MBE Advance Access originally published online on March 15, 2007
Molecular Biology and Evolution 2007 24(5):1269-1282; doi:10.1093/molbev/msm050
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Research Articles |
Age of Cichlids: New Dates for Ancient Lake Fish Radiations
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* Department of Biological Sciences, University of Hull, Cottingham Road, Hull HU6 7RX, United Kingdom
Institute of Zoology—Aquatic Ecology and Macroevolution, University of Bern, Baltzerstrasse 6, CH-3012 Bern, Switzerland
EAWAG Ecology Center, Fish Ecology and Evolution, CH-6047 Kastanienbaum, Switzerland
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, United Kingdom
|| School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, United Kingdom
E-mail: m.genner{at}hull.ac.uk.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Timing divergence events allow us to infer the conditions under which biodiversity has evolved and gain important insights into the mechanisms driving evolution. Cichlid fishes are a model system for studying speciation and adaptive radiation, yet, we have lacked reliable timescales for their evolution. Phylogenetic reconstructions are consistent with cichlid origins prior to Gondwanan landmass fragmentation 121–165 MYA, considerably earlier than the first known fossil cichlids (Eocene). We examined the timing of cichlid evolution using a relaxed molecular clock calibrated with geological estimates for the ages of 1) Gondwanan fragmentation and 2) cichlid fossils. Timescales of cichlid evolution derived from fossil-dated phylogenies of other bony fishes most closely matched those suggested by Gondwanan breakup calibrations, suggesting the Eocene origins and marine dispersal implied by the cichlid fossil record may be due to its incompleteness. Using Gondwanan calibrations, we found accumulation of genetic diversity within the radiating lineages of the African Lakes Malawi, Victoria and Barombi Mbo, and Palaeolake Makgadikgadi began around or after the time of lake basin formation. These calibrations also suggest Lake Tanganyika was colonized independently by the major radiating cichlid tribes that then began to accumulate genetic diversity thereafter. These results contrast with the widely accepted theory that diversification into major lineages took place within the Tanganyika basin. Together, this evidence suggests that ancient lake habitats have played a key role in generating and maintaining diversity within radiating lineages and also that lakes may have captured preexisting cichlid diversity from multiple sources from which adaptive radiations have evolved.
Key Words: molecular clock • Gondwanan fragmentation • fossil record • adaptive radiation • speciation
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Cichlid fishes are the only freshwater representatives of the suborder Labroidei and are naturally distributed across Africa, Madagascar, South and Central America, the Middle East, and the Indian subcontinent. Their species richness and diversity of morphology, color, and behavior has made them model organisms for the study of speciation and adaptive evolution (Kocher 2004
). However, dates of cichlid diversifications and major geological events associated with present distributions are poorly resolved. Molecular phylogenies have shown that cichlids are monophyletic (Streelman and Karl 1997; Sparks and Smith 2004) and that cichlid faunas of major biogeographical regions split in a chronological order congruent to the breakup of the Gondwanan landmass (Sparks and Smith 2004, 2005). Thus, it has been proposed that cichlids originated during the Late Jurassic or Early Cretaceous and had a Mesozoic Gondwanan distribution that started to fragment as Madagascar–India broke away from South America–Africa 121–165 MYA (Sparks and Smith 2005).
However, the oldest known fossil cichlids are Mahengechromis from Eocene Tanzania, dated to 
46 MYA (Murray 2001a), and Proterocara from Eocene Argentina (33.9–55.8 MYA) (Malabarba et al. 2006), whereas the earliest fossil labroids have been dated to around 65 MYA (Lundberg 1993). Moreover, few spiny-rayed teleost fish (Acanthomorpha) fossils are known between their first appearances in the Late Cretaceous and the Late Palaeocene/Early Eocene when they became common in the fossil record, exhibiting extensive diversity in morphology (Patterson 1993; Chakrabarty 2004). If the earliest cichlid fossils are approximately coincident with the origin of the family, their present distribution must be explained by intercontinental marine dispersal and not vicariance during the breakup of Gondwana (Vences et al. 2001).
The cichlid fishes of African lakes are particularly well known for their rapid speciation and extensive adaptive radiation. Reliable molecular clock estimates could help estimate the extent to which speciation and adaptive diversification have been dependent on the presence of long-lasting lacustrine habitats, if the onset of radiation occurred at around the time of entering a lacustrine habitat, whether divergence was initiated in surrounding drainages from where lakes were colonized multiple times, or whether recent desiccation events were associated with phases of diversification. Addressing these questions is of fundamental importance for understanding the causes and dynamics of adaptive radiation in this textbook evolutionary system.
Until now, attempts to date African cichlid evolutionary events have been predominantly based on rates of molecular evolution extrapolated from mammals (Meyer et al. 1990), assumptions of monophyletic origins of cichlid radiations within lake catchments of known age (Vences et al. 2001), or assumptions of lineage splitting being synchronized with lake-level changes (Sturmbauer et al. 2001; Verheyen et al. 2003). Although these published date estimates are informative, they do have the potential to be misleading because of inconsistencies in rates of molecular evolution among evolutionary lineages, uncertainties over the timing, extent and biological implications of lake-level changes, and the possibility that lake radiations may have been founded by multiple colonizers. As a consequence, many calibrations may be required. Here, we estimate the timing of divergence among the major cichlid taxa (subfamilies and tribes), employing calibration points first from the cichlid fossil record and second from the fragmentation of Gondwana. We then compare these with rates estimated using a range of relatively well-dated non-cichlid fish fossils. Finally, we use the dates derived from the cichlid fossil record and Gondwana fragmentation calibrations to estimate the timing of molecular divergence in radiating African cichlid clades.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Dating Global Cichlid Divergence
From published records, the earliest known fossils of extant cichlid taxa were identified, and from published phylogenies, extant sister lineages of fossil taxa were identified. Phylogenetic relationships of these, together with representatives of all major cichlid lineages and 2 outgroups from the related family Pomacentridae, were analyzed using DNA sequences from 2 mitochondrial genes (cytochrome b and 16S) and one nuclear gene (TMO-4C4).
Sequences not already available on GenBank were generated for this study. DNA was isolated from ethanol-preserved fin tissue using the CTAB–chloroform method. Primers used were TMO-4C4F and TMO-4C4R (Streelman et al. 2002), 16SAR and 16SBR (Palumbi et al. 1991), and L14724
[GenBank]
and H15149
[GenBank]
(Kocher et al. 1989). All polymerase chain reactions (PCRs) were performed in 25 µl reactions including 1 µl genomic DNA, 2.5 µl 10x PCR buffer, 2.5 µl dNTPs (1 mM), 1 µl each primer (10 mM stock), 1 µl MgCl2 (25 mM stock), 0.5 units Taq, and 14.9 µl double-distilled water. For all primers, PCR conditions were as follows: 1 min at 95 °C; then 34 cycles of 95 °C for 30 s, 43 °C for 30 s, and 72 °C for 1 min followed by 72 °C for 5 min. Cleaned PCR products were directly sequenced using a Beckman CEQ sequencer and Quickstart cycle sequencing kits (Beckman Coulter, Fullerton, CA) according to manufacturer's protocol. New sequences have GenBank accession numbers EF470866
[GenBank]
–EF470904
[GenBank]
(Supporting Information S1, Supplementary Material online).
Sequences were aligned using ClustalW in Dambe (Xia and Xie 2001). Phylogenetic trees were reconstructed using Neighbor-Joining (NJ) in PAUP*4.0b10 (Swofford 2002), maximum likelihood (ML) in PhyML (Guindon and Gascuel 2003), and Bayesian inference (BI) using MrBayes 3.04 (Huelsenbeck and Ronquist 2001). Prior to analyses, MrModeltest 1.1b (http://www.abc.se/~nylander) was used to determine the best-fitting model of sequence evolution for the concatenated data, and the GTR + 
+ I model was selected. NJ was undertaken using ML distances with parameters inferred from MrModeltest. Branch support for ML and NJ was calculated as bootstrap percentages of 1,000 replicates. BI analyses were performed with the GTR + + I model using 4 Markov chains and 5,000,000 generations, a burn-in of 10% was discarded.
Branches not supported by >70% in BI and >50% in ML and NJ were collapsed resulting in a conservative dating topology (fig. 1). First, we employed 3 Gondwanan fragmentation events as calibration points. Following Vences et al. (2001), we used the separation of East and West Gondwana 121–165 MYA, the separation of Africa from South America 86–101 MYA, and the separation of Madagascar and India 63–88 MYA. Second, we employed 8 cichlid fossil calibrations (table 1 and fig. 2). Calculations followed a Bayesian "relaxed-clock" approach using PAML (Yang 1997), Multidivtime (Thorne and Kishino 2002), and the procedures outlined by Rutschmann (2004). Briefly, we used the F84 + model with parameters estimated using BaseML in PAML, Paml2modelinf to generate the input files for Estbranches program, and Multidivtime to generate estimated dates of lineage divergence using all genes combined. The F84 + distances for each gene showed strong linear correlations with distances from the best-fitting models selected by MrModeltest (Supporting Information S3, Supplementary Material online), indicating the F84 + model was appropriate. The earliest and latest dates of all calibration nodes were treated as hard constraints. In all, 5,000 samples of the chain were taken with 10 cycles between each sampling following a 10,000-cycle burn-in. Expected time between tip and root was 1 (=100 MYA) (standard deviation [SD] = 1), and rate at the root was 0.5 (SD = 0.5). Largest possible time between tip and root was 3 (=300 MYA). Other parameters were as default.
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Comparing Rates of Molecular Evolution of Cichlids and Other Bony Fishes
Dating of cichlid divergence events using first Gondwanan fragmentation and second cichlid fossils produced 2 distinct scenarios of cichlid evolution (see Results and Discussion). Thus, we aimed to determine which set of evolutionary rates most closely matched those suggested by 6 independently dated phylogenies of bony fishes. One of these phylogenies was a topology with fossil calibrations (Benton and Donoghue 2007
), whereas the remaining 5 were fossil-dated phylogenies based on mitochondrial genomic data (Inoue et al. 2005; Yamanoue et al. 2006; Hurley et al. 2007) or nuclear sequence data (Steinke et al. 2006; Hurley et al. 2007). We incorporated our global cichlid topology (fig. 1) into the published topologies, and aligned cytochrome b and 16S mitochondrial DNA sequences from all taxa included in each phylogeny. We then dated cichlid divergence events using the published divergence times of non-cichlids as calibration points and the Bayesian "relaxed-clock" method described above. The priority for the largest possible time between tip and root was set to 5 (=500 MYA) for the data sets of Inoue et al. (2005), Yamanoue et al. (2006), and Hurley et al. (2007). For the Benton and Donoghue (2007) fossil dates, we treated both earliest and latest dates of each calibration node as hard constraints, and for the remainder of the dated phylogenies, we employed published 95% confidence or credibility intervals for each calibration node as hard constraints. Two sets of dates were available for each of the phylogenies of Inoue et al. (2005) and Yamanoue et al. (2006), so here we used each set separately. Finally, we compared rates of evolution between 1) cichlid and non-cichlid teleosts represented in the 6 previously dated phylogenies and 2) cichlids and other labroid families, by compiling published TMO-4C4, cytochrome b, and 16S sequences from GenBank and using RRTree (Robinson-Rechavi and Huchon 2000).
Dating African Cichlid Radiations
We compiled mitochondrial DNA sequences selecting widely sampled genes with broad within-radiation taxonomic coverage and including as many taxa in the global topology as possible (Supporting Information S5, Supplementary Material online). Three genes, NADH2, cytochrome b, and the control region (D-loop), were concatenated for the Lake Barombi Mbo and Tanganyika radiations, whereas control region alone was employed for the Lake Malawi, serranochromine, and Lake Victoria Region haplochromine radiations (Supporting Information S4, Supplementary Material online). Polymorphisms at individual nuclear genes tend to either be limited both within and between haplochromine radiations (Sültmann et al. 1995; Mayer et al. 1998; Booton et al. 1999; Won et al. 2006) or lack lineage sorting (Nagl et al. 1998; Terai et al. 2002). Hence, the more rapidly evolving and faster coalescing mitochondrial genes may provide the best available means of dating haplochromine divergence events. We aligned these "African sequence sets" in MUSCLE (Edgar 2004). Phylogenies were constructed using NJ methods in PAUP* using uncorrected P distances. Trees were rooted using the most ancient cichlid lineages identified from the global topology (fig. 1). Resultant topologies were highly congruent with published phylogenies.
We then applied a novel dating strategy to each African sequence set independently. First, we calculated mean P distances between pairs of taxa in the African sequence set that were also present in the dated global phylogeny. We then identified the nodes that each pair of taxa converged upon in the dated global phylogeny, enabling us to calculate mean P distance for these nodes using the African sequence set genes. Time dependency of rates of molecular evolution can influence calculations of divergence times, particularly of more recent divergences (Ho et al. 2005; Ho and Larson 2006). Thus, for the mitochondrial DNA control region, we also employed a series of 10 post-Pliocene calibration points from biogeographic events estimated to have occurred between 5,000 and 50,000 years ago (table 3). We next plotted genetic distances against estimated ages using dates estimated first from cichlid fossils and second from Gondwanan fragmentation (fig. 5). The power curves fitted to these relationships (all r2 > 0.95) enabled us to extrapolate dates from our global phylogeny to all pairwise divergences in the African sets. We then identified taxa converging at nodes in African sequence set topologies and calculated node dates. Species relationships and basal nodes proved difficult to identify within the haplochromine radiations, as a result of extensive incomplete mitochondrial DNA interspecific lineage sorting. In these cases, we examined mean differences among the unique haplotypes within clades (table 6).
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Mapping Lake Formation on African Cichlid Phylogeny
We reconstructed a phylogram of African cichlids using a 1,047 bp alignment of 438 mitochondrial NADH2 mitochondrial DNA sequences using PhyML. Branches not supported by >50% bootstrap support were collapsed. Node dates were then placed using combined information from first the global phylogeny and then the ages of the radiations calculated using the African sequence sets. Where dates for nodes were not available, these were estimated using the same power curve extrapolation procedure used for dating the main African sequence sets. For clarity, well-supported monophyletic clades were collapsed at basal nodes. Periods of lake formation were taken from the literature.
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Results and Discussion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Global Diversification
Our recovered topology of the deeper level evolutionary relationships of cichlids using 2 mitochondrial genes (16S and cytochrome b) and 1 nuclear gene (TMO-4C4) (fig. 1) was congruent with those of prior analyses (Sparks and Smith 2004
), except for the relationships among Hemichromis, Tylochromis, Pelvicachomis, and Pelmatochromis. Using calibrations based on the fragmentation of Gondwana, we calculated cichlid origins at 133.2 MYA (95% credibility interval: 122.5–151.8 MYA). This was well over twice the age estimate obtained from cichlid fossil calibrations, which indicated African Eocene origins 46.0 MYA (95% credibility interval: 45.7–46.3 MYA) (fig. 2 and table 2).
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Because the Gondwanan and cichlid fossil calibrations predict such radically different scenarios for the diversification of cichlids, we have also analyzed 6 independently calibrated data sets. All suggested cichlids originated during the Early Cretaceous and thus showed a much closer agreement with dates calculated using the Gondwanan-calibrated cichlid clock than with those estimated from cichlid fossils (table 2 and fig. 3). In light of this, we conclude there is good reason to reassess both the timescale and nature of the evolutionary divergence process in cichlids. Moreover, relative rate tests yielded no evidence of different rates of sequence evolution in cichlids compared with the teleosts included in the 6 published dated phylogenies or to other labroid families (Supporting Information S2, Supplementary Material online). These results, together with evidence that the Gondwanan landmass fragmented in the same chronological order as cichlid phylogenetic reconstructions, support Early Cretaceous cichlid origins. These results also strongly suggest that reliance on the known cichlid fossil record leads to substantial underestimation of divergence times.
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Divergence dates derived from the fragmentation of Gondwana imply a gap in the cichlid fossil record as wide as 90 Myr. Nevertheless, in principle, the fossil record is not inconsistent with Cretaceous cichlid origins. The time disparity may be explained by the incomplete nature of the fossil record. Fossils can only provide hard minimum divergence dates (Heads 2005
; Benton and Donoghue 2007), and the cichlid fossils we used as calibrations may represent late first occurrences of the taxon within the palaeontological record. In support of this hypothesis is the substantial decay in numbers of cichlid fossil discoveries with increasing age suggesting a low probability of finding pre-Oligocene cichlid fossils (fig. 4). Importantly, the reliability of cichlid fossil dates also depends upon accurate identification. We employed only fossils attributed to extant lineages as calibrations. It is possible that extinct fossil genera, such as Palaeofulu from the Early Miocene of Kenya and Macfadyena from the Oligocene of Somalia (Van Couvering 1982), actually belong to extant lineages that may have diverged earlier than would be supposed on the basis of more reliably assigned fossils. The use of non-cichlid bony fishes in dating divergence events has the potential to increase accuracy by providing more well-identified and dated fossils for use as calibration points, plausibly explaining why cichlid divergence dates calibrated using other bony fish fossils show a closer match to those suggested by the Gondwanan vicariance calibrations. Divergence dates estimated from cichlid fossils would be supported by convincing evidence for post-Oligocene marine dispersal, but none has been found (Chakrabarty 2004, 2006; Sparks and Smith 2005), despite saline-tolerant, brackish water taxa being known from a number of cichlid lineages, including the Etroplinae, Geophagini, Hemichromini, and Tilapiini. The presence of Etropline cichlids on both Sri Lanka and India provides no evidence of marine dispersal because terrestrial connections were present as recently as 10,000 years ago (Bossuyt et al. 2004).
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Accumulation of Genetic Diversity within African Radiations
Lake Tanganyika contains the highest diversity of ancient lacustrine cichlid lineages. The basin is believed to have started to form around 20 MYA, initially as an extensive swampland (Tiercelin and Mondeguer 1991
), but attaining deep-lake conditions some 6–12 MYA (Cohen et al. 1993). Calibrations based on cichlid fossils yielded dates of basal nodes (table 4, fig. 6) consistent with suggestions that the cichlids began radiating early during formation of deep-lake conditions into a few surviving taxa now represented as distinct tribes (Salzburger et al. 2002, 2005). By contrast, dates derived from Gondwanan fragmentation indicate that ancestors of every major tribe entered the lake independently and that molecular diversity within some tribes began to accumulate around the time of colonization, or indeed in 2 cases (Trematocarini and Ectodini) possibly before colonization (table 4 and fig. 6). The Gondwanan estimates also suggest that the Haplochromini may have been split into several lineages already prior to the formation of deep-water conditions in Lake Tanganyika and that at least 2 haplochromine lineages colonized the basin independently prior to initial rifting. The first lineage, Pseudocrenilabrus, has one species in the catchment. The second lineage contains Tropheini (a group endemic to the lake basin that has radiated into a large number of species/geographic races), Astatoreochromis (2 species in the lake) and Astatotilapia (2 species in the lake). The latter genus includes Astatotilapia burtoni, from a relatively deep East African haplochromine mtDNA lineage (fig. 2), and Astatotilapia stappersi, a member of the Lake Victoria Region superflock mtDNA lineage (fig. 2)(Salzburger et al. 2005).
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The tilapiine radiation of the crater lake Barombi Mbo comprises 11 known species in 4 genera. Gondwanan fragmentation and cichlid fossil calibrations indicate this radiation started to accumulate molecular diversity 0.47 (±0.19 SD) and 0.19 (±0.09 SD) MYA, respectively. The lake is estimated to be approximately 1 Myr old (Cornen et al. 1992
), and so both sets of dates are consistent with the hypothesis of within-catchment diversification (Schliewen and Klee 2004) (table 4 and fig. 6).
The southern Africa serranochromines, the haplochromines of Lake Malawi, and the haplochromines of the Lake Victoria region were dated using sequences from the rapidly evolving mitochondrial control region, including calibration from post-Pliocene divergence events (table 3). Like previous studies (Ho et al. 2005; Ho and Larson 2006), we found that rates of molecular change showed a sharp decline with increasing divergence time until a baseline substitution rate was reached after a divergence time of approximately 1,000,000 years (fig. 5). This pattern has been attributed to purifying selection and/or saturation at mutational "hot spots" along rapidly evolving sequences (Ho et al. 2005; Ho and Larson 2006).
The onset of accumulation of molecular diversity within the Lake Malawi haplochromine radiation, comprising 450–600 species in around 50 genera, was dated to 4.63 (±2.14 SD) and 2.44 (±1.01 SD) MYA using Gondwanan fragmentation and cichlid fossil calibrations, respectively. Both estimates support the hypothesis that the flock radiated within the timescale of the basin history. Rifting that formed Lake Malawi is estimated to have begun 8.6 MYA with deep-water conditions attained by 4.5 MYA (Ebinger et al. 1993; Delvaux 1995). The earliest known fossil freshwater assemblages in the catchment are from the Chiwondo beds laid down in lacustrine conditions 2.3 MYA (Van Damme and Pickford 2003), but taxonomic identities of cichlids within the deposits are unclear (Murray 2001b).
Of greater controversy is the age of the Lake Victoria region cichlid fishes. Over 500 closely related haplochromine cichlid species comprise the East African "superflock" inhabiting Lakes Victoria, Kyoga, Rukwa, Kivu, Albert, George, Edward, and surrounding water bodies. Diversity between unique haplotypes within the widespread superflock was dated on average to 0.273 (±0.216 SD) and 0.189 (±0.133 SD) MYA using the Gondwanan fragmentation and cichlid fossil calibrations, respectively. Within the region occupied by the superflock, Lake Victoria forms a phylogeographic zone largely distinct in mitochondrial DNA from the neighboring western rift lakes Albert, George, Edward, and Kivu (Verheyen et al. 2003). We dated divergence within the Lake Victoria catchment to 0.120 (±0.110 SD) and 0.089 (±0.074 SD) MYA using the Gondwanan fragmentation and cichlid fossil calibrations, respectively. Both estimates are within the age of the Lake Victoria catchment formation, approximately 400,000 years ago (Johnson et al. 2000).
The extant members of the serranochromine radiation comprise at least 24 species broadly distributed across southern Africa. It has been proposed that much of their species richness and morphological diversity arose in Palaeolake Makgadikgadi (Joyce et al. 2005), a vast lake that formed between 315,000 and 460,000 years ago and dried up within the last few thousand years, leaving the present-day Makgadikgadi salt pans (Moore and Larkin 2001). Mean interspecific mitochondrial divergence within the 2 radiating clades was dated, irrespective of calibration used, to the middle Pleistocene (Gondwana fragmentation means: Clade "I" 0.425 ± 0.36 MYA, Clade "III" 0.630 ± 0.43 MYA, cichlid fossil means Clade I 0.279 ± 0.22 MYA, Clade III 0.401 ± 0.25 MYA), approximately the period when the now-extinct lake was one of Africa's largest water bodies.
Timescale of Speciation and Adaptive Radiation within Lake Tanganyika
Since the pioneering molecular phylogenetic studies of African cichlids, numerous studies have presented estimates of divergence times. Although most studies have only tentatively calculated these dates, a consensus timescale of cladogenesis has emerged (see Kocher 2004; Salzburger and Meyer 2004). The development of this paradigm began with extrapolation of an estimated rate of mitochondrial DNA (cytochrome b) evolution from mammals (Meyer et al. 1990). Using this rate, haplochromine radiations of the Lake Victoria region and Lake Malawi were estimated at 200,000 years and 700,000 years, respectively, whereas the divergence of the Haplochromini and Lamprologini was dated to 4.5 MYA (Meyer et al. 1990). Subsequently, divergence dates have been calculated employing assumptions that the basal radiations of the Lake Tanganyika endemic cichlid tribes occurred between 6 and 12 MYA based on evidence that during this period deep lacustrine conditions began to form in Lake Tanganyika (Sturmbauer et al. 1994; Duftner et al. 2005; Koblmüller et al. 2005; Won et al. 2005, 2006). Additionally, the basal radiation of the Lake Malawi haplochromine flock has commonly been used for calibration with the assumptions that it occurred between 0.7 and 4 MYA (Sturmbauer and Meyer 1993; Nagl et al. 1998, 2000, 2001; Salzburger et al. 2005). Calibrations have also been based on assumptions of divergence events being synchronized with Lake Malawi surface-level changes. Sturmbauer et al. (2001) calculated sequence evolution rates assuming that the typically rock-associated "mbuna" and the often sand-associated "utaka" (=Malawi Hap dominated; table 4) mitochondrial DNA lineages of Lake Malawi haplochromines diverged during a rise in water level 0.59–1 MYA following a desiccation event identified by Delvaux (1995). Rates based on this calibration have subsequently been employed several times for both African and Neotropical cichlids (Baric et al. 2003; Sturmbauer et al. 2003; Verheyen et al. 2003; Barluenga and Meyer 2004; Brandstätter et al. 2005; Barluenga et al. 2006).
Notably, no molecular phylogenetic studies have presented dates that strongly conflict with the established timescale of African cichlid evolution presented by Kocher (2004) and Salzburger and Meyer (2004), and this evolutionary scenario is generally supported by the timescale derived from the fossil cichlid calibrations (fig. 6). Briefly, this scenario would suggest that a small number of colonizing species entered a proto-lake Tanganyika when swamp and shallow lake conditions were present between approximately 12 and 20 MYA. Upon entering this habitat, some of these species began to radiate forming the present-day Lake Tanganyika cichlid tribes (Salzburger et al. 2002). Next, during the formation of deep-water conditions approximately 6–12 MYA, these lineages diversified further and began to accumulate species richness and morphological diversity (Salzburger et al. 2002).
By contrast, the timescale of African cichlid evolution suggested by the Gondwanan calibrations is very different. It proposes Lake Tanganyika as a reservoir of more than 10 riverine lineages that subsequently underwent adaptation for deep-water lacustrine conditions and radiated but became extinct in river systems over a timescale of approximately 20 Myr. One possible mechanism driving such extinctions could be climatically driven cycles of desiccation and flooding of the shallow rivers and streams in the Lake Tanganyika catchment. There is substantial evidence that East Africa has been through dramatic climatic changes, for example, from substantial lake-level changes recorded in the sediments of the East African great lakes (Johnson et al. 2002) and in the East African molluscan fossil record (for discussion, see Genner et al. 2007). If the Gondwanan calibrations are reliable, then the molecular lineage diversity present in Lake Tanganyika must have formed elsewhere. It is alternatively possible that this took place in one or more palaeolakes that formerly existed in this region. Lake formation and extinction appear to be common phenomena within sub-Saharan Africa (Cahen 1954; Stewart 2001; Van Damme and Pickford 2003), and several large palaeolakes are known from the upper Congo basin (Cahen 1954). If so, then the emerging Lake Tanganyika may have captured existing molecular diversity from previous lacustrine radiations, which in turn seeded new radiations (Seehausen 2006).
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Conclusions |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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This study has provided 2 dichotomous timescales for the evolution of cichlid fishes and discussed the evidence surrounding both. In doing so, we hope to have contributed to the debate concerning timescales of cichlid evolution. Of particular interest are our results using the combined Gondwanan/post-Pliocene biogeographical calibrations that enabled us to reconcile global cichlid distributions and the comparatively young ages of endemic species flocks within lake basins without invoking scenarios of multiple intercontinental marine dispersal events. Further study is needed to investigate the implications of this calibration set for phylogeography and the mechanisms driving speciation and adaptive radiation. Nevertheless, with respect to African cichlid evolution, it is notable that both cichlid fossil and Gondwanan fragmentation calibrations indicate a strong association between onset of molecular radiation and the formation of lacustrine water bodies, in turn suggesting that colonization of lakes is associated with accelerated speciation and adaptive radiation. This may possibly be linked to the diversity of habitats that lakes provide as larger lakes have the largest cichlid flocks (Seehausen 2006
). As such, it is clear that ancient lakes have played a key role in generating and maintaining diversity within radiating lineages, and the role of ecological opportunity in generation and maintenance of biodiversity should not be underestimated. |
Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Supplementary materials are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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We thank A. Smith and R. Mohammed for securing cichlid tissue samples and S. Mwaiko for providing unpublished sequences of Lake Malimbe haplochromines. K. Young provided helpful comments on an early draft and A. Murray provided helpful information concerning fossil calibrations. This work has been improved with helpful comments from J. Hey and 2 anonymous reviewers. This work was funded by Natural Environment Research Council award NER/A/S/2003/00362.
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Footnotes |
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Jody Hey, Associate Editor
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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