Evolutionary Histories of Lake Tanganyika Cichlids Differ Between Habitats: Phylogenetic Evidence from Two Deepwater Tribes

Evolutionary histories of Lake Tanganyika cichlids differ between habitats: phylogenetic evidence from two deepwater tribes

Diploma thesis

Paul Kirchberger

Under the supervision of

Priv.-Doz. Dipl.-Ing. Dr.nat.techn. Kristina M. Sefc

Institute of Zoology

University of Graz

Graz, April 2012

Acknowledgements

I’d like to thank Kristina Sefc for giving me the opportunity to work in her group and for her excellent supervision of my research. I really appreciate the independence I was afforded during my work as well as the clear guidance and helpful comments from the beginning to the end of my thesis. I really enjoyed my time here!

I am equally thankful to Stephan Koblmüller for his great support, for answering my sometimes stupid questions and for his incredible helpfulness.

Furthermore, thanks to Karin Mattersdorfer for showing me the ropes in the lab and also to the rest of the research group for creating a nice environment to work in.

To my parents Wiltrud and Gerald, thank you for your love and support and for being such great parents! Keep up the good work.

Obligatory shout out to Thomas!

So long, and thanks to all the fish!

Abstract

Around 2000 fish species of the family Cichlidae (Perciformes, Teleostei) can be found in and around the three East African Great Lakes, the centers of cichlid biodiversity. Several adaptive radiations produced an abundance of species differing dramatically in their morphology, behavior and general ecology. Although Lake Tanganyika contains fewer species than Lakes Malawi and Victoria, it houses what is perhaps the most diverse assembly of cichlids. The lake’s shore habitats consist of alternating patches of rocky and sandy substrates which serve as geographic barriers and promote allopatric speciation. Due to the relative ease by which gene flow can be curbed along the structured shoreline, speciation often does not include the evolution of reproductive barriers. As a consequence, hybridization in secondary contact occurs in cichlids that inhabit the shallow waters of the lake. As opposed to the littoral habitat, the lake’s deepwater habitats are characterized by lower species diversity and fewer available niches. A lack of pronounced geographic barriers should have necessitated the early evolution of reproductive barriers between incipient species. As a consequence, we hypothesize that deepwater species should display no signs of hybridization. To test this, I constructed nuclear multi-locus AFLP phylogenies of two endemic deepwater tribes, the Bathybatini and Limnochromini, and compared them with mitochondrial phylogenies of earlier studies. Using a tree-based homoplasy excess test, I could not find any signs of hybridization in the history of these tribes, which confirmed our hypothesis. Furthermore, the nuclear phylogenies provided well supported reconstructions of the speciation patterns of the Bathybatini and Limnochromini. Both tribes underwent adaptive radiations and ecological segregation during speciation could be inferred. In both tribes, taxonomic uncertainties due to conflicting results of earlier studies could be resolved.

Abstract ...... 3

1. Introduction ...... 5

1.1 The Origin of Speciation ...... 5

1.2 East African Cichlids as model systems for evolutionary biology ...... 6

1.3 Lake Tanganyika - The ancient lake in the center of cichlid biodiversity ...... 9

1.4 Littoral and deepwater habitats of Lake Tanganyika ...... 11

1.5 Cichlids of the depths ...... 13

1.6 AFLP and its use in phylogenetics ...... 18

1.7 Goals of this thesis ...... 22

2. Material and Methods ...... 23

2.1 Sample collection ...... 23

2.2 DNA isolation ...... 26

2.3 AFLP analysis ...... 26

2.4 AFLP scoring...... 27

2.5 Phylogenetic inference ...... 28

3. Results ...... 29

3.1 Bathybatini ...... 29

3.2 Limnochromini ...... 32

4. Discussion ...... 35

4.1 Bathybatini ...... 35

4.2 Limnochromini ...... 37

4.3 Conclusions ...... 40

5. Literature ...... 42

1. Introduction

1.1 The Origin of Speciation

Despite its title, Charles Darwin’s The Origin of Species (1859) contains remarkably few thoughts on the actual process by which species arise and instead is more concerned with variation within species. Part of the reason for this absence in Darwin’s work might have been his belief that species are not actual, discrete entities but rather arbitrarily defined groups in a continuous flow of variation among organisms: “…I look at the term species, as one arbitrarily given for the sake of convenience to a set of individuals closely resembling each other, and … it does not essentially differ from the term variety, which is given to less distinct and more fluctuating forms” (Darwin, 1859, page 52). However, this is not what we generally observe in nature, and while the reality of species might seem obvious at first glance, it is not obvious that the process of incremental evolution that Darwin described would result in the clearly discernable groups of organisms that we see in day to day life. However, for practical and philosophical reasons, most biologists both from the past and present consider species (at least of eukaryotic organisms) to be real. Dobzhansky (1937a) pointed out that “formation of discrete groups is so nearly universal that it must be regarded as a fundamental characteristic of organic diversity”. Coyne & Orr (2004) noted that the study of how species arise, speciation, is one of the “few areas of evolutionary biology not overshadowed by Darwin’s immense achievements”. Maybe for this reason, speciation research remained a neglected sub-field of evolutionary biology in the pre-modern synthesis era. In the late 1930’s-early 1940’s, works by Theodosius Dobzhansky such as “Genetics and the Origin of species” (1937b) and later Ernst Mayr’s “Systematics and the Origin of Species” (1942) invigorated both theoretical and practical research on the true origin of species. Even in these early days of speciation research, the tropical freshwater fish family Cichlidae was of immense interest to biological science.

1.2 East African Cichlids as model systems for evolutionary biology

The extraordinary diversity of cichlid fishes found in the East African Rift Valley could not be explained by standard speciation models that saw vicariance events as the primary driver of the origin of species. It soon became apparent that this standard allopatric mode of speciation alone could not account for the diversity of this family. The primary question of cichlid research was, and still is, how could so many cichlids species evolve seemingly without geographic barriers and seemingly violating the principle of competitive exclusion (Salzburger and Meyer, 2004)? In 1984, according to eminent evolutionary biologist Ernst Mayr, the major obstacles to solving what was termed the “cichlid problem” were the unknown age of these species flocks as well as lack of clarity in taxonomy and in cladistic analyses (Mayr, 1984). By the 21st century, enormous advances have been made in all three points and cichlids have become model systems in the study evolutionary processes (see for example Sturmbauer, 1999; Kornfield and Smith, 2000; Kocher, 2004; Seehausen, 2006; Turner, 2007).

Phylogenetic analyses have shown that the closet relatives of cichlids are marine damselfishes (Pomacentridae) and surfperches (Embiotocidae) (Mabuchi et al., 2007). While the oldest cichlid fossil is dated 46 million years, the origin of the lineage can be traced back to the supercontinent Gondwana. The cichlids already seem to have been widely distributed when the continent fragmented into Indo-Madagascar and South America-Africa 120-160 million years ago. As a consequence, they now inhabit freshwater lakes and streams across India, the Middle East, Africa and Madagascar as well as Middle and South America in a typical Gondwana-distribution (Genner et al. 2007) (See Fig. 1)

Fig. 1: Current distribution of the Chichlidae. A) The Phylogenetic tree of the main cichlid lines show sister group relations between African and American as well as Indian and Madagascan Cichlidae. B) Gondwana supercontinent in the Late Triassic (around 200 million years ago) (From Salzburger and Meyer, 2004)

However, what makes the cichlids so interesting to evolutionary biology nowadays is their astonishing explosion of diversity after the breakup of Gondwana. As mentioned before, there are up to 3000 species of cichlids, with around 2000 present in and around the three East African Great Lakes, Victoria, Malawi and Tanganyika. These three lakes were the center of perhaps one of the most spectacular adaptive radiations of vertebrates (Kocher, 2004). Adaptive radiation, the rapid evolution of phenotypical and ecological diversity within a single lineage, essentially requires two factors: 1) Ecological opportunity and 2) evolutionary innovation (Schluter, 2000). The first was provided in the formation of the African Rift Valley lakes by the splitting of the African tectonic plate roughly 40 million years ago. These new environments provided a rich variety of unoccupied niches for the ancestors of modern cichlids (Fryer and Iles, 1972). But the cichlids were obviously not the only new colonists of these habitats, so why did the cichlids explode in diversity while other fish families such as evolved only a handful of species? One factor that is often attributed to the evolutionary success of the cichlids is their possession of pharyngeal jaws, formed by the derived 5th gill arch (Fig. 2) (Liem, 1973).

Fig. 2: Schematic representation a cichlid skull. Pharyngeal jaw apparatus drawn in red, black lines indicate pharyngeal jaw apparatus muscles. Numbers inidicate major features of cichlid pharyngeal jaw apparatus: 1) fusion of left and right lower jaw elements. 2) suspension of lower jaw element in muscular sling running from neurocranium to posterior muscular arms of lower jaw. 3) Diarthroric (freely movable) articulation of upper jaw with underside of neurocranium. From Mabuchi et al. (2007)

The possession of oral and pharyngeal jaws allows cichlids to separate the acts of collecting and processing food, tasks that are normally performed by the oral jaws alone. The two sets of jaws are thus free to specialize and allowed the cichlids to adapt to a large variety of food sources and niches (Liem, 1973). However, the (convergent) evolution of pharyngeal jaws in other groups of fish did not lead to adaptive radiations (Alfaro et al., 2009) and indeed there is a second key innovation that also might have been responsible for the cichlid’s evolutionary success: Their highly complex breeding behavior (Crapon de Crapona, 1986). Cichlids display a wide variety of mating and parental care strategies, ranging from single and cooperative substrate breeding to mono- and biparental mouthbrooding, from mono- to polygamy and a number of alternative reproductive strategies displayed within a single species. The high pressure of sexual selection by female mate choice is thought to have

contributed considerably to the radiations of these animals and to the often colorful mating dresses (see for example Salzburger, 2009; Sefc, 2011).

1.3 Lake Tanganyika - The ancient lake in the center of cichlid biodiversity

With an age of 9-12 Mya, Lake Tanganyika is by far the oldest of the East African Great Lakes, the centers of cichlid biodiversity. While Lake Tanganyika contains fewer species than the other two Great Lakes Lakes Malawi and Victoria, the roughly 250 Tanganyika cichlids surpass their relatives in diversity of morphology, ecology and behavior. These differences, coupled with the greater genetic diversity due to the age of the species assemblage and their polyphyletic origin make Lake Tanganyika cichlids the best understood in terms of phylogenetic relationships (Snoeks, 2000). Morphological cladistics divides them into 12 (Poll 1986) or 16 (Takahashi, 2003) monophyletic tribes derived from several initial seeding lineages that independently colonized the lake and radiated within it (Salzburger et al., 2002b) (Fig. 3).

Fig. 3: Phylogeny of Lake Tanganyika cichlids. Triangles indicate radiations, their size corresponds to the extent of the radiations. From Takahashi & Koblmüller, 2011

The species assembly is thought to have evolved in close concert with the geological activity that formed Lake Tanganyika. Sturmbauer (1998) gives an account of the lake’s formation in conjunction with the radiation of its fish fauna (see Fig. 4): 20 million years ago, a flat basin containing the slowly meandering Proto-Malagarazi river was formed by the drifting of the African tectonic plate. Further drift changed the river into a swamp and a system of small and shallow proto-lakes was created. With the loss of water outlets to the Congo river, the lakes deepened and ultimately merged into a single body of water some 5-6 million years ago. The establishment of deepwater conditions at this point is thought to have coincided

with the so-called “primary lacustrine radiation” that resulted in the diversification of the seeding lineages into the major trophic niches (Salzburger et al., 2002b). Water level fluctuations persisted up to the modern age and continually changed the shore habitats of the lake, which might have acted as triggers for additional smaller radiations. A second lacustrine radiation is thought to have happened 2,5-3 million years ago, leading to further diversification of most mouthbrooding lineages (Koblmüller et al., 2008).

Fig. 4: Coincidence of habitat changes due to the formation of modern Lake Tanganyika with periods of adaptive radiation. Initial seeding of the lake exemplified by 3 symbolic lineages, two of them radiating in response to different trigger events. Boxes indicate timescale of radiation, crosses extinctions. From Sturmbauer, 1999.

1.4 Littoral and deepwater habitats of Lake Tanganyika

The largest cichlid diversity of Lake Tanganyika can be found on the shorelines. The littoral habitat of Lake Tanganyika is composed of alternating patches of sandy and rocky habitats, which offers a great number of geographic barriers as well as opportunities for allopatric speciation and color pattern diversification at the onset of this process (Sefc et al., 2007;

Koblmüller et al., 2011). On the other hand, reduced species richness in the deep benthal and pelagial seems to be common in all East African Great Lakes (Turner, 1996; Seehausen et al., 1997a; Shaw et al., 2000; Koblmüller et al., 2008). At least three factors may have contributed to this pattern: i) reduced niche diversity in the pelagic and in deepwater benthic zones, ii) a narrow ambient light spectrum consisting only of short-wavelength blue light and hence less promotive of diversification mechanisms contingent on color perception than the shallow clear-water habitats (Seehausen et al., 1997b; Seehausen et al., 1999; Knight and Turner, 2004; Maan et al., 2004) and iii) the absence of strong barriers to gene flow. Indeed, deepwater cichlid species often have lake-wide distributions with very low, if any, population genetic structure over large geographic distances (Shaw et al, 2000; Genner et al., 2008; Genner et al, 2010); see also the Lake Tanganyika clupeid Limnothrissa miodon (Hauser et al., 1998), and the centropomid Lates stappersi (Kuusipalo et al., 1999). On the other hand, high levels of genetic differentiation, sometimes accompanied by phenotypic divergence on small geographic scales, are characteristic for the species-rich guild of stenotopic rock-dwelling cichlid species (see for example Wagner and McCune., 2009; Konijnendijk et al., 2011). However, allopatric diversification in the fragmented littoral zone was not necessarily accompanied by the evolution of pre- or post-zygotic isolation, so that secondary contact imposed by lake level fluctuations has often led to hybridization or introgression between previously allopatric taxa (for example Rüber et al., 2001). Even today, substrate breeders of the tribe Lamprologini can be found in mixed-species pairs (Sturmbauer et al, 2010), and interspecific fertilizations occur in communally nesting, shell- breeding lamprologines (Koblmüller et al., 2007b). Indeed, phylogenetic analyses of predominantly littoral cichlid lineages revealed that interspecific hybridization has played, and still plays, an important role in the evolution of these fish (Rüber et al., 2001; Schelly et al, 2006 Egger et al., 2007; Koblmüller et al., 2007b; Koblmüller et al., 2007c; Koblmüller et al., 2010; Nevado et al., 2009; Sturmbauer et al., 2010) Thus, in the majority of recent molecular studies on species relationships within littoral tribes, especially when comparing mitochondrial and nuclear phylogenies, the explanation for the tree topologies involved the claim of introgression and hybridization between established lineages in addition to incomplete lineage sorting (reviewed in Takahashi and Koblmüller, 2011).

Although it has not been formally investigated, tribes composed of deepwater species seem to differ in some aspects. Lacking the geographic structure introduced by littoral habitat heterogeneity, deepwater species may still be spatially separated by distance, by segregation of breeding grounds, by variable hydrological conditions (Coulter, 1991; Fryer, 2006; Genner et al., 2010) or by large-scale fragmentation of the lake basin during major droughts (Duftner et al, 2005; Koblmüller et al., 2005). Generally however, the potential barriers to gene flow for deepwater species are less insurmountable than those met by stenotopic littoral cichlids. Specifically in Lake Tanganyika, the evolution of stenotopy regarding depth, bottom type or light intensity may have been prohibited by the seasonal upwelling of anoxic waters (Eccles, 1986). Given the high potential for gene flow, is is likely that diversification of lineages will either be curtailed (as suggested by the relative species paucity) or be attended by strong reproductive isolation right from the start. This would imply that introgression following cladogenesis occurred at much lower rates, if at all, in the deepwater species than in littoral cichlids.

1.5 Cichlids of the depths

With a depth of 1470m, Lake Tanganyika displays one of the world’s richest deepwater communities. Deepwater habitats consist of flat or slightly sloped shelf areas as well as steep and rocky slopes and fish can be found up to a depth of around 200m, where the oxygenated zone ends (Coulter, 1991). Several cichlid tribes such as the Bathybatini and Limnochromini (Fig. 5) have populated these habitats and evolved a great variety of specific niche adaptations.

Fig. 5: Cladograms of the Bathybatini and Limnochromini as inferred from mtDNA data by Koblmüller et al.(2005) and Duftner et al. (2005). Solid lines represent the topology obtained by maximum likelihood algorithm, dotted lines strict consensus from neighbor- joining, maximum likelihood and maximum parsimony.

The Tribe Bathybatini

The tribe Bathybatini (sensu Takahashi 2003) comprises 17 species in three genera: (1) The genus Bathybates comprises six large (30-40 cm) piscivorous species hunting mainly pelagic freshwater clupeids (B. fasciatus and B. leo), benthic cichlids (B. graueri, B. vittatus and B. ferox) or undefined prey (the rare, elusive B. horni), in addition to the small (20 cm) B. minor, which is a specialized clupeid hunter. In accordance with their trophic niches, Coulter (1991) distinguished three morphotypes among the Bathybates species, the fast-swimming fusiform predators B. fasciatus, B. leo and B. horni, the generalized shape of the benthic feeders B. graueri, B. vittatus and B. ferox, and the small clupeid-mimicking B. minor, which mingles with its prey and accompanies the diurnal clupeid migrations. Based on trawl net and gill net catches, B. minor were classified as pelagic, B. fasciatus and B. leo as chiefly bathypelagic and the remaining four species (B. graueri, B. vittatus, B. ferox and B. horni) as chiefly benthic (Coulter 1991). Except for B. minor, which was never found below 70m, Bathybates species descend to depths of 150-200m. (2) The member of the monotypic genus Hemibates, H. stenosoma, is an abundant benthic species on the muddy bottom of southern Lake Tanganyika feeding on fish and shrimps mainly at depths between 100 and 200 m

(Coulter 1991). (3) The small-bodied (<15cm) species of the genus Trematocara (formerly assigned to the genera Trematocara and Telotrematocara, Poll 1986) comprise nine benthic and bathypelagic species feeding on a variety on invertebrate prey, fish larvae and phytoplankton. They are found at maximum depths of 75 to 200 m (Coulter 1991). Following the upward movement of zooplankton, many Trematocara species undertake nightly migrations along slopes into the littoral.

All members of the Bathybatini are maternal mouthbrooders. Some species release their fry in shallow areas, but overall, data on bathybatine breeding behaviour is anecdotal or lacking (Coulter 1991; Konings 1998). The species are sexually dimorphic, with males of Bathybates and Hemibates sporting species-specific patterns of dark stripes, bars and dots on a silver background and egg-spots on the anal fins, and males of the silvery Trematocara with dark dorsal fin markings. Females of all species show a uniformly silver/brown coloration.

A recent phylogenetic study based on three mitochondrial genes supported the monophyly of Bathybates as well as of the species therein, and indicated a polytomy of three equi- distant lineages representing Bathybates, Hemibates and Trematocara (Koblmüller et al. 2005). Within Bathybates, B. minor appeared ancestral to a radiation of the six large species, which showed a basal split of B. graueri and low statistical support for the branching order of the remaining species (see Fig. 5). The short internal branches among the large Bathybates species supported a rapid radiation at approximately 2.3 – 2.7 MYA, coinciding with the rapid diversification of other Lake Tanganyika cichlids (Koblmüller et al. 2004, 2007a; Duftner et al. 2005). Competition and resource partitioning as well as potential geographic isolation during an extreme low-stand of the lake were proposed as promoters of Bathybates speciation (Koblmüller et al. 2005).

The Tribe Limnochromini

The Limnochromini sensu Poll (1986) consist of 13 species in 8 genera: Benthochromis, Baileychromis, Gnathochromis, Greenwoodochromis, Limnochromis, Reganochromis, Tangachromis and Triglachromis, a grouping based on external morphological features and the pharyngeal apophysis. This classification has been repeatedly called into question both by morphological and biochemical studies. Kocher et al. (1995) noted that the maternal 15

mouthbrooder Gnathochromis pfefferi should be grouped within the Tropheini based on mitochrondrial NADH2 data, an assessment that was universally confirmed by other studies (Salzburger et al. 2002b, Sturmbauer et al. 2003, Takahashi 2003, Duftner et al. 2005). The remaining member of the genus Gnathochromis, G. permaxillaris, was placed ancestral to the tribe Ectodini based on allozyme data (Nishida, 1997). However, no other study so far confirmed this result. The genus Benthochromis was also removed from the Limnochromini (Salzburger et al. 2002b) and subsequently placed in its own tribe, the Benthochromini, by Takahashi (2003). Takahashi’s updated systematic classification of cichlids reduced the Limnochromini to 8 species in 6 genera by the erection of the tribes Benthochromini (Benthochromis tricoti and B. melanoides) and Greenwoodochromini (Greenwoodochromis christyi and G. bellcrossi) as well as the removal of Gnathochromis pfefferi. The most recent study of the Limnochromini, based on the mitochondrial NADH2 gene and control region confirmed the status of the tribe Benthochromini and the removal of G. pfefferi but firmly rejected the Greenwoodichromini (Duftner, 2005). This ultimately leaves the Limnochromini a tribe with 10 species in 6 genera, which, despite its shrinkage over time, is still an extraordinarily diverse group of cichlids. The tribe’s namesake genus Limnochromis consists of three species: L. abeelei, L. auritus and L. staneri. All three are believed to be generalist predators mostly inhabiting the deeper benthic zones (below 100m, up to 200m for L. abeelei) of Lake Tanganyika. They are characterized by their large heads and opercular spots. Limnochromis auritus, which is sometimes found in shallower parts of the lake, is said to dig interconnected tunnels several feet in depth into the muddy substrate. It feeds on small arthropods and snails. Due to the deepwater distribution of the larger L. abeelei and L. auritus, not much is known about their biology. All Limnochromis have very short intestinal tracts, indicating a generalized predatory lifestyle. The larger L. staneri seems to be restricted to soft prey, i.e. arthropods and small fish, while the thick pharyngeal teeth of L. abeelei also enable it to feed on mollusks. (Coulter, 1991; Konings, 1998). Greenwoodochromis bellcrossi and christyi represent another morphologically distinct genus of the Limnochromini. On the basis of their resemblance to the paedophagous Lake Malawi cichlid genus Diplotaxodon, Coulter (1991) speculated that G. chyristyi might ram other mouthbrooding cichlids and feed on the eggs and larvae released by the mothers. Konings (1998) notes that G. christyi is mainly found in the deeper rocky habitat of in the southeast. 16

Nothing is known about the distribution of G. bellcrossi, which is distinguished from G. christyi by morphometric characteristics only. The remaining member of the genus Gnathochromis, G. permaxillaris, is distinguished by its strongly enlarged upper lip and peculiar protractile mouth apparatus. The praemaxillae are rapidly extended, enlarging the mouth volume and effectively creating a vacuum that enables the fish to suck up zooplankton (Fryer & Iles 1972, Coulter 1991). G. permaxillaris is considered an inhabitant of the muddy depths of Lake Tanganyika, however, it is also frequently caught in shallow water (Pearce, 1985). Baileychromis centropomoides and Reganochromis calliurus are both predatory deepwater species dwelling on muddy bottoms (Bailey and Stewart, 1977). Noting their similarities, Bailey & Stewart (1977) originally placed both species into the genus Leptochromis, which unbeknownst to them already harbored a group of marine fishes (Burgess, 1926). The new genus Reganochromis was proposed in response but centropomoides was ultimately removed and placed into a new monotypic genus Baileychromis in Poll’s cichlid systematic (1986). B. centropomoides and R. calliurus share an elongated form with large eyes but B. centropomoides is distinguished from R. calliurus by its extremely flattened, pike-like snout and a the strongly elongated first five spines of the dorsalis (Fryer & Iles, 1972). R. calliurus is believed to be a more generalized predator of arthropods and small fish, B. centropomoides’ esociform appearance could indicate a greater reliance on fish (Coulter, 1991). Triglachromis otostigma shows perhaps the most peculiar adaptations to life in the depths. It dwells over muddy ground and uses its pectoral fin rays, which are not connected to a membrane, to comb through the ground. When potential food particles are detected, they are swallowed with large amounts of mud, which is regularly found in the guts of caught Triglachromis. (Coulter, 1991; Konigns, 1998).

As noted before, the phylogenetic relationships of Lake Tanganyika cichlids are relatively well known, with most major lineages clearly established (Snoeks, 2000). However, the rapid radiation of lineages still poses problems to efforts of accurate phylogenetic reconstruction. In cases such as the previously described Bathybatini and Limnochromini, morphology and biochemistry-based methods produce differing results with no way of deciding which or even whether one of them represents the accurate phylogenetic reconstruction. Furthermore, most biochemical data relies on analysis of few mitochondrial genes

(Koblmüller et al., 2008). However, phylogenetic inference based solely on mitochondrial markers is notoriously problematic. Mitochondrial phylogenies often do not accurately represent the evolutionary relationships of taxa due to the mtDNA’s propensity to introgression, subjection to selection and divergent rates of mutation (Ballard & Whitlock, 2004). These factors often prevent accurate tree reconstructions and thus the proper mapping of evolutionary transitions, testing of evolutionary hypotheses and proper assessment of biodiversity for conservation efforts. The assessment of phylogenetic relationships based on a whole genome approach would circumvent the problems posed by the analysis of mtDNA. Whole genome sequencing is still not financially feasible for projects that involve a large number of specimens such as the reconstruction of the phylogeny of an entire lineage (Meudt and Clarke, 2007). However, in lieu of that, other methods exist.

1.6 AFLP and its use in phylogenetics

AFLP is a whole genome fingerprinting method developed by Vos et al. in 1995. It is based on the cleavage of DNA by restriction enzymes at specific palindromic sites within the genome. Two enzymes, a “rare” and a “frequent” cutter, are used to digest the entire genome of an organism. This results in a large but manageable number of differently sized DNA fragments of unknown sequence but with enzyme-specific sticky ends. Linker DNA can then be ligated to these fragments and used as template for PCR-amplification. In a preselective amplification step, primers with a sequence homologous to the linker+restriction site and a single selective nucleotide are used. A second PCR uses more selective primers to amplify a fraction of the originally created fragments (around 1/4096, Luo et al., 2007). The fragments can then be separated by length and visualized on an electrophoresis-gel, or, when using fluorescence-labeled primers, as electropherograms by capillary-electrophoresis (Fig. 6).

Fig. 6: Schematic of basic AFLP reaction. Genomic DNA is cut by two restriction enzymes, ligation of linker DNA and two selective PCR amplifications produce a sequence-specific set of DNA fragments. Schematic from Meudt & Clarke, 2007)

The obtained data, usually converted into binary presence/absence matrices, can be used in a variety of applications. Although initially designed for the generation of high-density linkage maps, marker assisted plant breeding or strain identification, its use in inferring phylogenetic relationships became quickly apparent (Janssen et al., 1996). Early reviews of the AFLP technique noted its high reliability and reproducibility as well as its low cost and the ability to quickly produce a large number of phylogenetically informative markers even from small quantities of unknown sequences (Blears et al., 1998; Mueller and Wolfenbarger, 1999). In many studies AFLP has been shown to outperform similar dominant marker systems such as random amplified polymorphic DNAs (RAPD) or inter simple sequence repeats (ISSR) (see Meudt and Clarke, 2007). Even in some cases where sequence information is available on both nuclear and organelle DNA, whole genome analysis of AFLP can help resolve difficult phylogenies (for example Despres et al., 2003; Mendelson and Simons, 2006, Mendelson and Wong, 2010).

However, the use of AFLP in inferring phylogenies is not without problems. The independence of AFLP markers (=characters), a prerequisite of phylogenetic inference, is not always assured (Bussell et al., 2005). Furthermore, scoring of AFLP fragments is not entirely

objective. Assignment of fragment-length classes (bins) for homologous peaks cannot be reliably performed by computer programs alone and human scoring is a consistent source of error. Fortunately, measures to prevent excessive errors exist and the use of repeat samples can give an estimate of the reliability of the data/scoring (Whitlock et al., 2008). Perhaps the biggest problem of the AFLP technique is the erroneous scoring of homoplasious bands as homologous. The AFLP reaction creates a very large number of asymmetrically distributed bands and nonhomologous bands often occupy the same position in a gel or electropherogram due to their size similarity (Vekeman et al., 2002). These can neither be discerned by automated nor manual scoring methods and introduce a considerable source of error in phylogenetic studies. Dependent on the evolutionary distance of the study organisms, estimations of homoplasy can range from 2.5% up to 100% (O’Hanlon and Peakall, 2000). Generally, the resolution of AFLP datasets decreases with phylogenetic distance (see Garcia-Pereira et al., 2010) and for this reason, it has been suggested to restrict the use of AFLP technique to the analysis of closely related taxa (Althoff et al., 2007). However, it has been shown that it is indeed possible to reconstruct phylogenies at the family level and above (Dasmahapatra et al., 2009). Related to homoplasy is the problem of “collision”. Collisions occur when bands of similar size within a single genotype comigrate and form a single peak. The probability of such a collision to occur in a typical plant genome is already > 50% when only 19 AFLP bands are observed (Gort et al., 2006).

Another looming problem is inaccurate modeling for the process of AFLP evolution. The most common methods of inferring phylogenies from AFLP data are conversion into a distance matrix and the use of clustering algorithms such as neighbor-joining (NJ) or UPGMA to construct trees. Heuristic parsimony analysis is also employed, usually yielding similar results (Koopman, 2005). However, these methods are at least in theory not ideally fitted for the analysis of AFLP bands. Since generation of AFLP data is dependent on the presence of two restriction sites, loss of bands is more common than gain, which in turn should be more common in closely than in more distantly related taxa. This asymmetry is not considered in parsimony analysis (Luo et al., 2007). When using NJ algorithms for constructing phylogenies from AFLP data, it is common to use distance measures based on restriction site evolution models (for example Nei and Li, 1979). However, evolution of AFLP bands does not only

depend on mutations at these restriction sites, but is also affected by changes in the sites between them. Substitutions as well as insertions and deletions can create new restriction sites within the AFLP bands or cause them to shift their position due to altered size. Furthermore, distance matrices in general suffer a loss of information when converted from character matrices (Luo et al., 2007). There exists a character-based Bayesian model for phylogenetic inference from binary data such as AFLPs (Mau and Newton, 1997). This algorithm is increasingly being used for Bayesian inference (BI) using MrBayes software (Huelsenbeck and Ronquist, 2001). However, this two state presence-absence model is an oversimplification of AFLP evolution (Brouat et al., 2004), a fact that is often not taken into account when using BI on AFLP data. Two actual models for Bayesian inference using AFLP data exist but even the more simple algorithm that only takes into account marker length and substitutions between two restriction sites is already so computationally demanding that general use is unfeasible (Luo et al., 2007). A more complex algorithm that also incorporates insertions and deletion while still retaining a simple Jukes-Cantor substitution model requires even more computational power (Luo and Larget, 2009).

Despite these theoretical concerns, AFLP is widely used in practice and a variety of studies have successfully employed both distance and character based methods for inferring phylogenies (for example Koopman et al., 2008; Dasmahapatra et al., 2009). While AFLP has always remained relatively underused in zoology compared to other biological disciplines (Bensch and Akesson, 2005), it has long since proven its worth in uncovering the phylogenies of rapidly radiation lineages such as the East African cichlids (Albertson et al., 1999). Thanks to the invention of simple, AFLP-tree based tests for hybridization the technique has gained another great utility in investigating species with putative hybrid origin (Seehausen, 2004). Other novel methods of hypothesis testing using AFLP are also expanding the role of AFLPs from simple phylogenetic tools to investigating for example the role of selection at certain AFLP loci (Meudt and Clarke 2007). Of course, AFLP and other fingerprinting techniques are expected to eventually become obsolete with the increasing prevalence of next generation sequencing technology (Meudt and Clarke, 2007; Rieseberg et al, 2012). However, at least at the time, it is not economically and technically feasible to sequence the genomes of large numbers of non-model organisms. AFLP still offers a nearly unmatched quick and

inexpensive alternative for genotyping many individuals without prior sequence information and will therefore remain in use for quite some time (Meudt and Clarke, 2007).

1.7 Goals of this thesis

1.) Reconstruction of the phylogenetic relationships of of Lake Tanganyika deepwater cichlid tribes Bathybatini and Limnochromini using AFLP. Previous phylogenetic reconstructions based on morphology or the analysis of few genetic loci have oftentimes proven to be contradictory. As a result, the evolutionary history and taxonomic standing of both groups remain unclear. The use of nuclear multi-locus AFLP analysis could help clearing up these ambiguities and possibly provide better resolved phylogenetic trees than previous studies.

2.) Tests for signals of ancient hybridization in deepwater lineages. The allopatric diversification of cichlids in the fragmented littoral habitats is often not accompanied by reproductive isolation and thus, molecular genetic investigations have often found cases of introgression and hybridization. As opposed to the shoreline, deepwater habitats are characterized by weak geographic structure and reduced niche diversity. This would result in a low species number and necessitate the erection of intrinsic barriers for gene flows early in evolution. We expect that therefore, deepwater species should show largely congruent mt- and ncDNA trees as well as test negative for homoplasy excess as outlined by Seehausen (2004). We test this hypothesis in the two deepwater tribes Bathybatini and Limnochromini.

2. Material and Methods

2.1 Sample collection

Bathybatini

The Bathybatini dataset is based on a total of 38 specimens, representing all seven Bathybates species, Hemibates stenosoma as well as Trematocara unimaculata and T. macrostoma (Tab. 1). All specimens were obtained between 1999 and 2011 from local fishermen at Lake Tanganyika and identified to species by SK. Unfortunately, it was not possible to obtain a comprehensive taxon sampling for the genus Trematocara as, because of their small size and hence low market value, these fish (except for the largest species T. unimaculatum) are not caught by local fishermen.

Tab. 1: Bathybatini samples Extraktion Tube Species Location 12879 KS1/C3 Bathybates fasciatus Mpulungu Market 12885 KS1/C9 Bathybates fasciatus Mpulungu Market 12889 KS1/D7 Bathybates fasciatus Toby's Place 12890 KS1/D8 Bathybates fasciatus Toby's Place 12893 KS1/E1 Bathybates fasciatus Mpulungu Market 12917 T04 14/G10 Bathybates fasciatus Tanganyika Lodge 12913 T99 4/D8 Bathybates ferox Lufubu estuary 12877 KS1/C1 Bathybates graueri Mpulungu Market 12878 KS1/C2 Bathybates graueri Mpulungu Market 12883 KS1/C7 Bathybates graueri Mpulungu Market 12897 KS1/E5 Bathybates graueri Mpulungu Market 12901 KS1/E9 Bathybates graueri Mpulungu Market 12902 KS1/E10 Bathybates graueri Mpulungu Market 12911 T05 1/J9 Bathybates graueri Mpulungu Market 12912 T05 6/A1 Bathybates graueri Mpulungu Market 12919 T01 7/C3 Bathybates graueri North of Sumbu 13101 T07 25/E9 Bathybates horni Mpulungu Market 12907 T01 3/E7 Bathybates leo Mpulungu Market 12921 T03 9/I1 Bathybates leo Mpulungu Market 12923 T03 9/I3 Bathybates leo Mpulungu Market 12925 T03 9/I5 Bathybates leo Mpulungu Market 23

13100 T07 16/G2 Bathybates leo Mpulungu Market 12909 T99 4/E2 Bathybates minor Lufubu estuary 12910 T06 1/G5 Bathybates minor Kalambo 12933 T99 4/B2 Bathybates minor Sumbu 12882 KS1/C6 Bathybates vittatus Mpulungu Market 12924 T03 9/I4 Bathybates vittatus Mpulungu Market 12926 T03 9/I6 Bathybates vittatus Mpulungu Market 12929 T06 3/D7 Hemibates stenosoma Mpulungu Market 12930 T06 3/D8 Hemibates stenosoma Mpulungu Market 12931 T06 3/E5 Hemibates stenosoma Mpulungu Market 12932 T06 10/A8 Hemibates stenosoma Mpulungu Market 12880 KS1/C4 Trematocara unimaculata Mpulungu Market 12881 KS1/C5 Trematocara unimaculata Mpulungu Market Telotrematocara 12935 T06 3/E11 macrostoma Mpulungu Market Telotrematocara 12936 T06 3/E12 macrostoma Mpulungu Market Telotrematocara 12937 T03 4/J10 macrostoma Mpulungu Market

Coordinates of sampling sites (if known): Kalambo, S 8°37’ E 31°12’; Kalambo Lodge, S 8°37’ E 590 31°37’; Lufubu estuary, S 8°32’ E 30°44’; Sumbu, S 8°31’ E 30°29’; Tanganyika Lodge, S 8°47’ E 591 31°05’

Limnochromini

This Limnochromini dataset is based on 52 individuals of the Lake Tanganyika C-lineage (Clabaut et al. 2005) with the exception of the riverine Orthichromini (see Tab. 2). Fish were either caught on site using gill nets, acquired from aquarium trade or, in the case of deepwater-dwellers such as the Limnochromini, bought from local fishermen. For this reason, Tangachromis dhansi, a member of the Limnochromini, could not be included in the sampling as it is seldom caught due to its small size and low economic value.

Tab. 2: Limnochromini samples

Extraction Tube Species Location 13429 KS4/H4 Greenwoodochromis bellcrossi Aquarium trade 13430 KS4/H5 Greenwoodochromis bellcrossi Aquarium trade 13367 KS2/D7 Greenwoodochromis christyi Mpulungu fishmarket 13390 KS2/D8 Greenwoodochromis christyi Mpulungu fishmarket 13399 T03 3/B8 Greenwoodochromis christyi Mpulungu fishmarket 12951 T03 10/D1 Limnochromis staneri Mpulungu fishmarket

13366 KS2/D6 Limnochromis staneri Mpulungu fishmarket 13365 KS2/D5 Limnochromis staneri Mpulungu fishmarket 13483 KS2/D10 Limnochromis abeelei Mpulungu fishmarket 13369 KS2/D9 Limnochromis abeelei Mpulungu fishmarket 12943 T03 10/D9 Limnochromis abeelei Mpulungu fishmarket 13467 T01 7/H9 Gnathochromis permaxillaris Aquarium trade 13464 KS2/D4 Gnathochromis permaxillaris Mpulungu fishmarket 13473 T04 12/F9 Limnochromis auritus Chituta Bay 12953 T01 5/F9 Limnochromis auritus Mbita (West) 13373 KS2/C9 Limnochromis auritus Mpulungu fishmarket 12961 T06 3/A10 Baileychromis centropomoides Mpulungu fishmarket 13417 T06 3/E6 Baileychromis centropomoides Mpulungu fishmarket 13418 T06 3/E7 Baileychromis centropomoides Mpulungu fishmarket 13419 T06 3/E8 Baileychromis centropomoides Mpulungu fishmarket 13420 T06 10/A2 Baileychromis centropomoides Mpulungu fishmarket 12962 T06 3/E20 Baileychromis centropomoides Mpulungu fishmarket 13485 T10 10/A7 Reganochromis calliurus Aquarium trade 13484 T10 10/A6 Reganochromis calliurus Aquarium trade 13362 T10 10/A5 Reganochromis calliurus Aquarium trade 13359 T09 20/H6 Triglachromis otostigma Aquarium trade 13371 KS2/C7 Triglachromis otostigma Mpulungu fishmarket 13360 T09 20/H7 Triglachromis otostigma Aquarium trade 13372 KS2/C8 Triglachromis otostigma Mpulungu fishmarket 13434 KS4/H6 Triglachromis otostigma Aquarium trade 13435 KS4/H7 Triglachromis otostigma Aquarium trade 13052 KS1/G6 Xenotilapia ochrogenys Aquarium trade 13051 KS1/G5 Xenotilapia ochrogenys Aquarium trade 12981 T04 12/H3 Cyprichromis leptosoma Chituta Bay 12988 T02 8/F1 Cyprichromis microlepidotus Aquarium trade 13091 KS2/A5 Paracyprichromis nigripinnis Aquarium trade 13358 T99 4/H10 Benthochromis tricoti Kalambo Lodge 13357 T01 7/C4 Benthochromis melanoides Sumbu (North) 13356 T06 33/B3 Perissodus microlepis Kalambo Lodge T0631F5 T06 31/F5 Perissodus microlepis Mbita (East) 13438 T99 4/D5 Haplotaxodon microlepis Lufubu estuary 13437 KS4/G9 Ctenochromis benthicola Aquarium trade 13436 KS4/G8 Ctenochromis benthicola Aquarium trade 13439 T99 4/D4 Cyphotilapia frontosa Lufubu estuary 13440 T99 4/D3 Cyphotilapia frontosa Lufubu estuary KS4F10 KS4/F10 Tropheus moorii Murago KS4C1 KS4/C1 Tropheus moorii Murago 13361 T10 10/A4 Tropheus duboisi Aquarium trade Coordinates of sampling sites (if known): Kalambo Lodge, S 8°37’ E 590 31°37’; Lufubu estuary, S 8°32’ E 30°44’; Chituta bay S 8°43’71’’ E 31°09’35‘’, Mbita (West) S 8°45’52’’ E 31°05’84’’, Mbita (East) 8°45,20' S, 31°05,19' E Sumbu (North) S 8°31’ E 30°29’ 25

2.2 DNA isolation

Fin clips or white muscle tissue were persevered in ethanol and DNA isolated using a proteinase K digestion/high salt precipitation method described by Miller et al. (1988). DNA concentrations were measured using an IMPLEN NanoPhotometer. All samples were diluted in TE-buffer to a concentration of 6ng/µl.

2.3 AFLP analysis

AFLP analysis was performed as described by Vos et al. (1995), however, EcoRI and MseI were used as restriction enzymes as described by Egger et al. (2007). For restriction digestion of genomic DNA, 60ng DNA were incubated with 0.5µl MseI (10U/µl, New England Biolabs), 0.25µl EcoRI (20U/µl, New England Biolabs), 0.5µl 100xBSA and 5µl New England Biolabs enzyme buffer filled to a total volume of 50µl with HPLC water. Samples were first incubated at 37°C for 3 hours and then at 70°C for 15 minutes. Adapters for ligation to restriction product were 5’- CTC GTA GAC TGC GTA CC-3’ and 5’-AAT TGG TAC GCA GTC TAC-3’ for EcoRI adaptor (5pmol/µl) and 5’-GAC GAT GAG TCC TGA G-3’ and 5’TAC TCA GGA CTC AT-3’ for MseI adaptor (50pmol/µl). The adaptors were incubated for 5min at 95°C and then cooled for 10min at room temperature.

Adapter ligation was performed with the entire 50µl restriction digestion, 1µl EcoRI-adapter (5pmol/µl), 1µl MseI-adapter (50pmol/µl), 1µl T4 Ligase Buffer and 0,2µl T4 DNA ligase filled to a final volume of 60µl with HPLC water. The ligation mix was incubated over night at 22°C and subsequently diluted to a total volume of 180µl with HPLC water.

The PCR mix for preselective amplification was as follows: 3µl diluted restriction ligation product, 2µl 10xdNTP mix (10µM), 2µl 10x Buffer (MgCl2), 0,1µl Taq Polymerse (5U/µl, BioThermTM) and 0,4µl EcoRI-preA (5’-GAC TGC GTA CCA ATT CA- 3’; 10µM) and 0,4µl MseI- preC (5’-GAT GAG TCC TGA GTA AC- 3’; 10µM) filled with HPLC water to a volume of 20µl. PCR conditions were as follows: 1 cycle 72°C 2min followed by 20 PCR cycles 94°C 20sec melting, 56°C 30sec annealing and 72° 2min amplification and a 30min holding step at 60°C.

After confirmation of successful amplification using agarose gel electrophoresis, the amplificates were diluted 1:10 in HPLC water.

10 (Bathybatini) or 8 (Limnochromini) selective amplifications were performed using the following primer combinations: EcoRI-ACA/MseI-CAA, EcoRI-ACA/MseI-CAG, EcoRI- ACA/MseI-CAC, EcoRI-ACA/MseI-CAT, EcoRI-ACT/MseI-CAT, EcoRI-ACT/MseI-CAA, EcoRI- ACT/MseI-CAG, EcoRI-ACT/MseI-CAC, EcoRI-ACC/MseI-CAA and EcoRI-ACC/MseI-CAC. Primers were identical to adapters with two additional selective nucleotides at the 3’ end. The PCR mix for selective amplification was as follows: 1µl of preselective PCR product,

0,25µl of each primer (10µM), 0,8µl 10x dNTP mix (10µM), 1µl 10x Buffer (MgCl2), 0,1µl Taq Polymerse (5U/µl, BioThermTM) filled to a total volume of 10µl with HPLC water. The selective PCR protocol was as follows: 1 cycle of 94°C 2min; 10 cycles of 94°C 20sec melting, 65°C 30sec annealing (-1°C each cycle) and 72°C 2min amplification; 25 cycles of 94°C 20sec melting, 56°C 30sec annealing, 72°C 2min amplification and one final cycle of 72°C for 30sec. Selective amplification products were visualized using an ABI 3130 x 1 automated sequencer (Applied Biosystems) using Genescan-500 ROX size standard (Applied Biosystems).

2.4 AFLP scoring

Polymorphic positions were initially determined using GeneMapper 3.7 software in a range from 50-500bp for the Bathybatini and 50-300bp for the Limnochromini. Bins were first set automatically by the software and then manually checked. Individual samples that failed to produce peaks of sufficient quality were removed for the specific primer combination but retained for the complete dataset. Bins containing obvious PCR artifacts (detectable through negative controls containing no DNA or though presence/absence in repeats) were deleted. Bins containing obvious non-homologous peaks were also removed. When apparent, peaks that clearly belonged to certain bins but were not included due to sequencer-induced misalignment were manually added to the dataset. Final peak scoring and conversion of the AFLP data into a binary matrix was performed using AFLPScore 1.4 (Whitlock et al. 2008). For each dataset, 20 replicas were used to calculate the mismatch error rate for all unique loci.

2.5 Phylogenetic inference

For both Bathibatini and Limnochromini, neighbour joining (NJ) tree based on Nei and Li’s distances (Nei and Li 1979) were constructed in PAUP 4.0b5; Swofford, 2000). Bootstrap values from 1000 pseudo-replicates were used as standard measure of confidence in the reconstructed tree topologies. Although accurate models for Bayesian tree construction using AFLP datasets do exist (Luo et al. 2007), the high demands for processing power make them unfeasible to use (Luo et al. 2007; Koopman et al. 2008). Thus, Bayesian phylogenetic inference (BI) was conducted in MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001), employing the restriction site model with the ‘noabsencesites’ coding bias correction (Ronquist et al. 2005; Koopman et al. 2008). The Dirichlet prior for the state frequencies was set to (2.44, 1.00) for Bathibatini and (1.98, 1.00) for Limnochromini, matching the actual 0/1 frequencies in the dataset. Posterior probablilities were obtained from Metropolis-coupled Markov chain Monte Carlo simulations (2 independent runs; 10 chains with 8,000,000 generations each; chain temperature: 0.2; sample frequency: 1,000; burn-in: 4,000,000 generations). Chain stationarity and run parameter convergence were checked in Tracer 1.5 (Rambaut and Drummond, 2009). To test for homoplasy excess introduced by hybridization we conducted a tree-based method as outlined by Seehausen (2004) by removing single species from the dataset and observing the change in bootstrap values in the NJ tree (also see Egger et al. 2007, Koblmüller et al. 2010). In theory, the inclusion of a hybrid taxon in a multi-locus phylogeny introduces homoplasy with clades that contain its parental taxa. Hybrid taxa should be intermediate to the parental taxa since they carry a mosaic of parental characteristics. Thus, by decreasing the amount of homoplasy in the dataset, removing the hybrid taxon should increase the bootstrap support for the clades that include the parental taxa or their descendents, whereas removing non-hybrid taxa should have no effect on the statistical support of other nodes.

Testing for consistency between mtDNA- and AFLP-based tree topologies employed two different strategies. In a first test we evaluated whether our AFLP-NJ-topology can be

explained by the mtDNA data of Koblmüller et al. (2005, Bathybatini) and Duftner et al. (2005, Limnochromini). We inferred the log likelihood of the AFLP-NJ-topology based on the mtDNA datasets of Duftner et al. (2005) and Koblmüller et al. (2005) by constraining maximum likelihood (ML) tree search to a topology identical to the species tree suggested by the NJ analyses of our AFLP data, applying the substitution model used in this previous study (HKY+I+G) . To test for significant differences between the unconstrained (Koblmüller et al. 2005) and constrained mtDNA topology of the Bathybatini, we performed an ML-based Shimodaira-Hasegawa (SH) test (Shimodaira and Hasegawa 1999; full optimization, 1,000 bootstrap replicates) in PAUP. In a second test we evaluated by means of a Bayes factors approach (Kass and Raftery 1995) whether the mtDNA trees of the Limnochromini and Bathybatini (Duftner et al, 2005; Koblmüller et al. 2005) can be explained by our AFLP data and whether the AFLP-NJ and BI trees significantly differ from each other. We performed BI searches constraining the topology to that of the mtDNA-topologies (Duftner et al., 2005; Koblmüller et al. 2005) and the inter-specific relationships implied by the AFLP-NJ-tree in MrBayes 3.1.2 employing the same settings as above. Bayes factor comparison - using the harmonic means of the likelihood throughout different runs (Newton and Raftery 1994; Suchard et al. 2001) - among the three alternative phylogenetic hypotheses was performed in Tracer 1.5. Values of 2 x ln BF (two times the difference between the harmonic means of the two models) > 10 are considered strong evidence to support one model over another (Kass and Raftery, 1995). 3. Results

3.1 Bathybatini

The final AFLP dataset consisted of 659 unique loci with a mismatch error rate of roughly 3%, which falls within the acceptable limit for mismatch error rates as defined by Whitlock et al. (2008). Both, the NJ and BI analysis yielded largely congruent and well supported topologies with only minor differences between them (Fig. 7A, B).

Fig. 7: Phylogenetic relationships of the Bathybatini based on 659 polymorphic AFLP loci. (A) NJ tree (employing Nei and Li distances [65]) and (B) BI tree are shown. Only bootstrap values >50 and posterior probabilities >0.5 ares shown. Branches are labelled with the samples’ lab ID numbers. (C) The marginal density of posterior distribution of likelihood (LnL) for each of the two MrBayes runs of the unconstrained AFLP data (violet, grey), the AFLP-NJ- topology-constraint (blue, green) and the mtDNA-topology-constraint (orange, red). Fish were drawn after photographs in [60] to demonstrate the interspecific differences in male nuptial patterns.

Whereas all species resulted as monophyletic in the NJ tree, Bathybates graueri was not resolved as a monophylum in the BI tree, but as paraphylum including the well supported clade of the other large Bathybates species (Fig. 7B). Despite this minor topological difference, Bayes factor comparison strongly supports the BI tree over the NJ tree (2 x ln BF = 26.828; Fig. 7C). We note, however, that the two-state model implemented in MrBayes does not fully cover the complex genetic process of AFLP evolution and thus provides accurate phylogenetic inference less likely than distance methods [Suchard et al., 2001; Althoff et al., 2007; Luo et al. 2007). Hence, the observed differences between AFLP and NJ tree topologies might be attributed to this problem. Both, the NJ and BI analyses support the monophyly of all three genera with the genus Trematocara representing the most ancestral branch (Fig. 7A,B). Within the genus Bathybates, the small and morphologically most distinct member of the genus, B.minor, resulted as sister taxon to the remaining large Bathybates species. Branch length among the large Bathybates species are rather short and some received rather low statistical support, indicating a period of rapid cladogenesis. Nevertheless, both NJ and BI analyses revealed a largely consistent phylogenetic pattern within the large Bathybates species. Both, SH-test and Bayes factor comparison revealed significant differences between mtDNA and AFLP phylogenies (SH-test: -lnL of -9505.385 versus -9585.142 for mtDNA versus AFLP-NJ-topology, p < 0.001; Bayes factors: 2 x ln BF of 88.576 between mtDNA and AFLP-BI-topology and 61.748 between mtDNA and AFLP-NJ- topology, Fig. 2C). The homoplasy excess test provided no evidence for introgressive hybridization (Tab. 2). The observed increase in bootstrap support of the Bathybates leo- vittatus node upon removal of B. fasciatus is an expected result from the loss of a sister group which shares similarities with both B.leo and B. vittatus. The same can be observed with the removal of B. horni or B. ferox.

Tab. 3: Homoplasy excess test of Bathybatini Node Removed 1 2 3 4 5 6 Original 64 99 37 59 100 100 B. leo - 96 39 62 100 100 B. vittatus - 99 45 54 100 100 B. fasciatus 100 - 37 55 100 100 B. ferox 63 100 - 61 100 100 B. horni 66 99 76 - 100 100 B. graueri 59 96 44 100 100 100 B. minor 63 96 41 38 100 100 Nodes: 1 = B. vittatus/B. leo ; 2 = B. fasciatus / B. vittatus-leo; 3 = B. ferox /B. fasciatus- vittatus-leo; 4 = B. horni/B. ferox-fasciatus-vittatus-leo; 5 = B. graueri/B. horni-ferox-fasciatus-vittatus- leo; 5 = B. minor/B. graueri-horni-ferox-fasciatus-vittatus-leo

3.2 Limnochromini

AFLP analysis of 53 individuals in 21 species resulted in a dataset of 901 unique loci with a mismatch error rate of < 2% as determined by AFLPScore 1.4. Bayes factor comparison confirmed that AFLP phylogenies using the NJ as well as the BI algorithm differ significantly (2x ln BF = 19.07 for NJ and 33.456 for BI) from an earlier mtDNA topology by Duftner et al (2005). Bayes factor comparison also favors the BI over the NJ topology (2x ln BF = 14.386). However, it is important to remember that the restriction-site specific algorithm employed in MrBayes is an oversimplification of the process of AFLP evolution (see Luo et al., 2007; Koopman et al, 2008; Luo and Larget, 2009) and therefore might in fact not represent the most likely tree topology. Both employed algorithms clearly demonstrated the monophyly of the Limnochromini, the included genera with the exception of Limnochromis as well as the monophyly of all single species described by Koblmüller et al. (2008) (Fig. 8)

Fig. 8: Phylogenetic relationships of the Limnochromini and other C-lineage tribes based on 901 polymorphic AFLP loci. a) NJ tree employing Nei-Li distances (Nei and Li, 1979) b) BI tree. Only bootstrap support values and posterior probabilities above 50 and 0.5 respectively are shown. Branches are labeled according to the lab’s sample ID numbers. Limnochromini schematics were drawn based on live photographs and correspond with order of species in the NJ topology.

Both NJ and BI topologies show rather low statistical support values in ancestral nodes of the Limnochromini, indicating a period of rapid adaptive radiation. However, the tree topologies differ in some aspects: Bootstrap support for the NJ topology is low in all ancestral nodes of the Limnochromini, indicating early radiation of the entire tribe. The topology of the BI tree indicates ancient divergence of Baileychromis, Reganochromis and Triglachromis with later development of the rest of the tribe. Furthermore, the BI tree unites Limnochromis staneri and Limnochromis abeelei in a single clade positioned as a sister taxon to the Greenwoodichromis genus. The NJ tree indicates subsequential splits of Limnochromis abeelei and staneri. The homoplasy excess test did not find evidence for ancient hybridization events (Tab. 4). None of the changes in bootstrap values correspond to patterns expected with the removal of a hybrid species.

Tab. 4: Homoplasy excess test in the Limnochromini Node 1 2 3 4 5 6 7 8 Original 97 89 93 52 58 54 100 97 G. christyi - 95 96 42 50 56 100 97 G. bellcrossi - 65 90 51 55 56 100 96 L. staneri 100 - 91 53 56 57 100 96 L. abeelei 92 100 - 49 59 48 100 98 G. permaxillaris 96 85 94 - 42 45 100 96 L. auritus 96 91 91 85 - 70 - 96 B. 98 98 92 47 55 43 - 96 centropomoides R. calliurus 96 88 95 52 45 60 100 97 T. otostigma 96 92 91 50 65 52 100 - Nodes: 1 = G. christyi/G. bellcrossi; 2 = 1/L. staneri; 3 = 2/L. abeelei; 4 = 3/G. permaxillaris; 5 = 4/L. auritus; 6 = 5 /B. centropomoides-R.calliurus; 7 = B. centropomoides/R. calliurus, 8 = Limnochromini/Triglachromis otostigma

4. Discussion

4.1 Bathybatini

In the original classification of Lake Tanganyika cichlid tribes by Poll (1986), Bathybates and Hemibates were included in a tribe Bathybatini as sister group to the Trematocarini (equivalent to the genus Trematocara), a hypothesis supported both by lepidological (Lippitsch, 1998) as well as allozyme data (Nishida, 1997). In contrast, based on morphological characteristics, Stiassny (1991) and Takahashi (2003) proposed a sister group relationship of Bathybates and Trematocara. Currently, all three genera, Bathybates, Hemibates and Trematocara, are united in the tribe Bathybatini (Takahashi, 2003). A previous mtDNA phylogenetic study remained equivocal with regard to the two competing morphological classifications and suggested that Bathybates, Hemibates and Trematocara diverged rapidly from their common ancestor (Koblmüller et al., 2005). In the present AFLP phylogeny, the length differences between the most ancestral branches favour Poll´s original classification (Poll, 2003) with Trematocara as sister group to Hemibates + Bathybates. Consistent with the mitochondrial phylogeny (Koblmüller et al., 2005) the AFLP data confirm the split between Bathybates minor and the larger members of the genus Bathybates with B. graueri as their most basal representative and identifies a period of rapid cladogenesis at the onset of the diversification of the large Bathybates species. However, mtDNA and AFLP phylogenies differ significantly with respect to the branching pattern among the remaining large Bathybates species. Introgressive hybridization and ancient incomplete lineage sorting are two alternative sources of topological disagreement between nuclear and mitochondrial trees (Shaw, 2002; Machado and Hey, 2003; Holland et al., 2008; Nevado et al., 2009; Koblmüller et al., 2010), resulting in similar phylogenetic patterns difficult to resolve by strict hypothesis testing (Wilyard et al., 2009). Circumstantial inference can be based on the fact that lineage sorting is expected to lag behind rapid cladogenetic events, such that the rapid radiation of the large Bathybates species predisposes this clade to mitonuclear phylogenetic incompatibilities without implying post-cladogenetic introgression (Takahashi et al., 2001; Wilyard et al., 2009). Likewise, monophyly of species in both mitochondrial and nuclear trees 35

(excepting the paraphyly of B. graueri in Bayesian AFLP tree) and negative tests for homoplasy excess in the AFLP data do not provoke a conjecture of introgressive hybridization between the Bathybatini species. These findings support our hypothesis that deepwater species are less prone to introgressive hybridization than littoral species, but reservations arise on the one hand from the possibility that introgression occurred but was not recognized given our samples, data and the power of our analyses, and on the other hand from the small number of species in the phylogeny. Principally, rates of interspecific introgression may not differ between littoral and deepwater cichlids, but will nonetheless lead to higher incidences of introgression in the species-rich groups than in a less speciose clade. If this was the case, the lack of a signal of introgression in Bathybates and Hemibates would be fully explained by the low diversification rate of the lineage and hence limited opportunity for interspecific hybridization, without implying the evolution of complete reproductive isolation early on during diversification.

The branching order of Hemibates and the basal Bathybates species, which is reconstructed congruently by mtDNA and AFLP markers, suggests repeated transitions between benthic and bathypelagic feeding mode (ecological data from Coulter, 1991). The basal H. stenosoma represents a benthic generalist feeding on shrimps and various species of fish. The next split led to the specialized pelagic clupeid hunter B. minor, which mimics its prey in size and coloration and stages surprise attacks from within the sardine shoals. Then, B. graueri took a step back to the benthic habitat, specialized on cichlid prey and evolved a large body size. The chronology of the following radiation of the large bathypelagic clupeid hunters and benthic cichlid hunters remains unresolved, but involved at least one transition from benthic to bathypelagic habitat preferences. Depth preferences may vary between these species (Coulter, 1991), such that speciation may have involved both niche and spatial segregation. The apparent ecological differentiation among lineages may have reduced the fitness of hybrids [Grant and Grant, 1992; Hatfield and Schluter, 1999; McCracken and Sorenson, 2005) and may have promoted the evolution of a mate recognition system, perhaps based on the species-specific melanic patterns of male Hemibates and Bathybates. The efficacy of monochromatic black, silvery and white body and fin patterns in mediating assortative mating in the dark, short-wavelength dominated environment has recently been

demonstrated for deepwater cichlid species of Lake Malawi. These sympatric and morphologically similar species differ primarily in male nuptial patterns and their reproductive isolation is corroborated by genetic differentiation estimates (Genner et al, 2007b). However, there is increasing evidence that color pattern is not the only cue for mate recognition in cichlids fish and it is possible and likely that auditory (Verzijden et al., 2010) and olfactory cues (Plenderleith et al., 2005) play a role in mediating assortative mating in deepwater species, too.

4.2 Limnochromini

This AFLP-marker based study of the benthic deepwater tribe Limnochromini, along with Duftner et al.’s mtDNA based work, suggests a monophyletic association of at 9 (perhaps 10 since no samples were available for Tangachromis dhansi) species. Furthermore, we once again highlight the problematic nature of the name-giving genus Limnochromis. The genus has once been considered a “catch-all assemblage” (Coulter, 1991), formerly containing 11 species of which all but three were later assigned to different genera (Poll, 1986; Takahashi, 2003). Even the monophyly of the remaining species L. abeelei, auritus and staneri is very questionable, as Duftner et al.’s mitochondrial and our nuclear genomic studies suggest. While L. abeelei and staneri appear closely related, only our BI algorithm resolves them as a single clade. L. auritus, which unlike its two deepwater congeners, also lives in more shallow parts of the lake, clusters with neither. Greenwoodochromis bellcrossi and G. christyi however clearly form a monophylum within the Limnochromini, contradicting Takahashi’s Greenwoodichromini tribe (Takahashi, 2003), which was already questioned by Duftner et al. (2005). Incidentally, the clustering of our outgroup species Ctenochromis benthicola with Cyphotilapia frontosa also resolves Takahashi’s uncertain systematic placement of that species. When it comes to the evolution of taxa within the Limnochromini, our genomic study paints a different picture than Duftner et al.’s mitochrondrial data. Their study detected three sublineages within the tribe, one containing Triglachromis otostigma, a second Baileychromis, Reganochromis, Gnathochromis and Limnochromis auritus and a third Greenwoodochromis as well as Limnochromis abeelei and staneri (see Fig. 1). Both of our topologies support the Triglachromis sublineage, however, they also clearly separate Baileychromis and Reganochromis from the rest of the tribe. Short branch-lengths and low 37

bootstrap support in our neighbour-joining topology are also similar to Duftner et al.’s, who calculated a time of rapid genesis of all sublineages around 2.9-3.5 million years ago. Our Bayesian tree shows the split of the Baileychromis/Reganochromis and the Triglachromis sublineages occurring before a radiation event that resulted in the diversification of the rest of the tribe. Such cytonuclear discordance (contradictory nuclear and mitochrondrial topologies) is a common occurrence in cichlid phylogenies. Both ancient incomplete lineage sorting as well as ancient hybridization and introgression are possible explanations for the observed differences (for example Koblmüller et al., 2010; Nevado et al., 2011). The clear monophyly of all Limnochromini species in both nuclear and mitochrondrial trees as well as the negative result of the homoplasy excess test provide evidence against the occurrence of hybridization as the cause of the disagreement between the trees. Recent studies of other deepwater taxa such as the tribe Bathybatini (see above) and the genus Xenotilapia were similarly unable to find signs of hybridization (Kidd et al., 2012). Similar to the Bathybatini, ancient incomplete lineage sorting resulting from rapid speciation events remains as a possible explanation for the observed differences in mitochondrial and nuclear tree topologies. Together with the apparent lack of hybridization, the phylogenies obtained in our study allow us to speculate on how the Limnochromini might have evolved. The deepwater zone generally does not offer the same amount of geographical separation as the littoral zone and consequently, habitat overlap is frequent. In the case of the Limnochromini, the species live largely sympatric. Speciation might therefore have involved early ecological segregation with resulting reduced fitness for hybrids and stronger selective pressure on the development of reproductive barriers. Disregarding the differences in branching order, the ancestors of the modern Limnochromini indeed seem to have split in three ecologically distinct sublineages. One split, resolved as the first in the BI tree, resulted in the development of fish with elongated bodies and large eyes, Baileychromis and Reganochromis. Ecological data on these species is sparse due to their deepwater lifestyle, but it seems like both dwell on muddy bottoms as benthic predators, hunting shrimp and small fish (Coulter, 1991). Especially Baileychromis centropomoides’ flattened, almost pike- like snout gives it the appearance of a predominantly piscivorous hunter. It remains unclear whether this split occurred before the radiation of the rest of the tribe (as the BI topology suggests) or at the same time. A second very distinct sublineage, resolved both in 38

mitochondrial and nuclear phylogenies, is formed by Triglachromis otostigma. This fish is the only known sediment feeder among the Lake Tanganyika cichlids. Its habitat extends from the shores far into the depths of the lake. It uses the pectoral fin ray tips, which lie free from the fin membrane, as sensory organs to screen the muddy ground, which is swallowed in large quantities when possible food is detected (Coulter, 1991; Konings, 1998). The third sublineage consists of several species of rather similar appearance. Gnathochromis permaxillaris is the most morphologically distinct. It lives both in shallow and deep parts of the lake, its strongly enlarged upper lip and protractile mouth apparatus allow it to suck in zooplankton (Konings, 1998). Limnochromis auritus, which does not cluster with the other Limnochromis species, is also found both in shallow and deep waters but feeds on small arthropods and snails. The remaining four Limnochromini, Greenwoodichromis christyi and bellcrossi as well as Limnochromis staneri and abeelei are all true benthic deepwater fish. Both Limnochromis species are generalized predators of muddy flats, feeding on small fish and arthropods, the thick pharyngeal teeth of L. abeelei enable it to also crack the shells of mollusks. The two Greenwoodochromis species have conquered a slightly different deepwater habitat, the transition between sandy and rocky bottoms. Based on its resemblance to the paedophagous Lake Malawi cichlid genus Diplotaxodon, Coulter (1991), suggested a similar lifestyle for G. christyi. Together with L. staneri and L. abeelei, the genus Greenwoodochromis might represent a second transition into the benthic deepwater habitat.

In conclusion, this study provides a comprehensive, multi-locus phylogeny of the tribe Limnochromini. As a result of adaptive radiation, three sublineages including elongated predators, sediment-eaters and generalized invertebrate feeders seem to have evolved. No signals of ancient hybridization could be found, indicating early establishment of reproductive barriers and ecological segregation during radiation. In accordance with other studies, this apparently emerges as a common pattern among deepwater species in Lake Tanganyika and possibly elsewhere.

4.3 Conclusions

These two studies once again confirm the applicability of AFLP as an affordable and informative method for inferring phylogenies. The AFLP based topologies of both the Bathybatini and Limnochromini differed notably from earlier results based on mtDNA data and Bayes factor comparisons support the AFLP trees versus the alternative mtDNA topologies. Nonetheless, it was not possible to unambiguously resolve the radiations of the two tribes. However, the clear resolution of early divergences and strong support values for the monophyly of all species indicate that this might not be result of a weakness in the methodology. Rather, the observed polytomies might represent real evolutionary events. Polytomies and/or low statistical support resulting in “starburst” like topologies without strong internal structures are expected in rapidly radiating lineages and have already been noted in the first AFLP-based study of cichlids (Albertson et al., 1999). It is unlikely that a higher number of loci would have resulted in a significantly better tree resolution. In the case of the Bathybatini, a tree based on only half of the primer combinations produced the same tree topology as the final tree and bootstrap support values did not increase considerably with the inclusion of more loci.

The results of both studies support the hypothesis of differing evolutionary patterns in deepwater and littoral cichlids of Lake Tanganyika. Neither the Bathybatini and Limnochromini studied in this work nor a study of substrate breeding Xenotilapia (Ectodini), which included several deepwater species (Kidd et al., 2012), detected signals of ancient hybridization. On the other hand, the well studied cichlids of the shores of Lake Tanganyika show the opposite pattern, as hybridization is frequently inferred (see Koblmüller et al., 2011). Clearly, differences in deepwater and shore habitats are influencing the way these fish are evolving and as a consequence the number of species in these habitats. An important factor might be presence or absence of barriers for gene flow. In shore habitats, even small geographic barriers such as short stretches of sand can prevent gene flow for philopatric rock-dwelling cichlids (see Sefc et al., 2007; Koblmüller et al., 2010). This leads to the development of many geographic races (often differentiated by color only), and ultimately to allopatric speciation. As evidenced by high hybridization rates among shore-dwelling cichlids, such allopatric speciation is often not accompanied by the evolution of reproductive barriers. The 40

breakdown of geographic barriers coupled with hybridization and transgressive segregation could then provide a trigger for adaptive radiations from hybridogenic ancestral stock. (Salzburger et al., 2002; Seehausen, 2004). On the other hand, geographic barriers are scarce in the deepwater regions of Lake Tanganyika, in particular in the pelagic habitats. Populations might be rarely separated for a long enough time to develop notable genetic differences that could lead to transgressive segregation when reunited. The two studies presented here indicate that deepwater species probably arise primarily not because of geographic separation of populations but rather due to ecological segregation. The hybrid offspring of two ecologically separated species would lack their parents’ specific adaptations and probably suffer from reduced fitness (Hatfield and Schluter, 1999; Schluter, 2001), which in turn would put selective pressure on the early development of strong reproductive barriers. Lacking the boost in diversity due to fragmentation, hybridization and introgressive segregation, fewer species arise, and those which do, develop effective reproductive barriers early in their evolution. Indirectly, these studies therefore also highlight the importance of vicariance events and hybridization in the origin of species in Lake Tanganyika and perhaps elsewhere.

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