT 84 INTRODUCTION 85 MATERIAL AND METHODS 89 RESULTS 92 DISCUSSION 97 Xanthophores markers 98 Genes involved in actin related intracellular transport 98 Endosomal and Golgi transport 99 AKNOWLEDGMENTS 102

SUMMARY 103 ZUSAMMENFASSUNG 106 REFERENCES 109

5 Chapter I: Introduction

6 That cichlids are an ideal model system to study adaptive radiations has been written so many times that it is almost impossible to find an innovative way to say it once more. Writing an introduction on East African cichlids is equivalent to the way cichlids evolve: no real innovation, but rather tinkering from an already existing toolkit (Stiassny 1991a; Strauss 1984). However this thesis brings novelty in our understanding of the cichlids evolution through the work realized during my PhD and that I will develop in three chapters. These three projects can be seen as attempts to increase our knowledge on particular aspects of the cichlid radiations, supporting the fact that these are the ideal model for the study of radiations.

THE CICHLIDS

Cichlids around the world

The cichlids are teleosts fish belonging to the class of the (ray- finned fish). They are included in the Perciforms, which is with 150- 230 families the largest order of fishes. Most families in many suborders are not currently definable in terms of shared derived characters and thus may not be monophyletic (Froese and Pauly 2005) and Miya, personal communication). However, cichlids are believed to be closely related to the Embiotocidae (surfperches), the Pomacentridae (damselfishes) and the Labridae ( and parrotfishes) within the Labroidei suborder. Cichlids are found in South and Central America (400 species) including Cuba and Hispaniola (4 species); Africa (more than 2000 species); Madagascar (more than 18 species); Arabia and adjacent areas (Israel, Syria, Iran) (5 species) and India and Sri Lanka (3 species). While the South American and Central American cichlids, the African cichlids and the Indian cichlids are monophyletic, the Malagasy clade also nests the Indian species (Fig. I.1).

7

Fig. I.1A, B Distribution of the Cichlidae. A The distributional pattern of the cichlids, with the representatives from India, Sri Lanka, and Madagascar forming the most basal lineages and the reciprocally monophyletic African and American lineages as sister-groups, is consistent with an initially Gondwanaland distribution (Zardoya et al. 1996; Streelman et al. 1998; Farias et al. 2000, 2001; Sparks 2004). B The supercontinent of Gondwanaland some 200 million years ago (MYA). Figure and legend from (Salzburger and Meyer 2004)

The Gondwanian distribution of the cichlids favors the hypothesis of drift vicariance. An other hypothesis of dispersal across marine environments was also suggested based on molecular clock estimates of divergence (Kumazawa et al. 2000; Vences et al. 2001), the tolerance of some cichlids to salty water and the fact that all others Labroidei but one are marine fish. The fossil record was also use to defend the dispersal hypothesis. Indeed, the oldest fossil of cichlid found dates from the Eocen (54-38Ma) (Murray 2001) giving to the family a minimum age of 45 millions years. However, this fossil is morphologically indistinguishable from extant forms. Such a derived specimen indicates that cichlids are most likely a much older group than what the fossil implies (Sparks 2003). Also, the studies giving the divergence time estimated on molecular clock have been criticized (see Chakrabarty 2004). Therefore, the drift vicariance scenario remains the favored one, supported by phylogenies of numerous authors (Cichocki 1976; Farias et al. 2000; Farias et al. 1999; Schliewen and Stiassny 2003; Sparks 2003; Streelman et al. 1998; Zardoya and Meyer 1996).

8 Cichlids of East Africa

Among the cichlids, some clades from East Africa have been particularly prone to explosive speciation and morphological, ecological and behavioral radiations. Cichlids in East Africa are found in lakes and rivers that provide a huge sweet water system widespread on Burundi, Kenya, Rwanda, Tanzania, Zambia, Malawi and Uganda. The three lakes of the East African Rift valley – Lake Tanganyika, Lake Malawi and Lake Victoria - are considered as nest for adaptive radiations and explosive speciation. Lake Tanganyika is the oldest of the rift lakes. Its central basin began to form between 9 and 12 MYA, the northern (8-7 MYA) and the southern basin (2–4 MYA) began to fill at later periods (Cohen, 1997); deepwater conditions exist since about 5–6 MYA (Tiercelin and Mondeguer 1991), and that age has also been estimated for the lineages that have primarily seeded the lake (Nishida 1991). Indeed, the oligophylic assemblage from lake Tanganyika was probably seeded by seven (Nishida 1991) or eight (Salzburger et al. 2002b) riverine ancestral lineages. However, the subsequent radiation occurred within the lake basin: the Tanganyikan assemblage is composed of 197 endemic species in 49 endemic genera (Poll 1986), that are morphologically highly diverse (see Chapter II). The of this assemblage is reviewed in more detailed in the introduction of the Chapter III (or (Clabaut et al. 2005). Lake Tanganyika is believed to be an old reservoir (Nishida 1991) from which the haplochromine ancestor of the species flock of Lake Malawi and Victoria originated (Salzburger et al. 2005). Lake Malawi is 2 to 4 Millions years old, but the cichlids are thought to have invaded it 700,000 years ago only (Meyer et al. 1990). Literature provides different estimates for the cichlid species diversity in the Malawian flock: between 450 and 600 species (Genner and Turner 2005), more than 800 endemic species in 49 endemic genera for some other authors (Moran et al. 1994; Snoeks 2004). In this flock, five species are belonging to the Tilapiini tribe but the rest belongs to the Haplochromini (Turner 1996) and appears to be of monophyletic origin (Meyer 1993b). They include the mbuna, an important and highly studied group of cichlid fish to which Maylandia zebra, the model we use in the genomic study reported in Chapter IV belongs. The Lake Victoria is the youngest one of the three Great Lakes with an age of 250,000-750,000 years. Molecular phylogenetic studies agree that the age of Lake Victoria’s cichlid fauna is less than 200,000 years (Meyer et al. 1990; Nagl et al. 2000), probably about 100,000 years (Salzburger and Meyer 2004; Verheyen et al. 2003). It contains 300 endemics and originated from two

9 separate lineages (Verheyen et al. 2003). Its flock is, however, tightly associated with the species occurring in the surrounding lakes (Lakes Albert, Edward, George, Kyoga and Kivu and several smaller lakes in the region) and it is therefore referred to as a “superflock” (Greenwood 1973; Greenwood 1979; Greenwood 1980; Salzburger and Meyer 2004; Verheyen et al. 2003). Due to the extreme young age of this superflock, phylogenetic inference among the species is difficult because of shared mitochondrial haplotypes, as well as the persistence of ancestral polymorphisms in the non-coding region of the DNA (Nagl et al. 1998). Ecologically, the superflock can be divided in two clusters: the mpibi (rock dwellings) and the rest. Each of the cichlid species flock from the three East African lakes presents levels of endemicity reaching almost 100% (Greenwood 1984; Mayr 1984; Snoeks 2000) and remarkably, the lakes do not have a single cichlid species in common (Fryer and Iles 1972). The number of endemic species was found to be correlated with the size rather than the age of the lake (see Fig. I.2 or Salzburger and Meyer 2004). The Lake Malawi species flock is the exception, for it might be at a different stage in the radiation process than the other lakes (see also Danley and Kocher 2001).

Fig. I.2. The size, but not the age, of a lake roughly correlates with its species number of endemic cichlids (Mayr 1942). Figure and legend from (Salzburger and Meyer 2004).

10 ADAPTIVE RADIATION

Adaptive radiation is defined by Schluter (2000) as the evolution of ecological diversity in a rapidly multiplying lineage, i.e., the differentiation from a single ancestor into an array of species that inhabit a variety of environments and that differ in traits used to exploit those environments. The Galápagos finches, the Anolis lizards and the Hawaiian silverswords are, together with the East African cichlids, the most famous model systems to illustrate adaptive radiations. However, numerous other groups, including invertebrates, are representatives of this phenomenon. Schluter (2000) defines four criteria that characterize an adaptive radiation. I report these criteria thereafter and summarize for each of them the literature existing on cichlids. I also show how the work realized during my thesis provided more evidence that the East African cichlids are the ideal model system to study adaptive radiations.

Trait utility

This criteria implies that there is evidence that morphological and physiological traits of species are indeed increasing the fitness of the corresponding species in the corresponding environment. It demands to test - experimentally and/or theoretically - whether traits associated with particular environment consistently enhance performance there. The evolutionary success of the cichlids in their diversity, and the high speciation rates found within the radiating flock can only confirm the fitness advantages given by the different traits. However, very few quantitative data are available on the matter. The adaptive nature of feeding in cichlid was studied by electromyographic technics by (Liem 1974; Liem 1978; Liem 1979; Liem 1980a; Liem 1980b; Liem and Osee 1975). Studies on the advantages of feeding behavior and dental morphology on the scraping of algae in Lake Tanganyika (Yamaoka 1983) have also been performed.

11 Phenotype-Environment correlation

Non-adaptive radiation is a logical null hypothesis in any cases, and adaptive radiation cannot be assumed a priori. Therefore, the adaptive nature of a radiation needs to be tested through the establishment of correlations between phenotypes and environment. Numerous examples of convergent evolution between ecologically equivalent genera or species from different lakes support the existence of a correlation between environment and phenotype (Kocher et al. 1993). The convergence of body shape in some ecomorphologically equivalent cichlid species from Lakes Malawi and Tanganyika, hypothesized by Fryer and Iles (1972) was also tested using geometric morphometrics (Kassam et al. 2003b). Resource-based divergent selective regimes in cichlids led to resource partitioning and brought about similar trophic morphologies independently and repeatedly (Rüber et al. 1999). Evolution of body shape has been shown to be correlated with trophic related features, and therefore with feeding habits (Liem 1974; Liem 1978; Liem 1979; Liem 1980a; Liem 1980b; Liem and Osee 1975; Rüber and Adams 2001). However, although these studies establish a clear link between environment and phenotype, they do not offer statistical evidence of this correlation. This lack has been filled with the work shown in Chapter II. We first show the small influence of the phylogeny on the body morphology of 45 Tanganyikan cichlids species, using geometric morphometrics. We then reveal a significantly positive correlation of body shape with ecological traits such as feeding habits and depth of water column in which cichlids occur. This study is the first one providing quantitative evidence for the adaptive nature of the cichlid radiation.

Common ancestry

An adaptive radiation applies to a group defined by a common ancestor. The concept of common ancestry is different from monophyly, since all the descendants of one ancestor do not necessarily radiate. In this sense, the whole cichlid family could be seen as a unique radiation, with only some subgroups effectively radiating. However, the separation into several independent or replicate adaptive radiations is preferred, because the same sequence of adaptations to ecological niches evolved repeatedly in lineages that inhabit similar environments (see Bernatchez et al. 1999; Losos et al. 1998; Schluter and McPhail 1993). The phylogeny of haplochromines from the East African lakes

12 provides strong support for replicate adaptive radiations in East African cichlids (Clabaut et al. 2005; Salzburger et al. 2005). Even the Tanganyikan assemblage is probably falling under the concept of replicate adaptive radiations. First as a consequence of its oligophyly, one should consider the Trematocarini (8 species), the Bathybatini (8 species), the Eretmodini (4 species), the (~100 species), and the C-lineage (~80 species) as independent or parallel intralacustrine species flocks (Salzburger and Meyer 2004), of which only the Lamprologini and some tribes of the C-lineage radiated significantly, with similarities in morphological and ecological characteristics (see Chapter II).

Rapid speciation

This criteria implies the existence of one or more bursts in the emergence of new species around the time that ecological and phenotypic divergence is underway. The young ages of the East African lakes and the great number of endemic cichlid species they contain is characteristic of rapid speciation events. As consequence of the young age of lakes Malawi and Victoria species flocks for example, the reconstruction of the relationship between cichlids has been shown particularly complicated because of the persistence of ancestral polymorphisms across species (Moran and Kornfield 1993) for Lake Malawi). Although from an older age, phylogenetic reconstruction for the Tanganyikan cichlid species flock offered so far unsatisfying results. As will be detailed in Chapter III, the Tanganyikan cichlid tribes have been studied using different markers: allozyme data (Nishida 1991), SINE insertion patterns (see, e.g., Takahashi et al. 1998), mitochondrial DNA sequences (see, e.g., Baric et al. 2003; Koblmueller et al. 2004a; Kocher et al. 1995b; Salzburger et al. 2002b; Sturmbauer et al. 2003; Sturmbauer and Meyer 1992; Sturmbauer et al. 1994). These works were overall in agreement with the eco-morphologically defined tribes of Poll (1986). However, the relationships of these tribes with each other remained unclear, justifying the use of a slowly evolving molecular marker to resolve the Tanganyikan phylogeny. Phylogenetic trees and a discussion of the use of the molecular marker RAG1 in comparison to the mitochondrial DNA marker ND2 are shown in Chapter III. The phylogeny, still unresolved in some parts, is another illustration of the rapid (and probably recent) nature of the cichlids speciation in Lake Tanganyika.

13 RADIATION OF THE EAST AFRICAN CICHLIDS

Through recent phylogenetic and population genetics studies of radiating groups, it has been shown that different vertebrate radiations (Galapagos finches and East African cichlids for example) follow similar evolutionary trajectories (Streelman and Danley 2003). Groups diverge along axes of habitat, trophic morphology and communication, often in that order, defining three stages in the process of the radiation. Variation in this sequence can be observed in some groups, but the East African cichlids, offer again an ideal model system to illustrate this radiation in three stages.

Adaptive stages of the radiation

The primary radiation and the creation of macrohabitat clades An early ecological split appears to be common in cichlid radiations. The adaptation to two major macrohabitats resulted in two large benthic clades: the sand-dwellers and the rock-dwellers in Lake Malawi (Streelman and Danley 2003); (Danley and Kocher 2001; Moran et al. 1994), in Lake Victoria (Nagl et al. 2000), and in Lake Tanganyika (Sturmbauer 1998). Also West African cichlids have experienced sympatric divergence of benthic and pelagic forms in multiple crater lakes (Schliewen et al. 1994). Finally, this pattern occurs in a great number of other lacustrine fish groups as well.

The secondary radiation and the refinement of the trophic apparatus Cichlids are characterized by a restructuring of pharyngeal jaw and a decoupling of certain oral jaw element (Liem 1974; Liem 1980a). The jaws are exceedingly versatile and adaptable; for example, they can change in form even within the lifetime of a single individual (see discussion in Meyer 1990a; Meyer 1990b). Although this “key innovation” occurs also in Labroid that do not show any exceptional species diversity, it is though to have increased the diversity of trophic resources available to cichlids and permitted the diversification of trophic structures. The phenomenon has been documented for the mbuna and other cichlid lineages (Liem 1974) that diversified in response to competition for trophic resources with only minor changes in structures unrelated to trophic morphology.

14 Non adaptive stage of the radiation through sexual selection

Speciation on the basis of sexual selection in East African cichlids has been proposed on the basis of field observations (van Oppen et al. 1998) as well as mate choice experiments in the laboratory (Knight et al. 1998; Seehausen et al. 1999). Further evidence for sexual selection came from the observable breakdown of visual reproductive barriers under monochromatic light conditions or in turbid waters (Seehausen et al. 1997). These results suggest that male body hue is the primary discriminatory factor among a hierarchy of visual cues used by females (Seehausen and van Alphen 1998). Radiations in which phenotypic divergence is driven by secondary sexual is also qualified as non-adaptive radiation, since the evolution of preferences is not guided by environment. The non-adaptive nature of the speciation leads to a species divergence exceeding the one generated by the two first stages mentioned above: a comparison of the within to between genera variation in color pattern suggests that male reproductive coloration has diversified more rapidly than other characters such as depth preference, preferred substrate size and aggression (Deutsch 1997), see also (McKaye 1984; Ribbink et al. 1983). As a consequence, males are larger and are brightly colored, whereas females are generally smaller and cryptically colored (Ribbink et al. 1983). This is the case of Maylandia zebra, a cichlid belonging to the Lake Malawi species flock. The males of Maylandia zebra are blue (the more dominant being more brightly colored than the non-dominant) and female and juveniles are yellow. Since the divergence of male color patterns has significantly contributed to the rapid diversification of the sand-dwelling cichlids of Lake Malawi (McKaye et al. 1993; Taylor et al. 1998) we found relevant to assign Maylandia zebra as model fish to study genes involve in skin coloration. This genomic work is further detailed in Chapter IV.

15 Chapter II: Geometric Morphometric Analyses Provide

Evidence for the Adaptive Character of the Tanganyikan

Cichlid Radiations

Céline Clabaut, Paul M.E. Bunje, Walter Salzburger, Axel Meyer

16 ABSTRACT

The cichlids of East Africa are renowned as an important model system for the study of adaptive radiation. However, the relationships between ecology, morphological diversity, and phylogeny for these fish have not been satisfactorily quantified yet. We used geometric morphometric methods to describe the body shape of 45 species of East African cichlid fish, with a focus on the Lake Tanganyika species assemblage which is composed of more than 200 endemic species. The main differences in shape concern the length of the whole body and the relative sizes of the head and caudal peduncle. We investigated the phylogenetic signal contained in the morphometric data set with the Phylogenetic Mixed Model, as well as the importance of the influence of phylogeny on similarity of shape between closely related species with cluster analyses and Normalized Mantel statistics on the phylogenetic distances and distances in the morphospace. After concluding that the influence of phylogeny was small, we investigated the evolution of body shape in relation to known ecological traits using MANOVA. We found that body shape was strongly predicted by feeding preferences (i.e., trophic niches) and the water depths at which species occur. Calculation of the disparity within tribes indicates that even though the morphological diversification associated with explosive speciation has happened in only a few tribes of the Tanganyikan assemblage, the potential to evolve diverse morphologies exists in all tribes. Quantitative data support the existence of extensive parallelism in several independent adaptive radiations in Lake Tanganyika. Notably, Tanganyikan mouthbrooders belonging to the C-lineage and the substrate spawning Lamprologini have evolved a multitude of different shapes from elongated and Lamprologus-like hypothetical ancestors.

17 INTRODUCTION

The Great Lakes of East Africa (Victoria, Malawi and Tanganyika) are among the world’s most diverse freshwater ecosystems and contain unique species flocks of cichlid fish comprised of hundreds of endemic species each (Fryer and Iles 1972). The large number of species, the high degree of ecological and morphological specialization, and the rapidity of lineage formation make these fish ideal model systems for the study of adaptive radiation and explosive speciation (Fryer and Iles 1972; Kocher 2004; Kornfield and Smith 2000b; Meyer 1993c; Salzburger and Meyer 2004; Stiassny and Meyer 1999). Adaptive radiation was defined by Simpson (1953) as the “more or less simultaneous divergence of numerous lines from much the same adaptive type into different, also diverging adaptive zones”. In the case of the East African cichlids, this diversification process has indeed occurred within a short period of time over wide geographic areas, resulting in many short branch lengths and unresolved phylogenies (Kornfield and Smith 2000b). However, the occupation of diverging adaptive zones has not previously been analyzed quantitatively. In addition to the rapidity of speciation, (Schluter 2000) defines three other criteria of adaptive radiations: common ancestry, phenotype-environment correlation, and utility of the trait (performance or fitness advantages of the trait in its corresponding environment). Through the quantification of the morphological variation of the body shapes of many cichlid species, we tested the phenotype-environment correlation for the Tanganyikan cichlid species flock, a critical estimate of the breadth of adaptive zone occupation. Lake Tanganyika is the oldest of the Great Lakes with an estimated age of nine to twelve million years (Cohen et al. 1997; Cohen et al. 1993). Although lakes Victoria and Malawi are younger, they harbor more species of cichlid fish (~500 and ~1,000 species respectively). However, the estimated 250 cichlids species (Brichard 1989; Snoeks et al. 1994; Turner et al. 2001) in Lake Tanganyika are morphologically, ecologically and behaviorally the most diverse. The original assignment to tribes was established by Poll (1986). Recently, Takahashi (2003) suggested the erection of five additional tribes for genera previously assigned to one of Poll’s twelve tribes (Poll 1986). These taxonomic studies were based solely on morphological characters. Furthermore, it has been established with molecular markers that the lake was seeded by several ancient lineages (Kocher et al. 1995a; Nishida 1991; Salzburger et al. 2002b) which evolved in step with changes in the lake´s environment: the Tylochromini, Trematocarini, Bathybatini, Tilapiini, Boulengerochromini, Eretmodini, the

18 ancestor of the Lamprologini and the ancestor of the C-lineage (Clabaut et al. 2005). The C-lineage diversified further from a supposed Lamprologus –like ancestor (Koblmueller et al. 2004b; Salzburger et al. 2002b) into eight tribes, the Cyphotilapiini, the , the Cyprichromini, the Perissodini, the Orthochromini, the , and the Haplochromini including the Tropheini (Clabaut et al. 2005; Kocher et al. 1995a; Salzburger et al. 2005; Salzburger et al. 2002b; Sturmbauer and Meyer 1993; Verheyen et al. 2003). The cichlids - alone with the characids and the castomids (McCune 1981) - are one of the few fish families with extreme large variation in body shape. This degree of morphological diversity exists among all tribes of cichlids from Lake Tanganyika, but as well as within tribes. Interestingly, convergence in eco- morphological traits and coloration patterns appears to be common between groups that are distantly related (Kocher et al. 1993; Meyer 1993c; Rüber et al. 1999; Stiassny and Meyer 1999) and even closely related (Reinthal and Meyer 1997; Rüber et al. 1999). Variation in body form has important fitness consequences (Gatz 1979; Guill et al. 2003). It is therefore important to quantitatively describe differences in body shape that exist between cichlids within a phylogenetic framework to understand their occupation of different ecological niches within Lake Tanganyika. Morphometrics is the study of shape variation and its covariation with other variables of interests (Bookstein 1991; Dryden and Mardia 1998). Geometric morphometrics is a recently developed approach that explicitly retains information on spatial covariation among landmarks (Rohlf and Marcus 1993). These landmark-based techniques pose no restrictions on the directions of the variation and the localization of shape changes, and they are effective in capturing information about the shapes of organisms. Geometric morphometrics also allows the reconstruction of a group consensus shape and the hypothetical shape of a common ancestor. It is possible to visualize changes and transformations necessary to distinguish one shape from another. Multivariate statistical procedures are complementary to morphometric methods (Calvalcanti et al. 1999; Rohlf et al. 1996; Rohlf and Marcus 1993; Zelditch et al. 2004) since they allow the statistical characterization of the morphological variation itself. They are also used to test for significant correlations between body shape and ecological traits or to evaluate the importance of phylogenetic relationships to the similarity of shape among two closely related species. Closely related taxa are expected to be more similar to one another than they would be without shared evolutionary history (Felsenstein 1985; Guill et al. 2003; Rosenberg 2002; Rüber and Adams 2001). It is therefore important to include phylogenetic information

19 with geometric morphometric approaches to reliably establish the link between an observed pattern of morphological variation with the hypothesized underlying adaptive process (Coddington 1990; Linde et al. 2004) by removing co-variation due to common ancestry. In the last 15 years, although the use of geometric morphometrics methods has grown rapidly (Adams et al. 2004), studies on the shapes of cichlids have been mainly restricted to traditional morphometrics (Hanssens et al. 1999; Kassam et al. 2003a). Geometric morphometric methods have been used in only a few publications: Bouton et al. (2002) found that the head shapes of haplochromines of Lake Victoria are correlated with eight environmental variables; Rüber and Adams (2001) established a correlation between body shape and trophic morphology for the Tanganyikan tribe Eretmodini, features that turned out to be independent of their phylogenetic relationships. Studies on whole body shape variation have also helped to distinguish morphologically different species that belong to the same complex (Klingenberg et al. 2003) and to demonstrate morphological convergence between distantly related species (Kassam et al. 2003b). Recently, geometric morphometrics were used to test conjectures about the relative degree of morphological diversity among Lake Malawi and Tanganyika cichlid species flocks (Chakrabarty 2005). Here we present a geometric morphometric study based on the largest data set so far for Lake Tanganyika cichlids, including 1002 specimens from 45 species. At least one representative of each of the seventeen tribes (Takahashi 2003a) was included in this study, except for the monogeneric tribes Benthochromini, Boulengerochromini, Greenwoodochromini, and Ctenochromis benthicola, for which no specimens were available. Nine riverine species and specimens from Lake Malawi and surrounding lakes have been included as well (see Table II.1). We quantify the body shapes of these 45 cichlids species using 17 landmarks. We previously conducted a phylogenetic analysis of the Lake Tanganyika cichlids taxa based on available mitochondrial DNA sequences (the complete ND2 gene) (Clabaut et al. 2005; Salzburger et al. 2002b). We use several methods (the phylogenetic mixed model (Housworth et al. 2004), cluster analyses, Mantel statistics on genetic versus morphological distances) to test for the influence of phylogeny on body form evolution. Variation among species is also discussed with reference to several ecological traits (trophic preferences, habitat differentiation, various mating and breeding systems) using various statistical tools. Finally, we describe morphological disparity within and among tribes, as well as the structure of the morphospace with species assigned to ecological groups. These analyses

20 enable us to confirm the adaptive character of the radiation, as well as to discuss the role of body shapes and their potential for invading different adaptive zones, which ultimately led to the coexistence of a large number of cichlid species in Lake Tanganyika (Kassam et al. 2003a; Rüber and Adams 2001).

21 Table II.1. Characterization of the included species of Lake Tanganyika cichlids.

Taxonomy information sampling locality GenBank accession nr nr of specimens Tribe Taxon ND2 Bathybatini sp. Lake Tanganyika U07239 22 Cyphotilapiini frontosa Lake Tanganyika U07247 8 Cyprichromini Cyprichromis leptosoma Lake Tanganyika AF398224 29 Paracyprichomis brieni Lake Tanganyika AF398223 24 Ectodini Callochromis stappersi Lake Tanganyika AY337775 14 Cunningtonia longiventralis Lake Tanganyika AY337780 24 Cyathopharynx furcifer Lake Tanganyika AY337781 18 Ectodus descampsi Lake Tanganyika AY337790 15 Enantiopus melanogenys Lake Tanganyika AY337770 25 Grammatotria lemairii Lake Tanganyika AY337787 46 nasuta Lake Tanganyika AY337783 30 Xenotilapia ochrogenys Lake Tanganyika AY337767 32 Eretmodini Eretmodus cyanostictus Lake Tanganyika AF398220 20 erythrodon Lake Tanganyika AF398218 16 Haplochromini Astatoreochromis alluaudi Lake Kanyaboli AY930075 19 Haplochromis paludinosus Nanganga River AY930107 15 Melanochromis auritus Lake Malawi AY930069 28 Metriaclima zebra Lake Malawi U07263 25 Pseudocrenilabrus multicolor Lake Kanyaboli AY930070 16 Lamprologini compressiceps Lake Tanganyika AF398229 21 Julidochromis ornatus Lake Tanganyika DQ093111 18 Lamprologus congoensis Congo River AF317272 6 Lamprologus cylindricus Lake Tanganyika DQ093115 4 Lamprologus teuglesi Congo River AF398225 16 Telmatochromis vittatus Lake Tanganyika AY740396 25 leleupi Lake Tanganyika DQ093113 28 Julidochromis regani Lake Tanganyika AF398230 20 Neolamprologus calliurus Lake Tanganyika AF398227 30 Limnochromini Limnochromis auritus Lake Tanganyika AF398216 15 Orthochomini Orthochromis malagaraziensis Malagarazi River AF398232 30 Orthochromis uvinzae Malagarazi River AY930048 9 Orthochromis mazimeroensis Mazimero River AY930053 17 Perissodini Perissodus microlepis Lake Tanganyika AF398222 18 straeleni Lake Tanganyika AF398221 12 Tilapiini Tilapia rendalli East Africa AF317259 31 Oreochromis tanganicae Lake Tanganyika AF317240 29 Trematocarini unimaculatum Lake Tanganyika AF317268 30 Tropheini Ctenochromis horei Lake Tanganyika AY930100 18 Limnotilapia dardennii Lake Tanganyika DQ093109 35 Lobochilotes labiatus Lake Tanganyika U07254 16 Petrochromis polyodon Lake Tanganyika AY930068 29 Simochromis babaulti Lake Tanganyika DQ093110 23 Simochromis diagramma Lake Tanganyika AY930087 22 Tropheus duboisi Lake Tanganyika AY930085 37 Tylochromini Tylochromis polylepis Lake Tanganyika AF398215 37

22 MATERIALS AND METHODS

Specimens

Specimens included in this study, their origins, the number of specimens per species, their assignment to one of the seventeen tribes according to Takahashi (2003), and GenBank accession numbers of DNA sequences are listed in Table II.1. Voucher specimens are deposited in the Royal Museum for Central Africa in Tervuren, Belgium.

Data collection

Images of the left side of 1902 individuals were taken with a digital camera (Nikon Coolpix 995) in the Museum for Central Africa in Tervuren, Belgium. Specimens belong to 45 different species representing 14 of the 17 tribes to which Tanganyikan cichlids have been assigned (see Takahashi 2003). Some of the cichlids had their mouth open through a combination of head lifting (Liem 1991; Liem and Summers 2000; Westneat 1990) and the posession protusile jaws (Barel 1983). Since these mechanisms might introduce unwanted bias in the position of head landmarks, with an open mouth were eliminated from the following analyses. The x, y coordinates of 17 landmarks (Fig. II.1) were digitized as described elsewhere (Klingenberg et al. 2003). These were measured twice by the same person, and the mean of the two measurements was used as raw data for subsequent analyses.

23

7

5

6 13

3 14 2 8 1

9 17

10

16 15 4 11 12

Fig. II.1. Description of the landmarks: 1 = Tip of the snout at the fold anterior to the ethmoid/nasal bones, touching the upper lip when the premaxilla is retracted (mouth closed); 2 = Corner of the mouth, at the corner of the skin fold where the maxillary angle rests when the mouth is closed; 3 = Center of the eye; 4 = Base of the isthmus; 5 = Boundary between smooth and scaly skin; 6 = Dorsal end of the pre-occular groove; 7 = Anterior base of the dorsal fin; 8 = Opercular origin; 9 = Base of the leading edge (upper, anterior) of the pectoral fin; 10 = Base of the trailing edge (lower, posterior) of the pectoral fin; 11 = Anterior base of the pelvic fin; 12 = Anterior base of the anal fin; 13 = Posterior end of the dorsal fin base; 14 = Base of the caudal fin, dorsal; 15 = Posterior end of the anal fin base; 16 = Base of the caudal fin, ventral; 17 = Base of the caudal fin at the level of the lateral line.

24 Direct analysis of the coordinates would be inappropriate as the effects of variation in position, orientation and sizes of the specimens can introduce bias. Non-shape variation was therefore mathematically removed using tpsSuper (Rohlf 2004). This program performs a Generalized Procustes Analysis (GPA) that superimposes landmark configurations using least-squares estimates for translation and rotation. The centroid of each configuration is translated to the origin and configurations are scaled to a common unit size. Finally, the configurations are optimally rotated to minimize the squared differences between corresponding landmarks (Gower 1975; Rohlf and Slice 1990). Procustes analysis fits minimized least squared distances between each landmark in all specimens, while the relative distances of the 17 landmarks to each other remain constant. Principal component analyses (PCA) were performed for each tribe in PAST (Hammer et al. 2001) in order to identify outliers (specimens not belonging to the 95% ellipse). After discarding non-representative specimens, the matrix contained 1002 samples with 34 coordinates each. From this matrix we generated the consensus shape for each species and for each tribe using tpsSuper (Rohlf 2004). We collected information about ecological characteristics of each species from the literature, disregarding general information at the level of the tribe to avoid the influence of phylogeny on the calculation of correlations (Table II.2). We used the following references to complete the ecological character data-matrix for all taxa included in our analysis (Barlow 1991; Coulter 1991; Gerbrand 1998; Goodwin et al. 1998; Hori 1991; Kassam et al. 2003a; Konings 1988; Kuwamura 1997; Lowe-McConnell 2002; Nagoshi and Yanagisawa 1997; Nishida 1997a; Parsons 2003; Poll 1956; Ribbink 1991; Rüber and Adams 2001; Winemiller et al. 1995).

25 Table II.2. Ecological characters of included species species and tribe affiliation habitat parental care mating system Breeding feeding preferences depths type Bathybatini Bathybathes sp. bathypelagic maternal Mb fish 0-25-80 Cyphotilapiini Cyphotilapia frontosa rock maternal polygyny, harem Mb fish, large invertebrates 0-20-30 Cyprichromini Cyprichromis leptosoma rock maternal polygamous Mb pelagic zooplankton, copepods Paracyprichromis brieni littoral zone pelagic maternal polygamous Mb zooplankton and microbenthos Ectodini Ectodus descampsi sand maternal polygyamy Mb insects, invertebrates, plants 0-5-25 Callochromis stappersi sand maternal polygyamy Mb invertebrates Eniantiopus melanogenys sand maternal polygyamy Mb invertebrates Ophtalmotilapia nasuta intermediate maternal polygyamy Mb aufwuchs Cyathopharynx furcifer intermediate maternal polygamous Mb aufwuchs 0-5-15 Grammatotria lemairii sand maternal polygyny, harem Mb invertebrates and snails 0-15-60 Cunningtonia longiventralis intermediate maternal polygamy Mb filamentous algae Xenotilapia ochrogenys sand and mud biparental polygyamy; polyandry Mb copepods, larvae ostracods, 0-5-35 school benthos Eretmodini Eretmodus cyanosticus rock, surge water biparental monogamous Mb filamentous algae 0-5 Spathodus erythrodon rock, surge water biparental Mb diatoms, aufwuchs and insects 0-5 larvae Haplochromini Haplochromis paludinosus river maternal polygyny Mb generalist river Pseudotropheus zebra rock maternal Mb vegetarians Astatoreochromis alluaudi ubiquitous maternal polygyny Mb molluscs Pseudocrenilabus multicolor river maternal polygamy Mb generalist Melanochromis auratus rock maternal polygyny Mb omnivore Lamprologini Lamprologus mocquardii river maternal polygyny, harem Ss invertebrates river Lamprologus congoensis river maternal polygyny, harem Ss invertebrates river Julidochromis ornatus rock cooperative monogamy Ss zoobiocover 0-10 Lamprologus leleupi rock biparental monogamous Ss zooplankton, benthic arthropods, 0-5 invertebrates Lamprologus cylindricus rock biparental polygamous Ss invertebrates Altolamprologus calvus rock and sediment maternal monogamy Ss zoobiocover Telmatochromis vittatus intermediate maternal monogamous Ss aufwuchs and invertebrates 0-5 Julidochromis reganii intermediate biparental monogamy Ss zoobiocover 0-5 Lamprologus brichardi rock cooperative mono/polygamous/poly Ss zooplankton, algae gynous Limnochromini Limnochromis auritus mud biparental monogamous Mb invertebrates, snails, small fish 0-35- 115 Orthochromini Orthochromis malagaraziensis river maternal Mb plankton rsup Orthochromis uvinzae river maternal Mb algae river Orthochromis mazimeroensis river maternal Mb algae river Perissodini Perissodus microlepsis ubiquitous mat/biparental monogamy Mb scales and microbenthos 0-15-95 Plecodus straeleni rock biparental monogamy Mb scales and eggs 0-5 Tilapiini Tilapia rendalli river biparental monogamous Ss aquatic plants river Oreochromis tanganicae ubiquitous maternal polygyamy Mb detritus, phytoplankton 0-5-25 Trematocarini Trematocara unimaculatum mud maternal Mb diatoms, detritus, gastropodes, 0-50- crustaceans, zooplankton 120 Tropheini Simochromis diagramma mud maternal polygamy Mb aquatics weeds, algae rinf 0-15 Ctenochromis horei ubiquitous maternal polygyny Mb omnivore (benthos, fish, invertebrates) Lobochilotes labiatus rock maternal polygyny Mb gastropods, crabs, benthos 0-5-30 Petrochromis polyodon rock maternal polygamy aufwuchs, algae 0-5-25 Tropheus duboisi rock maternal polygamy Mb aufwuchs Limnotilapia dardennii rock and sand maternal polygyny Mb omnivore (algae, plants and 0-5-85 invertebraes) Simochromis babaulti mud maternal Mb vegetarian 0-30 Tylochromini Tylochromis polylepsis sand and mud maternal polygyny Mb molluscs, vegetal matters, water rinf 0-5- plants 75

26 Ecological data included differentiation of the habitat by “preferred depths in the water column” categories, i.e., the depths of the water column where the cichlids occur. This assignment (Poll 1956) was binned into four categories: shallow water (0 to 5m), medium water (0 to 30m), deep water (0m to more than 30m) and rivers. We also coded the substrate of the preferred habitat: mud, sand, rock, intermediate substrate between these categories or ubiquitous, a fifth category for rivers, and a sixth category for other habitats not involving any substrate (e.g., deep water species). Feeding preferences were assigned to six different categories: exclusively vegetal, zooplankton and detritus, benthic invertebrates, fish, scales, and a final category called “generalist” for species that are particularly opportunistic feeders. Another variable related to prey type, in which pelagic prey were divided into nektonic prey, represented by organisms that move actively in the water column, and planktonic prey, a category that grouped organisms suspended in the water column. The rest were coded as benthic prey, i.e., sessile prey or slow moving organisms living on the substrate (Linde et al. 2004). Finally, prey were coded as mobile or non-mobile. This characteristic might have an influence on the strategy used by a cichlid to capture the prey, and therefore on the shape of the fish predator. Information was also collected on the type of parental care given (maternal, bi-parental or involving helpers i.e., cooperative breeding), on mating system (monogamy, polygamy, polygyny and a fourth category for more complex mating behavior) and on breeding type (mouthbreeders or substrate guarders).

27 Phylogenetic analyses

Forty-five complete sequences of the mitochondrial NADH Dehydrogenase Subunit II gene (ND2; 1,047bp) (Clabaut et al. 2005; Koblmueller et al. 2004b; Salzburger et al. 2005; Salzburger and Meyer 2004; Salzburger et al. 2002b) were analyzed using maximum likelihood (ML) [with PAUP* 4.0b10 (Swofford 2002a)] methods. Tylochromis polylepis was declared as outgroup (according to (Farias et al. 2000; Lippitsch 1995; Salzburger et al. 2002b; Stiassny 1990). We ran the Modeltest 3.06 routine (Posada and Crandall 1998) to determine, with a hierarchical likelihood ratio test, the appropriate model of molecular evolution for the ML analyses. We used the GTR+I+Γ model (Rodriguez et al. 1990) with A=0.297; C=0.3795; G=0.0846; α =0.9192; I=0.3565 and κ A-C=0.4049; A- G=11.3001; A-T=0.6488;C-G=0.8348; C-T=4.2669; G-T=1. The ND2 gene contains enough informative characters to achieve a well supported topology for the Lake Tanganyika species assemblage (Clabaut et al. 2005; Salzburger et al. 2002b).

Statistical analysis

The morphometric data were reduced using a principal component analysis (PCA) implemented in PAST (Hammer et al. 2001) into three axes explaining 75% (in decreasing order 55.4%, 10.6% and 9%) of the variance. Using Mesquite (Maddison and Maddison 2004), we mapped our phylogenetic tree in the morphospace defined by the coordinates of the first three axes of the PCA. Mesquite also calculates the coordinates of the landmarks of the hypothetical ancestral shape at each node using a parsimony model. Additionally, we plotted the shape of the ancestor of the C-lineage in the morphospace to test the hypothesis that the ancestor of the C-lineage was Lamprologus-like as assumed by Salzburger et al. (2002) and Koblmüller et al. (2004). We used several methods to evaluate the influence of phylogeny on the various body shapes of the Tanganyikan cichlid assemblage. We calculated the Euclidean distances between each pair of taxa in the morphospace described by the three first components. Normalized Mantel statistics (Mantel 1967; Smouse et al. 1986) were then used to evaluate the correlation between the phylogenetic distances and the Euclidean distances in the morphospace between all pairs of taxa. The program “Test for Association Between Two Symmetric Distance Matrices with Permutation Iterations” (Calvalcanti 2005) was used to estimate the

28 probability of obtaining a correlation equal to or greater than the calculated value over 10,000 random matrix permutations. A cluster analysis using different algorithms (UPGMA, Ward’s method, and single linkage with Euclidean distances) was performed in PAST. These multivariate algorithms group species based on their overall morphological similarities, and enabled us to visualize which groups are congruent with those defined by the phylogeny. Statistical methods in comparative analyses must take into account the lack of independence of traits due to phylogeny. The method most often used to correct for phylogenetic effects is the Felsenstein Independent Contrasts (FIC) analysis (Felsenstein 1985). This method is based on a Brownian motion model in which the amount of change in a trait is proportional to the branch lengths. For adaptive radiations, interspecific variation may be best explained by rapid evolutionary changes rather than by slow gradual changes. We therefore performed, instead of the classic FIC, a comparative analysis of our morphometric coordinates using the Phylogenetic Mixed Model (PMM) (Housworth et al. 2004). With this model, in addition to the gradual accumulation of evolutionary changes, traits are allowed to evolve so rapidly that their current states are essentially unconstrained by their phylogenetic past. The measured correlation of the trait values between two species results partially from the phylogenetic relationship between species and partially from an independent, species-specific contribution. The PMM may be particularly effective for large clades that might exhibit considerable morphologic diversity at the tips of the phylogeny (Housworth et al. 2004), as is the case for East African cichlids. In an attempt to clarify the relationships between body shapes and different ecological characters, a MANOVA (Multivariate Analysis of Variance) was used in PAST. It evaluates whether predefined group centroids differ significantly in their position, i.e., we tested the alternative hypotheses that the multivariate Euclidean distances among means of our ecology based groups are not equal to 0. The significance is computed by a permutation of group membership, with 5000 replicates. Differences in shape among objects can be described in terms of differences in the deformation grids depicting these objects, following the principle of d’Arcy Thompson’s transformation grids (Thompson 1917). The shape differences of one specimen to another are represented as a bent grid superimposed over the coordinates of the initial specimen. For normalized shape coordinates, we used the thin-plate spline (Bookstein 1991; Dryden and Mardia 1998) to map the deformation in shape along axes of the morphospace. For statistically significant

29 multivariate analyses, we calculated the average shape of each ecological cluster, as well as the consensus shape of these averages [consensus calculated with tpsSuper (Rohlf 2004)]. We then depicted the transformation grid of each ecological group to this consensus, highlighting the principal differences in morphology existing between the significantly different ecological clusters defined by the MANOVA.

Disparity within and among lineages

In order to estimate the distribution of morphological diversity among tribes, we calculate values of morphological disparity for each of the tribes, as a test of the constraint on morphological diversification of the Lake Tanganyika cichlids. We performed independent Procrustes analysis on each tribe with PAST (Hammer et al. 2001) using the coordinates of all specimens included in the tribe. We then estimated, with IMP (Sheets 2005), the disparity existing within tribes and calculated the correlation between disparity and number of specimens or species contained in the tribe in our data set. We also bootstrapped the morphological disparity of the whole group, the contribution of each tribe to the overall disparity and the correlations of those disparity values with the number of species and specimen per tribe. An alternative approach developed by Foote (1993) was also used, comparing the morphological disparity of the 14 tribes included in our study to the disparity of this group after exclusion of one of the tribes at a time. This indicates which of the tribes have a significant effect on total morphological disparity. Finally, using species consensuses landmark coordinates, we computed within-group variability, among-group variability, and discreteness (Foote 1991; Smith and Bunje 1999) and performed 10,000 bootstrap replicates of the pairwise distances between species. We performed these calculations for species grouped by tribe (fourteen groups), but also by their feeding preferences (six groups), and by depth of habitat where the species occur (four groups) (see Materials and Methods; data collection). This method is used for the first time to assess the relationships (distance and overlap) between taxonomically and ecologically pre- defined groups (tribe assignment, habitat and feeding preferences). This constitutes a test of which characteristics significantly structure variation in the morphospace.

30 RESULTS

Phylogenetic analysis

The ML tree is shown in Figure II.2. Results are similar to previous studies (Clabaut et al. 2005; Kocher et al. 1995a; Salzburger et al. 2002b) with Tilapia rendalli (it does not belong to the Tanganyiakan assemblage) occupying the most ancestral position, followed by a clade formed by Bathybates minor, Oreochromis tanganicae, and Trematocara unimaculatum. These tribes are basal to two major clades, a clade formed by the Eretmodini sister group of the Lamprologini and a clade including all remaining taxa called the C-lineage (Clabaut et al. 2005). In the C-lineage, Limnochromis auritus and Cyphotilapia frontosa form the most ancestral lineages and are the sister groups to the Cyprichromini, the Perissodini, the Orthochromini, the Ectodini, and the Haplochromini including the tribe Tropheini. The Haplochromini are represented in our study by specimens from Lake Malawi, Lake Kanyaboli and from the Nanganga river. Species from Lake Malawi are the sister group to the Tropheini.

31

Fig. II.2. Maximum likelihood phylogram of Lake Tanganyika cichlids based on ND2 of 45 taxa. Tylochromis polylepsis was designated as the outgroup.

32 Description of the morphospace

In the PCA representation (Fig. II.3), the first two axes explain 55.4% and 10.6% of the variation respectively. The first axis is strongly loaded by components that represent the length and height of the body. Specimens on the left side of the graph are elongated and thin, while the ones on the right side are stouter and deeper. The second axis mostly describes variation in head shape. The lower the specimens are in the graph, the smaller and shorter their head is, the most posterior is their anal fin at the body, and the shorter is their caudal peduncle. The third axis (not shown) explains 8.96% of the variation and this axis is loaded by the position of the mouth and the caudal peduncle, shifted up to the rest of the landmarks and therefore expanding the ventral part of the body. The superimposition of the phylogenetic tree on the morphospace shows no directional trend in the evolution of body shape at the Tanganyikan assemblage level, nor at the tribe level. Basal tribes present a wide range of shapes: some have a wide and round body and head shape (like Oreochromis tanganicae), while others have a thin body and head shape (like Bathybates minor and Trematocara unimaculatum). The Lamprologini also occupy a large portion of the morphospace because some species have extreme shapes (e.g., the deep-bodied Altolamprologus calvus and the elongated short-headed Telmatochromis vittatus). Between these two species, a range of intermediate forms exists. The eight species that represent the Ectodini in our study are, in general, elongated fish but with a more pointed head than the majority of Tanganyikan cichlids. Variation in this tribe follows the transformation of the first axis, i.e., changes in the length of their body. On the other hand, the Haplochromini (including the Tropheini) have a deeper body, and are differentiated also on the second axis, i.e., they show an extensive range of head shapes. Interestingly, the pattern of shape evolution within tribes seems to be, based on the PCA, different for each of them. For example, the derived species of the Ectodini tend to be more elongated than ancestral ones, whereas the inverse pattern is observed for the Tropheini. The other species-rich groups (Haplochromini, Lamprologini) do not seem to show any discernable trend at all (at least within the morphospace defined by the first two axes of the PCA). Sister species in our phylogeny are often rather distant from each other in the morphospace: Perissodus microlepsis has a shape similar to the average Lamprologini, and Plecodus straeleni is found nested among the Haplochromini/Tropheini. On the other hand, some species are placed in the area of the morphospace occupied by species of a different tribe. The Cyprichromini,

33 for example, are found within the morphospace occupied by the Ectodini, the Orthochromini with the Lamprologini and the Eretmodini with Haplochromini/Tropheini. Phylogenetically unrelated species are characterized by similar coordinates in the space defined by the two first axes: Tilapia rendalli, Cyphotilapia frontosa and Petrochromis polyodon, for example. The coordinates of the hypothetical ancestor of the C-lineage, reconstructed by Mesquite, are found within the subspace occupied by the Lamprologini and close to the coordinates defining the position of the hypothetical ancestor of the Lamprologini (Fig. II.3).

34

PC2

PC1

Fig. II.3. (A) PCA plot (origin in the center of the plot) with phylogenetic relationships among species. The pictures of the most extreme shapes of this morphospace are shown to illustrate the differences. The deformation vectors from individuals showing minimal scores to individuals showing maximal scores along each the (B) horizontal axis (PC1) and (C) vertical axis (PC2) are also given.

35 This is consistent with the assumption that the ancestor of the C-lineage was probably a Lamprologus-like cichlid (Koblmueller et al. 2004b; Salzburger et al. 2002b), albeit somewhat shorter and wider than the hypothetical ancestor of the Lamprologini (Fig. II.4).

Fig. II.4. Deformation grid describing the differences between the body shape of the hypothetical ancestor of the Lamprologini (represented by points) and the hypothetical ancestor of the C-lineage.

Influence of phylogeny on body shape evolution

The assignment to tribe of the species contained in our data set was found to be significant with the non-parametric MANOVA of Euclidean distances (F= 3.794 and p<0.0001). However, none of the cluster analyses managed to recover the taxonomy at the tribe level. Only two tribes were recovered consistently with the three algorithms (i.e., with UPGMA, Ward’s method, and single linkage with Euclidean distances): the Eretmodini and the Tilapinii. All other tribes were morphologically too diverse to be clustered statistically. Some species that belong to the same tribe were consistently found to cluster together: among the Tropheini, the two Simochromis (S. diagramma and S. babaulti) with Limnotilapia dardenni; among the Ectodini: Callochromis stappersi, Ectodus melanogenys and Grammatotria lemarii; and Cyathopharynx furcifer and Cunningtonia longiventralis together. This latter pair of Ectodini is always found clustered with three Haplochromini/Tropheini (Haplochromis paludinosus, Astatoreochromis alluaudi and Ctenochromis horei). The Orthochromini are not clustered, since Orthochromis uvinzae is always assigned to a different part of the cluster analysis

36 space. However, the Orthochromini are always clustered with some lamprologine representatives: Orthochromis uvinzae with Telmatochromis, and O. mazimeroensis and O. malagarasiensis with N. calliurus, L. congoensis and L. leleupi. L. cylindricus is associated with Paracyprichromis brieni; Bathybates minor with Perissodus microlepsis. The assumption that sister species are not necessarily close morphologically is also confirmed by the calculation of the distances existing between species in the 3D morphospace: the smallest distances are not even found between two species that belong to the same tribe. Furthermore, the Mantel test (Mantel 1967) revealed no significant correlation between morphological distances and phylogenetic distances for the total data set (p=0.97), nor for any of the sub sets tested: the C-lineage (p=0.98), the Ectodini (p=0.97), the Lamprologini (p=0.90), the Haplochromini (p=0.76) or within the ancestral lineages (p=0.20). These results suggest that the Brownian motion model of FIC is not the optimal choice in the case of the rapid radiation of cichlids, and we used the PMM instead in order to account for covariance in landmarks resulting from phylogenetic relatedness. COMPARE (Martins 2004) calculated 561 correlations between the 34 coordinates. 216 of these correlations had a significant probability (i.e., p>0.5), but only six pairs of landmarks had both of their coordinates significantly correlated to each other. And only two of the landmark pairs (11 and 12; 11 and 14) were highly significantly correlated, (i.e., with p values >0.75 for correlation of xi to xj and yi to yj and >0.50 for xi to yj and xj to yi, i and j being two landmarks).

Influence of ecology on body shape evolution

The non-parametric MANOVA found a significant correlation with between shapes and four of the ecological traits tested. In the case of habitat depth distributions, the MANOVA revealed, for the data available, a significant correlation with the coordinates describing body shape (p=0.0232). We calculated the average shapes of each cluster (shallow water, medium water, deep water and rivers), and the consensus of these four averages. The deformation grids applied from the overall consensus to each of the cluster averages are shown in Figure II.5. Fish in shallow water tend to have a shorter but wider caudal peduncle, a short pelvic fin and a more pointed head than average. The boundary of the scaly area and the position of the dorsal fin are, on the other hand, at a more anterior position. In deep water we find the reverse pattern. The caudal

37 peduncle is longer and the pelvic fin has a more anterior position than average. The boundary of the scaly area, as well as the position of the dorsal fin, are posterior to the average. In our data set, fish living in medium depths have a short and wide body shape whereas fish from rivers have a slender body (Fig. II.5).

38

A

B

C

D

Fig. II.5. Deformation grids from the consensus shape of all habitat depth pre- defined ecological groups to the consensus shape of each of these groups individually: (A) shallow water, (B) medium water, (C) deep water, and (D) riverine habitats.

39 With regard to feeding preferences (Fig. II.6), we found a significant correlation between the categories of prey and body shape in all three different coding strategies tried. The separation into six categories resulted in a significant correlation (p=0.0012) as did the separation into planktonic, nektonic and benthic prey types (p=0.0022; and p=0.0048 if we included the generalists species) and the binary coding on whether the prey is mobile or not (p=0.0214). Fish feeding on passive prey items such as plants and plankton have a shape close to the average shape, but with a short caudal peduncle. Benthic prey feeders are slender and elongated. Among the nektonic prey feeders, we can distinguish between scale eaters and piscivorous fish. The Perissodini, a tribe of scale eaters, have a mouth shifted up compared to the rest of the landmarks describing their anterior body, the latter being shorter than average. Bathybates minor and Cyphotilapia frontosa, both piscivorous fish, have a much larger anterior body. Their dorsal fin starts quite far from the boundary of the scaly area, and although their body can be somewhat reduced in length, their caudal peduncle is still quite long. Finally, breeding types are also correlated with body shape (p=0.0316), but we do not show these deformation grids (see discussion).

40

A

B

C

D

E

Fig. II.6. Deformation grids from the consensus shape of the four ecological groups of different feeding habits to the consensus shape of each of these groups individually: (A) planktonic prey, (B) benthic prey, (C) nektonic prey: scales eaters, (D) nektonic prey: piscivores, (E) generalists.

41 Disparity within and among species

The disparity of tribes is slightly correlated with the number of specimens per tribe (R²=0.533), and even more to the number of species per tribe (R²=0.6706). Figure II.7 shows that the Tropheini and the Perissodini are particularly far from the regression line. The Tropheini presents a low disparity value for such a high number of species represented in our study, whereas the Perissodini, a bi-generic tribe, shows very high disparity. When these two tribes are eliminated from the correlation analysis, we observe an increase in the correlation factor (R²=0.8231 and 0.8993 respectively). The Lamprologini (D=0.006196) and the Ectodini (D=0.006102) are the most morphologically diverse tribes whereas the Limnochromini (D=0.002076) and the Bathybatini (D=0.002052) are the least.

Fig. II.7. Correlation (A) between the morphological disparity within a tribe and the number of specimens included in this tribe and (B) between the morphological disparity in a tribe and the number of species included in this tribe.

42 The results of the disparity analysis are shown in Table II.3 and in Figure II.8.

Table II.3. Disparity values for the whole morphospace, excluding each of the tribes one by one, and 95% confidence interval values. Only the morphospace without Cyphotilapiini is significantly smaller than the total morphospace (0.0038674 < 0.0041523).

tribe disparity interval < interval > Cyphotilapiini 0.0036715 0.0035446 0.0038674 Tilapini 0.0040038 0.0036678 0.0043812 Bathybatini 0.004122 0.0038186 0.0045294 Cyprichromini 0.0042069 0.0039232 0.0046724 Perissodini 0.0043901 0.0041625 0.0048317 Trematocarini 0.0044809 0.0042187 0.0049759 Lamprologini 0.0045247 0.0043039 0.0050552 Tylochromini 0.0045396 0.0043053 0.0050782 Ectodini 0.0045667 0.0042628 0.0049475 Orthochromini 0.0045677 0.0043034 0.0049753 Tropheini 0.0045788 0.0043232 0.0050894 Eretmodini 0.0046057 30043766 0.0051149 Limnochromini 0.004619 0.0043837 0.0050913 Haplochromini 0.0046946 0.0044449 0.0050744 morphospace 0.004398 0.0041523 0.0048315

Fig. II.8. Disparity values, number of species and number of specimens within each of the 14 tribes.

43 Cyphotilapia frontosa is found to have the largest influence on the disparity of the whole morphospace. The value of its contribution to the disparity of the whole morphophace is the highest, and after the exclusion method (Foote 1993), only it significantly affects the disparity of the morphospace outside of the 95% confidence interval. It also implies that all the other tribes cannot be statistically discriminated in the morphospace (Foote 1993). No correlation was found between disparity values of the different subgroups and the number of specimens used for the calculation of the disparity (R²=0.1642), nor with the number of different species comprised in the tribe in our study (R²=0.1688). Even though within group variability for our pre-defined groups (tribes and ecological groups) seems to be similar (Fig. II.9A), the assignment to tribes shows lower values. This is probably a consequence of having more tribes than ecological distinct groups. However, we observe a dramatic increase of the variability among groups (Fig. II.9B) and therefore of the discreteness (Fig. II.9C) when species are grouped by feeding preferences. This result enables us to reject, in the case of the grouping by feeding preferences, the null hypothesis under which groups are equally overlapping in the morphospace (i.e., non discrete). This means that feeding types structure the morphological variation better than grouping of the species by tribe or preferred habitat depth.

44

A

0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 Tribe Depth Feeding Type

B

3

2.5

2

1.5

1

0.5

0 Tribe Depth Feeding Type

C

45 40 35 30 25 20 15 10 5 0 Tribe Depth Feeding Type

Fig. II.9. Graphs showing values of morphological diversity (A) within groups and (B) among groups for the different groupings. (C) Discreteness values for those groupings (Discretness = variability among groups /variability within groups).

45 DISCUSSION

The cichlids of East Africa are viewed as an ideal model system for the study of adaptive radiations (Fryer and Iles 1972; Kocher 2004; Kornfield and Smith 2000b; Salzburger and Meyer 2004; Stiassny and Meyer 1999). We used geometric morphometric methods to describe the body shape of 45 species of East African cichlid fish, with a focus on the Lake Tanganyika species assemblage. This assemblage contains the largest degree of morphological variation of all East African cichlid radiations as well as their ancestral lineages (e.g., (Salzburger et al. 2005). We presented quantitative data supporting the parallel evolution of several adaptive radiations within Lake Tanganyika. Indeed, the Tanganyikan mouthbrooders (C-lineage) and the substrate spawning Lamprologini have evolved a multitude of different shapes from two very similar hypothetical ancestors. We confirm the adaptative character of these independent radiations through the presentation of statistically significant correlations between body shape and ecological characters. Finally, the impressive morphological diversity contained in the tribes included in our study, even those represented by very few specimens or species, illustrates the singular potential for radiation that characterizes cichlid fish.

No influence of phylogeny on body shape evolution of the Tanganyikan cichlid assemblage

In previous studies on cichlids and other organisms (Guill et al. 2003; Rosenberg 2002; Rüber and Adams 2001) phylogenetic relationships between species were found to be of great importance to the evolution of shape, resulting in closely related species that resemble each other more than more distant relatives species. As expected in the case of rapid morphological diversification, however, cluster analyses of the Tanganyikan cichlid assemblage showed little similarity with phylogenetic assignment. We therefore investigated correlations between phylogenetic distances and Euclidian distances in the morphospace using Mantel statistics. However, no statistically significant correlation could be found, highlighting the surprisingly small influence of phylogeny on the shapes of cichlids. These results were predictable at the scale of the whole Tanganyikan assemblage since Lake Tanganyika does not comprise a monophyletic flock (Kocher et al. 1995a; Nishida 1991; Salzburger et al. 2002b). Even so, when we tested homogeneity within a monophyletic entity such as the whole C-lineage, the Haplochromini, the Ectodini or the Lamprologini, we could still find no significant

46 correlations. This absence of correlation between morphologic and genetic distance, as well as the results of the cluster analyses, suggest that extensive intra-lake parallelism and rapid morphological diversification exist. We also calculated the quantity of phylogenetic signal contained in the morphometric data set using the Phylogenetic Mixed Model. The PMM is, in comparison to a traditional FIC (Felsenstein 1985), particularly effective with clades exhibiting considerable morphological diversity at the tips of the phylogeny (Housworth et al. 2004). Although the sample size of 45 taxa is probably not large enough to obtain trustworthy estimates of the heritable and non-heritable correlation components, good estimates of the total phenotypic correlation can be obtained. One of the disadvantages of the PMM is that small sample sizes can lead to negative variance estimates, and therefore to heritabilities and correlations outside of their natural range. But this was not the case in our study, validating its use for the Tanganyikan cichlid species flock. The PMM showed that our data set is characterized by a dearth of phylogenetic inertia. The absence of phylogenetic inertia in our morphometric data is particular to adaptive radiations (Schluter 2000). In general, smaller distances in the morphospace are expected to be found within a family of closely related organisms (Gatz 1979). In the darters (Percidae), for example, a significant correlation between phylogenetic distances and distances in the morphospace indicated that body shape is greatly influenced by phylogenetic history (Guill et al. 2003). Our results statistically support the assumption that there is no correlation between the degree of molecular and morphological variation among cichlid adaptive radiations (see also Clabaut et al. 2005; Sturmbauer and Meyer 1992; Verheyen et al. 2003).

The adaptive character of the Lake Tanganyika radiations

In the case of adaptative radiation, body shapes are similar among species not only because of shared evolutionary history, but also because of common ecological characteristics (Claude et al. 2004). These characteristics are of course indirectly and to a certain extend linked to shared evolutionarily history, but it has been shown that these types of characters are also evolutionary more labile than others (e.g. body size, morphology, life-history, physiology) (Blomberg et al. 2003; deQueiroz and Wimberger 1993). If shared evolutionary history is not the main cause of similarity in shape then parallel evolution of a multitude of

47 geographically isolated founder populations (Sturmbauer et al. 2003) might lead to morphological due to equivalent ecological conditions. Lake fish are distributed according to water depth and the nature of the substrate. Species have a particular depth range, which may be extremely narrow (less than 5m) or broad (up to 100m) (Ribbink 1991). Divergent selection for fish inhabiting near-shore, littoral zones and off-shore, open water habitats might arise from two major differences between these environments: water velocity and the difference in resource composition and availability. Hydrodynamic theory posits that a more fusiform body shape reduces drag, and hence reduces the energetic expenditure necessary to maintain position in flowing water (reviewed in Langherans 2003). In our study, the most important global differences in body shape are related to body length. This is particularly the case for the transformation from one shape to another within the Ectodini which have colonised different habitats during their radiation, from shallow water to more open water. Furthermore, we observe a correlation in the PCA between the length of the body of Grammatotria lemairii, Ectodus descampsi and Cyathopharynx furcifer and their preferred water depth (Poll 1956). Specifically, the deeper they live, the more elongated is their body. Habitat preferences of species range from highly specific to extremely broad (Ribbink 1991). In the cluster analysis, Cyathopharynx furcifer and Cunningtonia longiventralis were always grouped with Haplochromis paludinosus, Astatoreochromis alluaudi (Lake Victoria and surroundings) and Ctenochromis horei. The latter three species cited are riverine cichlids, a fact that could explain why they are morphologically close to each other. They are characterized by a deep body shape, while on the other hand, the Orthochromini, also riverine, have a more elongated body shape. They are grouped into two morphological clusters, which is consistent with the findings of De Vos and Seegers (1998) who place O. mazimeroensis and O. malagarasiensis in a monophyletic group. These two Orthochromis species are riverine, as well as Lamprologus congoensis, and they are grouped together in our cluster analyses. Two other Lamprologini, N. calliurus and L. leleupi are also found in this group. Orthochromis uvinzae, on the other hand, is clustered apart from the rest of the Orthochromini tribe with Telmatochromis vittatus. We therefore suggest the existence of two types of riverine cichlid body shape. A deep-bodied morphotype adapted to river with slow water current (Haplochromini), and an elongated type living in surge water (Lamprologini and Orthochromini). The elongated type may be more generalist in terms of habitat, enabling such fish to colonize new types of water and

48 eventually, in the case of the Lamprologini, become precursors of the lake radiation as suggested by Schelly and Stiassny (2004). Another characteristic change revealed by the second axis of the PCA (Fig. II.3) concerns the proportion of sizes of the different body parts (head and caudal peduncle). This is particularly the case for the Haplochromini, generally deep- bodied fish but whose head and caudal peduncle contain a large amount of shape variation compared to the rest of the body. The Haplochromini, and especially the nested Tropheini, show little variation in their habitat preferences since they are mainly restricted to rocky shore areas and they diversified primarily in their feeding habits. As a consequence, the Tropheini also present a surprisingly low disparity value even when the number of species and specimens analyzed is taken into account. The examples above highlight the adaptive character of Tanganyikan cichlid body shape evolution, even though specific interpretations of the results are difficult as various ecological parameters are acting simultaneously on body shape evolution. For example, in the PCA, the scales-eaters Plecodus straeleni and Perissodus microlepsis show different body shapes but are similar in the position of the mouth and the size and characteristic shape of their head (Fig. II.3). Their very specific feeding habit (scale eating) may constrain the features involved in the feeding behavior, while the rest of the body is evolving independently - as shown by the high disparity value of this tribe. One possible explanation for the evolution of overall body shape is mimicry of the body shape of their preys, as observed in P. straeleni preying on Cyphotilapia frontosa (Brichard 1978; Coulter 1991). The resemblance of these species is supported by the cluster analysis using Ward’s method and the UPGMA algorithms. Perissodus microlepsis on the other hand is long and thin, characteristics probably related to the fact that these fish need to be good open water swimmers in order to attack prey easily (Winemiller 1991), as does the piscivore Bathybates minor. Other ecological characteristics might render the interpretations of our results difficult. Cichlids consume abundant prey when available, even outside their speciality (Langerhans et al. 2003; McKaye and Marsch 1983; Winemiller 1990), leading Liem to call them “jacks of all trades” (Liem 1980b). This opportunism in their feeding habits, though some species are extremely specialized, might also have an influence on body shape, constraining it to a more average form. However, the analysis of morphospace variability shows that feeding type is the main characteristic structuring the morphospace (Fig. II.9). In other words, species with similar feeding behavior occupy discrete regions of the morphospace, leading to the conclusion that morphological variation is strongly related to what

49 a species specializes on trophically. A geometric morphometric analysis more focused on the head shape would probably strenghten this result and enable a more explicit interpretation of the changes in shape in relation to ecology. The significant correlation between shapes and reproductive behavior (mouthbrooders versus substrate spawners) is difficult to explain. The category “substrate spawning” contains all Lamprologini and just one species from another tribe (Tilapia rendalli). The significantly positive correlation obtained in the MANOVA corresponds to the significantly positive correlation found with the MANOVA when the predefined clusters are tribe affiliations.

Potential for diversification

The Lamprologini and the Ectodini are the most morphologically diverse tribe of Lake Tanganyika, which can be explained by the great amount of trophic and habitat diversity that characterize the fish of these two tribes (Barlow 2000; Chakrabarty 2005; Stiassny 1997; Sturmbauer and Meyer 1993). The influence of the Cyphotilapiini on morphospace disparity is characteristic of the peripheral position of this tribe. The fact that this subgroup´s position in the overall morphospace is far from the centroid is probably due to the pronounced bump on their forehead (Fig. II.10). This very peculiar feature has a strong influence on the description of shape after Procustres analysis, since the displacement of the landmarks located on the head will be carried through to all other landmarks. The fact that no other subgroup really influences the size of the total morphospace as much as the Cyphotilapiini indicates that all other tribes strongly overlap in the morphospace. Therefore, we can assess that morphology of the body shape of the Tanganyikan cichlids is not constrained, since even species-poor tribes with few specimens tend to occupy large portions of the entire morphospace (Fig. II.3 and Fig. II.8). This implies that even though only a few tribes (Haplochromini, Ectodini, Lamprologini) are extremely species-rich, the other tribes, irrespective of whether they are ancient or young lineages, and irrespective of whether they contain many or few species, still contain an impressive disparity in their morphology. This illustrates the potential for radiation that exists in cichlid lineages. However, the absence of evolutionary novelties in the cichlid body shape has been noted before (Stiassny 1991b), the Cyphotilapiini and their hormonal bump being the exception and consequently highlighted by the results of the disparity analysis. The disparity analysis gives

50 compelling quantitative evidence for the fact that cichlids are tinkering with an ancestral toolkit of shape.

Fig. II.10. Cyphotilapia frontosa and the characteristic bump on its head.

Independent adaptive radiations in Lake Tanganyika

Our study reveals that for the adaptive radiations of the Lake Tanganyika cichlids, the influence of phylogeny on the evolution of form is small when compared to other fish clades. Body shape evolution is more strongly affected by ecology. We suggest the presence of two general patterns of diversification within the lake´s species flock. One trend in diversification is constrained by habitat preferences, and is characterized by body shape being more or less elongated (Ectodini, Lamprologini, see Fig. II.5). The other evolutionary trajectory is correlated with trophic habits, leading to changes in the different proportions of sizes of the different parts of the body (Haplochromini, see Fig. II.6). The ancestor of the C-lineage was Lamprologus-like as indicated by our analysis and could have undergone first an expansion in different habitat types, and later a specialization in feeding behaviors. In the case of the mouthbrooders of the C- lineage as well as in the case of the lamprologine substrate spawners, sexual selection would then be a speciation mechanism happening only after morphological diversification (Danley and Kocher 2001). In either case, it is apparent from these analyses that ecology plays a remarkably strong role in generating morphological diversity. This fact confirms the presence of a strong correlation between phenotype and environment, as expected for an adaptive radiation (Schluter 2000).

51 ACKNOWLEDGMENTS

We thank J. Snoeks, from the Royal Museum for Central Africa in Tervuren, Belgium and E. Verheyen, from the Royal Belgian Institute of Natural Sciences in Brussel, Belgium for sharing their collections. R. Getta and the other members of the Meyer-lab are thanked for providing technical assistance. We are grateful to C. Klingenberg for defining the landmarks and writing the landmark taking routine, as well as for valuable advice at the beginning of the project. This study was supported by a visiting grant from the Royal Belgian Institute of Natural Sciences for C. Clabaut, by the Landesstiftung Baden-Württemberg, the Center for Junior Research Fellows (University Konstanz) and the EU (Marie Curie fellowship) to W. Salzburger, and grants of the Deutsche Forschungsgemeinschaft to A. Meyer.

52 Chapter III: Comparative Phylogenetic Analyses of the

Adaptive Radiation of Lake Tanganyika Cichlid Fishes:

Nuclear Sequences are Less Homoplasious but also Less

Informative than Mitochondrial DNA

Céline Clabaut, Walter Salzburger, Axel Meyer

53 ABSTRACT

Over two hundred described endemic species make up the adaptive radiation of cichlids in Lake Tanganyika. This species assemblage has been viewed as both an evolutionary reservoir of old cichlid lineages as well as an evolutionary hotspot from which the modern cichlid lineages arose, seeding the adaptive radiations in Lakes Victoria and Malawi. Here, we report on a phylogenetic analysis of Lake Tanganyika cichlids combining the previously determined sequences of the mitochondrial ND2 gene (1,047 bp) with newly derived sequences of the nuclear RAG1 gene (~700 bp of intron 2 and ~1100 bp of exon 3). The nuclear data – in agreement with mitochondrial DNA – suggest that Lake Tanganyika harbors several ancient lineages that did not undergo rampant speciation (e.g., Bathybatini, Trematocarini). We find strong support for the monophyly of the most species-rich Tanganyikan group, the Lamprologini, and we propose a new taxonomic group that we term the C-lineage. The Haplochromini and Tropheini both have an 11bp deletion in the intron of RAG1, strongly supporting the monophyly of this clade and its derived position. Mapping the phylogenetically informative positions revealed that, for certain branches, there are six times fewer apomorphies in RAG1. However, the consistency index of these positions is higher compared to the mitochondrial ND2 gene. Nuclear data therefore provide on a per basepair basis less, but more reliable phylogenetic information. Even if in our case RAG1 has not provided as much phylogenetic information as we expected, we suggest that this marker might be useful in the resolution of the phylogeny of older groups.

54 INTRODUCTION

The Great Lakes of East Africa are among the world’s most diverse freshwater ecosystems. In particular, Lakes Victoria, Malawi and Tanganyika (Fig. III.1) contain unique species flocks of cichlid fishes counting hundreds of endemic species each (Fryer and Iles 1972). The shear number of species, the degree of ecological and morphological specialization, and the rapidity of lineage formation characterize the East African cichlid assemblages and make them well-known model systems for the study of adaptive radiations and explosive speciation (Fryer and Iles 1972; Kocher 2004; Kornfield and Smith 2000a; Salzburger and Meyer 2004; Stiassny and Meyer 1999).

Fig. III.1. Map of East Africa showing the lakes and the main river systems of that region.

Lake Tanganyika is the oldest of the Great Lakes and – with an estimated age of nine to twelve million years (Cohen et al. 1997; Cohen et al. 1993) – the second oldest lake in the world surpassed only by Lake Baikal. An estimated number of 250 endemic species of cichlids occur in Lake Tanganyika (Snoeks et al. 1994; Turner et al. 2001). Although this number lies below the estimates for Lake Victoria (~500) and Lake Malawi (~1,000), the Lake Tanganyika cichlids are morphologically, ecologically and behaviorally the most diverse. Poll (1986) recognized twelve distinct tribes based on morphological characters. Eight of these tribes are endemic to the lake and – compared to other cichlid species flocks – relatively species poor: Eretmodini (4 species), Cyprichromini (6), Perissodini (9), Bathybatini (8), Trematocarini (10), Limnochromini (13), Tropheini (24), and Ectodini (32). The remaining four tribes (Haplochromini,

55 Tilapiini, Lamprologini and Tylochromini) have representatives that occur in other locations in Africa and three of them are the most species-rich tribes of cichlids. Poll (1986) assigned four cichlid genera of Lake Tanganyika and its surrounding rivers to the Haplochromini (~1,800 species), which are distributed throughout the African continent but virtually absent from West-Africa (Greenwood 1981) and include the species flock of Lake Malawi and the Lake Victoria region superflock (Greenwood 1973; Greenwood 1980; Salzburger et al. 2005; Verheyen et al. 2003). Also, two species of Tilapiini are found in Lake Tanganyika. The most species rich tribe of cichlids from Lake Tanganyika, the Lamprologini (79 species), includes at least five representatives that are found outside the lake in the Congo River system and in the Malagarazi River (Schelly et al. 2005; Schelly and Stiassny 2004; Sturmbauer et al. 1994) (see Fig. III.1). Finally, the Tylochromini, which have their center of diversity in West Africa (Stiassny 1990) are represented in Lake Tanganyika by a single species. Recently, Takahashi (2003) suggested the erection of five additional tribes for genera so far assigned to one of Poll’s twelve tribes (Poll 1986). Based on internal and external morphological features, Takahashi (2003) elevated the endemic genera Benthochromis, Boulengerochromis, Cyphotilapia, “Ctenochromis” benthicola, and Greenwoodochromis to tribal status. The reconstruction of the phylogeny of the East African cichlids in general, and that of the species flocks of the three Great Lakes in particular, remains a challenge, despite considerable effort by both morphological as well as molecular phylogeneticists (see e.g., Albertson et al. 1999; Allender et al. 2003; Kocher et al. 1995b; Nishida 1991; Nishida 1997b; Poll 1986; Salzburger et al. 2005; Salzburger et al. 2002b; Shaw et al. 2000; Takahashi 2003b). Due to the extreme pace of lineage formation and the relatively young age of at least some of these species flocks, the analysis of commonly used markers such as mitochondrial DNA (mtDNA) sequences was still not able to resolve all phylogenetic issues. For example, the persistence of ancestral polymorphisms as a consequence of incomplete lineage sorting has been suggested to occur in particular in the extremely young and therefore closely related cichlid species of Lakes Malawi (Moran and Kornfield 1995) and Victoria (Abila et al. 2004; Nagl et al. 1998; Verheyen et al. 2003). Recent attempts to overcome this problem include the analysis of AFLP markers, which have successfully been applied to a subset of species from Lakes Malawi and Victoria (Albertson et al. 1999; Allender et al. 2003; Seehausen et al. 2003). The phylogenetic problems are different in the older Lake Tanganyika, where shared haplotypes between species are not

56 known (Koblmueller et al. 2004a; Sturmbauer et al. 2003) – except as a result of hybridization (Rüber et al. 2001; Salzburger et al. 2002a). For Lake Tanganyika´s species assemblage, morphological characters have, generally, proven to be useful for the assignment of species into (Poll 1986; Takahashi 2003b). However, the use of the same characters for phylogenetic purposes may be misleading. On one hand, a large amount of variation exists within tribes while, on the other hand, convergence in eco-morphological traits and coloration patterns appears to be common between groups that are only distantly related (Kocher et al. 1993; Meyer 1993a; Meyer 1993b). These potential problems and difficulties with morphological data argue for the usage of molecular data to resolve the phylogenetic relationships within Lake Tanganyika’s cichlid assemblage. Several phylogenetic hypotheses exist for the Lake Tanganyika species assemblage based upon molecular markers. Nishida (1991) used allozyme data to distinguish several more ancient Tanganyikan tribes (Tylochromini, Tilapiini, Bathybatini, Trematocarini, Lamprologini) from the derived “H-lineage” – a clade combining the tribes Perissodini, Limnochromini, Ectodini, Eretmodini, Cyprichromini, Haplochromini and Tropheini. These allozyme data (Nishida 1991) already revealed a close relationship between the Tropheini and Haplochromini, which was confirmed in all later DNA sequence-based studies (see e.g., Kocher et al. 1995b; Salzburger et al. 2002a; Salzburger et al. 2005; Sturmbauer and Meyer 1993; Verheyen et al. 2003) and combined analyses of allozyme and mtDNA data sets (Nishida 1997b). Many studies on the phylogeny of Lake Tanganyika cichlids are based on mitochondrial DNA sequences – e.g., Kocher et al. (1995) focused on the ND2 gene and Salzburger et al. (2002a) on combined DNA sequences of the control region, the cytochrome b and the ND2 gene – confirmed the existence of several ancient groups in Lake Tanganyika with the placement of representatives of the Tylochromini, Tilapiini, Bathybatini and Trematocarini (only in Salzburger et al. 2002a) as sister group to all remaining tribes. In contrast to Nishida (1991), however, the Eretmodini were not resolved within the “H-lineage” (sensu Nishida 1991) but were placed as sister group to the Lamprologini (Kocher et al. 1995) or as sister group to the Lamprologini plus the remaining tribes of Nishida’s (1991) original “H-lineage” instead (Salzburger et al. 2002a; see also Takahashi 2003). Kocher et al. (1995), Nishida (1997) and Salzburger et al. (2002a) already found that Cyphotilapia frontosa [originally assigned to the Tropheini by Poll (1986)] should be assigned to its own new tribe (see Takahashi 2003). Also the insertion patterns of short interspersed nuclear elements (SINEs) in Lake Tanganyika cichlids have been used as phylogenetic

57 markers (Takahashi et al. 1998; Takahashi et al. 2001; Terai et al. 2004), which confirmed the prior phylogenetic hypotheses based on mitochondrial DNA. In an extensive analysis, Takahashi et al. (2001) found incongruent insertion patterns in some of the SINEs, which they interpreted as signature of ancient incomplete lineage sorting in the course of the primary radiation of the Lake Tanganyika cichlids. However, since SINE insertions are evolutionarily rare events in rapidly evolving lineages, the resulting phylogenies often lack sufficient resolution. So far, no phylogenetic study on Lake Tanganyika´s cichlid species flock based upon nuclear DNA sequences has ever been done. We conducted a comparative phylogenetic analysis of Lake Tanganyika cichlids based both on mitochondrial (1,047 bp of the ND2 gene) and nuclear (~1,800 bp of the RAG1 gene including exon and intron sequences) DNA. The analyses were performed on 43 ingroup taxa. At least one representative of each of the sixteen tribes (Takahashi 2003) were included in this study, except for the Benthochromini, the Greenwoodochromini, and Ctenochromis benthicola for which no tissue was available. Several riverine species and species of the Lake Malawi species flock have been included (see Table III.1).

Table III.1. Characterization of the studied species of Lake Tanganyika cichlids. Species names follow the fishbase website nomenclature (www.fishbase.org), tribe assignment follow the nomenclature of Takahashi (2003). Voucher specimens are available from (1) The Royal Museum of Central Africa, Tervuren, Belgium; (2) Axel Meyer, Lehrstuhl für Zoologie und Evolutionsbiologie, Department of Biology, University of Konstanz, Germany ; and (3) Christian Sturmbauer, Institute of Zoology, University Graz, Austria *The Malagarazi River Orthochromis are classified as Haplochromini here, although they should be placed into a new tribe (see this study and Salzburger et al. 2005) **Note that the newly described L. teugelsi (Schelly and Stiassny 2004) was in previous studies (e.g. Salzburger et al. 2002a) identified as L. mocquardi.

58

GenBank Taxonomy information sampling locality Voucher nr. (Institution) accession nr

Tribe Taxon ND2 RAG1 INTRON2 RAG1 EXON3

Heterochromis multidens West Africa n/a AF398214 - -

Bathybatini Bathybates sp. Lake Tanganyika T1992-270B (1) U07239 DQ012183 DQ012225

Cyphotilapiini Cyphotilapia frontosa Lake Tanganyika AM-139 (2) U07247 DQ012176 DQ012219

Cyprichromini Cyprichromis leptosoma Lake Tanganyika T1995-1822B (1) AF398224 DQ012192 DQ01223

Paracyprichomis brieni Lake Tanganyika AM-53E (2) AF398223 - DQ012212

Ectodini Aulonocranus dewindti Lake Tanganyika AM-8.3 (2) AY337782 DQ012198 DQ012240

Cunningtonia longiventralis Lake Tanganyika AM-90-16VIII (2) AY337780 DQ012201 DQ012243

Cyathopharynx furcifer Lake Tanganyika AM-90-16VIII14 (2) AY337781 DQ012199 DQ012241

Ectodus descampsi Lake Tanganyika AM-90-II7 (2) AY337790 DQ012190 DQ012232

Enantiopus melanogenys Lake Tanganyika AM-90-IV (2) AY337770 DQ012196 DQ012238

Grammatotria lemairii Lake Tanganyika AM-71 (2) AY337787 DQ012200 DQ012242

Ophthalmotilapia nasuta Lake Tanganyika T1992-414B (1) AY337783 DQ012197 DQ012239

Xenotilapia ochrogenys Lake Tanganyika T2004-4D8 (2) AY337767 DQ012179 DQ012221

Eretmodini Eretmodus cyanostictus Lake Tanganyika AM-75 (2) AF398220 DQ012194 DQ012236

Spathodus erythrodon Lake Tanganyika T1992-740 (1) AF398218 DQ012175 DQ012218

Haplochromini Astatoreochromis alluaudi Lake Kanyaboli R173 (2) AY930071 - DQ012217

Astatotilapia burtoni Lake Tanganyika T2004-4I9 (2) AF317266 DQ012203 DQ012245

Haplochromis paludinosus Nanganga River AM-T2 (2) AY930107 DQ012191 DQ012233

Melanochromis auritus Lake Malawi AM-29T (2) AY930069 DQ012177 DQ012220

Metriaclima zebra Lake Malawi AM-30T (2) U07263 DQ012207 DQ012249

Orthochromis malagaraziensis* Malagarazi River AM-T5 (2) AF398232 DQ012187 DQ012229

Orthochromis uvinzae* Malagarazi River AM-TZ94-112b (2) AY930048 DQ012172 DQ012214

Pseudocrenilabrus multicolor Lake Kanyaboli R082 (2) AY930070 DQ012173 DQ012215

Lamprologini Altolamprologus compressiceps Lake Tanganyika AM-90-16VIIIN (2) AF398229 DQ012171 DQ012213

Julidochromis ornatus Lake Tanganyika AM-31T (2) AF398230 DQ012195 DQ012237

Lamprologus congoensis Congo River AM-307 (2) AF317272 DQ012188 DQ012230

Lamprologus cylindricus Lake Tanganyika AM-Lc (2) DQ093115 DQ012209 DQ012251

Lamprologus teugelsi** Congo River AM-Lt (2) AF398225 DQ012184 DQ012226

Neolamprologus calliurus Lake Tanganyika AM-Nc (2) AF398227 DQ012205 DQ012247

Neolamprologus leleupi Lake Tanganyika AM-Nl (2) DQ093113 DQ012206 DQ012248

Limnochromini Limnochromis auritus Lake Tanganyika CS-2320 (3) AF398216 DQ012204 DQ012246

Perissodini Perissodus microlepis Lake Tanganyika T1995-1449B (1) AF398222 DQ012202 DQ012244

Plecodus straeleni Lake Tanganyika T2004-4H9 (2) AF398221 DQ012178 -

Tilapiini Boulengerochromis microlepis Lake Tanganyika T2004-1H1a (2) U07240 DQ012193 DQ012235

Oreochromis tanganicae Lake Tanganyika T2004-3F4 (2) AF317240 DQ012181 DQ012223

Trematocarini Trematocara unimaculatum Lake Tanganyika T2004-3G3 (2) AF317268 DQ012185 DQ012227

Tropheini Ctenochromis horei Lake Tanganyika T1995-547B (1) AY930100 DQ012208 DQ012250

Limnotilapia dardennii Lake Tanganyika T2004-1A6 (2) DQ093109 - DQ012211

Lobochilotes labiatus Lake Tanganyika T1995-1403B (1) U07254 - DQ012210

Petrochromis polyodon Lake Tanganyika T2004-1D8 (2) AY930068 DQ012174 DQ012216

Simochromis babaulti Lake Tanganyika T1992-1153 (1) DQ093110 DQ012182 DQ012224

Simochromis diagramma Lake Tanganyika T1992-312 (1) AY930087 DQ012186 DQ012228

Tropheus duboisi Lake Tanganyika AM-T77 (2) AY930085 DQ012180 DQ012222

Tylochromini Tylochromis polylepis Lake Tanganyika AM-341 (2) AF398215 DQ012189 DQ012231

59 Lately, nuclear DNA (ncDNA) sequences have increasingly been used for phylogenetic studies of more closely related taxa (see e.g., Koepfli and Wayne 2003). The combination of ncDNA with mtDNA is advantageous as these two kinds of markers belong to different linkage groups (Brower et al. 1996) and are inherited in different ways. One advantage of ncDNA compared to mtDNA is the generally observed reduced level of homoplasy among more distantly related taxa as a consequence of a slower rate of evolution (see e.g., Avise 1984; Brown et al. 1982). Due to those differences in mutation rates between ncDNA and mtDNA data sets, we decided to apply two different phylogenetic algorithms that take those differences into account: Maximum likelihood (ML) and Bayesian inference (BI). Bayesian phylogenetic inference has recently been shown to deal efficiently with complex models of molecular evolution (Nylander et al. 2004). We also applied three strategies to quantify and qualify the phylogenetic signal present in the three partitions (ND2, intron 2 of RAG1 and exon 3 of RAG1): (i) the likelihood mapping method (Strimmer and von Haeseler 1996; Strimmer and von Haeseler 1997) implemented in the program TREE-PUZZLE 5.0 (Schmidt et al. 2002) (ii) the mapping of phylogenetic informative positions and autapomorphies on the obtained phylogenies and the determination of the consistency index (Kluge and Farris 1969) for each variable position, and (iii) the calculation of the sequences divergence between pairs of taxa, to visualize and quantify the rate of molecular evolution, as well as to establish the relative contribution of the different partitions of the data set to the pairwise distances.

60 MATERIAL AND METHODS

Molecular Methods

We determined DNA sequences of a ~1,800 bp segment of the nuclear RAG1 gene of 43 specimens of Lake Tanganyika cichlids representing 14 tribes (according to Takahashi et al. 2003). The RAG1 segment included 692 bp of intron 2 and 1,111 bp of exon 3 (Fig. III.2).

Fig. III.2. Organization of the RAG1 gene showing the positions of the primers used in this study. The primer HBF3 has been published previously (Brinkmann et al. 2004), the primers CF1, CR1, CR5 and Kali F1 have been designed for this study.

DNA has been extracted from ethanol preserved fin-clips or muscle tissue using a high-salt extraction followed by an ethanol precipitation (Brufford et al. 1998). Polymerase chain reaction (PCR) amplification was performed according to standard methods (12 µL of HPLC water, 2.5 µL of each primer at 10µM, 2.5 µL of the reaction mix and 2.5 µL of dNTP at 10µM). Two pairs of primers have been used in order to obtain two overlapping PCR products. For the sequence containing the second RAG1 intron the primers were (forward) HBF3 5’AARGGGGGACGICCNCHICAGC3’ (Brinkmann et al. 2004) or KaliF1 5’AAGGGTTTATGTTCAATCAA’ and (reverse) CR1 5’AGGGCTGGAATATCTGGCGG3’ (both designed for this study). For the sequence exclusively situated in the exonic region, the primers used were (forward) CF1 5’ GCCGCCAGATCTTCCAGCCCT 3’ and (reverse) CR5 5’ TGCGGGCGTAGTTTCCATTCA 3’ (both newly designed). The PCR products were purified with Qiaquick spin columns (Qiagen) following the manufacturer’s protocol. If necessary, PCR products were cloned using the TA cloning kit (Invitrogen). Sequencing reactions were performed according to standard methods for all five primers using the Big Dye sequencing chemistry v3.0 (Applied Biosystems). The DNA sequences were determined on an ABI 3100 capillary sequencer (Applied Biosystems). A list of specimens included in this study, their assignment to one of the sixteen tribes according to Takahashi (2003), as well as GenBank accession numbers of the DNA sequences are listed

61 in Table III.1. Voucher specimens are deposited in the Royal Museum for Central Africa in Tervuren, or are available from the authors.

Phylogenetic analyses

DNA-sequences were aligned with the computer programs Sequencher (GeneCodes) and ClustalX. Seventeen gaps encoded as indels had to be included in the intron region of RAG1 (including an 11 bp long deletion), and two triplets of gaps had to be included in the exon. We also analyzed 44 sequences of the mitochondrial NADH Dehydrogenase Subunit II gene (ND2; 1,047bp) available from GenBank (see Table III.1) (Koblmueller et al. 2004a; Salzburger et al. 2005; Salzburger and Meyer 2004). Phylogenetic analyses were performed on several data partitions: ND2, RAG1, and a combined data set. For four specimens it was not possible to amplify the intron of RAG1, and for two specimens we encountered the same problem with the exon of RAG1. Since the resulting missing data represent less than a third of the combined sequence, we decided to include these taxa in the phylogenetic analyses. Also, this decision seems justified based on the observation that characters that are complete (i.e., scored across all taxa, as here for the ND2 sequence) increase accuracy much more effectively in highly incomplete taxa than do characters that are scored in only some taxa (Wiens 2003). For phylogenetic reconstruction we used maximum likelihood (ML) [with PAUP* 4.0b10 (Swofford 2002b)] and Bayesian inference (BI) [with Mr. Bayes (Ronquist and Huelsenbeck 2003)] methods. Heterochromis multidens and Tylochromis polylepis were declared as outgroup (according to (Farias et al. 2000; Lippitsch 1995; Salzburger et al. 2002a; Stiassny 1990) for the analyses of the combined and the ND2 data set resulting in a total of 44 taxa; for the RAG1 data set, only Tylochromis polylepis was used as single outgroup, resulting in a total of 43 taxa. This was necessary because Heterochromis was among the taxa for which no RAG1 sequence could be amplified. We ran the Modeltest 3.06 routine (Posada and Crandall 1998) to determine, with a hierarchical likelihood ratio test, the appropriate model of molecular evolution for ML analyses. For each data set, a model was chosen from a set of 56 models. We used the GTR+I+G model (Rodriguez et al. 1990) for the ND2 and for the RAG1 data set, and the HKY+I+G model (Hasegawa et al. 1985) for the combined data set (see Table III.2 for model parameters).

62

Table III.2. Models defined by ModelTest for the different partitions and the model used for the combined data set after an iterative optimization (see Materials and Methods for further details).

data set ND2 alone RAG1 alone combined iterative best fitting model/estimates GTR+I+G GTR+I+G HKY+I+G HKY+I+G frequency A 0.297 0.2462 0.2525 0.2517 frequency C 0.3795 0.2279 0.2945 0.2943 frequency G 0.0846 0.2412 0.1878 0.1873 frequency T 0.2389 0.2847 0.2652 0.2667 transition/transversion ratio 2.9883 3.0086 gamma shape parameter 0.9192 0.7920 0.5625 0.5689 proportion of invariable sites 0.3565 0.3225 0.4416 0.4396 Number of substitution types 6 6 2 2 kappa A - C 0.4049 0.8280 n/a n/a A - G 11.3001 1.5762 n/a n/a A - T 0.6488 0.7101 n/a n/a C - G 0.8348 1.4702 n/a n/a C - T 4.2669 2.5227 n/a n/a G - T 1 1 n/a n/a

The ML tree of the combined data set was calculated with PAUP* 4.0b10 (Swofford 2002b) using an iterative approach. After an initial heuristic search (default parameters) the model parameters were re-estimated based on the obtained phylogeny with the “likelihood score” option in PAUP* and used for a new cycle of heuristic searches. This procedure was repeated until the -ln likelihood did not change anymore in two consecutive runs (see Swofford and Sullivan 2003). We then performed a bootstrap analysis with 100 replicates. Bayesian inference (BI) analyses were performed with MrBayes 3.0 (Huelsenbeck and Ronquist 2001), which uses a Markov Chain Monte Carlo (MCMC) approach for sampling the joint posterior probability distributions. We ran four MCMCs in parallel for 10,000,000 generations, sampling trees every 10 generations, and excluding 10% of all trees as burn-in. The different partitions were taken into account in the combined data set. For the ND2 and the RAG1 data sets, we performed a single ML heuristic search and the BI was run for 1,000,000 generations (tree sampling every 10 generations; burn in = 10%). To assess the support for crucial branches in the RAG1, we used the one- tailed Shimodaira-Hasegawa (SH) topology test using RELL bootstraps with 1,000 replicates as implemented in PAUP* (Shimodaira and Hasegawa 1999); see also

63 (Felsenstein 1985). We constructed a tree in which the monophyly of the Orthochromis species was enforced, and compared it to the best unconstrained tree. Finally, we assessed the heterogeneity in phylogenetic signal (i.e., incongruence) among the individual data sets and partitions. We used the level of bootstrap support (BS) in ML trees to measure the degree of conflict among topologies recovered in the separate analyses as well as the posterior probabilities (PP) values estimated by the Bayesian analysis. We also performed a likelihood mapping analysis with TREE-PUZZLE 5.0 (Schmidt et al. 2002), to visualize the phylogenetic signal in the combined data set as well as in the mitochondrial and nuclear partition independently.

Analysis of Character Evolution

For the reconstruction and comparison of character evolution in the nuclear and mitochondrial gene segments we used the maximum likelihood phylogeny of the combined data set and mapped the parsimonious informative positions and autapomorphies on the respective branches. We mapped those positions by using the “list of apomorphies” command in the “describe tree” menu in PAUP* (Swofford 2002) using the accelerated transformation (ACCTRAN) character-state optimization under default settings, with gaps handled as missing data. We differentiated between the source DNA segment of each informative position (ND2, intron 2 of RAG1 or exon 3 of RAG1), in order to quantify the relative contribution of each DNA segment to the phylogenetic signal in the entire combined data set. We also calculated the consistency index [CI (Kluge and Farris 1969)] for each informative position. We then compared the number of parsimonious informative positions and the CI at the level of deep nodes to those in the Ectodini and the Lamprologini clade, which were chosen as examples for monophyletic groups derived from these deep nodes.

Pairwise Genetic Distances Comparison

We calculated with PAUP* (Swofford 2002) the pairwise distances in the different partitions (ND2, exon 3 of RAG1, intron 2 of RAG1 and the combined RAG1 sequences) as well as for the entire combined data set, using the HKY+G+I model of molecular evolution and the model parameters estimated from the

64 combined data set topology. After excluding those pairs of taxa involving a segment of missing data, we plotted for each taxon pair the corrected pairwise distances of ND2 vs. RAG1 and intron 2 of RAG1 vs. exon 3 of RAG1 in order to test for differences in the rate of molecular evolution between the different partitions. The pairwise distance ratios of RAG1/ND2 and intron 2 of RAG1/exon 3 of RAG1 for each pairwise comparison were then used to calculate the average pairwise distance ratios for both types of comparisons. From these ratios we estimated the length of nuclear DNA segment that would have to be sequenced in order to reach a similar level of divergence that is present in the mtDNA. We also performed such estimations for the two partitions of the ncDNA. We then divided our data set into five taxon clusters according to the results of the phylogenetic analyses (see Figs. III.3-5) in order to highlight differences in the ND2 vs. RAG1 pairwise distances. This partition into five taxon clusters was used to compare distances within each of the five clusters (closely related taxa) to those among the five clusters (distantly related taxa). Finally, we grouped taxon clusters I, II, and III (H-lineage sensu Salzburger et al. 2002a or ‘C-lineage’ as defined here) and compared their ND2 vs. RAG1 pairwise distances with those of groups IV, V plus Tylochromis polylepis (‘remaining taxa’) in order to delineate the relative contribution of mtDNA vs. ncDNA variation at different phylogenetic “depths”.

65 RESULTS

In the RAG1 sequences, 154 heterozygous sites in the 1,803 bp alignment of the 43 species were scored as polymorphisms on the basis of equal peaks heights in electropherograms (27 as Y, 31 as M, 38 as R, 16 as K, 34 as S and 8 as W). We also noticed that the intron contains a deletion of 11 bp, found in Astatotilapia burtoni, Metriaclima zebra, Haplochromis paludinosus, Ctenochromis horei, Melanochromis auratus and Pseudocrenilabus multicolor (Haplochromini) and Simochromis diagramma, Simochromis babaulti , Tropheus duboisi and Petrochromis polyodon (Tropheini). The exon of M. auratus shows an additional codon (CAA between position 106 and 108 in our alignment), whereas Cyphotilapia frontosa lacks a codon (AGG between positions 544 and 546). The four-cluster likelihood mapping analyses revealed a percentage fraction of 18.6% unresolved quartet topologies for the RAG1 data set (64.2% fully resolved), and 1% unresolved quartet topologies for the ND2 data set (95.7% fully resolved). In the combined data set only 0.2% of the quartet topologies were found to be unresolved (97.8% fully resolved).

Results of the Phylogenetic Analysis

RAG1 For this analysis, Tylochromis polylepis was chosen as outgroup. The maximum likelihood analysis (Fig. III.3A) identified a clade containing Orthochromis uvinzae and Oreochromis tanganicae as sister group to all remaining ingroup taxa. However, the SH test did not reject the monophyly of the two Orthochromis representatives when comparing the maximum likelihood phylogeny to a tree in which these two taxa have been constrained as a clade (p = 0.097). The remaining taxa were clustered into five clades forming a polytomy: Bathybates sp. plus Trematocara unimaculatum, Limnochromis auritus plus Cyphotilapia frontosa, the Lamprologini, a clade containing the Ectodini plus Boulengerochromis microlepis and the Perissodini, and a poorly supported clade including the Haplochromini, the Tropheini, the Eretmodini, the Cyprichromini, and Orthochromis malagaraziensis (note that in this analysis, the synapomorphic 11bp insertion of the Haplochromini was not considered). Only the monophyly of the Lamprologini (BS=97; PP=100) was strongly supported; the monophyly of Bathybates sp. plus Trematocara unimaculatum received moderate support (BS=64; PP=100) as well as the monophyly of Limnochromis auritus plus

66 Cyphotilapia frontosa (BS=56; PP=98). The monophyly of the Ectodini/Boulengerochromis microlepis/Perissodini clade (PP=70) and the monophyly of the clade comprised by the Haplochromini the Tropheini, the Eretmodini, the Cyprichromini, and Orthochromis malagaraziensis (PP=70) were supported only in the Bayesian inference phylogeny.

Fig. III.3. Maximum likelihood (GTR+I+G) phylogram of Lake Tanganyika cichlids based on RAG1 alone (A) (42 ingroup taxa; Tylochromis polylepsis as outgroup) and ND2 alone (B) (43 ingroup taxa; Heterochromis multidens as outgroup). Numbers above the branches are ML bootstrap estimates (100 replicates), numbers below the branches are Bayesian posterior probabilities (1,000,000 generations; 10% burn in), (values <50% are not shown).

ND2 For the analysis of the ND2 sequences, Heterochromis multidens was used as outgroup. In the ML (Fig. III.3B) and BI trees, Tylochromis polylepis occupied the most ancestral position, followed by a clade formed by Boulengerochromis microlepis, Bathybates sp., Oreochromis tanganicae, and Trematocara unimaculatum. These form the sister group of two major clades, a clade formed by the Eretmodini plus the Lamprologini and a clade including all remaining taxa. The sister group relationship of Eretmodini and Lamprologini was supported by low BS and PP. In the second clade, Limnochromis auritus and Cyphotilapia

67 frontosa form the most ancestral lineages, sister group to the Cyprichromini, the Perissodini, the Orthochromis species, the Ectodini, and the Haplochromini including the Tropheini. The monophyly of the tribes Eretmodini, Lamprologini, Cyprichromini, Perissodini, Ectodini, Tropheini, and Haplochromini (but excluding Orthochromis; see Salzburger et al. 2002a; 2005) was supported by high BS and PP.

Combined Data Set The combined analysis with Heterochromis multidens as outgroup, led to congruent results in ML and BI analyses (Fig. III.4). We obtained Tylochromis polylepis as most ancestral split, placed as sister group to Oreochromis tanganicae, a clade constituted by Boulengerochromis microlepi, Bathybates sp. and Trematocara unimaculatum and a group including all remaining taxa. In this group, the Eretmodini and Lamprologini were placed as sister group to a clade formed by Cyphotilapia frontosa plus Limnochromis auritus, a clade including the Perissodini sister to the Ectodini, a clade formed by the Cyprichromini, the Orthochromis species and the Haplochromini/Tropheini assemblage (see Fig. III.4 for BS and PP).

68

Fig. III.4. Maximum likelihood (HKY+I+G) phylogram of Lake Tanganyika cichlids based on ND2 and intron 2 of RAG1 and exon 3 of RAG1 of 44 taxa (declaring Heterochromis multidens as outgroup). Numbers above the branches are ML bootstrap estimates (100 replicates), numbers below the branches are Bayesian posterior probabilities (10,000,000 generations; 10% burn in), (values <50% are not shown). Names of the corresponding tribes are depicted on the right. Representatives of some tribes are shown on the right.

69 Mapping of Molecular Character Evolution

Of the 1,047 bp of ND2, 415 characters were parsimony informative (39.64%). In the ncDNA (1,803 bp), only 119 characters were parsimony informative (6.60%). Thus, about six times more informative positions were contained in the mtDNA compared to the ncDNA data set (length corrected). However, the CI of the tree of the combined data set was higher when only the ncDNA positions are taken into account (CI=0.751) than when the changes of the mtDNA alone are mapped on the combined tree (CI=0.379). The numbers of parsimonious informative positions and autapomorphies are depicted on relevant branches in Fig. III.5. At the level of deeper nodes (Fig. III.5A), PAUP* assigned 9.82 times more apomorphic characters in the mtDNA partition (211 of 1,047bp) than in the ncDNA partition (37 of 1,803bp). However, by calculating the mean of the consistency index of these apomorphies, we found that the nuclear positions (CI=0.57) are less homoplasious than the mitochondrial ones (CI=0.29). At the level of terminal nodes, 315 apomorphies were found in the mtDNA partition of the Ectodini compared to 101 in the ncDNA (Fig. III.5B), and 262 apomorphies were found in the mtDNA of the Lamprologini compared to 84 in the ncDNA (Fig. III.5C). Here, the mtDNA provides 5.37 (for the Ectodini and for the Lamprologini) times more informative positions compared to the ncDNA (length corrected). Again, the nuclear positions are less homoplasious (CI=0.79 for the Ectodini; 0.79 for the Lamprologini) than the mitochondrial ones (CI=0.38 for the Ectodini and CI=0.39 for the Lamprologini).

70

Fig. III.5. Mapping of the parsimony informative position contained in the sequences of ND2 (black box), intron 2 of RAG1 (white box) and exon 3 of RAG1 (grey box) on the obtained ML tree (A) (see Fig. III.4) as well as on the Ectodini (B) and the Lamprologini clade (C). The values on terminal branches in (B) and (C) represent autapomorphies of the respective taxon.

71 Pairwise Distances Comparisons

The corrected pairwise distances in the ND2 partition were on average 10.17 times higher than those in RAG1. In RAG1, the corrected pairwise distances were found to be about 1.11 times higher in the exon than in the intron. This contradicts the expectation that intron sequences – because of the presumed absence of evolutionary constraints – would be characterized by higher rates of evolution compared to exon sequences.

Fig. III.6. Pairwise distance comparisons. (A) Pairwise distances of ND2 (y- axis) versus pairwise distances of RAG1 (x-axis) of all possible taxon pairs among 43 cichlid species analyzed (Heterochromis multidens was not used). (B) Pairwise distances of RAG1/intron 2 versus RAG1/exon 3 of all possible taxon pairs among the 43 cichlids. (C) Definition of five clusters of taxa based on the ML topology of the combined data set (see Fig. III.4). Cluster I-III are referred to as ‘C-lineage’, clusters IV and V as ‘ancient tribes’. (D) Pairwise distance comparison (ND2 vs. RAG1) among closely (within groups I-V) and distantly (between groups I-V) related taxa. The trend-lines for the two groups of plots are depicted. (E) Pairwise distance comparison (ND2 vs. RAG1) among ‘ancient tribes’ and ‘young lineages’. The taxon pairs involving two lamprologine representatives are highlighted. This cloud of plots, although belonging to a more ancient tribe, is similar to those of the C-lineage pairwise comparisons.

72 The plot of corrected pairwise distances of RAG1 vs. ND2 (Fig. III.6A) showed clearly that for each taxon pair the corrected pairwise distance is higher in ND2. Pairwise distances in RAG1 ranged from 0.13% (Ctenochromis horei – Limnotilapia dardennii) to 4.16% (Petrochromis polyodon – Altolamprologus compressiceps), while they ranged from 0.29% (Metriaclima zebra – Melanochromis auritus) to 20.39% (Tylochromis polylepis – Simochromis babaulti) for the ND2 gene. The plot of corrected pairwise distances of intron 2 of RAG1 vs. exon 3 of RAG1 (Fig. III.6B) showed a more equal distribution around the bisecting line. Pairwise distances in intron 2 of RAG1 ranged from 0% (Cyathopharynx furcifer – Aulonocranus dewindti) to 5.5% (Julidochromis ornatus – Trematocara unimaculatum), and in the exon 3 of RAG1 from 0.18% (Metriaclima zebra – Melanochromis auritus) to 5.85% (Plecodus straeleni – Spathodus erythrodon). The ND2 vs. RAG1 pairwise distance comparisons between closely and distantly related taxa (as defined in Fig. III.6C) revealed that, on the whole, the ncDNA partition contributed to a similar extent to the pairwise genetic distances at both levels. By calculating the average of the relative contribution of the RAG1 partition to the total genetic distances over all pairwise comparisons, we found that in the group of closely related taxa RAG1 contributed on average to 14.99% of the total distance, and in the distantly related taxa we found that RAG1 contributed on average to 14.65% of the total distance. When looking at the trend-lines of those two clusters of plots, we noticed that the bigger the total distance was, the higher was the contribution of RAG1 to it. This observation is accentuated for the distantly related taxon pairs (see trend-lines in Fig. III.6D). In the pairwise comparisons of the ancestral lineages, RAG1 contributes 39.7% to the total distance (mtDNA plus ncDNA), in the Lamprologini 17.1% and in the C- lineage taxa only 14.5%. When comparing the C-lineage taxa to the remaining ones (Fig. III.6E) we observed a similar trend. The distances among taxon-pairs of the C-lineage are generally smaller compared to those among taxon-pairs in the remaining taxa. Also, the pairwise distances within the Lamprologini (highlighted in Fig. III.6E), which – as a tribe – does not belong to the C-lineage, are equivalent to those found within the C-lineage.

73 DISCUSSION

Missing Data and Support Values

Support values such as BS and PP are generally considered to be good indicators of the robustness of branches in phylogenetic trees. Unfortunately, real data sets do not always result in phylogenies with excellent support values, and biological phenomena such as the rapid lineage formation in adaptive radiations or explosive speciation events are particularly problematic to resolve due to the pace of cladogenesis within a short period of time (see e.g., Sturmbauer et al. 2003). When nodes are not supported by high bootstrap values (or when branches are not significantly different from a length of zero), they may have arisen by chance alone (Flynn and Nedbal 1998) or reflect the biological reality of an adaptive radiation. In the absence of real contradiction between different topologies obtained from different data sets (e.g., mtDNA and ncDNA) and different phylogenetic algorithms, the resulting topology may be considered to be robust despite only moderate support values. In our case, the topologies obtained from separate as well as combined data sets, and including mtDNA and ncDNA are congruent for most relevant branches, although some support values are low (Fig. III.3 and III.4). The analyses presented were based on the combined mtDNA and ncDNA sequences data set from 42 ingroup taxa. Six of those were partially incomplete for the ncDNA partition (four were missing the intron sequence, and two the exon sequence). Including incomplete taxa in an analysis is unlikely to be problematic as long as there is a sufficient amount of phylogenetic information in at least one broadly sampled data set to allow the resolution of these taxa in a phylogeny (Bininda-Edmonds and Sanderson 2001). As the ND2 tree is not incongruent with the trees obtained from the combined data set, we suppose that ND2 by itself contains enough informative characters to achieve a strongly supported topology (Salzburger et al. 2002b). Wiens (2003) stressed that the estimated relationships among complete sets of taxa are seemingly unaffected by the inclusion of incompletely sampled taxa. This is again confirmed in our analyses by comparing the tree obtained with ND2 alone to the one constructed from the combined data set. In the combined analysis, the six taxa, for which no complete RAG1 sequence was available, were placed at the same position (or at least within the same clade) as they were in the ND2 tree. What might be affected by the inclusion of incomplete taxa are support values rather than the overall topology.

74 The Phylogeny of the Lake Tanganyika Cichlid Assemblage

The assemblage of cichlid fishes in Lake Tanganyika is older than the species flocks of Lakes Malawi and Victoria (Fryer and Iles 1972; Salzburger et al. 2005), which is reflected in larger genetic differences facilitating the phylogenetic reconstruction of its cichlid lineages. Lake Tanganyika cichlids have so far been included in several phylogenetic studies using different sets of molecular markers. Nishida (1991) used allozymes and identified several ancient lineages of cichlids in Lake Tanganyika, as well as the derived mouthbrooders, which he termed “H- lineage” (see also Nishida 1997b for an analysis combining allozyme and mtDNA data). Later studies based on mtDNA alone (Kocher et al. 1995; Salzburger et al. 2002a) established a relatively clear scenario for the evolution of the Lake Tanganyika cichlid assemblage. The lake seems to harbor several older lineages that colonized the lake after its formation as well as younger tribes that are likely to have evolved in the course of a primary lacustrine radiation within Lake Tanganyika (Salzburger et al. 2002a). Among these tribes are the Haplochromini, which also include the species flocks of Lakes Malawi and Victoria, and it has been suggested, that Lake Tanganyika is the cradle of all haplochromine cichlids (Fryer and Iles 1972; Salzburger et al. 2002a; 2005). Also, ncDNA sequence data suggested that haplochromine cichlids are derived from Lake Tanganyika stocks (Mayer et al. 1998), albeit with low bootstrap support. However, the exact relationships among several tribes remained unclear and only a relatively small number of Tanganyikan cichlid species had been analyzed so far, particularly for ncDNA, although phylogenies based on several loci belonging to different linkage groups appear advantageous (see e.g., Brower et al. 1996; Seehausen 2004; Takahashi et al. 2001). In our analyses of the mtDNA data set (Fig. III.3B) and combining mtDNA and ncDNA (Fig. III.4), we corroborate that Tylochromis polylepis represents the most ancestral lineage of the species assemblage in Lake Tanganyika, which might have seeded the lake independently (probably only recently) from its presumed West-African center of origin where still most of its congeners can be found (Stiassny 1990). Consistently, a weakly supported Lake Tanganyika endemic clade consisting of Boulengerochromis microlepis, Oreochromis tanganicae, Bathybates sp. and Trematocara unimacultum forms the most ancestral group in the phylogeny of the combined data set. Despite the apparently ancestral position of O. tanganicae in our phylogeny, this species is likely to have colonized the lake only recently, since it shows a high level of sequence identity with its sister species O. niloticus (Klett and Meyer 2002). This distance is similar to those found

75 between sister species within the Lamprologini or the Ectodini. It seems interesting that this species was able to adapt to an ecological niche in a lake in which a highly specialized species assemblage of cichlids was already present (see also Salzburger et al. 2005). When considering only ncDNA (Fig. III.3A), Oreochromis tanganicae and Orthochromis uvinzae were placed as most basal sister group to all remaining taxa. The SH test, however, could not reject the monophyly of the two Orthochromis species. Trematocara unimaculatum and Bathybates sp. form one of the five clades of the basal polytomy, and Boulengerochromis microlepis was resolved in another of these clades in the ncDNA partition, together with the Ectodini and Perissodini. While the Trematocara unimaculatum/Bathybates sp. clade received moderate bootstrap support, the latter clade was not supported by the maximum likelihood bootstrap analysis. Our results thus corroborate the morphology-based assignment of Boulengerochromis microlepis in its own tribe, Boulengerochromini (Takahashi 2003), since it has never been found to form a monophyletic clade with Oreochromis tanganicae (Klett and Meyer 2002). In the combined and in the ND2 data set alone, the Eretmodini were – just as found by Kocher et al. (1995) and Salzburger et al. (2002a) – not resolved within the “H-lineage” (sensu Nishida 1991), but were placed as sister group of the Lamprologini instead. In the Bayesian inference phylogeny of the combined data set, the relative position of the Lamprologini and Eretmodini is poorly supported (PP=57). In the maximum likelihood and Bayesian inference analyses of the ncDNA partition, in which an11 bp insertion characteristic to the Haplochromini and Tropheini was not taken into account (see below), the two eretmodine taxa were resolved in a clade containing the Haplochromini, Tropheini and Cyprichomini (see Nishida 1991; Sturmbauer and Meyer 1993). The monophyly of that clade was weakly supported in the Bayesian inference phylogeny (PP=70). Therefore, further data and analyses will be necessary in order to assess the exact phylogenetic placement of the Eretmodini with respect to the Lamprologini and the C-lineage. In all analyses the Lamprologini were recovered as a monophyletic group (BS=98 and PP=100 for the combined data sets; BS=97 and PP=100 for RAG1 alone). Interestingly, in the topologies based on ncDNA, the riverine lamprologines (L. teugelsi and L. congoensis) were placed as sister group to all remaining taxa – unlike in all previous mtDNA analyses (Salzburger et al. 2002a; Sturmbauer et al. 1994) as well as in our trees based on the combined data set and ND2 alone (see Figs. III.3, III.4). If this seeming conflict would hold after sequencing of additional ncDNA segments, this finding would suggest that the

76 ancestors of riverine lamprologines presently found in the Congo drainage gave rise to the radiation of the most species-rich tribe in Lake Tanganyika, rendering those Congo taxa as ancestral relict species rather than, as suggested before (Salzburger et al. 2002a; Sturmbauer et al. 1994), a lineage that emigrated from Lake Tanganyika. Our combined analyses revealed strong support for the monophyly of what we propose as the ‘C-lineage’, the clade containing the Cyphotilapiini, Limnochromini, Perissodini, Ectodini, Cyprichromini, the Orthochromis, Haplochromini and Tropheini (i.e. the “redefined H-lineage” of Salzburger et al. 2002a). Within the C-lineage, Limnochromis auritus and Cyphotilapia frontosa, which were also grouped together in the ncDNA partition alone, occupied the most ancestral position in all analyses. Therefore, Cyphotilapia frontosa finds its place at the root of to the C-lineage, and not within the Tropheini, where it has been placed originally (Poll 1956) based on its Tropheus-like pharyngeal apophysis. These results, again, support the exclusion of Cyphotilapia from the Tropheini (see also Lippitsch 1998; Nishida 1997b; Salzburger et al. 2002a) and the creation of a new tribe, the Cyphotilapiini (see also Salzburger et al. 2002a; Takahashi 2003). In the maximum likelihood analysis, as well as in the BI phylogeny of the combined data set, the Perissodini were grouped together with the Ectodini, sister to the Cyprichromini, the two species of Orthochromis and the Haplochromini/Tropheini. In the ND2 partition, the Cyprichromini were placed as sister group to the Perissodini, the two Orthochromis species, the Ectodini and the Haplochromini/Tropheini. The topology of the Ectodini is similar to the one found by Kolbmüller et al. (2004) based on mtDNA only. The monophyly of the Haplochromini/Tropheini was supported by high BS and PP. The results of these analyses support the inclusion of Ctenochromis horei within the Tropheini, an assignment already proposed by Takahashi et al. (2003) and Sturmbauer et al. (2003). We furthermore identified an 11bp synapomorphic deletion in intron 2 of RAG1 of all Haplochromini/Tropheini, which strongly supports the monophyly of this group, and its derived position (the lower support in the ncDNA partition is most likely due to the fact that this 11 bp partition was not taken into account in the maximum likelihood analyses). The placement of the two species of Orthochromis as sister group to the Haplochromini in the combined tree (Fig. III.4) contradicts the topology found for the ND2 alone tree (Fig. III.3B) and previous findings based on mtDNA (Salzburger et al. 2002a; 2005). However, the Orthochromini do not contain the haplochromine-specific 11bp-deletion in the intron region RAG1 and their sister

77 group relationship to the Haplochromini is only poorly supported. More data would be necessary to clarify that issue. We note that Orthochromis polyacanthus from the Congo drainage was previously shown to be a member of the Haplochromini clade and, hence, not related to O. malagaraziensis from the Malagarazi drainage (Salzburger et al. 2002a; 2005; see also (De Vos and Seegers 1998). It is thus likely that the genus Orthochromis is polyphyletic and in need of a taxonomic revision (see also Salzburger et al. 2005). In the C-lineage, a large number of species arose within a short period of time, and are thus separated by only small genetic distances. Clearly, there is no correlation between the degree of molecular and morphological variation among the cichlid adaptive radiations (see also Sturmbauer and Meyer 1992; Verheyen et al. 2003). This is the reason why morphology-based phylogenetic studies are unlikely to resolve the relationships among cichlids. Furthermore, this independence of evolution makes it more likely that the propensity for explosive speciation in this group is likely based on genomic features of cichlids rather than on a simple function of time or mutation rate. Therefore, in the future, comparative evolutionary genomic analyses should be facilitated (Santini et al. 2003). The proposed sequencing of cichlid genomes (Kocher 2004) would certainly contribute to a better understanding of the “cichlid problem”.

Contribution of Nuclear DNA

Several previous studies on the phylogeny of East African cichlid species flocks were based upon the ND2 gene (Kocher et al. 1995; Shaw et al. 2000; Salzburger et al. 2002a; 2005). This mtDNA segment has been chosen because, in mammals, it has been known to evolve more rapidly than other mitochondrial proteins coding segments (Anderson et al. 1982; Kocher et al. 1995b). In cichlids it has been shown to be adequate for resolving genus-level phylogenies (Kocher et al. 1995), and it has a higher rate of molecular evolution than, for example, the widely used cytochrome b gene (Cummings et al. 1995; Russo et al. 1996; Salzburger et al. 2002b; Zardoya and Meyer 1996). Here we determined, for the first time, a ncDNA segment (about 1,800 bp of the RAG1 gene, including intron 2 and exon 3) in an effort to add the phylogenetic information provided by more slowly evolving DNA fragments in order to gain resolution at the level of deeper nodes of the Lake Tanganyika cichlids phylogeny. This gene has been widely used in phylogeny reconstruction of other groups of animals (e.g., (Brinkmann et al. 2004; Rüber et al. 2004; Van der Meijden et al. 2004; Venkatesh et al. 1999), so

78 that primers were available and the gene has been well characterized (e.g., no paralogs have been found so far). Another advantage of using ncDNA in addition to mtDNA is the possibility to combine molecular information provided by independent linkage groups (Brower at al. 1996). The four-cluster likelihood mapping analysis already indicated that, as expected, the RAG1 segment provided less phylogenetic signal compared to the ND2 sequences. However, the amount of about 81.4% resolved quartet topologies in RAG1 justified its use for phylogenetic reconstructions of these taxa. Also, when combining the mtDNA and ncDNA sequences, the phylogenetic signal improved so that in the combined data set only 0.2% of the quartet topologies remain unresolved. Thus, using the RAG1 partition alone might not lead to the resolution of the phylogeny as accurately as the combined data set can do (see also Rüber et al. 2004). For phylogenetic analyses in general, fast evolving (often mtDNA) genes are useful markers of evolutionary history and particularly for the resolution of rapid speciation events. Alternatively, very long stretches of slowly evolving DNA have been used as well. Gene segments that evolve relatively fast are likely to contain more homoplasious mutations, whereas more slowly evolving genes have fewer informative sites. Because of the accelerated rate of molecular evolution in mtDNA, a discrepancy in the numbers of informative positions was observed between the two partitions (on average there were six times more informative positions in the mtDNA partition). However, this difference is at least partly compensated for by the higher reliability of the phylogenetic informative positions in the ncDNA, which were less homoplasious (ncDNA: CI=0.75; mtDNA: CI=0.38). For mtDNA it has been known already that only a relatively small number of informative sites provides resolution on the relationships among different tribes of Lake Tanganyika cichlids (see e.g., Kocher et al. 1995; Salzburger et al. 2002a). A similar pattern was found in our analysis of the ncDNA partition, where only few apomorphies at the deeper node level could be identified. However, these showed a relatively high CI, and, thus, seem to provide a relatively robust phylogenetic signal. In particular, a relatively rare mutational event, an 11bp deletion, unambiguously supported the monophyly of the Haplochromini/Tropheini clade (see Salzburger et al. 2005 for a detailed phylogenetic analysis of the haplochromines). Also, within a cluster of the C- lineage (the Ectodini) and in the Lamprologini (not belonging to the C-lineage), relatively more apomorphies could be detected compared to the level of deeper nodes, with even higher consistency indices (Fig. III.5B, C). Again, the ncDNA seems to provide more phylogenetic information within the younger lineages

79 compared to the deeper nodes. This can be explained by the fact that apomorphies accumulated after the radiation that gave rise to the Lake Tanganyikan tribes. The contribution of the ncDNA relative to mtDNA to the pairwise distances tends to be higher for the pairwise distances calculated among distantly related species (Fig. III.6D), suggesting that the ncDNA is actually more informative for resolving the relationship among ancient clades. The trend line in Fig. III.6D shows that the bigger the distances between two sequences are, the more RAG1 is contributing to the total distance. RAG1 might thus be more efficient for resolving older radiations than the one of the Lake Tanganyika cichlids. ND2 evolves about 10 times faster than RAG1. This lies in the upper range of the differences in evolutionary rates usually accepted for these two types of DNA markers (Meyer 1993b). It also means that, in order to get the same quantity of phylogenetic information with nuclear DNA as with the ND2 gene, one would have to sequence 10,647 bp of RAG1. In RAG1 of Lake Tanganyikan cichlids exon 3 evolves equally fast as intron 2. This seems to be inconsistent with the general view that introns might be more informative since they are not as evolutionary constrained as exons, and therefore would tend to evolve more freely. Usually introns evolve around three times faster than exons since they are expected to evolve approximately as fast as third positions of codon-triplets do (Li 1997). In our case, the first and second codon positions in the exon 3 of RAG1 evolved more slowly compared to intron 2, while the third codon positions evolved faster than intron 2. The pairwise distance comparison in Fig. III.6E highlights an interesting position of the pairwise distances within the Lamprologini, which are situated in the range of the C- lineage pairwise comparison. This illustrates that, although the ancestor of the Lamprologini is likely to have evolved before the radiation of the C-lineage, their diversification might have happened more recently, in parallel to that of the C-lineage tribes. In general, the ncDNA improved support values in ML and BI, for example the monophyly of the Lamprologini and the Haplochromini/Tropheini. Future studies with sequences of an intermediate pace of molecular evolution such as unconstrained introns would seem to hold promise. The development of different kinds of markers (such as the 11bp deletion in the intron of all the Haplochromini, Tropheini included) should also be followed up, since they might contain phylogenetic information that would not be dependent on problems related to the rate of evolution. Thus, even if in the case of the Lake Tanganyika cichlid species

80 flock the use of other markers might be more appropriate, we believe that RAG1 is suitable for the resolution of phylogenies of older groups.

81 ACKNOWLEDGMENTS

We thank S. Koblmüller, J. Snoeks, C. Sturmbauer, E. Verheyen and L. De Vos for providing and identifying some of the specimens, and R. Gueta, I. Eistetter, T. Mack, and the other members of the Meyer-lab for technical assistance. We are grateful to M. Cummings and L. Rüber and the second anonymous reviewer for discussions and valuable comments on the manuscript. This study was supported by the Landesstiftung Baden-Württemberg, the Center for Junior Research Fellows (University Konstanz) and the EU (Marie Curie fellowship) to W. S, and grants of the Deutsche Forschungsgemeinschaft to A. M.

82 Chapter IV: Characterization of genes involved in cichlid coloration using microarrays and RT-PCR techniques

Céline Clabaut, Axel Meyer and Walter Salzburger

83 ABSTRACT

Cichlids of the Great Lakes of East Africa are a prime model system for the study of explosive speciation and adaptive radiations. In has been suggested that speciation on the basis of sexual selection is an important mechanism for the generation of new species, with male body coloration as primary discriminatory factor among a hierarchy of visual cues used by females in mate choice. We used cDNA microarray techniques to identify genes involved in coloration of cichlid fish. We compared skins of a male and female of the species Maylandia zebra that shows a dimorphism in body coloration, with males being blue while females are brightly yellow colored. Microarray experiments identified 46 genes that exhibited differential expression between the two sexes, of which 34 were confirmed to be differentially expressed by relative quantitative RT-PCR. Among them, we identified numerous genes involved in intracellular mobility of pigments granules related to the actin filament transport, as well as genes involved in the endosomal-to-Golgi vesicles trafficking. We also tested the differential expression in the two sexes of the paralogs fms a and fms b genes, that are already known to be good candidate genes for the study of coloration in teleost because of their role as precursors of xanthophores, the yellow pigment cell. Our results add evidence to the hypothesis of neo-functionalization of the paralog fms b. All these genes were also tested for their relative expression in a female albino of a closely related species of cichlid, Pseudotropheus callainos. With the support of microscopic images of the skin of these three specimens, we interpret the difference of expression of the selected genes between a blue male, a yellow female and an albino female. This study provides insights into the putative functional diversification of genes involved in the coloration of cichlids, and by extension on the evolution of coloration in teleost fishes more generally.

84 INTRODUCTION

Over 3,000 species belong to the teleost family Cichlidae, which makes them the most species-rich family of vertebrates. Alone 2,000 endemic species live in the Great Lakes of Eastern Africa. Because of their unique biodiversity, their extremely fast speciation rates and their species-richness they have been established as a prime model system for the study of evolutionary processes, especially for explosive speciation and the formation of adaptive radiations (Fryer and Iles 1972; Kocher 2004; Kornfield and Smith 2000a; Salzburger and Meyer 2004; Stiassny and Meyer 1999). The evolutionary success of the East African cichlids has been attributed to a combination of ecological opportunities (after the colonization of large lakes) as well as behavioral (maternal mouthbrooding) and morphological innovations (egg-spots, color polymorphisms, pronounced sexual dichromatism) (Braasch et al. submitted; Salzburger et al. 2005). For East African cichlids, speciation on the basis of sexual selection has been proposed as an important mechanism on the basis of field observations (van Oppen et al. 1998) as well as mate choice experiments in the laboratory (Knight et al. 1998; Seehausen et al. 1999). Further evidence for sexual selection came from the observable breakdown of visual reproductive barriers under monochromatic light conditions or in turbid waters (Seehausen et al. 1997). These results suggest that male body hue is the primary discriminatory factor among a hierarchy of visual cues used by females (Deutsch 1997; Seehausen and van Alphen 1998), resulting in cichlids being among the most colorful species assemblages in the kingdom. It has been shown that species with highly similar colors have evolved repeatedly within and among lakes with a predominance of blue and yellow colours (Allender et al. 2003; Seehausen et al. 1999; Seehausen and van Alphen 1999) resulting in a phenotypical diversity despite of their general genetic uniformity (Allender et al. 2003; Sturmbauer and Meyer 1992). Furthermore, colorational differences evolved intraspecifically and many color polymorphisms are known. These color forms can be so strikingly different that the casual observer would assign them to different species (Fryer and Iles 1972). Finally, many cichlid species, and in particular all modern haplochromines (Salzburger et al. 2005) are sexually dichromatic, with the male being the colorful sex, while females typically only have a dull body color. Coloration in vertebrates is based on the number and distribution of three types of pigment cells: dark melanophores, yellow to orange xanthophores, and

85 reflective iridophores responsible for a white or silvery coloration (Bagnara and Hadley 1973; Fujii 1993b; Fujii 2000; Mellgren and Johnson 2002). In teleost fishes, at least two more classes of chromatophores have been identified: the white leucophores and the blue cyanophores (Bagnara 1998). All three types originate from neural crest cells that migrate over the embryo during early ontogeny (Bagnara et al. 1979). Each chromatophore has its characteristic pigments that reside in special pigmentary organelles (Bagnara 1998; Fujii 1993a; Fujii 1993b). Cells with dispersed organelles give rise to the general color pattern of the animal, whereas chromatophores with aggregated organelles appear less conspicuously colored (reviewed by Luby-Phelps and Schliwa 1982; Obika 1986). These organelles are described as melanosomes, which contain melanin in melanocytes (Birbeck 1963; Charles and Ingram 1959; Drochmans 1960); pterinosomes, which contain pteridines in xanthophores (Bagnara 1976; Kamei-Takeuchi and Hama 1971; Matsumoto 1965b); and the reflecting platelets which contain purines in iridophores (Bagnara et al. 1979; Bagnara and Stackhouse 1961). Pterinosomes, like melanosomes, are derived from the Golgi complex (Obika 1993). They contain a species-specific set of pteridines that appear simultaneously with the differentiation of xanthophores (Hama 1963; Matsumoto 1965a; Matsumoto et al. 1960; Obika 1963). Sepiapterins, drosopterins and several colourless pteridines can be detected as yellow pigmentation that first becomes visible within pterinosomes (Kamei-Takeuchi and Hama 1971; Obika 1963). The understanding of the genetic mechanisms that drive the evolution of cichlid color patterns might aid in the understanding of explosive speciation. Information on the cichlid genome can also provide insights into the genome evolution of teleost fishes more generally. The use of the cichlid genome as a model perciform increased in recent years with the development of genetic linkage maps (Albertson et al. 2003; Kocher et al. 1998; Lang et al. 2006), BAC libraries (Katagiri et al. 2001; Watanabe et al. 2003), cDNA and EST libraries (e.g. Salzburger et al., unpublished) and microarrays (Renn et al. 2004). The sophistication of DNA microarray technology has also recently increased and now includes comparisons of genome-wide gene expressions in different tissues, as well as work on developmental and physiological regulatory pathways (Crawford et al. 1999; Gasch et al. 2000; Michaut et al. 2003; Segal et al. 2003). Here, we investigated the genetics of coloration in a cichlid using a microarray approach, and deciphered further differences in gene expression using relative quantitative Real Time PCR (RT-PCR).

86 We compared the RNA expression of ~ 6000 genes from the blue skin of a Maylandia zebra (MZ) male (Fig. IV.1a) versus the yellow skin of a female (Fig. IV.1b) from the same species. Maylandia zebra, also called Pseudotropheus zebra or Metriaclima zebra by some authors (Albertson et al. 2005; Kocher 2004), belongs to the ~ 1,000 haplochromine cichlid species flock of Lake Malawi. The adults are differentiated by a strong color dimorphism, but both sexes show black stripes on their flanks. The microarray was constructed using sequenced clones from a cDNA library of the African cichlid Astatotilapia burtoni (AP). This species is a suitable model for haplochromine cichlids as it has a phylogenetic intermediate position between the superflocks of Lakes Malawi and Victoria. AP and MZ are therefore closely related, and it has been shown that heterologous microarray hybridization can yield biologically meaningful data even in somewhat distantly related species (Renn et al. 2004). After identification of those genes that showed difference in expression between skins of different color morphs of MZ, we used relative quantitative RT- PCR in order to further investigate the results of the microarray experiments. We added to the list of those genes the fms a and fms b genes, good candidate genes for the study of coloration in fish. The fms gene is indispensable for xanthophore development as it is required for recruiting pigment cell precursors to xanthophore fates (Braasch et al. submitted; Williams et al. 2002a; Williams et al. 2002b). One of the main differences between the two types of tissues utilized is the presence of xanthophores in the yellow skin of females, and its absence in the blue skin of males. we therefore expect the fms paralogs to show different levels of expression which will be assessed with the RT-PCR experiments. The relative quantifications from the RT-PCR experiments were also performed using the skin of albino females from Pseudotropheus callainos (PC) as probes (Fig. IV.1c). PC belongs to the Lake Malawi flock, and is closely related with AP and particularly with MZ. The gene expression levels found in females albino PC enables us to decide on the possible cause of over-expression of genes in the female of MZ compared to the male of MZ. If the over-expression of a particular gene is also observed in the albino female of PC as well as in the yellow female MZ, the gene is probably related to the female state of the specimens. If the over-expression is not observable in the albino female PC whereas it is for the yellow female MZ, then the gene is probably involved in the yellow coloration.

87

a b c

d e f

g h i

Fig. IV.1. Images of fish and their skin under transmitted light and UV light. a- MZ male b- MZ female c- PC albino female (fish on top). The other fish from the picture are males, as can be seen from the white egg spots present on the anal fin d- skin of MZ male under transmitted light e- skin of MZ female under transmitted light f- skin of PC albino female under transmitted light g- skin of MZ male under UV light h- skin of MZ female under UV light i- skin of PC albino female under UV light.

88 MATERIAL AND METHODS

Colored fish of MZ were from the same brooding and maintained under standard conditions in the same tank (12h light; 12h dark, water at 25°C). Albino females of PC can be recognized from albino males since they do not possess the characteristic egg spot males harbor. Fish were sacrified by overdose of the anesthetic tricain (3-aminobenzoic acid ethyl ester methanesulfonate; Sigma) and were then frozen at –80°C. Skin pieces of 0.5cm² were removed from the individuals at the jonction between a yellow and a dark band. Fluorescence pictures were done of the skin of a blue male, a yellow female of MZ and a PC albino. The skin was taken in a zone containing the border of a black stripe (for MZ) and scales were left since their removal would destroy most of the chromatophores (data not shown). The skin was mounted in methyl cellulose with 1 drop of 0.2% 3-aminobenzoic acid ethyl ester (Sigma), 1 drop of dilute ammonia, 0.1% b-mercaptoethanol, pH 10.0. Pictures were taken under transmitted light and UV light (DAPI-filter) with a Axiophot 2 microscope (ZEISS). The dilute ammonia liberates pteridines from their protein carriers at high pH. These are then visualized as light green fluorescence (Epperlein and Claviez 1982; Marino et al. 2003; Odenthal et al. 1996). Total RNA was purified from skin using a TRIZOL LS (Life Technologies) (1mL) and chloroform (0,2mL) protocol, with an isopropanol and ethanol precipitation step. Purified RNA was suspended in RNase-free water, and the RNA quality was assessed after first strand cDNA synthesis using SUPERSCRIPT III Reverse Transcriptase (Invitrogen). cDNA was directly used for PCR amplifications of gapdh and actin genes that spanned at least one intron (for primers see Table IV.1). Thus the quality of the RNA extraction can be controlled, and clean cDNA pooled in order to decrease individual bias in following experiments.

89 Table IV.1. cDNA primer sets used as positive controls in RT-PCR and their annealing temperature.

primer sequence (5’ to 3’) length TA Ab-GAPDH-F25 CAC GAG ACC CAT TCC TCC ATC 21 bp 60 °C Ab-GAPDH-R509 AGT TGG AAT AGT TCT GCC TCG C 22 bp 60 °C Tilapia-Actin-F258 AAC TGG GAY GAY ATG GAG AA 20 bp 60 °C Tilapia-Actin-R369 TTG AAG GTC TCR AAC ATG AT 20 bp 60 °C SP6 Promotor ATT TAG GTG ACA CTA TAG 18 bp 52 °C T7 GTA ATA CGA CTC ACT ATA CGG 21 bp 52 °C

The microarray was built by Sciencion, Berlin, using clones from a cDNA library for the African cichlid AB. Clones are mainly anonymous, but 96 of them were replicates of candidate genes for coloration. In total, the array had 6192 genes. Probes were pooled from cDNA synthesized from RNA extracted from three different individuals for each sex and experiment. The hybridization of the probe was done using the Labelstar Array kit (Qiagen) and FluoroLink Cy 3-dCTP and Cy 5-dCTP (Amersham Biosciences) according to the manufacturer’s protocol. The array was read with Genepix 4000B (Axon Instruments). Three arrays (one that we will use as reference array and two dye flip replicates) were used to cover all the spotted genes since on certain slides background was covering several spots. The dye-flip (also known as dye swap or reverse labelling) technique generates paired slides where, on the first slide, one mRNA sample is labelled by Cy5 and the other mRNA sample is labelled by Cy3, while, on the second slide, the labels for the two samples are exchanged. This technique removes gene specific dye effect (Sartor et al. 2003; Yongxiang Fang et al. 2003). After normalization (performed with the Genepix Pro 4.0 software on the basis of the mean of the intensities ratios), 2196 of the genes showed a difference in expression in at least one dye-flip experiment. We isolated from this list the genes showing a difference in their log ratios of intensities superior at 2 folds between the reference array and at least one dye-flip array. We added to this list the gene fms a and fms b, for which a high intensity spot was detected on the reference array but with however no confirmation on any dye-flip array because of high background intensity on that particular spot. For the selected genes, sequences were obtained using the original insert from the cDNA library amplified with primers SP6 and T7 (see table IV.1) using an ABI PRISM 3100 sequencer (Applied Biosystems). Specific primers for each clone and for fms a and fsm b were then designed using Primer Express (Applied Biosystems) and tested for dimer formation in a classic PCR step. Relative

90 quantitative RT-PCR experiments were performed using the Quantitative SYBR Green PCR Master MIX (Applied Biosystems) and different dilutions of different combination of cDNA for each replicates. Amplification and detection of products were performed using the 7500 Real Time PCR system (Applied Biosystems) and with an annealing and elongation step of 34’’. Two housekeeping genes - Glyceraldehyde-3-phosphate dehydrogenase (or GAPDH) and actin - were chosen as internal control. Since the tissues under study are similar and the difference lies only in color and sex between the two samples, we estimated that two housekeeping genes would be sufficient to enable an accurate relative quantification of the difference of expression of a gene. However, a minimum of six replicates were performed per clones for males and females each, enabling a routine precision of the RT PCR of +/- 0.1 cycle (Marino et al. 2003). Three replicates minimum were performed for the albino females controls. Sequences of ESTs showing statistically significant differences in expression were then elongated using the forward primer designed for RT PCR, or new designed internal primers when the insert was long. These sequences were blasted in NCBI (using the tblastx function), and gene ontologies were searched using different data-bases and literature sources.

91 RESULTS

A picture of a specimen, of its skin under transmitted light and under UV are shown for MZ male and female as well as for the PC female albino (Fig. IV.1). We observe on the male skin under transmitted light (Fig. IV.1d) the presence of melanophores with the melanosomes dispersed in the cells. Less melanophores are observable on the skin of the yellow female of MZ (Fig. IV.1e), but they look very similar to the ones present in the males’ skin. Furthermore, pterinosomes are also seen as yellow vacuoles under transmitted light, the whole xanthosome remaining difficult to distinguish. In the picture of the skin of the female albino some dark stains of different shape are seen under transmitted light (Fig. IV.1f). No fluorescence is seen on the picture of the male’s skin under UV (Fig. IV.1g). For the female’s skin under UV light (Fig. IV.1h), we observe bright stains that autofluoresce against the dark green background. Some of those stains follow the same pattern as the yellow vacuoles in the Figure IV.1e, but overall we observe much more of these stains in the picture under UV light than of yellow vacuoles in Figure IV.1e. All of the stains observable on the Figure IV.1f of the female albino’s skin are autofluorescing when illuminated by UV light (Fig. IV.1i). After elongation, genes could be characterized by blast, the result of which is shown in Table IV.2. For the blasts where the identification is < than 1e-15, the name of the gene identified will be subtracted to the clone number in the rest of the manuscript. Genes listed as “similar to” show sequence similarities with E < 1e-5 (Altschul et al. 1990; Nadler et al. 2000). All gene selected present a difference from log ratios between the reference array and one dye-flip arrays were superior at 2 folds. However, we listed the selected genes in 3 categories depending on the robustness of the data of the second dye-flip array: when this data showed a difference between log ratios of the intensities superior at 1.5 folds, genes were grouped in category A. In the reverse case, i.e. if the difference between log ratios of the intensities of the second array was inferior at 1.5 folds, clones were included in category B. Finally category C contains genes selected in absence of data for the second dye-flip experiment. This classification of the genes highlight the robustness of the microarray data for each clone selected and tested for difference in expression with RT-PCR. Indeed, for some genes, microarray data and results of RT-PCR experiments are in conflict. This classification enables us to discriminate which of the signal seems more robust.

92 Table IV.2. Clones selected after normalization of the microarrays and their identification after blast on NCBI.

clone log intensity values Primers over-expression

reference dye-flip a dye flip b Forward GAPDH Actin length E value blast identification

Category A

02A F10 -3.444 0.314 -2.687 CTGGAGCCCTGCAGTTCCT - - 611 1E-60 defender against death cell

TGAATGGAAAGGTGCCGAC

02B A11 -4.092 0.77 -2.847 CCATGTCTTTCATCCAGGCC - - 848 1E-78 annexin max 3

GCATGCCCAAGTAAACGCTC

03A A11 3.929 0.603 3.395 GAAGGGCGGTATAGTGCAAAAA x x 672 0.13 similar to hypothetical protein

GCCCTTGAAGTGGAGCTGTG (GLP-177-10068)

03A B02 -2.797 0.567 -1.841 TCGAGGTGACAGGTGACGAG - x 691 1E-106 60S rib prot L5

CTGGCTGACCATCAATGCTCT

03A G01 -1.793 1.232 -2.872 TACTCGATCACTGTCTTGCTCCA x - 953 7E-173 COL1A2

GGACGGCTGCACTACACACA

03B B02 -2.381 0.084 -1.78 CCAGAAATGCCCCTTCACTG - - 680 3E-102 Tetraodon nigroviridis full-length cDNA

GAGAGAATACGGCCACGGAG

04A F07 1.187 -0.702 3.237 CCTGCTGCTGTCCAGTTTCA - x 480 1E-34 parvalbumin

CCCAGTGAGATCGTGGGTAAGA

04C E05 -2.626 0.541 -1.172 GCTTAAGCTGTTCCTCCAGAACTT - x 398 4E-35 parvalbumin

TCGTTCAGGACGCGTGC

11A G01 -2.685 0.504 -2.517 AGGTGGCCAACTCTGCCTTT - x 1183 3E-158 heat shock protein 90 beta

CCTCAAAACCACGCTTACGG

11B A05 1.827 -0.216 2.131 CCATCGTCCTCAGCTCCTTCT x x 1516 4E-22 leucine zipper-EF-hand

TCATTTCACACCCAGCTCACTG

11B A10 -3.121 -0.036 -1.774 AGTGGCAGAGGCGAACTTCA - - 896 5E-131 40S rib

TCTTCCAGAAACACTGGCCAG

12B B04 -3.767 -0.519 -3.6175 AAACGGACTCGAAGCACTGC - - 968 9E-20 Tetraodon nigroviridis full-length cDNA

CCTGTGAAAAGGATGGCCTCT

12B E12 -2.729 0.483 -1.971 GCGTTGGCAGTACCCTTTCTC - - 721 1E-89 L19 rib

TCGCAAGGGAAGACACACAG

12C D12 -2.67 0.535 -2.022 TAGTGTCCACAAGCTCCCATGA - x 950 0.002 similar to ITS

TGCCTTAGTCTCCCATGGAGAG

13B D03 -2.561 0.67 -2.979 CCAAGACCAGCCAAAAGCAG - x 1082 0.7 similar to Homo sapiens

TCCACCCCACTAGTACAAAAAGTG BAC RP11-295G12

13D H05 -1.564 1.222 -1.507 CAGGAAGCCTGGCAGAAGTT x x 645 7E-103 beta hemoglobinB-like

TCTACCCAGGGCAGAAACGA

14D D11 -1.171 1.102 -2.159 GACAGACATGAGGTGATGGGC - x 1116 2E-30 L29 rib

GGAAGGCCGCTATGGAGAA

18B H11 -1.835 0.279 -2.903 CGGCCGCGAATTCACTAGT x x 1046 3E-28 similar to allatropin

CGCGTACTCTGCGTTGTTACC fmsa CCTGCCTACTGACCAACCCA x x

TGTTCATGCGCAGGCTTATG fmsb TGGCCGTCTTGAGGTTTTGTAT x x

CCCACTGTCACCGAATGCTT

Category B

01A G09 -2.354 0.169 -1.404 AAGATGGCTCCTCGTAAAGGG - x 584 2E-86 S14

GCTGATCACCTGCTCCTCCT

01B B11 -2.951 -0.161 -0.5185 GTGGCCGGTCACTAGCCTC - x 601 2E-34 myosin light chain 2

GGGACGCGATTCATTCACAC

01C G09 -1.793 0.321 -1.285 CCCAGGGATGAAAGCTCCA - - 593 1E-105 beta actin

93 TCATCCTGACCTCACTTCTTCTCC

03B B07 -2.027 0.819 -1.404 GGCTCCACCTCAAGGGTGAT - x 511 1E-83 Tetraodon nigroviridis full-length cDNA

GATTTTCGTGAAGACGTTGACG

03C F01 -2.252 0.291 -1.221 TGCTTGAAGTTCATGCCGAC - x 769 1E-83 Tetraodon nigroviridis full-length cDNA

GCCTGTCCAACCTGCAGG

03C H09 -1.706 0.998 -1.001 GGAAAACAAGGAGGCCAAGAC - x 559 2E-77 fruYP1

GACGTTTTCTCTGCCGATGTG

03D A07 -1.521 0.967 -0.4895 TCCACAGCCCGCATCACTA - x 335 6E-47 Tetraodon nigroviridis full-length cDNA

TGCAAGTTCCTTGGCCTTTG

11B E02 -1.637 0.631 -0.95 CCCCTGGCAGAGGTGTAGC - x 493 5E-93 Tetraodon nigroviridis full-length cDNA

GAAGCTGCTCATGATGGCTG

12A H03 -1.834 1.439 -1.406 CTGGGAGATGCGGTGAAAAT - x 386 2E-7 beta thymosin

CAACAAACGAAAAAGGGCCA

12D F03 -1.678 0.89 -0.827 ACTTGCGAGAGGGCTTCAGA - - 670 1E-110 60S prot L13

TGGTTGTCCCAGCTGCTCTTA

13C E01 0.237 0.155 2.443 GGCTTCTTCTGAGCTAAAACCTTTG - - 693 2E-46 Tetraodon nigroviridis full-length cDNA

TGCCTGATGAAGCAATGCC

14C E03 -0.396 -0.742 1.903 CAATGGCATTCGGAGGTGTAC - x 645 2E-69 beta 2 parvalbumin

CCAGGGCTGCAGTGATGTTAG

14C H09 -1.08 -1.102 -2.476 GGGACAGAAACTGACTGGATTGT - x 1299 6E-101 copz1

CCTTCCACCTCCTCCACATTACT

15A B05 -1.112 -0.422 -2.451 TTTACGTCCAGATCCCTGACG - x 1057 6E-76 COL10A1

TCTCTGCAGCAAAGACGCC

15A D07 -1.196 -0.458 -2.1025 CACCAAAGGACAGGAGATGGA x x 1165 4E-8 similar to 16S

CCCTCCTCCTTTTTTGTGTGG

17A E10 1.019 -0.23 2.139 AGCATTCACAGGACGCCATC - x 1035 3E-5 similar to solute carrier

AAACGCAGACGCTCCCAA

19A D07 -1.787 0.255 -0.402 AGATGGTCCGTGAATGTGCA - - 694 7E-10 Tetraodon nigroviridis full-length cDNA

GGTGGTCAAGGCGGTGTTAA

Category C

03C D08 1.194 x -2.514 CCAGCAGGATGGGTACTACCA x x 451 0.018 similar to ta1 gene

CCGAAGGATCACCAGTGTGG

13C B02 1.096 x -1.869 AAGACACCTTTGGCAGAACCA x x 582 1E-11 Line

GCACGACAGATAGCTGCCTCT

15B H02 -1.387 x 1.247 GCCGCTCTGATGACAACGA - x 265 3E-21 adenylate kinase 5

AATACAGGTCCAGGCGCTTCT

15D G03 -1 x 1.236 ATTCAGGTGGACGGCAAAAC x x 1590 2E-138 RAB 11B

CCCAGCTGTGTCCCAGATCT

15D G09 -1.548 x 1.231 TTCATGCCATGTTTCACCCTC - x 391 2.3 similar to Plasmodium falciparum

TGGATTTTGGGTCAATTGCC chromosome 2 section 32

16D B11 -1.452 x 1.026 GCACGAAGCCTTACAGACGG - x 612 0.76 similar to clone RP23-21J10

ACAGGCGTCCATCACAGTCA

16D D05 -1.444 x 1.308 GGGAGGCAACAGGAATGACA - x 581 1E-13 similar to ITS1

ACGAGGTGCTCAAGACCTGAA

19B E11 -2.352 0.809 x CTTCAACGTCGAGAACGTCTCC - - 865 7E-174 elongation factor 1a

GCAACGTATCCACGACGGAT

19C D03 1.08 x -1.503 CTTGTTTTCCCCACAACCGA - x 588 1E-11 Tetraodon nigroviridis full-length cDNA

TGTTAGCACCCTGGGATCTGAC

20C F08 -1.936 x 2.032 CTCGTCCGCTTCTGGAACA - - 535 7E-13 similar to ITS1

TTTGTGGATGGAGCGATGC

21A E10 1.096 -3.454 x TAAGCAGTGGTAACAACGCAGAGT - x 611 5E-6 solute carrier family 2

GAGGTAGACTGAGTGGGAATCCC

94 Out of these 46 ESTs that showed the largest differences in the microarray experiments, 34 showed a difference in expression after RT PCR experiments. For eight of them and for the fms paralogs, this difference in expression was confirmed by normalization with both housekeeping genes (Fig. IV.2). For those, results of the expression profile of the same gene in the female albino are shown, enabling conclusion on the eventual function of the gene under study. Figure IV.2 also presents the level of expression of some candidate genes discussed hereafter. The 26 other genes were shown differentially expressed with actin as endogenous control but for one gene (clone 03A G01) found differentially expressed while normalized with GAPDH as housekeeping gene.

95

Fig. IV.2. Relative logarithmic expression of the clones showing statistically significant difference with both endogenous controls A. after normalization with actin. We also show values for genes of interest discussed in this paper. B. After normalization with gapdh. The values for the MZ male are fixed to 0. They are in dark gray for the MZ female andin light gray for the PC albino female.

96 DISCUSSION

By assessing the level of expression of thousands of genes, we sought to gain insight into the molecular pathways important for coloration in cichlid fish. The microarray experiment highlighted 48 genes exhibiting differential expression between the two sexes. Not all of the genes selected by the array were confirmed as differentially expressed by relative quantitative RT-PCR. Interestingly, the statistically significant results of the RT-PCR experiments were all but one biased toward over-expression in females. This might be due to the recruitment of more genes for the female coloration pathway, but we also cannot exclude the possibility of an artefact in the selection of genes from the microarray results, due to the fact that we used a single array as reference for the selection of differentially expressed genes. We also noticed a difference in results of RT PCR depending on the housekeeping gene used for normalization. Actin confirmed many more genes as differentially expressed with a statistical significance, whereas GAPDH seems to be a more sensitive endogenous control. On the other hand, GAPDH confirms the one gene found under-expressed in female (clone 03A G01, blast collagen with E = 1e-170). This result is not supported by normalization with actin. Under- expression of actin in males of PZ could explain the slight bias observed. Finally, microarray data and results of the RT-PCR experiments happen to be in some cases in conflict. In most cases, this seemingly conflict is due to the lack of robustness of the microarray data. For clone 18BH11, we observed (Fig. IV.2) relatively long errors bar, which put a doubt on the over-expression of this gene in female. In the case of the four other clones (03AB02, 12CD12 and 13BD03), this conflict might be explained by individuals variance, the genes concerned coding for proteins indicating a high metabolism (like the ribosomal protein L5). Still, genes that were found differentially expressed using both endogenous controls for normalization or with one endogenous control and the microarray data can be considered as very good candidates for the study of the genetics of coloration in cichlids. Some of these genes are encoding for ribosomal subunits, others are characteristic of signal transduction. The over expression of these genes are representative of a high metabolism in the corresponding specimens, males or females, but they cannot be directly related to difference in coloration between sexes of MZ. The genes highlighted as good candidate genes by the data and their putative relation to coloration are described thereafter (see also Fig. IV.3).

97 Xanthophores markers

The fms gene is indispensable for xanthophore development since it is required for recruiting pigment cell precursors to xanthophore fates. Additionally, temporal modification of fms-expression can lead to a variety of color pattern changes, especially in the zebrafish fins (Parichy and Turner 2003). Fms was likely duplicated early in the teleost lineage (Williams et al. 2002a) and the branches of fms b gene in AB are found longer compared to those of fms a gene, implying an accelerated rate of molecular evolution in this paralog (Braasch, unpublished). Williams and co-workers (2002) investigated the gene expression of pufferfish fms paralogs in different tissues. Fms paralogs show reverse sex- specific gene expression in the pufferfish’s gonads. Braasch et al. (unp.) show that fms a but not fms b is expressed in the anal fin of male AB. Thus, it is possible that the fms paralogs represent a case of sexual sub-functionalisation. Williams et al (2002) also reported that both fms paralogs are expressed in the pufferfish in a wide range of tissues, e.g. in the skin, which implicates that both could act in pigment cells. However, it is not yet clear whether the duplicated copies perform the same or different functions (Williams et al. 2002b). One of the selected gene after the microarray experiment and tested by RT PCR was the receptor tyrosine kinase fms b; we added to the study relative quantitative RT-PCR tests for fms a. These two genes were shown overexpressed in the MZ female after RT-PCR experiments, and with both endogenous control. However, a significant difference in expression of fms b in the yellow female of MZ compared to the PC female albino was found, while no difference was detected for fms a. This supports the hypothesis of a sub-functionalization of the two paralogs. This also confirm that fms b could be involved in coloration, its down regulation in the albino then responsible of the absence of coloration.

Genes involved in actin related intracellular transport

The translation elongation factor 1 complex (eEF1) plays a central role in protein biosynthesis, delivering aminoacyls-tRNAs to the elongating ribosome. Furthermore, its over-expression affects the organization and therefore function of the actin cytoskeleton in yeast (Munshi et al. 2001). Although the results were not confirmed by RT-PCR (which is slightly biased over genes over-expressed in females anyway), this gene is over-expressed in males with the microarray data. The gene coding for myosin light chain was also selected as one putative candidates for coloration metabolism in cichlid. These proteins are situated

98 around the neck of the heavy chains that form the myosins and have a regulatory role on the function of the myosins. In particular, myosins V are involved in vesicle movement into dendritic cell extensions in skin chromatocytes by binding to the filaments of F-actin. These myosins V also have heavy chains with longer necks than the other myosins, and therefore possess 6 light chains instead of one. The gene coding for the myosin light chain was found over-expressed in females compared to the male MZ. Beta thymosins are genes belonging to a small peptid family that bind with the cytoplasmic G-actin and inhib the polymerization of actin filaments. An increase in the concentration of the ß thymosins may promote the depolarization of F-actin and therefore reduce the movement of the myosins V. As for the myosin light chains genes, the ß thymosins are found to be over- expressed in females up to RT-PCR normalized with actin housekeeping gene. The fact that these genes are in vivo closely involved with actin can leave doubt on the significance of the high expression level found using actin for normalization. Furthermore, they were also both found over-expressed in males in the microarray experiments. However, the data of the arrays are not the most robust since the difference of expression of the second array was inferior at 1.5 folds. These contradictions in the results as well as the lack of robust data must be overcome in order to elucidate the exact influence of these genes on cichlid coloration.

Endosomal and Golgi transport

Rab11b is a low molecular weight GTP binding protein belonging to the larger branch of the Ras superfamily of GTPases. Rab11b can be found on the cytoplasmic face of organelles and vesicles, and is involved in intracellular membrane fusion reactions. This gene is a known marker for protein trafficking, sorting and recycling of the endosomal pathway. It has been found over- expressed in females with both GAPDH and actin used as endogenous control of the RT-PCR. Copz is a subunit of a coatomer protein complex. This protein complex is a precursor of non-clathrin coated Golgi transport vesicles (Waters et al. 1991). The presumed coat complex may be involved in membrane trafficking in these pathways, perhaps also performing sorting functions such as the delivery of biosynthetic enzymes or pigment/precursor transporters (Ooi et al. 1997). Copz is over-expressed in females, result confirmed by the microarray and the RT PCR

99 with actin as endogenous control. However, the RT-PCR experiment is showing that this gene is under-expressed in the female albino. Precedent exists for a connection between pigmentation and intracellular transport defects. Two human diseases, the Chediak–Higashi syndrome and the Hermansky–Pudlak syndrome, are characterized by albinism as well intracellular traffic disorders. In both diseases, cells exhibit abnormalities in endosomal/lysosomal trafficking various cytoplasmic organelles including melanosomes (Scriver 1995). The altered expression of a coat adaptor was also shown to influence quantitatively and qualitatively the pigment granules of the eye in Drosophila (Ooi et al. 1997). Since this is through transport between the endosome and the Golgi that pigments granules are thought to be synthesized, we can assume that RAB11B and copz are involved in the yellow coloration of the female of MZ. The production of xanthophores recquires necessarily a large amount of vesicules formation for synthesis and transport of the pigments. Furthermore, the downregulation of copz in the female albino of PC matches with the absence of color. It can be speculated that the absence of copz in female albino (or even its down regulation up to GAPDH endogenous control) prevents the formation of the pterinosome. As a consequence of the absence of this organelle, the colorful form of pteridins - sepiapterins and drosopterins - would not become visible as yellow pigment under transmitted light (Odenthal 1996; Obika, 1963; Kamei and Hama 71). Therefore, the absence of color in albino would be due to the absence of the colorful form of the pteridins, rather than the absence of xanthophores. This speculation also fits with the pattern of gene expression we found for fms a. Indeed, since fms a is equally expressed in female yellow and albino, it might be that the albino morph possesses xanthophores as much as yellow female does, without however displaying the colorful form of the pigment. This speculation is corroborated by previsous findings of Lin et al. (1992), in which it was shown that unpigmented melanophores were present and behaved normally in an albino zebrafish (alb-1/alb-1). Pictures of skin under UV light also support our hypothesis. Yellow coloration is due to the colorful form of pteridins, i.e., sepiapterins (Matsumoto and Obika, 1968; reviewed by Bagnara, 1966). Four other forms are colorless under visible light but show fluorescent colors under UV light: biopterin and neopterin (light blue fluorescence), xanthopterin (green fluorescence) and isoxanthopterin (dark blue fluorescence) (Odenthal et al. 1996). The fluorescence seen on the female albino skin (Fig. IV.1i) is likely produced by the presence of biopterin or neopterin. Indeed, the alternative assumption that it would be caused by iridophores (which also fluoresce, Bolker

100 et al. 2005) is invalid cause the fluorescent stains are not found in the male skin, skin that is although also silvery.

By publishing a list of candidate genes having a role in coloration in cichlids, our study validate the use of microarray techniques and RT-PCR experiments to gain insights in the molecular pathways leading to coloration in cichlids. We observed the over-expression of fms a and fms b paralogs in the skin of females, confirming the role of both of these paralogs in the yellow coloration. Their expression is however found to be different in the female albino, providing new evidence for a sub-functionalization of fms b. Our results also confirm the important role played by intracellular mobility on the actin filaments, as well as the endosome-Golgi trafficking. This publication can therefore be seen as a basis for numerous future projects, since the exact role and regulation of the candidates genes need now to be further investigated from a cellular and biochemical perspectives.

101 AKNOWLEDGMENTS

We thank R. Bahulikar and A. Materna from P. Kroth’s lab for providing material and technical support for RT-PCR experiments, and E. Hespeler, J. Haugg and the other members of the Meyer-lab for technical assistance. This study was supported by the Landesstiftung Baden-Württemberg, the Center for Junior Research Fellows (University Konstanz) and the EU (Marie Curie fellowship) to W. S, and grants of the Deutsche Forschungsgemeinschaft to A. M.

102 SUMMARY

Lakes Victoria, Malawi and Tanganyika in East Africa contain unique species flocks of cichlid fish, counting hundreds of endemic species each. The shear number of species, the degree of ecological and morphological specialization, and the rapidity of lineage formation characterize the East African cichlid assemblages and make them well-known model systems for the study of adaptive radiations and explosive speciation. The work presented in this thesis intends to increase our knowledge on particular aspects of the cichlid radiations. Adaptive radiation is defined as the evolution of ecological diversity in a rapidly multiplying lineage. Chapter II and III are describing the evolution of the Tanganyikan cichlids species flock for two of the criteria implied by the definition above: the adaptation of body shape to environment and the rapid diversification of the ancestral lineages within the lake. Also, the process of radiation in the case the East African cichlids is believed to happen in three stages. While the first two steps are truly adaptive (adaptation to new habitats followed by trophic specialization), the third stage is driven by sexual selection and therefore considered as non-adaptive. Chapter IV focus on the non-adaptive part of the radiation by identifying genes involved in coloration, since body color is believed to drive sexual selection in cichlids. Even though a link between environment and phenotype of East African cichlids could be established by previous works, this correlation was so far not statistically supported. I used geometric morphometric methods to describe the body shape of 45 species of East African cichlid fish, with a focus on the Lake Tanganyika species assemblage which is composed of more than 200 endemic species. Chapter II states that the main differences in shape concern the length of the whole body and the relative sizes of the head and caudal peduncle. I investigated the phylogenetic signal contained in the morphometric data set with the Phylogenetic Mixed Model, as well as the importance of the influence of phylogeny on similarity of shape between closely related species with cluster analyses and Normalized Mantel statistics on the phylogenetic distances and distances in the morphospace. After concluding that the influence of phylogeny was small, I investigated the evolution of body shape in relation to known ecological traits using MANOVA. I found that body shape was strongly predicted by feeding preferences (i.e., trophic niches) and the water depths at which species occur. Calculation of the disparity within tribes indicates that even though the morphological diversification associated with explosive speciation has happened in only a few tribes of the Tanganyikan assemblage, the potential to evolve diverse morphologies exists in all tribes. Quantitative data support the existence of extensive parallelism in several independent adaptive radiations in

103 Lake Tanganyika. Notably, Tanganyikan mouthbrooders belonging to the C- lineage and the substrate spawning Lamprologini have evolved a multitude of different shapes from elongated and Lamprologus-like hypothetical ancestors. In chapter III, I report on a phylogenetic analysis of Lake Tanganyika cichlids combining the previously determined sequences of the mitochondrial ND2 gene (1,047 bp) with newly derived sequences of the nuclear RAG1 gene (~700 bp of intron 2 and ~1100 bp of exon 3). The nuclear data – in agreement with mitochondrial DNA – suggest that Lake Tanganyika harbors several ancient lineages that did not undergo rampant speciation (e.g., Bathybatini, Trematocarini). I find strong support for the monophyly of the most species-rich Tanganyikan group, the Lamprologini, and I propose a new taxonomic group that I term the C-lineage. The Haplochromini and Tropheini both have an 11bp deletion in the intron of RAG1, strongly supporting the monophyly of this clade and its derived position. Mapping the phylogenetically informative positions revealed that, for certain branches, there are six times fewer apomorphies in RAG1. However, the consistency index of these positions is higher compared to the mitochondrial ND2 gene. Nuclear data therefore provide on a per basepair basis less, but more reliable phylogenetic information. Even if in our case RAG1 has not provided as much phylogenetic information as I expected, I suggest that this marker might be useful in the resolution of the phylogeny of older groups. Finally, it has been suggested that speciation on the basis of sexual selection is an important mechanism for the generation of new species, with male body hue considered as the primary discriminatory factor among a hierarchy of visual cues used by females. In chapter IV, I describe the use of the cDNA microarray technique to identify genes involved in coloration in cichlid fish. I compare skins of a male and female of the species Maylandia zebra that shows a dimorphism of body coloration, with the male being blue while the female is bright yellow. The microarray experiment highlighted 46 genes exhibiting differential expression between the two sexes, of which 34 were confirmed differentially expressed by relative quantitative RT-PCR. Among them, I identified numerous genes involved in intracellular mobility of pigments granules related to the actin filament transport, as well as genes involved in the endosomal to Golgi vesicles trafficking. I also tested for the differential expression in the two sexes of the paralogs fms a and fms b, already known as good candidate genes for the study of coloration in teleosts because of their role as precursors of xanthophores, the yellow pigment cell. Our results add evidence to the neo-functionalization of the paralog fms b. All these genes were also tested for relative expression in a female albino of a closely related species of cichlid, Pseudotropheus callainos. With the support of

104 microscopic pictures of the skin of these three specimens, I interpret the difference of expression of the genes selected between a blue male, a yellow female and an albino female. This study provides insights on the putative function of the genes in cichlids coloration, and by extension on the evolution of coloration in teleost fishes.

105 ZUSAMMENFASSUNG

Die Seen Victoriasee, Tanganyikasee und Malawisee in Ost-Afrika enthalten einzigartige Ansammlungen an Buntbarschenarten, wobei die Zahlen der jeweils enthaltenen endemischen Arten in die Hunderte geht. Die schiere Zahl an Arten, der Grad an ökologischer und morphologischer Spezialisierung und die Schnelligkeit der Artbildungsprozesse sind Charakteristika der Ostafrikanischen Buntbarsch-Artenschwärme und machen diese so zu einem wohlbekannten Modellsystem für Studien über adaptive Radiation und explosive Artenbildung („Explosive Speciation“). Die im Rahmen dieser Doktorarbeit vorgestellten Arbeiten haben zum Ziel unser Wissen über bestimmte Aspekte der Buntbarsch- Radiationen zu erwetiern. Unter adaptiver Radiation versteht man die Ausbildung einer ökologischen Vielfalt in einer sich schnell multiplizierenden Abstammungslinie. Kapitel II und III beschreiben die Evolution von Buntbarsche des Tanganyikasees in zwei der oben beschriebenen Kriterien ein: der Anpassung der Körperform an die Umwelt; und die schnelle Diversifizierung der Abstammungslinien innerhalb des Sees. Es wird angenommen, dass der Prozess der Radiation im Fall der ostafrikanischen Buntbarsche in drei Schritten geschieht. Während es sich in den beiden ersten Schritten wahrhaftig um adaptive, also um Anpassungsvorgänge handelt (Anpassung an neue Habitate gefolgt von trophischer Spezialisierung), wird das dritte Stadium von sexueller Selektion angetrieben und gilt daher als nicht adaptiv. Kapitel IV behandelt jenen nicht adaptiven Teil der Radiation. Hier werden Gene identifiziert, die für die Färbung der Buntbarsche eine Rolle spielen, da davon ausgegangen wird, dass die Ausbildung der Körperfärbung der Buntbarsche von sexueller Selektion angetrieben wird. Obwohl der Zusammenhang zwischen Umwelt und Phänotyp der ostafrikanischen Buntbarsche bereits in früheren Arbeiten hergestellt wurde, fehlten bisher signifikante statistische Analysen, die diesen Zusammenhang stützen. Um die Körperform von 45 Arten ostafrikanischer Buntbarsche zu beschreiben, wurden hier geometrisch-morphometrische Methoden angewendet. Der Fokus lag hierbei auf der Artenansammlung des Tanganyikasees, die von mehr als 200 endemischen Arten gestellt wird. In Kapitel II wird beschrieben, dass die Hauptunterschiede zwischen den Körperformen die Gesamtkörperlänge, die relative Größe des Kopfes sowie den Abstand von Afterflosse zur Schwanzflosse betreffen. Ich untersuchte das in dem morphometrischen Datensatz enthaltene phylogenetische Signal anhand des „Phylogenetic Mixed Model“. Zur Untersuchung der Stärke des Einflusses der Phylogenie auf Ähnlichkeiten der Gestalt nahe verwandter Arten wurden Cluster-

106 Analysen und „Normalized Mantel Statistics“ auf die phylogenetischen Abstände und die Anstände innerhalb des „Morphospace“ angewendet. Aus der Erkenntnis, dass der Einfluss der Phylogenie nur klein ist folgten Untersuchungen der Körperformevolution in Relation zu bekannten ökologischen Charakteristika („ecological traits“) unter Anwendung von MANOVA. Ich fand heraus, dass die Körperform in einem starken Maße durch die bevorzugte Art und Weise der Nahrungsaufnahme (z.B. trophische Nischen) und der Wassertiefe, in welcher die jeweilige Art vorkommt, vorhergesagt wird. Berechnungen der Disparität innerhalb von Unterfamilien weisen auf das Potential zur Ausbildung diverser Morphologien innerhalb aller Unterfamilien hin, obwohl die morphologische Diversifizierung einhergehend mit explosiver Artenbildung in nur wenigen Unterfamilien der Tanganyika-Artenemeinschaft stattfand. Quantitative Daten unterstützen die Existenz von zahlreichen Parallelismen zwischen verschiedenen voneinander unabhängig auftretenden adaptiven Radiationen innerhalb des Tanganyikasees. Bemerkenswerterweise haben die derselben Abstammungslinie (C-Linie, siehe unten) zugehörigen Maulbrüter des Tanganyikasees und die auf dem substrat-laichenden Lamprologini, ausgehend von verlängerten Lamprologus-artigen hypothetischen Vorfahren, eine Vielfalt verschiedener Formen ausgebildet. In Kapitel III stelle ich eine Phylogenetische Analyse der Buntbarsche des Tanganyikasees vor, die die bereits bekannten Sequenzen der mitochondrial codierten ND2 Gene (1047 bp) mit neu generierten Sequenzen des nukleären RAG1 Gens (~700 bp eines des Introns II und ~1100 bp des Exons 3) kombiniert. Die Daten aus kerncodierter DNA - in Übereinstimmung mit denen aus mitochondrialer DNA - legen nahe, dass der Tanganyikasee mehrere alte Abstammungslinien beherbergt, die keine explosive Artenbildung durchlaufen haben (z.B. Bathybatini, Trematocarini). Des Weiteren weist diese Arbeit stark auf die Monophylie der artenreichsten Gruppe im Tanganyikasee, den Lamprologini hin. Außerdem legt sie die Existenz einer neuen taxonomischen Gruppe nahe, welche ich die C-Linie nenne. Die Haplochromini und die Tropheini - beide weisen eine 11 bp Deletion in dem Intron des RAG1 Gens auf - deuten auf die Monophylie dieser Gruppe hin und auf ihre abgeleitete Position. Das „Mapping“ der phylogenetisch informativen Positionen zeigt, dass in bestimmten Ästen des Stammbaumes sechsmal weniger Apomorphien in RAG1 vorzufinden sind als im ND2. Jedoch ist der Consistency Index dieser Positionen im Vergleich zum mitochondrialen ND2 Gen größer. Daten aus kerncodierten Sequenzen stellen auf Nukleotidebene verglichen weniger aber verlässlichere phylogenetische Information bereit als mitochondriale Sequenzen. Obwohl in diesem Fall das

107 RAG1 Gen nicht so viel phylogenetische Information bot wie erwartet, so schlage ich doch vor, dass dieser Marker nützlich zur Auflösung der Phylogenie älterer Gruppen sein könnte. Anhand männlicher Körperfärbung, die als primärer diskriminierender Faktor in der Hierarchie der visuellen, von Weibchen benutzten Charakteristika gilt, wurde bereits vorgeschlagen, dass Artenbildung auf der Grundlage sexueller Selektion ein wichtiger Mechanismus für die Bildung neuer Arten ist. In Kapitel IV schließlich, beschreibe ich die Verwendung der cDNA Microarray Technik zur Identifikation von Gene, die in die Färbung von Buntbarschen involviert sind. Es wurde Haut von Männchen und Weibchen der Art Maylandia zebra, die einen geschlechtsabhängigen Dimorphismus der Körperfarbe aufweist, verglichen. Die Männchen dieser Art sind blau, während die Weibchen hell gelb in Erscheinung treten. Die Microarray Experimente hoben 46 Gene hervor, die unterschiedlich starke Expression in den beiden Geschlechtern aufwiesen. Von 34 dieser Gene wurde deren geschlechtsabhängig verschieden starke Transkription per RT-PCR bestätigt. Unter diesen konnten zahlreiche Gene identifiziert werden, die in die intrazelluläre Mobilität von Pigment-Granulae involviert und verwandt zu Genen des Actin Filament Transports sind, sowie Gene des endosomalen Vesikeltransports zum Golgi Apparat. Weiterhin wurde auf differentielle Expression der paralogen Gene fms a und fms b in den beiden Geschlechtern getestet. Diese Gene sind bereits wegen ihrer Rolle als Vorläufer der Xanthophoren - der gelben Pigmentzellen - als gute Kandidatengene zu Studien der Färbung von Teleostiern bekannt. Meine Ergebnisse unterstützen die Hinweise zur Neofunktionalisierung des fms b Paralogs. All diese Gene wurden auch auf ihre relative Expression in weiblichen Albinos der nahe verwandten Buntbarschart Pseudotropheus callainos hin untersucht. Die Daten zur unterschiedlichen Expression der ausgewählten Gene bei blauen Männchen, gelben Weibchen und Albino Weibchen wird weiterhin durch Mikroskopie Aufnahmen der Haut dieser drei Proben unterstützt. Diese Studie bietet neue Einsichten in die putativen Funktionen dieser Gene bei der Färbung von Buntbarschen, sowie Perspektiven zum Studium der Evolution von Färbung in Teleostiern.

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121 Results produced by collaborators

Chapter II

Figure 8 produced by Paul Bunje (University of Konstanz)

Chapter III

Figures formatted by Walter Salzburger and myself (University of Konstanz)

Chapter IV

Figure 1 produced by Walter Salzburger and myself (University of Konstanz)

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