Molecular Phylogenetics and Evolution 49 (2008) 153-169

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Molecular Phylogenetics and Evolution

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Age and spread of the haplochromine cichlid fishes in Africa

Stephan Koblmullera,b, Ulrich K. Schliewenc, Nina Duftnera,d, Kristina M. Sefca, Cyprian Katongoe, Christian Sturmbauera'* a Department o f Zoology, Karl-Franzens-University Graz, Universitatsplatz 2, A-8010 Graz, Austria b Department o f Evolutionary Biology, Evolutionary Biology Centre (EBC), Uppsala University, Norbyvagen 18D, SE-752 36 Uppsala, Sweden c Bavarian State Collection o f Zoology, Department o f Ichthyology, D-81247 Munich, Germany d Section o f Integrative Biology, University o f Texas at Austin, 1 University Station #C0930, Austin, TX 78712, USA e Department o f Biological Sciences, University o f Zambia at Lusaka, Zambia

ARTICLE INFO ABSTRACT

Article history: The Haplochromini are by far the most species-rich cichlid fish tribe that originated along with the so- Received 7 March 2008 called primary radiation of the Lake Tanganyika cichlid species flock, i.e. at the same time during which Revised 29 May 2008 the majority of the endemic Lake Tanganyika cichlid tribes emerged. Unlike the other tribes, the haplo- Accepted 30 May 2008 chromines are not restricted to Lake Tanganyika but distributed throughout Africa, except for the north­ Available online 7 June 2008 western part of the continent. Haplochromine cichlids seeded the adaptive radiation of cichlid fishes in Lakes Malawi, Kivu, Victoria, Turkana, as well as in the now extinct paleo-Lake Makgadikgadi. Here we Keywords: present a comprehensive phylogenetic and phylogeographic analysis of haplochromine cichlids that is Biogeography based upon DNA sequences of two mitochondrial gene segments of riverine taxa covering all major Afri­ Phylogeography River capture can biogeographic regions where haplochromines are found. Our analysis revealed that six lineages of Dispersal haplochromines originated within a short period of time, about 5.3-4.4 MYA. These haplochromine lin­ Cichlidae eages show a highly complex phylogeographic pattern, probably severely influenced by climate- and/or Phylogeny geology-induced changes of the environment, with river capture events most likely playing an important role for species dispersal. © 2 0 0 8 Elsevier Inc. All rights reserved.

1. Introduction 2000; Salzburger et al., 2005; Seehausen et al., 2003; Verheyen et al., 2003). Previous molecular phylogenetic studies suggested With an estimated number of 2500 species, the family of cichlid strongly that the Haplochromini arose almost simultaneously with fishes is the most speciose family of vertebrates comprising about the majority of other Tanganyikan cichlid lineages (Duftner et al., 10% of today’s teleost diversity (Snoeks, 2001; Turner et al., 2001). 2005; Koblmuller et al., 2004, 2005, 2007a) in the course of a pri­ Especially the cichlid species flocks of the East African Great Lakes, mary Tanganyika radiation, presumably about 5 -6 MYA (Salzburg­ Tanganyika, Malawi and Victoria, are well known for their rapid er et al., 2002, 2005), possibly contradicting the traditional view rates of speciation and thus serve as model systems for studying that riverine haplochromines founded the sub-flock of Lake Tang­ mechanisms underlying the phenomenon of adaptive radiation. anyika mouthbrooding lineages (Fryer and Iles, 1972; Nishida, With about 500 and 1000 cichlid species, respectively, Lakes Victo­ 1991). All the mouthbrooding tribes—including the Haplochro- ria and Malawi harbor the most diverse species flocks known to mini—that originated in the course of the primary Tanganyika radi­ date (Kocher, 2004; Kornfield and Smith, 2000; Meyer et al., ation have been included in the so-called C-lineage (Clabaut et al., 1990; Salzburger and Meyer, 2004; Turner et al., 2001; Verheyen 2005). Probably as a consequence of their younger evolutionary et al., 2003). Although Lake Tanganyika is the oldest of the East age, the species flocks of Lake Malawi and the Lake Victoria region African Great Lakes, its polyphyletic species flock contains only exhibit less phenotypic, behavioral and genetic diversity than the about 200-250 species, which are assigned to 12, or alternatively Lake Tanganyika flock (Greenwood, 1979, 1980; Meyer et al., 16, eco-morphologically distinct tribes (Poll, 1986; Takahashi, 1990; Verheyen et al., 2003). Nevertheless, haplochromine cichlids 2003). Unlike the Lake Tanganyika cichlid species flock, those of occupy almost all ecological niches in Lakes Malawi and Victoria Lake Malawi and the Victoria region are of mono- or diphyletic ori­ that are occupied by representatives of other tribes in Lake Tang­ gin, comprising only species assigned to a single tribe, the Haplo­ anyika, a result of convergent evolution of morphologies and chromini (Meyer et al., 1990; Moran et al., 1994; Nagl et al., behavioral strategies (Kocher et al., 1993; Kassam et al., 2006; Sugawara et al., 2005). Most haplochromine cichlids are maternal * Corresponding author. Fax: +43 316 380 5595. mouthbrooders, whereby females incubate their eggs and fry in E-mail address: [email protected] (C. Sturmbauer). the buccal cavity. With few exceptions (e.g. the endemic Lake

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Tanganyika genus Tropheus) haplochromines exhibit clear sexual by haplochromine cichlids. Thus, we do not intend to provide a dimorphism with large and brightly colored males. Usually, it is al­ complete phylogeny of the tribe Haplochromini, but rather aim most impossible to distinguish between females of closely related to put the pattern of diversification in a temporal and comparative haplochromine species, whereas the males show very distinct color context, including some taxa that have not been included in any patterns. Consequently, sexual selection is believed to play and to phylogenetic analysis before, but which turned out to be crucial have played an import role in the diversification of haplochromine for the interpretation of evolutionary pathways in this most spe­ cichlids, particularly in Lakes Malawi and Victoria (Turner, 1994; cies-rich cichlid lineage. Turner and Burrows, 1995; Seehausen and Witte, 1998; Seehausen, 2000; Danley and Kocher, 2001; Lande et al., 2001). 2. Materials and methods Aside from the impressive diversity in lakes, haplochromines also represent the vast majority of riverine cichlids in northern, 2.1. Taxonomic sampling and molecular biological methods eastern and southern Africa. Although the species flocks of the Great East African Lakes have been studied extensively for a long Our study is based on 60 specimens belonging to the cichlid time, only recently efforts have been made to elucidate the phylo­ tribe Haplochromini [including the Tropheini from Lake Tanganyi­ genetic relationships among African riverine cichlids and between ka; Salzburger et al. (2005)]. Based on Salzburger et al. (2002, the riverine and lacustrine cichlid faunas (Nagl et al., 2000; Joyce et 2005), five members of the Lake Tanganyika cichlid species al., 2005; Katongo et al., 2005, 2007; Salzburger et al., 2002, 2005; flock— Ophthalmotilapia ventralis (tribe Ectodini), Limnochromis Seehausen et al., 2003; Verheyen et al., 2003; Terai et al., 2004). auritus (Limnochromini), Cyprichromis leptosoma (Cyprichromini), The relative paucity of species in rivers compared to lakes was ex­ Plecodus straeleni (Perissodini) and Cyphotilapia frontosa (formerly plained by the temporal instability of riverine ecosystems provid­ assigned to the tribe Tropheini, later elevated to tribe status, Cyph- ing little opportunity for speciation via niche partitioning (Joyce otilapiini, by Takahashi, 2003)—were used as outgroup taxa. The et al., 2005). Thus, diversification appears generally driven by complete NADH dehydrogenase subunit 2 gene (ND2; 1047 bp) vicariance and geographic isolation (Joyce et al., 2005; Katongo and a 402 bp segment of the cytochrome b gene (CYTb) were ob­ et al., 2005, 2007) although there is some indication that sympatric tained from all 65 individuals. When available, we used previously diversification processes might be more likely than previously as­ published ND2 and CYTb sequences (Appendix A). Most of the sumed (Koblmuller et al., 2008). specimens were sampled during several field expeditions from A previous molecular phylogenetic study (Salzburger et al., 1995 to 2005, while some additional samples were obtained from 2005) has shown that the Haplochromini comprise several distinct the aquarium trade (Appendix A). Sampling sites are shown in Fig. lineages: (i) the Serranochromis-like haplochromines, which repre­ 1. Voucher specimens are stored at the Bavarian State Collection of sent a lineage of predominantly riverine, mostly rather large Zoology (ZSM), the Department of Zoology, University of Graz, and (sometimes even up to >40 cm) cichlids, distributed from the Con­ the Department of Biological Sciences, University of Zambia at Lu­ go system to South Africa; (ii) the genus Pseudocrenilabrus, which is saka. Of all specimens, fin clips were taken and preserved in 96% currently comprised of three valid species (but there are many ethanol. Taxonomy follows CLOFFA(van Oijen et al., 1991) by plac­ morphologically distinct populations, probably deserving species ing all haplochromine cichlids formerly placed in the genera status; see Twentyman-Jones et al., 1997; Katongo et al., 2005; Astatotilapia, Ctenochromis, Orthochromis, Schwetzochromis (par- Koblmuller et al., 2008), distributed from the Nile system to South tim), Thoracochromis and Xystichromis in Haplochromis. However, Africa; (iii) the genus Astatoreochromis, which includes three spe­ we retain the highly distinct type species of Schwetzochromis, Sch- cies, occurring in the Lake Victoria region and northern confluences wetzochromis neodon in that genus, as well as the Orthochromis of to Lake Tanganyika; (iv) the so-called ‘‘modern haplochromines” the Lake Tanganyika drainage (Malagarazi system and two isolate (sensu Salzburger et al., 2005), which include the Lake Victoria rivers east of Lake Tanganyika) in Orthochromis, as they are not clo­ superflock (cichlid species flock of Lake Victoria plus the surround­ sely related to the remaining haplochromini (Salzburger et al., ing lakes), the Lake Malawi species flock, the Tropheini from Lake 2005; de Vos and Seegers, 1998). Tanganyika and several riverine taxa, encompassing a tremendous For DNA extraction we applied a proteinase K digestion fol­ diversity accounting for more than 70% of the world’s cichlid spe­ lowed by protein precipitation with ammonium acetate. As prim­ cies (Salzburger et al., 2005). However, despite the clear reciprocal ers for amplification and sequencing of the CYTb we used L14724 monophyly of these haplochromine lineages, the phylogenetic and H15149 (Kocher et al., 1989). For amplification of the ND2 interrelationships among them could not be resolved with confi­ we used the primers MET and TRP (Kocher et al., 1995), whereas dence. Nevertheless, Salzburger et al. (2005) proposed a monophy­ for sequencing we additionally employed the internal primers lum including the genus Astatoreochromis and the ‘‘modern ND2.2A (Kocher et al., 1995) and ND2.T-R (Duftner et al., 2005). haplochromines” based on the presence of highly developed egg- The PCRs were prepared for a total volume of 17 i l containing spots in both lineages. Based on evidence from mitochondrial 0.085 il of Taq DNA polymerase (U/il; BioTherm”), 1.7 i l of each DNA sequences, the species of the genus Orthochromis found in primer (10 iM ), 1.7 i l 10x dNTP mix, 1.7 il 10x MgCl2 buffer, the Malagarazi and neighboring river systems (De Vos and Seegers, 7.62 i l high performance liquid chromatography (HPLC) water 1998), originally assigned to the Haplochromini, represent a dis­ and 2.5 i l of the extracted DNA. Amplification was performed on tinct lineage that originated in parallel to the remaining haplochro- a GeneAmp PCR system 9700 (Applied Biosystems) under the fol­ mines sensu stricto, roughly at the same time as the Tanganyika lowing conditions: an initial denaturation phase at 94 °C for radiation (Clabaut et al., 2005, 2007; Salzburger et al., 2005). 3 min followed 45 cycles with denaturation at 94 °C for 30 s, pri­ Here we present a phylogenetic analysis of the haplochromine mer annealing at 50 °C for 30 s and extension at 72 °C for 1 min cichlids, with representatives of all major lineages known to date, 30 s, with a final extension phase at 72 °C for 10 min. PCR-products with a focus on lineages that have been under-represented in pre­ were purified with ExoSAP-lT (Exonuclease I and Shrimp Alkaline vious studies, to discuss the evolutionary history and biogeo­ Phosphatase in buffer; Amersham Biosciences) prior to being graphic affinities of the Haplochromini. Unlike previous added as template for chain termination sequencing following phylogenetic studies on haplochromine cichlids that focused either the protocol described in Duftner et al. (2005). DNA fragments on the lacustrine species flocks or on single lineages, often re­ were purified with Sephadex” G-50 (Amersham Biosciences) fol­ stricted to particular regions, our samples cover all but one (Cuan- lowing the manufacturer’s instruction and subsequently visualized za) African freshwater bioregions (Thieme et al., 2005) inhabited on a 3130 capillary sequencer (Applied Biosystems). All sequences S. Koblmiiller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169 155

are available from GenBank under the accession numbers listed in positions and 77:22 for first codon positions. Likewise we obtained Appendix A. a proper weighting scheme for the CYTb gene. Based on the esti­ mated ti/tv ratio of 11.17 for third codon positions of 2- and 3-fold 2.2. Phylogenetic analyses degenerate amino acids, 1.71 for third codon positions of 4-fold degenerate amino acids, 0.97 for second codon positions and 2.02 DNA sequences were individually aligned by eye using the SE­ for first codon positions we applied the following weighting QUENCE NAVIGATOR software (Applied Biosystems). To assess scheme (tv/ti): 77:7 for third codon positions of 2- and 3-fold the overall phylogenetic signal we performed a likelihood mapping degenerate amino acids, 12.6:7 for third codon positions of 4-fold analysis (Strimmer and von Haeseler, 1997), using TREE-PUZZLE degenerate amino acids, 77:77 for second codon positions and 5.1 (Schmidt et al., 2002). For phylogenetic inference maximum 77:38.5 for first codon positions. In both genes, C/T substitutions parsimony (MP), neighbor joining (NJ), maximum likelihood (ML) at the first codon position of leucine were treated as a fifth base and Bayesian inference (BI) were applied using PAUP* 4.06b (Swof- and were down-weighted to the same weight as transitions at ford, 2000) and MrBayes 3.0b4 (Huelsenbeck and Ronquist, 2001). the third codon positions. To evaluate an appropriate substitution To assess the degree of saturation of transition (ti) and transver­ model for NJ and ML analysis, we calculated hierarchical likelihood sion (tv) mutations at each codon position of the ND2 and CYTb ratio test statistics using the program Modeltest 3.06 (Posada and gene, we plotted the number of mutations against pairwise uncor­ Crandall, 1998). The best-fitting model was TrN+I+r (Tamura and rected percentage distances (not shown). Based on the estimated Nei, 1993) with nucleotide frequencies A, 0.2748; C, 0.3482; G, ti/tv ratio inferred from these pairwise comparisons we derived a 0.1107; T, 0.2663, proportion of invariable sites (I), 0.4463, gamma weighting scheme for a weighted MP analysis. Due to the esti­ shape parameter (a), 1.0450, and R-matrix A m G, A m T, C m G mated ti/tv ratio of 7.04 for third codon positions of 2- and 3-fold and G m T, 1.0000; A m G, 19.0017 and C m T, 8.3617. For obtain­ degenerate amino acids of the ND2 gene, 2.78 for third codon posi­ ing MP and ML topologies we applied heuristic search procedures tions of 4-fold degenerate amino acids, 3.65 for second codon posi­ with random addition of taxa and TBR branch swapping (1000 rep­ tions and 3.55 for first codon positions we applied the following licates for MP; 100 replicates for ML). As standard measures of con­ weighting scheme (tv/ti): 77:11 for third codon positions of 2- fidence we applied bootstrapping (1000 pseudo-replicates for NJ and 3-fold degenerate amino acids, 27.5:11 for third codon posi­ and MP, 100 for ML) and quartet puzzling (Strimmer and von Has- tions of 4-fold degenerate amino acids, 77:22 for second codon eseler, 1996; 25,000 random quartets for ML). For the reconstruc- 156 S. Koblmiiller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169

Table 1 posterior probability distribution of lengths for all branches Comparison of alternative phylogenetic hypotheses was obtained by saving the branch lengths for 100 trees sampled Tree -ln L A -ln LP during the Bayesian tree search (due to computation limits only NJ 11268.2935 14.6159 0.185 every twentieth tree was sampled). For each sampled tree, the MP 11269.1694 15.4918 0.161 distance from the most recent common ancestor (MRCA) of the BI 11253.7268 0.0492 0.832 ingroup to each of the terminal taxa was calculated with Ca­ ML 11253.6776 Best dence v.1.0 (T.P. Wilcox; available at http://www.biosci.utex- Shimodaira-Hasegawa tests (Shimodaira and Hasegawa, 1999) were used to as.edu/antisense/). Means and standard deviations were determine whether NJ, MP and BI topologies differed significantly from the ML tree calculated and plotted in the program SPSS for Windows, version under a likelihood criterion. 14. Whenever the distance estimates from the MRCA to a termi­ nal taxon did not overlap with the estimates for another taxon, we concluded that the rate of evolution between these two taxa tion of phylogenetic relationships by Bayesian inference, rate het­ differed significantly. erogeneity was set according to a gamma distribution with six rate After discovering significant heterogeneity of branch lengths, categories (GTR model; Yang, 1994). Posterior probabilities were and hence of substitution rates among lineages, the penalized like­ obtained from a 3,000,000-generation Metropolis-coupled Markov lihood method (PL; Sanderson, 2002) with an optimization via the chain Monte Carlo simulation (two independent runs; 10 chains; Truncated Newton method (TN; Sanderson, 2002) as implemented chain temperature, 0.2; trees sampled every 100 generations), with in the program r8s v.1.7 (Sanderson, 2003) was used to obtain age parameters estimated from the dataset. We applied a burn-in of estimates for the major cladogenetic events in haplochromine 1.000.000 generations to allow likelihood values to reach stationa- cichlids, using the BI phylogram as input tree. PL allows for differ­ rity. To assess whether the topologies obtained by the different ent rates of evolution on every branch of a phylogenetic tree but tree building algorithms differed significantly we performed a Shi- applies a penalty function that smoothes rate variation between modaira-Hasegawa test (Shimodaira and Hasegawa, 1999; Table neighboring branches (Sanderson, 2002). The optimal smoothing 1) in PAUP. A four-cluster likelihood mapping analysis (Strimmer parameter to apply in PL analyses was determined by a cross-val­ and von Haeseler, 1997) using the program TREE-PUZZLE 5.1 idation approach as suggested by Sanderson (2002). The degree of (Schmidt et al., 2002) was applied to evaluate the support of dis­ uncertainty attached to the date estimates was assessed by apply­ tinct internal branches that are critical for the interpretation of ing the same procedure to 100 bootstrapped datasets (created with evolutionary pathways. the seqboot module in the PHYLIP package; Felsenstein, 1993), fol­ lowing the protocol described in the r8s-bootkit manual, provided 2.3. Estimation of divergence times by T. Eriksson at http://www.bergianska.se/index_forsk- ning_soft.html. The analyses were performed with three calibra­ Estimation of divergence times was based on a Bayesian tree, tion points: (i) the maximum age of about 200,000 years for the computed with MrBayes 3.0b4 (Huelsenbeck and Ronquist, radiation of the Lake Victoria superflock (Meyer et al., 1990; Nagl 2001), including the above-mentioned taxa plus additional repre­ et al., 2000; Verheyen et al., 2003); (ii) a maximum age of 0.57­ sentatives of the C-lineage and the tribe Lamprologini. As outgroup 1 million years for the split between the Lake Malawi mbuna and we used three species of the tribe Eretmodini, justified by the anal­ utaka cichlids, based on the age of the truly lacustrine habitat of ysis of Salzburger et al. (2002, 2005). Rate heterogeneity was set Lake Malawi (Delvaux, 1995; Sturmbauer et al., 2001) and (iii) according to a gamma distribution with six rate categories (GTR the maximum age of about 5-6 million years for the MRCA of the model; Yang, 1994) and posterior probabilities were obtained from cichlids assigned to the C-lineage (Clabaut et al., 2005), based on 3.000.000-generation Metropolis-coupled Markov chain Monte the age of the truly lacustrine habitat of Lake Tanganyika (Tiercelin Carlo simulations (two independent runs; 10 chains; chain tem­ and Mondeguer, 1991; Lezzar et al., 1996; Cohen et al., 1997). To perature, 0.2; trees sampled every 1000 generations), with param­ investigate the validity of the used calibration points, we per­ eters estimated from the dataset. A burn-in of 1,000,000 formed four analyses with different combinations of these calibra­ generations was applied to allow likelihood values to reach tion points (Table 2). Alternative hypotheses concerning the age of stationarity. haplochromine cichlids were tested by running three additional A Bayesian relative rates test (Wilcox et al., 2004) was con­ analyses (Table 2 ). In the first one, we assumed a maximum age ducted to test for significant differences in branch lengths. The of 15,000 years for the Lake Victoria species flock, based on a recent

Table 2 Validation of calibration points used for dating diversification events in the haplochromine cichlids

Calibrations used LT MRCA Haplochromini LM LV LT(5-6)/LM(0.57-1)/LV(0.2) 6.00 (6.00-6.00) 5.29 (4.91-5.70) 1.00 (0.73-1.00) 0.14 (0.01-0.20) Lt(5-6)/Lm (0.57-1) 6.00 (6.00-6.00) 5.29 (4.91-5.90) 1.00 (0.73- 1.00) 0.14 (0.01-0.20) LT(5-6)/LV(0.2) 6.00 (6.00-6.00) 5.32 (4.97-5.90) 1.28 (0.73-1.80) 0.15 (0.01-0.20) LT(5-6) 6.00 (6.00-6.00) 5.32 (4.97-5.90) 1.28 (0.73-1.80) 0.15 (0.01-0.21) Lt(5-6)/LM(0.57-1)/LV(0.015) 6.00 (6.00-6.00) 5.28 (4.90-5.90) 1.00 (0.72-1.00) 0.01 (0.01- 0.01) Lt(9-12) 12.00 (12.00-12.00) 10.40 (9.75-11.79) 2.75 (1.55-4.59) 0.29 (0.03-0.43) Haplochromini (22.72) 28.54 (23.46-31.72) 22.72 7.17 (2.75-9.21) 0.64 (0.07-1.24)

We ran four independent penalized likelihood analyses in r8s (Sanderson, 2003) using combinations of different sets of calibration points: the maximum estimated age for the lacustrine habitat in Lakes Tanganyika (LT; 5 -6 MY; Lezzar et al., 1996; Cohen et al., 1997; Tiercelin and Mondeguer, 1991) and Malawi (LM; 0.57-1.00 MY; Delvaux, 1995) and the maximum estimated age of 0.2 MY for the Lake Victoria superflock (Meyer et al., 1990; Nagl et al., 2000; Verheyen et al., 2003). In a fifth run we assumed a maximum age of 15,000 years for the LV species flock (Stager and Johnson, 2008) and combined this calibration point with the 5 -6 and 0.57-1 MY for LT and LM, respectively. In a sixth run we applied an age of 9-12 MY for the LT radiation. In a seventh run we used the estimated age of 22.72 (19.22-26.22) MYA for the MRCA of the Haplochromini (Genner et al., 2007), as calibration point. The numbers indicate the estimated value (in MY). Numbers in parentheses are minimum and maximum values obtained from 100 bootstrap replicates. PL, penalized likelihood (as described in Sanderson, 2002). S. Koblmuller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169 157 paper by Stager and Johnson (2008), who report that geophysical 14.01% in ND2 and from 0.00% to 13.21% in CYTb. In the combined and paleoecological data indicate that Lake Victoria dried out at dataset, pairwise differences ranged from 0.14% to 12.99%. least once between 18,000 and 14,000 years ago and that it was Our analyses, based on the combined dataset, yielded highly highly unlikely that the LV species flock could have survived this consistent results. Only slight differences were observed with re­ period in remnant ponds or marshes within the desiccated basin spect to the tree building algorithm used (Fig. 3 ). An evaluation or anywhere else. We combined this calibration point with the of the phylogenetic hypotheses obtained from NJ, MP, ML and Bl above-mentioned calibration points for Lake Tanganyika and Lake by means of a Shimodaira-Hasegawa test (Shimodaira and Hase- Malawi. As second alternative, we constrained the primary Tang­ gawa, 1999) revealed no significant differences between the alter­ anyika radiation to an age of 9-12 million years. This was based native topologies (Table 1). on the assumption that the primary Tanganyika radiation had ta­ A strict consensus tree of the NJ-, ML- and Bayesian tree and of ken place at the onset of the lake formation 9-12 MYA as a conse­ MP-trees [96 most parsimonious trees; tree length, 33,939 steps; quence of allopatric diversification in a series of small shallow consistency index (Cl) excluding uninformative characters, 0.46; lakes in the area currently occupied by Lake Tanganyika and the retention index (Rl), 0.77; rescaled consistency index (RC), 0.41], Congo River system. The assumption implies that the secondary is shown in Fig. 4. Six distinct lineages could be consistently iden­ radiation observed in the majority of the Lake Tanganyika cichlid tified: I, Haplochromis pectoralis; ll, Astatoreochromis alluaudi; Ill, a tribes (Koblmuller et al., 2004, 2005, 2007a,b; Duftner et al., new undescribed species from the upper Lufubu River; lV, the 2005) coincided with the formation of a real lacustrine habitat genus Pseudocrenilabrus (including Haplochromis machadoi); V, with deep-water condition about 5-6 MYA, compatible with the the ‘‘modern haplochromines” (sensu Salzburger et al., 2005); fossil calibrated diversification scenario discussed by Genner et Vl, the Central and South African Serranochromis-like cichlids al. (2007). In the third alternative, we used the age estimate of (sensu Joyce et al., 2005; Katongo et al., 2007). The branching or­ 22.72 MYA for the Haplochromini as fixed calibration point, as der among these lineages differed slightly depending on the tree was recently proposed by Genner et al. (2007) based on a pre­ building algorithm used. Whereas H. pectoralis represented the sumed Gondwanan origin of the family Cichlidae. most ancestral branch in ML and Bl, the serranochromine cichlids constituted the most ancestral split in NJ. ln MP, four major 3. Results branches were identified: the serranochromine cichlids, the mod­ ern haplochromines, the undescribed haplochromine species from 3.1. Phylogeny o f the Haplochromini the upper Lufubu River plus the Pseudocrenilabrus-assemblage, as well as H. pectoralis plus Astatoreochromis alluaudi. The sister Likelihood mapping yielded 97.0% fully resolved quartets for group relationship between H. pectoralis and A. alluaudi in MP is the combined dataset (Fig. 2), indicating a strong phylogenetic most likely due to long-branch attraction (Felsenstein, 1973; Hen- signal. Pairwise sequence divergence (uncorrected p-distance) be­ dy and Penny, 1989), given that all other analyses placed the split tween species within the haplochromines ranged from 0.00% to of the A. alluaudi lineage ancestral to the modern haplochromines.

Fig. 2. Results from the likelihood mapping analysis of the combined dataset (entire ND2, partial cytb), pointing to a strong phylogenetic signal. 158 S. Koblmuller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169

Fig. 3. Phylogeny of the Haplochromini based on the entire ND2 gene and part of the cytb gene reconstructed with different tree building algorithms: (A) NJ tree using the TrN+I+r (Tamura and Nei, 1993) model; (B) Strict consensus tree of 96 most parsimonious trees (33,939 steps; CI excluding uninformative characters, 0.46; RI, 0.77; RC, 0.41); (C) ML tree using the TrN+I+r model; (D) BI tree. Ophthalmotilapia ventralis, Limnochromis auritus, Cyprichromis leptosoma, Plecodus straeleni and Cyphotilapia frontosa were used as outgroup. Bootstrap values (for NJ and MP), quartet puzzling values (ML) and posterior probabilities (for BI) >50 are shown.

Furthermore, the ancestral position of H. pectoralis and the place- 5A and B). While the undescribed Lufubu haplochromine was ment of A. alluaudi as a separate lineage were also supported by placed ancestral to the species of the Pseudocrenilabrus-clade in the results of the four-cluster likelihood mapping analysis (Fig. MP, ML and BI, it was resolved as sister group to H. pectoralis in S. Koblmuller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169 159

Fig. 4. Strict consensus tree of 96 most parsimonious trees, the NJ, ML and BI tree, representing the phylogenetic relationships of the Haplochromini based on the entire ND2 gene and part of the cytb gene. Bootstrap values for NJ and MP are shown above the branches, quartet puzzling values (for ML) and posterior probabilities (for BI) below. Only values >50 are shown. Bars and roman numerals to the right indicate the six major haplochromine lineages: I, Haplochromis pectoralis; II, Astatoreochromis alluaudi; III, a new undescribed species from the upper Lufubu River; IV, the Pseudocrenilabrus-clade; V, the ‘‘modern haplochromines”; VI, the Central and South African Serranochromis-like cichlids. Note that the Tropheini from Lake Tanganiyka are included in the ‘‘modern haplochromines” (see also Salzburger et al., 2005).

NJ, albeit with low bootstrap support. High posterior probabilities, et al., 2005) were consistently placed as sister group to the quartet puzzling values and MP bootstrap values, as well as the remaining taxa. Among those, four distinct lineages—Haplochromis results of the four-cluster likelihood mapping analysis (Fig. 5C) bloyeti, Haplochromis burtoni, the Lake Malawi species flock and indicate that this species might indeed represent an ancestral the Lake Victoria superflock, including the North African Haplochr­ lineage within the genus Pseudocrenilabrus. Within the Pseudocre- omis sp. ‘‘El Fayoum”—could be identified, albeit with different nilabrus-clade, P. multicolor and P. nicholsi were resolved as sister branching orders depending on the tree building algorithm used. group to the taxa representing the Pseudocrenilabrus philander However, four-cluster likelihood mapping clearly indicated a sis­ species assemblage (see Katongo et al., 2005), also including Hap­ ter group relationship between A. burtoni and the Lake Victoria lochromis machadoi. Within this species assemblage, a clear bio­ superflock (Fig. 5D). Among the modern haplochromines sister geographic pattern becomes evident in that taxa sampled from to the Tropheini, the Lake Malawi species flock apparently repre­ rivers of the Congo drainage system were clearly separated from sents the most ancestral lineage, albeit with comparatively low taxa sampled in other river systems further south. Within the support by the four-cluster likelihood mapping analysis (Fig. 5E). modern haplochromines, Tropheini (including Gnathochromis pfef- Finally, the branching order of the three major lineages within feri; see Sturmbauer et al., 2003; Salzburger et al., 2005; Duftner the Congolese/southern African serranochromine clade was iden­ 160 S. Koblmuller et ai. f Molecular Phylogenetics and Evolution 49 (2008) 153-169

{a,d)-(b,c) (a,c)-(b.d) (a.dHb.c) (a,c)-(b,d) (ad)-(b,c) {a,c)-(b,d)

Fig. 5. Results from the four-cluster likelihood mapping analysis to evaluate the support of the three alternative branching orders of four predefined groups. (A) a, Haplochromis pectoralis; b, Lufubu haplochromine; c, genus Pseudocrenilabrus; d, remaining haplochromines; (B) a, outgroup; b, Haplochromis pectoralis; c, genus Pseudocrenilabrus + Lufubu haplochromine; d, remaining haplochromines; (C) a, genus Pseudocrenilabrus + Lufubu haplochromine; b, Astatoreochromis alluaudi; c, ‘‘modern haplochromines"; d, serranochromines sensu lato; (D) a, non-serranochromine haplochromines; b, Haplochromis brauschi, Cyclopharynx fwae, Schwetzochromis neodon; c, Haplochromis polyacanthus; d, serranochromines sensu stricto; (E) a, Haplochromis bloyeti; b, LM species flock; c, Astatotialpia burtoni; d, LV superflock; (F) a, Tropheini; b, Haplochromis bloyeti; c, LM species flock; d, Haplochromis burtoni + LV superflock.

tical in all analyses. The most ancestral branch was represented discussion see below). Thus, we favor divergence times derived by a lineage comprising Schwetzochromis neodon and the species from the three calibration points (LT, 5 -6 MY; LM, 0.57-1 MY; pair Haplochromis brauschi and Cyclopharynx fwae. The next ances­ LV, <0.2 MY) as the most plausible age estimates (Fig. 6). tral split was occupied by Haplochromis polyacanthus, followed by Our analysis suggested an age of 5.3 (4.9-5.7) MY for the most the remaining Congolese/southern African taxa, including several ancestral split within the tribe Haplochromini, when H. pectoralis species assigned to the genera Haplochromis, Serranochromis, Che- branches off the remaining members of the clade. All major lin­ tia, Sargochromis and Pharyngochromis. However, bootstrap sup­ eages were established 4.4 (3.8-5.0) MYA. The most recent com­ port was rather low for the sister group relationship of H. mon ancestor (MRCA) of the Pseudocrenilabrus-clade was polyacanthus to the latter taxa. Furthermore, four-cluster likeli­ estimated to an age of 2.1 (1.7-2.6) MY. For the MRCA of the ‘‘mod­ hood mapping favored the grouping of H. polyacanthus with S. ern haplochromines" we obtained an age of 3.4 (3.0-4.0) MY and neodon, H. brauschi and C. fw ae, although the percentage of fully for that of the serranochromines sensu lato the age was estimated resolved quartets was rather low (Fig. 5F). Within the third serr- to 4.1 (3.7-4.9) MY. The MRCA of the serranochromines sensu stric- anochromine lineage, no clear phylogeographic pattern became to was dated to an age of 2.1 (1.5-2.6) MY. evident in contrast to the situation in the Pseudocrenilabrus philan­ der species assemblage. Also, several species within this clade 4. Discussion were resolved as polyphyletic. 4.1. Phylogeny o f the Haplochromini 3.2. Divergence times Previous studies showed that haplochromines originated at the Analyses using different combinations of calibration points for time of the primary Tanganyika radiation, roughly in parallel to the major diversification events in different cichlid lineages—LT, 5­ remaining tribes of the C-lineage in the newly formed lacustrine 6 MY; LM, 0.57-1 MY; LV, <0.2 MY—indicate that age estimates ob­ environment of proto-Lake Tanganyika (Salzburger et al., 2002, tained with different subsets of calibration points were consistent 2005; Clabaut et al., 2005). Based on mtDNA data, the Orthochromis with each other (Table 2). Using the alternative age of species from the Lake Tanganyika drainage system (De Vos and <15,000 years for LV did not considerably alter the age estimates Seegers, 1998), originally assigned to the tribe Haplochromini, (Table 2 ). Contrary to these results, alternative hypotheses of Gen- emerged as a distinct lineage during the primary Tanganyika radi­ ner et al. (2007) concerning the age of the Lake Tanganyika flock ation (Salzburger et al., 2002, 2005; Clabaut et al., 2005, 2007). and the tribe Haplochromini were not compatible with currently However, there are to date no nuclear data supporting the place­ supported hypotheses of the evolutionary history of East African ment of the Lake Tanganyika drainage Orthochromis outside the cichlids, in particular the LM species flock (Table 2; for a detailed Haplochromini. Ancient incomplete lineage sorting (Takahashi et S. Koblmuller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169 161

Fig. 6. Chronogram of the haplochromine evolution as reconstructed using the penalized likelihood approach (Sanderson, 2002), implemented in r8s (Sanderson, 2003), based on a Bayesian tree, including representatives of all major tribes of the C-lineage (Clabaut et al., 2005) and the Lamprologini. The tree was rooted with three species of the tribe Eretmodini (Eretmodus cyanostictus, Spathodus erythrodon, Tanganicodus irsacae; only ingroup is shown in the figure). Grey bars show the range of time estimates obtained by an analysis of 100 bootstrap datasets; arrows indicate calibration points. Roman numbers refer to the six main lineages within the Haplochromini: I, Haplochromis pectoralis, II, Astatoreochromis alluaudi; III, a new undescribed species from the upper Lufubu River; IV, the Pseudocrenilabrus-dade; V, the ‘‘modern haplochromines"; VI, the Central and South African Serranochromis-like cichlids. Cichlid tribes other than the Haplochromini represented in this chronogram: Ect, Ectodini; Ort, Orthochromis from the Malagarazi River system (originally assigned to the Haplochromini but representing a distinct lineage within the Tanganyika radiation; Salzburger et al., 2005); Ben, Benthochromini; Per, Perissodini; Cypr, Cyprichromini; Lim, Limnochromini; Cyph, Cyphotilapiini; Lam, Lamprologini. Assignment of species to particular tribes follows Takahashi (2003) and is supported by molecular data (reviewed by Koblmuller et al., in press). al., 2001) might as well explain the separate position of these Orth- tribe Haplochromini, all major lineages originated almost simulta- ochromis species, but this needs more rigourous testing. Within the neously. Six distinct lineages, well supported by high bootstrap and 162 S. Koblmuller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169 quartet puzzle values as well as high posterior probabilities, can be cichlid radiations re-emerged. In the past, it had been widely ac­ identified within the haplochromine cichlids: I, H. pectoralis; II, A. cepted that the establishment of a real lacustrine environment alluaudi; III, a new undescribed species from the upper Lufubu Riv­ with deep-water conditions had induced the radiations of the er; IV, the Pseudocrenilabrus-clade; V, the ‘‘modern haplochro­ LT and LM cichlid species flocks 5 -6 and 0.57-1 MYA, respec­ mines”; VI, the Central and South African Serranochromis-like tively (see e.g. Sturmbauer et al., 2001; Salzburger et al., 2002). cichlids. Our findings largely agree with those of Salzburger et al. Recently, Genner et al. (2007) explored calibrations based on a (2005), and extend their hypothesis by identifying two additional hypothesized Gondwanan origin of the family Cichlidae, and on lineages within the haplochromine cichlids, namely the H. pectoral- the fossil record. The authors rejected the fossil calibration in fa­ is lineage and that containing the undescribed haplochromine spe­ vor of the Gondwana calibration because of a worse match with cies from the Lufubu River. substitution rates in other fishes. Both approaches, however, sug­ These new insights have relevant consequences for the recon­ gested a much older age for the East African cichlid radiations struction of the evolutionary history of the Haplochromini. Among than was previously assumed, and are in conflict with existing the six major lineages within the tribe, H. pectoralis is a strong can­ hypotheses on the timing and pattern of cichlid speciation in LT didate for being the most ancestral species in relation to the and LM (Koblmuller et al., in press). These inconsistencies may remaining haplochromines [Note that in a poorly resolved nuclear be associated, on the one hand, with the temporal distance be­ phylogeny, H. pectoralis clusters with haplochromines from LM and tween the Gondwana break-up and the lacustrine cichlid radia­ East African rivers (Mayer et al., 1998).]. The phylogenetic relation­ tions, and on the other hand, with the very distant relationship ships among the remaining lineages could not be resolved with between the fossil taxa available for calibration and the East Afri­ high confidence, indicating that they originated within a short time can species flocks. The main concerns are: (1) The MRCA of the span in evolutionary terms (see also Salzburger et al., 2005). Nev­ Haplochromini was dated to an age of 22.72 (19.22-26.22) MY ertheless, high posterior probabilities and the results of the four- by Gondwana calibration, which is considerably older than the cluster likelihood mapping analysis suggest that the new haplo- previously assumed age for this cichlid tribe (5 -6 MY, Salzburger chromine from the Lufubu River represents the sister group to et al., 2002). Several other studies have provided evidence that the Pseudocrenilabrus-clade. This placement is also corroborated the Haplochromini arose simultaneously with other tribes as­ by a shared phenotypic trait, the presence of a distinct orange spot signed to the so-called C-lineage (sensu Clabaut et al., 2005) at at the tip of the male’s anal fin, a feature exclusively found in mem­ the same time as the primary Lake Tanganyika radiation (Takah- bers of the genus Pseudocrenilabrus. The clustering of Haplochromis ashi et al., 2001; Salzburger et al., 2002, 2005; Terai et al., 2003; machadoi with Pseudocrenilabrus sp. ‘‘Olushandja” within the Clabaut et al., 2005; Sugawara et al., 2005). (2) By using Genner et Pseudocrenilabrus philander species complex is also supported by al.’s age estimate of 22.72 MY for the MRCA of the Haplochromini the presence of a distinct orange spot at the tip of the male’s anal as the only calibration point, we obtain an age of 7.2 (2.8-9.2) MY fin. Within the ‘‘modern haplochromines”, the Tropheini from LT for the LM species flock. This is in general agreement with the clearly form the sister group to the remaining haplochromines of Gondwana-based estimate of 4 MY (Genner et al., 2007), which this lineage, including the LM and LV species flocks, as already sug­ correlates well with the formation of the LM basin. Geological gested by Salzburger et al. (2005). Within the Serranochromis-like evidence, on the other hand, points to a complete desiccation of cichlids, the branching order among the three main lineages was LM about 0.57-1 MYA (Delvaux, 1995). It is, however, still con­ not resolved with confidence. While our analysis revealed the line­ tended whether or not the lake indeed dried out completely, age consisting of Schwetzochromis neodon, Cyclopharynx fwae and and moreover, whether the cichlid flock could have survived in Haplochromis brauschi as the basal sister group followed by H. poly­ surrounding rivers, where it became subsequently extinct after acanthus and the serranochromines sensu stricto, Salzburger et al. LM had refilled. Even the more recent, fossil-based estimate for (2005) proposed a sister group relationship between H. polyacan­ the LM species flock [2.8 (1.6-4.6)M Y] is still too old to allow thus and H. stormsi (not included in this study) and a lineage con­ for an origin of the LM species flock after the proposed desicca­ sisting of C. fw ae, H. brauschi and an undescribed haplochromine tion of the lake 0.57-1 MYA (Delvaux, 1995). (3) The extremely (not included in this study) species. It must be noted here that old age of the LT cichlid species flock implied by the Gondwana there is a taxonomic problem with H. polyacanthus, which was pre­ calibration (Genner et al., 2007) would impose a scenario where, viously synonymized with H. stormsi. This was reviewed by Green­ after the distinct tribes originated in different river systems or wood and Kullander (1994), who revealed that H. polyacanthus is small lakes, further diversification (within tribes) has happened limited to Lake Mweru. The geographic origin of our sample is un­ in a single lake about 10-20 million years ago (also see Koblmul- known, and it might be a different species of the genus Haplochr- ler et al., in press). Although the existence of such a large paleo- omis, most likely H. stormsi. The same sample was used in Lake in the Congo region has been postulated based on biogeo­ Salzburger et al. (2005). However, in both our analysis and that graphic patterns (Beadle, 1981; Coulter, 1991), there is no geolog­ of Salzburger et al. (2005), the phylogenetic position of H. polyacan­ ical evidence for this hypothesis. Instead, geological data suggest thus gained relatively low bootstrap support. Within the serrano- the presence of a series of small shallow lakes in the current chromines sensu stricto, several species resulted as non- Tanganyika basin for that time period period (Cohen et al., monophyletic. This phenomenon had already been observed in re­ 1997). In fact, the fossil-based (in contrast to the Gondwana- cent studies focusing on this particular cichlid lineage (Joyce et al., based) calibration of Genner et al. (2007) would place the ‘‘pri­ 2005; Katongo et al., 2007) and was attributed to the effects of mary Tanganyika radiation” (Salzburger et al., 2002) at 9 ­ incomplete lineage sorting and introgressive hybridization. 12 MYA, when a series of small shallow lakes was present in the area currently occupied by LT and the Congo River system 4.2. Justification of calibration points for estimating divergence times (Cohen et al., 1997). During the primary radiation, several ende­ mic lineages diversified and segregated with respect to their eco­ Despite potential pitfalls (Gillespie, 1991; Page and Holmes, logical niches, to become occupants of the shallow and deep 1998), time estimates from molecular data can provide an benthic habitats (Ectodini and Limnochromini, respectively), as approximate framework to put diversification events in a tempo­ well as the shallow and deep pelagic habitats (Cyprichromini, ral context. Our divergence time estimates were based on the fol­ Trematocarini and Bathybathini). The observed niche segregation lowing calibration points: LT, 5-6 MY; LM, 0.57-1 MY; LV, among tribes is more readily expected when the tribes emerged <0.2 MY. Recently, a new discussion about the age of East African in the niche-rich lacustrine environment provided by LT at 5 - S. KoblmiiUer et al. f Molecular Phylogenetics and Evolution 49 (2008) 153-169 163

6 MYA, rather than in isolated shallow and presumably swampy rently draining into LT, but apparently connected to the Upper lakes (see Koblmuller et al., in press, for a more detailed discussion). Congo system in the past (Katongo et al., 2005), and the third in­ In view of these considerations, the commonly assumed age of cludes samples from the Zambezi and the Cunene systems. The 5 -6 MY for the primary Tanganyika radiation remains the most split into these three lineages happened roughly 1.8 (1.4­ parsimonious scenario, yielding reasonable age estimates for both 2.3) MYA [alternative dates: 7.4 (5.2-12.0) and 3.3 (2.6­ the LM and LV species flock (see Table 2). In our discussion below 4.6) MYA]. Colonization of the Cunene system apparently we discuss age estimates obtained by applying the assumed cali­ happened only recently, about 0.4 (0.2-0.6) MYA [alternative bration points of 5-6, 0.57-1.0 and <0.2 MY for the LT, LM and dates: 2.0 (0.9-6.4) and 0.7 (0.2-1.2) MYA]. Although we cannot LV cichlid species flocks, respectively, but we also present the propose a detailed scenario for the colonization of other southern alternative estimates based on Genner et al.’s (2007) calibrations African river systems, it is very likely that they have been popu­ [Note that assuming an alternative age of <15,000 years for the lated by representatives of the P. philander species complex in LV species flock (Stager and Johnson, 2008) did not affect the dat­ the recent past. This is also supported by another study that found ing of other cladogenesis events (Table 2 )]. only limited DNA restriction enzyme variation among allopatric, morphologically distinct populations in southern Africa (De Villiers 4.3. Phylo-chronology and biogeographic implications et al., 1992). Thus, the Pseudocrenilabrus philander species complex exhibits a pronounced phylogeographic structure in northern Zam­ 4.3.1. Haplochromis pectoralis bia, with several genetically distinct lineages (see also Katongo et Our phylo-chronological reconstruction of the evolutionary his­ al., 2005), and seems to have colonized southern Africa only re­ tory of the Haplochromini suggests an age of 5.3 (4.9-5.7) MY for cently (De Villiers et al., 1992). The split between P. nicholsi and the branching of H. pectoralis, a species distributed in southeastern P. multicolor happened merely 0.7 (0.4-1.0)MYA [alternative Kenya and northeastern Tanzania, close to the Usambara dates: 2.6 (1.5-3.9) and 1.2 (0.7-1.8) MYA] indicating a rather Mountains, an area considered to be a pleistocene refugium during recent spread towards northern Africa. periods of drier climate (Burgess et al., 1998). Alternative datings for the split between H. pectoralis and the remaining haplochro- 4.5. Astatoreochromis and the ‘‘modern haplochromines" mines would imply an age of 10.4 (9.8-11.8) MY according to the Gondwana calibration, or 7.4 (6.2-8.5) MY assuming an age of 9­ The genus Astatoreochromis, represented by A. alluaudi, has been 12 MY for the primary Tanganyika radiation (fossil calibration). suggested to be the sister group to the ‘‘modern haplochromines" Although a colonization route via the Malagarazi and/or the Lake (Salzburger et al., 2005), since only in these two lineages true Rukwa system might have been possible, we cannot propose an ex­ egg-spots on the males’ anal fins with a yellow, orange or red cen­ act scenario due to a lack of geographically intermediate samples. ter and a colorless or transparent outer ring are found (Greenwood, Thus, a more comprehensive sampling in the area of the Eastern 1979). However, based on DNA data neither Salzburger et al. Arc Mountains, in particular of Tanzanian and Kenyan catchments, (2005) nor this study can unambiguously propose the sister group would be required to elaborate more detailed hypotheses concern­ relationship between these two lineages. Instead, both lineages ing the dispersal of this haplochromine lineage. emerged during a period of rapid lineage formation at the onset of diversification of the Haplochromini, but the presumably syna- 4.4. The undescribed haplochromine from the Lufubu River and the pomorphic egg-spots support the hypothesis of a common origin. Pseudocrenilabrus-clade Currently, three species of Astatoreochromis are known: A. alluaudi occurs in the LV region, A. straeleni in the Lukuga and Ruzizi Rivers, The undescribed haplochromine caught in Lufubu River (discov­ and A. vanderhorsti in the Malagarazi system. This distribution pat­ ered and introduced to the ichthyological community by the late L. tern indicates that the genus Astatoreochromis originated in the De Vos) constitutes a distinct lineage and most likely represents greater area of northern LT and further colonized associated river the sister group to the genus Pseudocrenilabrus. Judged from a pho­ systems. Moreover, there remains the possibility that river capture tograph of Balon and Stewart (1983), this fish is highly similar to a events between the Malagarazi system and the LV drainage have cichlid found in the Luongo River belonging to the Upper Congo facilitated faunal exchange between these two drainages, as indi­ River drainage. Should these ancestral Pseudocrenilabrus-like fish cated by the presence of the squeaker catfish Synodontis victoriae turn out to be the same species, the Lufubu River and the Upper in both systems. Congo system must have been connected with each other very re­ Within the ‘‘modern haplochromines" the split between the cently. Whether or not the ancestral Pseudocrenilabrus-like species tribe Tropheini from Lake Tanganyika and the remaining lineages originated in the Lufubu or the Luongo River, or in any other neigh­ was dated to 3.4 (3.0-4.0) MYA [alternative dates: 16.4 (13.9­ boring catchment, cannot be determined at this stage. The split be­ 20.0) and 7.0 (6.1-9.0) MYA]. It has been suggested that the ances­ tween the new haplochromine species from the Lufubu River and tor of the Tropheini was a riverine haplochromine that re-entered the ‘‘true" Pseudocrenilabrus was dated to 4.4 (3.8-5.0) MYA [alter­ LT to subsequently radiate into several distinct species that now native dates: 18.5 (16.1-21.2) and 8.4 (7.2-10.0) MYA]. Within the dominate the shallow rocky habitat (Salzburger et al., 2005). This Pseudocrenilabrus-clade, the ancestor of P. multicolor and P. nicholsi scenario implies that ancestral members of the tribe Tropheini, branched off from the P. philander species complex, including originally adapted to a riverine environment, would have been able Pseudocrenilabrus machadoi, 2.1 (1.7-2.6)MYA [alternative dates: to out-compete or complement an already established cichlid com­ 8.6 (6.5-12.2) and 3.9 (3.1-4.9) MYA]. This split represents the munity in this lacustrine habitat. We consider this scenario rather divergence of both the northern species P. multicolor, which occurs unlikely, and it seems at least equally plausible, that the Tropheini in the Lake Victoria region and the Nile system up to Egypt, and P. originated in LT. This is supported by an almost simultaneous ori­ nicholsi, which has a very restricted distribution in central eastern gin of all major haplochromine lineages, typical for lacustrine radi­ Congo, from the southern species (or variants) of the P. philander ations, but atypical for riverine assemblages (Joyce et al., 2005), species complex, which are widely distributed from the Upper which could well have taken place within LT. Salzburger et al. Congo system to South Africa. Within the P. philander species com­ (2005) proposed that the Malagarazi River and possibly also the plex, three distinct lineages become evident (see also Katongo et Ruzizi played an important role for a north- and westward dis­ al., 2005): one consists of samples from Lake Mweru, the second persal of the ‘‘modern haplochromines". This hypothesis is also comprises fish from the Lunzua and Lufubu Rivers, two rivers cur­ supported by our data, in that Haplochromis burtoni, which is found 164 S. Koblmuller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169

in LT and associated rivers as well as in Lake Kivu, was placed as bezi drainages is believed to have had a lively geo-morphological sister group to the LV superflock. It is still unclear, how LT cichlids history (Dixey, 1944), where rivers currently connected to the Con­ managed to populate Lake Kivu, but a suggested reversed water go system are assumed to have drained into the Zambezi wa­ flow of the Ruzizi prior to the uplift of the Virunga volcanoes north tershed in the past (Balon and Stewart, 1983), allowing for a of Lake Kivu (Beadle, 1981; Coulter, 1991) might have facilitated southward dispersal of the proto-serranochromines. A recent study this colonization route (Salzburger et al., 2005). It has been men­ (Joyce et al., 2005) provides evidence for an explosive adaptive tioned that Lake Rukwa might have acted as a link between LT radiation of serranochromines into an eco-morphologically diverse and LM (Coulter, 1991), a hypothesis we cannot confirm or reject species flock—comparable to those of the East African Great with our data. Indeed, Coulter’s hypothesis would be difficult to Lakes—that supposedly emerged in Lake paleo-Makgadikgadi, a test without fossils, since it is assumed that Lake Rukwa dried desiccated lake north of the Kalahari Desert, and subsequently out completely in the past, entailing the extinction of its original spread through the southern African rivers. There, a substantial fauna (Coulter, 1991). However, its present fish fauna mainly be­ number of these species has persisted, now representing the rem­ longs to the modern haplochromines, in particular to a lineage of nants of this species flock after the lake dried up about 2000 years East African riverine cichlids and the LV species flock (Seegers, ago, apparently still retaining a considerable portion of the Lake 1996; Nagl et al., 2000; Verheyen et al., 2003), implying a series paleo-Makgadikgadi cichlid diversity (Joyce et al., 2005). Further­ of river capture events that enabled the colonization of Lake Rukwa more, it has been suggested that several species re-entered the by species belonging to the LV superflock. Haplochromis bloyeti is Congo drainage very recently (Katongo et al., 2007) facilitated by probably a species complex of multiple closely related species a recent link between the Zambezi and Congo systems (Leveque, and occurs from the Niger system in the north-west to Tanzanian 1997; Key et al., 2004). Based on our data, a picture congruent with rivers in the south-east (van Oijen et al., 1991), appears to be a previous studies emerges (Joyce et al., 2005; Katongo et al., 2007): widespread representative of a lineage of East African riverine Two lineages that diverged at the onset of the serranochromine cichlids (Werner and Mokady, 2004; Salzburger et al., 2005), and diversification 2.1 (1.5-2.6)MYA [alternative dates: 10.0 (9.8­ exhibits great genetic diversity (Werner and Mokady, 2004). How­ 14.3) and 4.0 (3.0-6.3) MYA] dispersed southwards, diversified fur­ ever, it remains to be shown whether all individuals identified as H. ther and finally seeded the Makgadikgadi radiation. This radiation bloyeti in those studies indeed belong to this species, which was is assumed to have taken place no more than about 0.4 MYA (Joyce originally described from Tanzania. Interestingly, Haplochromis et al., 2005), as indicated by the emergence of a series of haplo- flaviijosephi, a species endemic to the central part of the Jordan sys­ types within a timeframe consistent with the formation of Lake pa- tem, is nested within this group of East African riverine cichlids, leo-Makgadikgadi. Haplochromis oligacanthus and H. polli pointing to a northward dispersal of this lineage via the Nile sys­ constitute a lineage that does not belong to the Makgadikgadi radi­ tem into the Jordan Rift valley between the late Pliocene and the ation. These species are distributed in the Lower Congo River and middle Pleistocene (Werner and Mokady, 2004). Another very re­ in the Sangha and Ubanghi River (tributary to the Lower Congo Riv­ cent dispersal to the north [0.12 (0.00-0.13) MYA; alternative er), respectively, suggesting a northward dispersal of the lineage dates: 0.6 (0.0-0.6) and 0.3 (0.00-0.3)] is indicated by the cluster­ possibly right after diverging from the remaining serranochro- ing of Haplochromis sp. ‘‘El Fayoum”, a species found in northern mines 1.9 (1.4-2.4)MYA [alternative dates: 9.0 (8.8-13.8) and Egypt, with H. sp. ‘‘Mburo Black” and H. squamipinnis from the Lake 3.6 (3.0-6.0) MYA]. Two species of Serranochromines that have Edward/Lake Albert region, again pointing to a colonization via the not been included in any of the phylogeographic studies so far Nile. Assuming a maximum age of 15,000 years for the LV species would be of great importance to get a deeper insight into the evo­ flock, this split is dated to an age of 0.11 (0.00-0.13) MY. The bio­ lutionary history of these cichlids: Serranochromis spei from the diversity in the Nile is assumed to have been severely reduced dur­ eastern Kasai and Lualaba drainages and S. janus from the Malagar- ing the dry period of the late Pleistocene. During the wet period in azi River. In particular, the phylogenetic placement of S. janus early Holocene, immigration of fishes from refuge areas such as would be of great interest; to date it is not clear how this species Lake Kivu (Verheyen et al., 2003) took place, facilitated by high managed to enter the Malagarazi River. Since S. robustus, which water levels in lakes and drainage connections between rivers that is closely related to S. thumbergi (Katongo et al., 2007), is currently are now completely separated (Livingstone et al., 1982; Stewart, found also in LM, and several Serranochromis species occur in the 2001). Chambeshi River (currently Upper Congo system), colonization scenarios via LM or the Chambeshi River and the Lake Rukwa re­ 4.6. The Serranochromis-like haplochromines gion into the Malagarazi River seem most plausible.

Within the serranochromines sensu lato, the three main lineages 4.7. Comparison with previous haplochromine divergence time arose almost simultaneously about 4.1 (3.7-4.9) MYA [alternative estimates dates: 18.1 (16.5-21.1) and 8.0 (7.2-9.9) MYA]. Schwetzochromis neodon, Cyclopharynx fw ae and Haplochromis brauschi, which con­ Salzburger et al. (2005) reported considerably younger ages for stitute one lineage, are restricted to the Fwa River (tributary to the ancestral splits in the Haplochromini. This might be due to the Sankuru River; Kasai system). The representative of the second their choice of calibration points: they did not use the ‘‘primary lineage, H. polyacanthus (or H. stormsi; see above), is mainly found Tanganyika radiation” as a calibration point, but instead con­ in the Upper Congo basin between Kisangani and Lake Mweru but strained the age of the serranochromines with the time window has also been reported from the Lower Congo River as well. The for the Lukuga connection between LT and the Congo system species of the third lineage are distributed in whole southern Afri­ (3.5-1.1 MYA; Lezzar et al., 1996; Cohen et al., 1997). Our data, ca, including the Congo system, LM and the Malagarazi River. Our however, indicate an older origin for the serranochromine clade data indicate that the ancestor of the Serranochromis-like cichlids (see above). Furthermore, Salzburger et al. (2005) assumed a has occurred in the Congo system. More samples, especially from strictly clock-like model of evolution, whereas we detected a sig­ the upper reaches of rivers belonging to the upper Kasai system, nificant deviation from clock-like evolution in the haplochromines are needed to determine an exact colonization scenario. and thus used the penalized likelihood approach to obtain age esti­ Recent studies have shown a complex evolutionary history for mates. Indeed, when applying the molecular-clock method to our the serranochromine cichlids sensu stricto (Joyce et al., 2005; Kat- data and using the constraints 0.57-1 MY and <0.2 MY for the ongo et al., 2007). The border zone between the Congo and Zam­ LM and LV species flocks, respectively, we obtained age estimates S. Koblmuller et al. / Molecular Phylogenetics and Evolution 49 (2008) 153-169 165 similar to those presented by Salzburger et al. (2005) (data not tions and re-colonization from refuge areas (Livingstone et al., shown). 1982; Stewart, 2001). River capture events might have facilitated the colonization of new water bodies, which would explain the 5. Conclusions occurrence of certain species in different rivers systems (see e.g. Waters and Cambray, 1997; Giddelo et al., 2002; Koblmuller Our phylogenetic analysis of the haplochromine cichlids re­ et al., 2006 for discussion on the impact of river capture events vealed the existence of six major mitochondrial lineages, two of on the distribution of African non-cichlid fishes). We emphasize which were not included in the previous study of Salzburger the urgent need of denser geographic sampling, especially from et al. (2005): H. pectoralis (distributed around the East African different drainage systems in the south eastern Congo basin, An­ arc mountains), and an undescribed haplochromine species from gola and around the East African Arc Mountains, in order to the upper Lufubu River, which currently drains into Lake Tang­ reconstruct the biogeographic history and evolution of the most anyika. All six major haplochromine lineages originated in a per­ speciose cichlid lineage. iod of rapid cladogenesis about 5.3-4.4 MYA. Nevertheless, our mtDNA data suggest H. pectoralis as the most ancestral haplochro- Acknowledgments mine lineage. A monophylum including the genus Astatoreochr- omis and the ‘‘modern haplochromines”, as suggested by We thank W. Salzburger, E. Schraml, L. Seegers, O. Seehausen Salzburger et al. (2005) and indicated by the well developed and F. van der Bank for providing DNA samples, and the team egg-spots on the anal fin displayed in both lineages could not at the Mpulungu Station of the Ministry of Agriculture, Food be confirmed. Within the ”modern haplochromines”, the Trophe- and Fisheries, Republic of Zambia for their cooperation during ini from Lake Tanganyika form the sister group to the species fieldwork. We thank W. Wickler and Max-Planck-Society for sup­ flocks from Lake Malawi and the lake Victoria region as well as porting a fieldtrip of U.K.S. This study was funded by the Austrian some riverine taxa. Interestingly, the serranochromines and the Science Foundation (Grant P17680 to C.S.). S.K. received a DOC genus Pseudocrenilabrus share a similar dispersal pattern. Both fellowship and N.D. a DOC-FFORTE (Women in Research and lineages started to diversify in the upper Congo system, and sub­ Technology) fellowship, both provided by the Austrian Academy sequently expanded their distribution range to the south (south­ of Sciences. K.M.S. was supported by the Austrian Science Foun­ ern Africa) and north (serranochromines: lower Congo and dation (Grant P17380) and N.D. by an Erwin-Schrodinger scholar­ associated rivers; Pseudocrenilabrus: central Congo and Nile sys­ ship, also from the Austrian Science Foundation. C.K. was tem). Our analysis implicates a complex phylogeographic history, supported by a scholarship from the OAD, Austrian Ministry of which was probably affected by climatically and/or geologically Foreign Affairs and also by funds from the Royal Museum of Cen­ induced changes of the environment, associated with local extinc­ tral Africa, Belgium.

Appendix A

List of samples examined in this study, with distribution range, sampling locality (if known) and GenBank Accession Numbers

Species Distribution range Sampling locality Accession No. ND2 cytb Astatoreochromis alluaudi Lake Victoria, Kyoga, Edward, ? AF398234 AF428157 George—region Lake Kanyaboli, Kenya EU753923 EU753872 Chetia brevicauda Buzi River system Mussopa River near Zomba, South Africa EU753924 EU753873 Chetia brevis Incomati River system Driekoppies Dam near Komatipoort, South EU753925 EU753874 Africa Chetia flaviventris Limpopo River system Roodeplaat Dam near Pretoria, South EU753926 EU753875 Africa Roodeplaaat Dam near Pretoria, South EU753927 EU753876 Africa Cyclopharynx fwae Lake Fwa Lake Fwa, DRC AY930099 AF428158 Gnathochromis pfefferi Lake Tanganyika LT U07248 AF428166 Haplochromine sp. nov. ? Lufubu River, Zambia EU753928 EU753877 Haplochromis albolabris Cunene system Cunene River at Ruacana, Namibia EU753929 EU753878 Haplochromis bloyeti Rivers, streams and certain lakes in Nyumba ya Mungu, Tanzania EU753930 EU753879 Kenya, Uganda, Tanzania; Niger system in Mali; Lake Chad; Nile Haplochromis brauschi Lake Fwa Lake Fwa, DRC EU753931 EU753880 Haplochromis burtoni Lake Tanganyika and associated Kalambo River (above falls), Zambia EU753932 EU753881 rivers; Aquarium stock AF317266 Z21773 Lake Kivu Haplochromis buysi Cunene River system Cunene River at Ruacana, Namibia EU753933 EU753882 Haplochromis calliptera Lake Malawi region; Lower Zambezi, Lake Kisiba, Masoko, Tanzania EU753934 EU753883 Buzi, Pungwe and Save River systems Haplochromis horei Lake Tanganyika LT EU753935EU753884 Haplochromis machadoi Cunene River system Cunene River at Ruacana, Namibia EU753936 EU753885 (continued on next page) 166 S. Koblmuller et at./Molecular Phylogenetics and Evolution 49 (2008) 153-169

Appendix A (continued) Species Distribution range Sampling localityAcc. Nr. Accession No. ND2 cytb Haplochromis oligacanthus Ubanghi River system (tributary Ngoko River at Moloundou, Congo EU753937 EU753886 to the lower Congo River) Haplochromis pectoralis south eastern Tanzania; possibly Nyumba ya Mungu, Tanzania EU753938 EU753887 also Kenya Nyumba ya Mungu, Tanzania EU753939 EU753888 Haplochromis phytophagus Lake Kanyaboli Lake Kanyaboli, Kenya EU753940 EU753889 Haplochromis polli Lower Congo River Commercial import from Kinshasa, DRC EU753941 EU753890 Haplochromis polyacanthusa Upper Congo basin (Lake Mweru) Commercial import from Kinshasa, DRC AF398231 AF428159 Haplochromis rudolfianus Lake Turkana Lake Turkana, Loyangalani, Kenya EU753942 EU753891 Haplochromis squamipinnis Lakes Edward and George and Lake Edward, Uganda EU753943 EU753892 Kazinga Channel Haplochromis sp. Lake Kanyaboli Lake Kanyaboli, Kenya EU753944 EU753893 Haplochromis sp. ‘‘El El Fayoum Oasis El Fayoum Oasis, Egypt EU753945 EU753894 Fayoum" Haplochromis sp. ‘‘Mburo Lake Mburo Lake Mburo, Uganda EU753946 EU753895 Black" Labidochromis caeruleus Lake Malawi Aquarium stock AY740383 EU753896 Lobochilotes labiatus Lake Tanganyika Lake Tanganyika U07254 AF428170 Nimbochromis livingstoni Lake Malawi Aquarium stock EU753948 EU753897 Nimbochromis venustus Lake Malawi Aquarium stock EU753947 EU753898 Pharyngochromis acuticeps upper/middle Zambezi River system Sinazongwe, Zambia EF393692 EU753899 Rundu, Namibia EU753949 EU753900 Pseudocrenilabrus multicolor Nile River system; Lake Albert, Aquarium stock AY602992 AY600141 George, Kyoga, Victoria - region Pseudocrenilabrus nicholsi East central Congo basin Aquarium stock AY602994 AY600143 Pseudocrenilabrus philander Members of the Pseudocrenilabrus Kabala, Zambia AY602993 AY600142 Pseudocrenilabrus sp. philander species complex are Lufubu, Zambia EU753950 EU753901 Pseudocrenilabrus sp. distributed Lunzua, Zambia EU753951 EU753902 Pseudocrenilabrus sp. from the Upper Congo River system Lake Mweru; Zambia EU753952 EU753903 Pseudocrenilabrus sp. southwards to South Africa Olushandja, Namibia EU753953 EU753904 Pseudotropheus tropheops Lake Malawi Aquarium stock AY740384 EU753905 Sargochromis carlottae Okavango, upper/middle Zambezi Kafue Flats, Zambia EF393682 EU753906 River systems Sargochromis coulteri Cunene River system Olushandja, Namibia EU753954 EU753907 Olushandja, Namibia EU753955 EU753908 Sargochromis giardi Cunene, Okavango, upper/middle Mongu, Zambia EF393714 EU753909 Zambezi River systems Sargochromis mellandi Upper Congo River system Mifimbo, Lake Mweru, Zambia EF393701 EU753910 (Chambeshi and Luapula River, Lake Mweru) Sargochromis sp. aff. ? Okavango River, Namibia EU753956 EU753911 carlottae Schwetzochromis neodon Fwa River Lake Fwa, DRC EU753957 EU753912 Serranochromis altus Okanvango, upper Zambezi River Mambova, Zambia EF393696 EU753913 systems Serranochromis angusticeps Cunene, Okavango, upper/middle Mongu, Zambia EF393709 EU753914 Zambezi, upper Congo (Luapula- Cunene River at Ruacana, Namibia EU753958 EU753915 Mweru) Cunene River at Ruacana, Namibia EU753959 EU753916 Rivers systems Serranochromis Cunene, Okavango, upper/middle Mongu, Zambia EF393705 EU753917 macrocephalus Zambezi, upper Congo (Luapula- Mweru, Lulua, Kasai) River systems Serranochromis stappersi Lake Mweru, Luapula River Mifimbo, Lake Mweru, Zambia EF393698 EU753918 Mifimbo, Lake Mweru, Zambia EU753960 EU753919 Serranochromis thumbergi Cunene, Limpopo, Okavango, Samfya, Lake Bangweulu, Zambia EF393703 EU753920 upper/middle Zambezi, upper Congo ? EU753961 EU753921 River systems Tropheus moorii Lake Tanganyika Aquarium stock U07267 Z12044 S. Koblmuller et at. / Molecular Phylogenetics and Evolution 49 (2008) 153-169 167

Appendix A (continued) Species Distribution range Sampling localityAcc. Nr. Accession No. ND2 cytb Outgroups Callochromis macrops Lake Tanganyika Lake Tanganyika AY337795 AY337851 Ophthalmotilapia ventralis Lake Tanganyika Lake Tanganyika U07257 Z21771 Xenotilapia sima Lake Tanganyika Aquarium stock U07270 AY337802 Orthochromis Malagarazi River system Malagarazi River, Tanzania AF398232 AF428161 malagarazensis Orthochromis mazimeroensis Upper Malagarazi River system Mazimero River, Burundi AY930053 AF428162 Benthochromis melanoides Lake Tanganyika Lake Tanganyika AY682512 EU753922 Benthochromis tricoti Lake Tanganyika Lake Tanganyika EU753962 AF428164 Perissodus microlepis Lake Tanganyika Lake Tanganyika AF398222 AF428167 Plecodus straeleni Lake Tanganyika Lake Tanganyika AF398221 Z21777 Cyprichromis leptosoma Lake Tanganyika Lake Tanganyika AY740336 AY740185 Paracyprichromis brieni Lake Tanganyika Lake Tanganyika AF398223 Z21776 Limnochromis auritus Lake Tanganyika Lake Tanganyika AY337766 Z21775 Triglachromis otostigma Lake Tanganyika Lake Tanganyika AF398217 Z30004 Cyphotilapia frontosa Lake Tanganyika Aquarium stock U07247 AF428169 Altolamprologus Lake Tanganyika Lake Tanganyika EF191107 AF428163 compressiceps Lamprologus callipterus Lake Tanganyika Lake Tanganyika AF398226 Z29992 Lamprologus teugelsib Lower Congo River system Aquarium stock AY740385 Z29993 Neolamprologus brichardi Lake Tanganyika Lake Tanganyika AF398227 Z29997 Palaeolamprologus toae Lake Tanganyika Lake Tanganyika AY682543 Z30003 Variabilichromis moorii Lake Tanganyika Lake Tanganyika DQ055016 AF438795 Eretmodus cyanostictus Lake Tanganyika Lake Tanganyika DQ055010 AF428155 Spathodus erythrodon Lake Tanganyika Lake Tanganyika AF398218 AF428156 Tanganicodus irsacae Lake Tanganyika Lake Tanganyika DQ055007 Z21779

Note. That the taxonomy follows CLOFFA (van Oijen et al., 1991) by placing all haplochromine cichlids formerly placed in the genera Astatotilapia, Ctenochromis, Orthochromis, Schwetzochromis (partim), Thoracochromis and Xystichromis in Haplochromis. However, we retain the highly distinct type species of Schwetzochromis, S. neodon in that genus, as well as the Orthochromis of the Lake Tanganyika drainage in Orthochromis, as they are not closely related to the remaining Haplochromini (Salzburger et al., 2005; de Vos and Seegers, 1998). Sequences not generated in the framework of this study were obtained from GeneBank from following publications: Sturmbauer and Meyer (1992,1993), Sturmbauer et al. (1994), Kocher et al. (1995), Klett and Meyer (2002), Salzburger et al. (2002,2005), Koblmuller et al. (2004,2007b), Brandstatter et al. (2005), Duftner et al. (2005), Katongo et al. (2005, 2007), Schelly et al. (2006). a It must be noted, that there is a taxonomic problem with H. polyacanthus, which was previously synonymized with H. stormsi. This is reviewed by Greenwood and Kullander (1994), who revealed that H. polyacanthus is limited to Lake Mweru. The geographic origin of our sample is unknown, and it might be a different species of the genus Haplochromis, most likely H. stormsi. The same sample was used in Salzburger et al. (2005). b In previous publications (Sturmbauer et al., 1994; Salzburger et al., 2002) this species was identified as L. congoensis; description of L. teugelsi by Schelly and Stiassny (2004).

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