Phylogeny and evolutionary origin of haplochromines
Mitochondrial DNA (mtDNA) sequence data have a long and successful history in the study of East African cichlid evolution (see e.g., [1, 4, 9, 17, 24, 25]). Limitations with mtDNA have only been encountered when focusing on the phylogeny among extremely closely related species due to the possibility of the persistence of ancestral polymorphism (see e.g., ), or because of hybridization events (see e.g., [27–29]). Nevertheless, mtDNA sequences proved to be particularly suitable for the reconstruction of the East African cichlid phylogeny at the tribal level and for tribal assignments [9, 15]; for phylogenetic reconstruction within older tribes [30, 31]; and for phylogeographic analyses [4, 32]. Also, because of the extremely fast rate of lineage formation in cichlids, nuclear and even some mitochondrial genes  are too slowly evolving to contain phylogenetic information (reviewed in ).
The different phylogenetic algorithms, with which we analyzed our data, revealed largely congruent results. In all analyses, and in agreement with previous results , we found that the Eretmodini are placed as sister group to the substrate spawning Lamprologini – with an estimated number of up to 100 species the most species-rich tribe in LT – plus several LT tribes and all haplochromine representatives (see Fig. 3 for the maximum likelihood tree). The molecular phylogenies thus corroborate that all haplochromines are ultimately derived from LT cichlids and that their ancestor(s) are likely to have left LT secondarily.
We consider all species that belong to the monophyletic group descending from this ancestor (asterisk in Fig. 3), to being haplochromines sensu lato. These are further divided into four distinct groups, a Congolese/Southern African lineage (CSA), the genera Pseudocrenilabrus (Ps.) and Astatoreochromis (As.), and the modern haplochromines (MH). While the respective monophyly of these four lineages was supported by high bootstrap values and Bayesian posterior probabilities, our analyses could not unambiguously resolve the exact relationships between these four lineages [maximum likelihood and Bayesian inference (see Fig. 3): (CSA, (Ps., (As., (MH)))); maximum parsimony: (As., ((Ps., CSA), (MH))); neighbor joining: (Ps., (CSA, (As., (MH))))]. An evaluation of these alternative hypotheses by means of a Shimodaira-Hasegawa test  and a four-cluster likelihood mapping analysis  indicated that none of these alternative branching orders receives significantly more support than the others. This suggests that the four lineages of the haplochromines sensu lato evolved almost contemporaneously from a common ancestor. This is further supported by the observation of relatively short branches interrelating these four lineages and the generally low bootstrap values and Bayesian posterior probabilities supporting the corresponding relationships proposed by the different algorithms.
The CSA lineage is composed of several widespread and moderately species-rich groups of the Congo drainage and Southern Africa and consists of two main clades: A clade with a species from the Congo drainage (Ctenochromis oligacanthus) ancestral to Southern African genera (Serranochromis, Sargochromis, Pharyngochromis) was resolved as sister group to a clade comprised by solely Congo drainage taxa (Orthochromis, Haplochromis sp. nov., Cyclopharynx and Thoracochromis). Within the Pseudocrenilabrus - and Astatoreochromis -clades, branch lengths were relatively short and a more detailed phylogeographic sampling would be necessary to resolve the relationships between the different geographic lineages.
The modern haplochromines consist of species flocks of an unparalleled diversity. They include the endemic LT tribe Tropheini (~25 species) sister-group to a clade comprised by the entire species flock of LM (~1,000 species) and several East African riverine and lacustrine lineages (~200 species) plus the LV region superflock (~600 species). With approximately 1,800 species this – here phylogenetically defined – monophyletic lineage makes up about 7% of all known teleost fish.
Astatotilapia, Thorachochromis, and Orthochromis are polyphyletic genera
Our phylogenies show that several genera are in fact polyphyletic, and major taxonomic revisions will be required in the future to take our phylogenetic results into consideration. For example, and in agreement with previous studies (see e.g., [4, 14, 33]), Astatotilapia emerges as polyphyletic genus, with representatives assigned to both the East African riverine clade in the modern haplochromines and the LV region superflock.
The genus Thoracochromis, represented in our analysis by T. brauschi, has also been shown to be polyphyletic before , with T. brauschi from the Congo drainage as a more ancestral lineage, and T. petronius and T. pharyngealis from the Nile drainage with affinities to the LV region superflock (note that the Nile River Thoracochromis of  are consequently listed as Haplochromis in ). The placement of T. brauschi as sistergroup to T. petronius and T. pharyngealis plus the remaining representatives of the LV region superflock in the AFLP based phylogeny of  seems to contradict our mtDNA based results in which T. brauschi was identified as member of the CSA lineage. However, the reported branching order did not receive considerable bootstrap support in . Also, the choice of Astatoreochromis alluaudi (mislabeled as Astatotilapia alluaudi in ) as single outgroup species seems problematic, as our present analyses (see above) and former results  indicate that A. alluaudi and not T. brauschi is more closely related to the modern haplochromines (and, thus, also to the LV region superflock). Further analyses including nuclear DNA sequence data and more taxa assigned to the genus will be necessary to address this problem.
Based on the phylogeny it is apparent that the Orthochromis lineage, which is confined to the Malagarasi River system and two isolated rivers, East of LT , is not part of the radiation of haplochromine cichlids. This is further supported by the breeding behavior of these fish: While the Malagarasi River Orthochromis are biparental caregivers , the haplochromines sensu lato are all maternal mouthbrooders. Thus, the Orthochromis -species from the Malagarasi area, a group of exclusively riverine fish, should be placed into its own tribe. The name Orthochromis is, however, also used for riverine species from the Congo drainage. In our analyses, Orthochromis polyacanthus and O. stormsi from the Congo River system fall – according to their distribution – into the CSA-clade leaving also the genus Orthochromis polyphyletic (see also ). We suggest to using Orthochromis for the CSA lineage representatives, since O. polyacanthus was the first species of the genus to be described , and Schwetzochromini (as tribe name) and Schwetzochromis (as genus name) for the Malagarasi area species, since this name was repeatedly used for some species of that complex (see e.g., [9, 34, 35]).
Phylogeography and phylochronology
We note that the application of a molecular clock for estimating divergence times has the potential of not being without problems for several reasons (see e.g., [37, 38]). However, a molecular-clock-based time estimate does surely provide an approximate framework for phylogeographic inferences. Our phylogeographic scenario (Fig. 4a), which is derived from the maximum likelihood phylogenetic and molecular clock analyses, suggests that several lineages independently left LT to colonize surrounding river systems and consequently other lakes in East Africa. The molecular clock calibration based on the chronogram generated with r8s  (Fig. 4b,c) yielded about 2.4 MYA (1.22 – 4.02 MYA) for the split of the CSA lineage from the common ancestor of the haplochromines sensu lato, and about 2 MYA (1.15 – 3.89 MYA) for the first branching events within the CSA clade. The spread of Congo drainage taxa into southern river systems occurred at a later stage, most likely at the relatively shallow watershed between upper branches of the Congo River and the Zambezi River – a scenario that is also supported by the placement of Serranochromis sp. (from Lake Mweru in the upper Congo) as sister group to the Zambezi/Southern African genera Sargochromis and Pharyngochromis in our phylogenies. However, further sampling in that area would be necessary to reconstruct the southward spread of the CSA lineage.
At essentially the same time as the CSA lineage, the ancestors of the Pseudocrenilabrus- and the Astatoreochromis- lineage diverged from their common ancestor. Despite their large distributional ranges – they also colonized the LV (both lineages) and LM (Pseudocrenilabrus) region – the genera Astatoreochromis and Pseudocrenilabrus never underwent considerable speciation. The three described Astatoreochromis species occur in the LV region including Lakes Edward and George (A. alluaudi), in the rivers Rusizi and Lukuga (A. straeleni), and in the Malagarasi River (A. vanderhorsti). The three species of Pseudocrenilabrus occur from the Nile system to the LV region (P. multicolor), in Eastern- and Southern Africa including LM (P. philander), and in the central Congo basin (P. nicholsi). All analyzed representatives are relatively closely related suggesting a recent spread of these lineages in East Africa. However, we did not include P. nicholsi, which is morphologically different from P. multicolor and P. philander and would – if it really belonged to the Pseudocrenilabrus -lineage – represent the only haplochromine in the Congo drainage that is not a member of the CSA lineage.
The most recent common ancestor of the modern haplochromines was dated to have existed about 1.8 MYA (0.66 – 3.78 MYA) (Fig. 4). This ancestral lineage forms the crucial phylogenetic and biogeographic link between the species flocks of all three East African Great Lakes, and its discovery documents the existence of much earlier hypothesized fish-accessible waterways between these waterbodies [40, 41]. Apparently, the Malagarasi River (and possibly the Rusizi) played a major role for the dispersal of these fishes, since many modern haplochromine lineages occur in these drainages and in lakes South-Eastern and North of LT exclusively, which argues against the view that LM haplochromines originated from Zambezi River stocks . Whether or not Lake Rukwa has ever acted as link between the faunas of LT and LM  cannot be answered by our data. Lake Rukwa seems to have overflowed at its maximum levels into LT several times. However, Lake Rukwa has also become very shallow in recent geological times and it might have dried up completely  eradicating its original fauna. At present, Lake Rukwa harbors haplochromines that belong to the East-African riverine clade in Figs. 2, 3 [4, 25].
Our analyses also recovered another closely related lineage to the LV region superflock in the East-African riverine clade, in addition to Haplochromis gracilior form Lake Kivu . This lineage includes H. paludinosus that occurs in the Malagarasi (which was already suggested by ), as well as undescribed species from Tanzania and Lake Edward (Figs. 2, 3). It is, however, unclear by which waterway haplochromine cichlids once colonized Lake Kivu. The flow of the Rusizi, presently from Lake Kivu into LT with the Panzi falls as strong barrier for fish migration, might actually have been reversed before the uplift of the Virunga volcanoes north of Lake Kivu as suggested by deposits of fossil LT mollusks and fluviatile sands in the upper Rusizi valley [41, 42]. This connection could possibly explain how haplochromines of LT origin were able to colonize Lake Kivu about 1.5 million years ago (Fig. 4).
Evolutionary key-innovations of haplochromines
One of only few synapomorphies of the haplochromines sensu lato is the particular type of cranial apophysis for the upper pharyngeal bones . The distinctive organization of the pharyngeal apophysis, a second set of jaws that is functionally decoupled from the oral ones , is characteristic to all cichlids and has been interpreted as prominent feature that – because of its adaptability – contributes to the cichlids' evolutionary success [2, 3, 10, 43]. It is, however, not evident how the relatively minor morphological modification of part of that structure in the haplochromines  might function as an evolutionary key-innovation. Interestingly, however, all haplochromines sensu lato are maternal mouthbrooders with the females alone incubating the eggs in their buccal cavities [10, 12]. Mouthbrooding, which is regarded as an adaptation to predation pressure [44–46], has evolved several times independently and in diverse behavioral modes in cichlids [10, 22, 47, 48]. The characteristic maternal mouthbrooding behavior displayed by haplochromines is believed to being a derived character state [35, 46, 47]. Mouthbrooding strongly limits the number of eggs and fry that can be raised and might have led to generally much smaller population sizes, which has, for example, population genetic implications on fixation of alleles, and might result in smaller effective population sizes. Furthermore, mouthbrooding species may be considered to being promising colonizers of new habitats, since only a single mouthbrooding female is necessary for the founding of a new population.
An eminent feature of several female mouthbrooding cichlid genera is the occurrence of egg-spots on the anal fins of males. In some species also females show such ovoid markings, but these are smaller and much less conspicuous than in males. Also, some species of the modern haplochromines, e.g., some deep-water lineages of LM, have lost their egg-spots secondarily. In mimicking real eggs to attract females, these egg-spots function as natural releasers [22, 23], or intra-specific sexual advertisement , apparently serving to ensure a greater fertilization success of the eggs by bringing about greater proximity of the female's mouth to the male's genital opening. Based on the molecular phylogeny, we could trace the origin of the characteristic egg-spots (ocelli) [10, 23] to the common ancestor of the Astatoreochromis -lineage and the modern haplochromines. There are other cichlid species in which males display yellow or red marks on their pelvic, dorsal or anal fins, but only in these lineages true egg-spots on the males' anal fins with a yellow, orange or red center and a colorless/transparent outer ring [10, 22] are found. Interestingly, the branch leading to the Astatoreochromis -lineage and the modern haplochromines is the one with a pronounced potential for an increased rate of speciation (see Fig. 4c). Based on the character state reconstructions (Fig. 5) it seems likely that this ancestor was riverine. Thus, it may be concluded that the egg-spots first evolved in a haplochromine cichlid that inhabited a turbid riverine environment, where these conspicuous markings would seem to be particularly effective and necessary for intra-specific communication.
Another innovation that further distinguishes the exceptionally species-rich modern haplochromines from all other cichlids is the occurrence of numerous color morphs, often accompanied by sexual color dimorphism. Inter- and intra-specific polychromatism combined with maternal mouthbrooding involving egg-spots as releasers can be hypothesized to being permissive for sexual selection through female choice and, hence, the haplochromines' propensity for species formation, as sexual selection is probably a major causal factor in the origin of isolating mechanisms and the maintenance of reproductive isolation [18, 49–53]. These distinctive features of the modern haplochromines, that have arisen just in their ancestor, in combination with the numerous ecological niches that are provided by the large East African lakes might thus have induced a considerable increase of the haplochromines' evolutionary potential. The importance of large waterbodies for the evolution of the modern haplochromines is reflected by the fact that these cichlids only radiated in lakes (and species number rather correlates to the size, but not to the age, of a lake), whereas the riverine lineages are all species-poor albeit often widespread (Figs. 1, 3).
Replicate adaptive radiations of the 'modern haplochromines'
A common feature of many adaptive radiations is that their founders are believed to have had a more generalist's lifestyle, while adaptive radiations themselves are defined by being composed of highly specialized species with narrower niche widths [54, 55]. Theory predicts that generalists more likely have better dispersal abilities and are expected to be able to adapt readily to novel environmental settings . A generalist ancestor scenario fits well with the diversification of haplochromine cichlids. Generalist riverine species of the genera Astatoreochromis, Astatotilapia, Pseudocrenilabrus, and Haplochromis (e.g., bloyeti), are ancestral to the adaptive radiations of the Tropheini of LT, and/or the radiations of LM and the LV region superflock. These genera are widely distributed and not confined to Eastern Africa, and they are the only ones that could inhabit the waterways that – over geological time spans – connected the lakes of Eastern Africa.
The phylogeny presented here (Fig. 3) reveals that modern haplochromines gave rise to several major adaptive radiations; the most prominent ones are those of LM and LV. Interestingly, it uncovers that also the radiation of the Tropheini from LT  must now be considered as an additional radiation of the modern haplochromines, corroborating the much older perception that LT accommodates several independent species flocks . It is further suggested by mapping the fishes' lifestyle onto our molecular phylogeny that the highly specialized Tropheini are descendents of a river-living species. This implies that the ancestor of the Tropheini successfully re-entered the lake habitat and evolved into the presently dominant group in the rocky littoral zone of LT. Thus, this lineage of modern haplochromines managed to occupy "empty niches" in an apparently "full" ecosystem, as all remaining tribes, which now account for about 200 species, had already been established when the ancestor of the Tropheini secondarily entered LT (Figs. 2, 3). The observation that these fish underwent an independent adaptive radiation in LT underlines the haplochromines' propensity for speciation.
In an apparent contrast to most other known examples of adaptive radiations  is the finding that the generalist ancestors of the haplochromine species flocks were derived from already highly diverse and specialized LT endemics (Fig. 3). Therefore, specialization may not be an "evolutionary one-way street", but rather some lineages have reversed their level of specialization, i.e., generalists arose from highly specialized lineages, yet, apparently retained their high propensity for speciation and level of evolvability (see ). The faunal revolution of LT's radiation of cichlids was thus not confined to the lake habitat itself (see also [8, 9, 58]), but it effectively involved large parts of Africa via the intermediate step of repeatedly evolving generalist riverine lineages – in much the same way as the adaptive radiation in LV produced haplochromine species that secondarily colonized surrounding rivers .
Our phylogeny of haplochromines provides strong support for replicate adaptive radiations in East African cichlids. The concept of replicate radiations, in which the same sequence of adaptations to ecological niches evolved repeatedly in lineages that inhabit similar environments, has been developed based on sympatric species pairs of fishes in postglacial lakes and on the Anolis lizard ecomorphs on different islands of the Greater Antilles [59–61]. Our inclusive phylogenetic and phylogeographic study shows that similar ecological types of cichlids in the different East African lakes evolved independently (see also [2, 17]), yet it also shows that the convergent ecotypes in the species flocks of LM, LV, Lake Kivu as well as in the Tropheini [2, 10, 12, 13, 17] arose from the same ancestral phenotype in the ancestor of the modern haplochromines. We suggest that a combination of behavioral (maternal mouthbrooding) and morphological innovations (egg-spots, color polymorphisms, pronounced sexual dichromatism) as well as ecological opportunities (after the colonization of large lakes) might have predestined this particular lineage to give rise to these replicate adaptive radiations.
It has been noted before that lineages of LT origin have left the lake secondarily (there are, for example, about five lamprologine species that are found in the Congo and Malagarasi Rivers) [9, 58]. Here, we show that the entire haplochromine diversity has its origin in LT corroborating the view that ancient lakes not only preserve biodiversity but also act as biodiversity hotspots, genetic reservoirs and cradles from which new lineages evolve [4, 8, 9]. What remains to be answered is where the LT cichlids originated and to what extent a proposed and meanwhile desiccated Pliocene lake in the Congo plains [41, 42] was the source of the ancient LT lineages, pushing back even further the onset of replicate adaptive radiations in East African cichlids.