Phylogenomics uncovers early hybridization and adaptive loci shaping the radiation of Lake Tanganyika cichlid fishes

Tanganyika is the oldest and phenotypically most diverse of the three East African cichlid fish adaptive radiations. It is also the cradle for the younger parallel haplochromine cichlid radiations in Lakes Malawi and Victoria. Despite its evolutionary significance, the relationships among the main Lake Tanganyika lineages remained unresolved, as did the general timescale of cichlid evolution. Here, we disentangle the deep phylogenetic structure of the Lake Tanganyika radiation using anchored phylogenomics and uncover hybridization at its base, as well as early in the haplochromine radiation. This suggests that hybridization might have facilitated these speciation bursts. Time-calibrated trees support that the radiation of Tanganyika cichlids coincided with lake formation and that Gondwanan vicariance concurred with the earliest splits in the cichlid family tree. Genes linked to key innovations show signals of introgression or positive selection following colonization of lake habitats and species’ dietary adaptations are revealed as major drivers of colour vision evolution. These findings shed light onto the processes shaping the evolution of adaptive radiations. Tanganyika’s cichlid radiation is the main source of East African cichlid diversity. Irisarri et al. resolve its phylogenetic backbone using anchored phylogenomics and identify trans-lineage hybridization prior to major speciation bursts and adaptive loci underlying ecological innovations.

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title: The evolutionary genomics of cichlid fishes: explosive speciation and adaptation in the postgenomic era.
abstract: With more than 1,500 species, cichlid fishes provide textbook examples of recent and diverse adaptive radiations, rapid rates of speciation, and the parallel evolution of adaptive phenotypes among both recently and distantly related lineages. This extraordinary diversity has attracted considerable interest from researchers across several biological disciplines. Their broad phenotypic variation coupled with recent divergence makes cichlids an ideal model system for understanding speciation, adaptation, and phenotypic diversification. Genetic mapping, genome-wide analyses, and genome projects have flourished in the past decade and have added new insights on the question of why there are so many cichlids. These recent findings also show that the sharing of older DNA polymorphisms is extensive and suggest that linage sorting is incomplete and that adaptive introgression played a role in the African radiation. Here, we review the results of genetic and genomic research on cichlids in the past decade and suggest some potential avenues to further exploit the potential of the cichlid model system to provide a better understanding of the genomics of adaptation and speciation.

title: Evaluating genomic divergence and parallelism in replicate ecomorphs from young and old cichlid adaptive radiations
abstract: Comparative genomic studies of closely related species typically focus on single species pairs at one given stage of divergence. That makes it difficult to infer the continuum of evolutionary process during speciation and beyond. Here, we use whole‐genome resequencing to examine genomic patterns of divergence in three sympatric cichlid species pairs with very similar functional and ecological differentiation, but different ages. We find a strong signature of increasing genomic divergence with time in both the mitochondrial genome and the nuclear genome. In contrast to many other systems, we find that in these cichlids, regions of elevated relative differentiation also exhibit increased absolute differentiation. We detect a signature of convergent evolution in a comparison of outlier regions across all three species pair comparisons, but the extent of it is modest, and regions that are strongly divergent in any one pair tend to be only slightly elevated in the other pairs, consistent with a repeatable but polygenic basis of traits that characterize the ecomorphs. Our results suggest that strong functional phenotypic differentiation, as seen in all three species pairs, is generally associated with a clear signature of genomic divergence, even in the youngest species pair.

title: Age and spread of the haplochromine cichlid fishes in Africa.
abstract: The Haplochromini are by far the most species-rich cichlid fish tribe that originated along with the so-called primary radiation of the Lake Tanganyika cichlid species flock, i.e. at the same time during which the majority of the endemic Lake Tanganyika cichlid tribes emerged. Unlike the other tribes, the haplochromines are not restricted to Lake Tanganyika but distributed throughout Africa, except for the northwestern 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 present a comprehensive phylogenetic and phylogeographic analysis of haplochromine cichlids that is based upon DNA sequences of two mitochondrial gene segments of riverine taxa covering all major African biogeographic regions where haplochromines are found. Our analysis revealed that six lineages of haplochromines originated within a short period of time, about 5.3-4.4 MYA. These haplochromine lineages show a highly complex phylogeographic pattern, probably severely influenced by climate- and/or geology-induced changes of the environment, with river capture events most likely playing an important role for species dispersal.

title: A robust phylogeny among major lineages of the East African cichlids.
abstract: The huge monophyletic group of the East African cichlid radiations (EAR) consists of thousands of species belonging to 12-14 tribes; the number of tribes differs among studies. Many studies have inferred phylogenies of EAR tribes using various genetic markers. However, these phylogenies partly contradict one another and can have weak statistic support. In this study, we conducted maximum-likelihood (ML) phylogenetic analyses using restriction site-associated DNA (RAD) sequences and propose a new robust phylogenetic hypothesis among Lake Tanganyika cichlid fishes, which cover most EAR tribes. Data matrices can vary in size and contents depending on the strategies used to process RAD sequences. Therefore, we prepared 23 data matrices with various processing strategies. The ML phylogenies inferred from 15 large matrices (2.0×10(6) to 1.1×10(7) base pairs) resolved every tribe as a monophyletic group with 100% bootstrap support and shared the same topology regarding relationships among the tribes. Most nodes among the tribes were supported by 100% bootstrap values, and the bootstrap support for the other node varied among the 15 ML trees from 70% to 100%. These robust ML trees differ partly in topology from those in earlier studies, and these phylogenetic relationships have important implications for the tribal classification of EAR.

title: Out of Tanganyika: Genesis, explosive speciation, key-innovations and phylogeography of the haplochromine cichlid fishes
abstract: BackgroundThe adaptive radiations of cichlid fishes in East Africa are well known for their spectacular diversity and their astonishingly fast rates of speciation. About 80% of all 2,500 cichlid species in East Africa, and virtually all cichlid species from Lakes Victoria (~500 species) and Malawi (~1,000 species) are haplochromines. Here, we present the most extensive phylogenetic and phylogeographic analysis so far that includes about 100 species and is based on about 2,000 bp of the mitochondrial DNA.ResultsOur analyses revealed that all haplochromine lineages are ultimately derived from Lake Tanganyika endemics. We find that the three most ancestral lineages of the haplochromines sensu lato are relatively species poor, albeit widely distributed in Africa, whereas a fourth newly defined lineage – the 'modern haplochromines' – contains an unparalleled diversity that makes up more than 7% of the worlds' ~25,000 teleost species. The modern haplochromines' ancestor, most likely a riverine generalist, repeatedly gave rise to similar ecomorphs now found in several of the species flocks. Also, the Tanganyikan Tropheini are derived from that riverine ancestor suggesting that they successfully re-colonized Lake Tanganyika and speciated in parallel to an already established cichlid adaptive radiation. In contrast to most other known examples of adaptive radiations, these generalist ancestors were derived from highly diverse and specialized endemics from Lake Tanganyika. A reconstruction of life-history traits revealed that in an ancestral lineage leading to the modern haplochromines the characteristic egg-spots on anal fins of male individuals evolved.ConclusionWe conclude that Lake Tanganyika is the geographic and genetic cradle of all haplochromine lineages. In the ancestors of the replicate adaptive radiations of the 'modern haplochromines', behavioral (maternal mouthbrooding), morphological (egg-spots) and sexually selected (color polymorphism) key-innovations arose. These might be – together with the ecological opportunity that the habitat diversity of the large lakes provides – responsible for their evolutionary success and their propensity for explosive speciation.

title: Do estimated and actual species phylogenies match? Evaluation of East African cichlid radiations.
abstract: A large number of published phylogenetic estimates are based on a single locus or the concatenation of multiple loci, even though genealogies of single or concatenated loci may not accurately reflect the true history of species diversification (i.e., the species tree). The increased availability of genomic data, coupled with new computational methods, improves resolution of species relationships beyond what was possible in the past. Such developments will no doubt benefit future phylogenetic studies. It remains unclear how robust phylogenies that predate these developments (i.e., the bulk of phylogenetic studies) are to departures from the assumption of strict gene tree-species tree concordance. Here, we present a parametric bootstrap (PBST) approach that assesses the reliability of past phylogenetic estimates in which gene tree-species tree discord was ignored. We focus on a universal cause of discord-the random loss of gene lineages from genetic drift-and apply the method in a meta-analysis of East African cichlids, a group encompassing historical scenarios that are particularly challenging for phylogenetic estimation. Although we identify some evolutionary relationships that are robust to gene tree discord, many past phylogenetic estimates of cichlids are not. We discuss the utility of the PBST method for evaluating the robustness of gene tree-based phylogenetic estimations in general as well as for testing the clade-specific performance of species tree estimation methods and designing sampling strategies that increase the accuracy of estimated species relationships.

title: A tribal level phylogeny of Lake Tanganyika cichlid fishes based on a genomic multi-marker approach

title: Ancient hybridization fuels rapid cichlid fish adaptive radiations
abstract: Understanding why some evolutionary lineages generate exceptionally high species diversity is an important goal in evolutionary biology. Haplochromine cichlid fishes of Africa's Lake Victoria region encompass >700 diverse species that all evolved in the last 150,000 years. How this ‘Lake Victoria Region Superflock' could evolve on such rapid timescales is an enduring question. Here, we demonstrate that hybridization between two divergent lineages facilitated this process by providing genetic variation that subsequently became recombined and sorted into many new species. Notably, the hybridization event generated exceptional allelic variation at an opsin gene known to be involved in adaptation and speciation. More generally, differentiation between new species is accentuated around variants that were fixed differences between the parental lineages, and that now appear in many new combinations in the radiation species. We conclude that hybridization between divergent lineages, when coincident with ecological opportunity, may facilitate rapid and extensive adaptive radiation.

title: Phylogeny and biogeography of cichlid fishes (Teleostei: Perciformes: Cichlidae)
abstract: Family level molecular phylogenetic analyses of cichlid fishes have generally suffered from a limited number of characters and/or poor taxonomic sampling across one or more major geographic assemblage, and therefore have not provided a robust test of early intrafamilial diversification. Herein we use both nuclear and mitochondrial nucleotide characters and direct optimization to reconstruct a phylogeny for cichlid fishes. Representatives of major cichlid lineages across all geographic assemblages are included, as well as nearly twice the number of characters as any prior family‐level study. In a strict consensus of 81 equally most‐parsimonious hypotheses, based on the simultaneous analysis of 2222 aligned nucleotide characters from two mitochondrial and two nuclear genes, four major subfamilial lineages are recovered with strong support. Etroplinae, endemic to Madagascar (Paretroplus) and southern Asia (Etroplus), is recovered as the sister taxon to the remainder of Cichlidae. Although the South Asian cichlids are monophyletic, the Malagasy plus South Asian lineages are not. The remaining Malagasy lineage, Ptychochrominae, is monophyletic and is recovered as the sister group to a clade comprising the African and Neotropical cichlids. The African (Pseudocrenilabrinae) and Neotropical (Cichlinae) lineages are each monophyletic in this reconstruction. The use of multiple molecular markers, from both mitochondrial and nuclear genes, results in a phylogeny that in general exhibits strong support, notably for early diversification events within Cichlidae. Results further indicate that Labroidei is not monophyletic, and that the sister group to Cichlidae may comprise a large and diverse assemblage of percomorph lineages. This hypothesis may at least partly explain why morphological studies that have attempted to place Cichlidae within Percomorpha, or that have tested cichlid monophyly using only “labroid” lineages, have met with only limited success.

title: Reconciling fossils and molecules: Cenozoic divergence of cichlid fishes and the biogeography of Madagascar
abstract: The biogeographical origins of the extant vertebrates endemic to Madagascar are largely unsolved, but have often been related to vicariance in the context of fragmentation of the supercontinent Gondwana in the Mesozoic. Such hypotheses are especially appealing in the case of cichlid fishes, which show phylogenetic relationships reflecting the temporal successions of the breakup of Gondwana. We used molecular clock data to test this assumption. Location Fragments of the 16S rRNA gene and of the nuclear Tmo-4C4 locus, partly obtained from Genbank from South American, African, Malagasy and Indian cichlids were analysed.  Based on monophyletic cichlid radiations in African lakes, we calibrated a molecular clock. The obtained rates were used to estimate the age of divergence of the major cichlid clades.  The results agreed better with a Cenozoic than with a Mesozoic divergence, and were in accordance with the fossil record. Sequence divergences of the 16S and 12S rRNA genes of most lineages of Malagasy terrestrial and freshwater vertebrates from their non-Malagasy sister groups were below saturation and many were relatively similar to those of cichlids.  A Cenozoic dispersal from continental landmasses may explain the origin of most extant Malagasy vertebrate groups better than a Jurassic/Cretaceous vicariance.

title: Total evidence: molecules, morphology, and the phylogenetics of cichlid fishes.
abstract: We present a most comprehensive phylogenetic analysis of the family Cichlidae. New data analyzed include mitochondrial 16S rRNA sequences and two nuclear loci (Tmo-M27 and Tmo-4C4) for a large taxonomic sampling with emphasis on South American species. We also incorporate a published morphological data set for a total evidence analysis. Character congruence among mitochondrial (74 taxa) and nuclear data (50 taxa) was high. However, partition-homogeneity tests suggest significant heterogeneity among molecular and morphological data. In agreement with results obtained from molecular data alone, total evidence analysis (1,460 characters for 34 taxa) supports a robust phylogenetic hypothesis for the family Cichlidae that is congruent with drift-vicariance events associated with the fragmentation of Gondwana. Our analyses confirm the placement of Malagasy/Indian cichlids as the most basal lineages, with a sister-group relationship to the monophyletic African and Neotropical clades. Total evidence suggests that the controversial African genus Heterochromis is at the base of the African radiation. Among more than 50 Neotropical genera analyzed, Retroculus is identified as the basal taxon, with successive branching of Cichla, Astronotus, geophagines (including crenicichlines) + chaetobranchines, and cichlasomines + heroines. Relative rate tests applied to mitochondrial DNA suggest significantly higher rates of genetic variation in Neotropical than in African taxa, and both mitochondrial and nuclear sequences show that rate heterogeneity among Neotropical lineages is confined to the geophagine cichlids.

title: Disentangling incomplete lineage sorting and introgression to refine species-tree estimates for Lake Tanganyika cichlid fishes
abstract: Adaptive radiation is thought to be responsible for the evolution of a great portion of the past and present diversity of life. Instances of adaptive radiation, characterized by the rapid emergence of an array of species as a consequence to their adaptation to distinct ecological niches, are important study systems in evolutionary biology. However, because of the rapid lineage formation in these groups, and the occurrence of hybridization between the participating species, it is often difficult to reconstruct the phylogenetic history of species that underwent an adaptive radiation. In this study, we present a novel approach for species-tree estimation in rapidly diversifying lineages, where introgression is known to occur, and apply it to a multimarker dataset containing up to 16 specimens per species for a set of 45 species of East African cichlid fishes (522 individuals in total), with a main focus on the cichlid species flock of Lake Tanganyika. We first identified, using age distributions of most recent common ancestors in individual gene trees, those lineages in our dataset that show strong signatures of past introgression. This led us to formulate three hypotheses of introgression between different lineages of Tanganyika cichlids: the ancestor of Boulengerochromini (or of Boulengerochromini and Bathybatini) received genomic material from the derived H-lineage; the common ancestor of Cyprichromini and Perissodini experienced, in turn, introgression from Boulengerochromini and/or Bathybatini; and the Lake Tanganyika Haplochromini and closely related riverine lineages received genetic material from Cyphotilapiini. We then applied the multispecies coalescent model to estimate the species tree of Lake Tanganyika cichlids, but excluded the lineages involved in these introgression events, as the multispecies coalescent model does not incorporate introgression. This resulted in a robust species tree, in which the Lamprologini were placed as sister lineage to the H-lineage (including the Eretmodini), and we identify a series of rapid splitting events at the base of the H-lineage. Divergence ages estimated with the multispecies coalescent model were substantially younger than age estimates based on concatenation, and agree with the geological history of the Great Lakes of East Africa. Finally, we formally tested the three hypotheses of introgression using a likelihood framework, and find strong support for introgression between some of the cichlid tribes of Lake Tanganyika.

title: The root of the East African cichlid radiations
abstract: BackgroundFor decades cichlid fishes (Perciformes: Cichlidae) of the East African cichlid radiations (Teleostei: Cichlidae) have served as natural experimental subjects for the study of speciation processes and the search for potential speciation key factors. Despite numerous phylogenetic studies dealing with their intragroup relationships, surprisingly little is known about the phylogenetic placement and time of origin of this enigmatic group. We used multilocus DNA-sequence data from five nuclear and four mitochondrial genes and refined divergence time estimates to fill this knowledge gap.ResultsIn concordance with previous studies, the root of the East African cichlid radiations is nested within the so called "Tilapias", which is a paraphyletic assemblage. For the first time, we clarified tilapiine intragroup relationships and established three new monophyletic groups:"Oreochromini", "Boreotilapiini" and a group with a distribution center in East/Central Africa, the "Austrotilapiini". The latter is the founder lineage of the East African radiations and emerged at the Miocene/Oligocene boundary at about 14 to 26 mya.ConclusionOur results provide the first resolved hypothesis for the phylogenetic placement of the megadiverse East African cichlid radiations as well as for the world's second most important aquaculture species, the Nile Tilapia, Oreochromis niloticus. Our analyses constitute not only a robust basis for African cichlid phylogenetics and systematics, but provide a valid and necessary framework for upcoming comparative phylogenomic studies in evolutionary biology and aquaculture.

title: Reticulate phylogeny of gastropod-shell-breeding cichlids from Lake Tanganyika – the result of repeated introgressive hybridization
abstract: BackgroundThe tribe Lamprologini is the major substrate breeding lineage of Lake Tanganyika's cichlid species flock. Among several different life history strategies found in lamprologines, the adaptation to live and breed in empty gastropod shells is probably the most peculiar. Although shell-breeding arose several times in the evolutionary history of the lamprologines, all obligatory and most facultative shell-breeders belong to the so called "ossified group", a monophyletic lineage within the lamprologine cichlids. Since their distinctive life style enables these species to live and breed in closest vicinity, we hypothesized that these cichlids might be particularly prone to accidental hybridization, and that introgression might have affected the evolutionary history of this cichlid lineage.ResultsOur analyses revealed discrepancies between phylogenetic hypotheses based on mitochondrial and nuclear (AFLP) data. While the nuclear phylogeny was congruent with morphological, behavioral and ecological characteristics, several species – usually highly specialized shell-breeders – were placed at contradicting positions in the mitochondrial phylogeny. The discordant phylogenies strongly suggest repeated incidents of introgressive hybridization between several distantly related shell-breeding species, which reticulated the phylogeny of this group of cichlids. Long interior branches and high bootstrap support for many interior nodes in the mitochondrial phylogeny argue against a major effect of ancient incomplete lineage sorting on the phylogenetic reconstruction. Moreover, we provide morphological and genetic (mtDNA and microsatellites) evidence for ongoing hybridization among distantly related shell-breeders. In these cases, the territorial males of the inferred paternal species are too large to enter the shells of their mate, such that they have to release their sperm over the entrance of the shell to fertilize the eggs. With sperm dispersal by water currents and wave action, trans-specific fertilization of clutches in neighboring shells seem inevitable, when post-zygotic isolation is incomplete.ConclusionFrom the direct observation of hybrids we conclude that hybridization between distantly related gastropod-shell-breeding cichlids of Lake Tanganyika follows inevitably from their ecological specialization. Moreover, the observed incongruence between mtDNA and nuclear multilocus phylogeny suggests that repeated hybridization events among quite distantly related taxa affected the diversification of this group, and introduced reticulation into their phylogeny.

title: Mitogenomic evaluation of the historical biogeography of cichlids toward reliable dating of teleostean divergences
abstract: BackgroundRecent advances in DNA sequencing and computation offer the opportunity for reliable estimates of divergence times between organisms based on molecular data. Bayesian estimations of divergence times that do not assume the molecular clock use time constraints at multiple nodes, usually based on the fossil records, as major boundary conditions. However, the fossil records of bony fishes may not adequately provide effective time constraints at multiple nodes. We explored an alternative source of time constraints in teleostean phylogeny by evaluating a biogeographic hypothesis concerning freshwater fishes from the family Cichlidae (Perciformes: Labroidei).ResultsWe added new mitogenomic sequence data from six cichlid species and conducted phylogenetic analyses using a large mitogenomic data set. We found a reciprocal monophyly of African and Neotropical cichlids and their sister group relationship to some Malagasy taxa (Ptychochrominae sensu Sparks and Smith). All of these taxa clustered with a Malagasy + Indo/Sri Lankan clade (Etroplinae sensu Sparks and Smith). The results of the phylogenetic analyses and divergence time estimations between continental cichlid clades were much more congruent with Gondwanaland origin and Cretaceous vicariant divergences than with Cenozoic transmarine dispersal between major continents.ConclusionWe propose to add the biogeographic assumption of cichlid divergences by continental fragmentation as effective time constraints in dating teleostean divergence times. We conducted divergence time estimations among teleosts by incorporating these additional time constraints and achieved a considerable reduction in credibility intervals in the estimated divergence times.

title: Convergent Evolution within an Adaptive Radiation of Cichlid Fishes
abstract: The recurrent evolution of convergent forms is a widespread phenomenon in adaptive radiations (e.g., [1-9]). For example, similar ecotypes of anoles lizards have evolved on different islands of the Caribbean, benthic-limnetic species pairs of stickleback fish emerged repeatedly in postglacial lakes, equivalent sets of spider ecomorphs have arisen on Hawaiian islands, and a whole set of convergent species pairs of cichlid fishes evolved in East African Lakes Malawi and Tanganyika. In all these cases, convergent phenotypes originated in geographic isolation from each other. Recent theoretical models, however, predict that convergence should be common within species-rich communities, such as species assemblages resulting from adaptive radiations. Here, we present the most extensive quantitative analysis to date of an adaptive radiation of cichlid fishes, discovering multiple instances of convergence in body and trophic morphology. Moreover, we show that convergent morphologies are associated with adaptations to specific habitats and resources and that Lake Tanganyika's cichlid communities are characterized by the sympatric occurrence of convergent forms. This prevalent coexistence of distantly related yet ecomorphologically similar species offers an explanation for the greatly elevated species numbers in cichlid species flocks.

title: Comparative Phylogenetic Analyses of the Adaptive Radiation of Lake Tanganyika Cichlid Fish: Nuclear Sequences Are Less Homoplasious But Also Less Informative Than Mitochondrial DNA
abstract: Over 200 described endemic species make up the adaptive radiation of cichlids in Lake Tanga-nyika. This species assemblage has been viewed as both an evolutionary reservoir of old cichlid lineages and an evolutionary hotspot from which the modern cichlid lineages arose, seeding the adaptive radiations in Lakes Victoria and Malawi. Here we report on a phylogenetic analysis of Lake Tanganyika cichlids combining the previously determined sequences of the mitochondrial ND2 gene (1047 bp) with newly derived sequences of the nuclear RAG1 gene (∼700 bp of intron 2 and ∼1100 bp of exon 3). The nuclear data—in agreement with mitochondrial DNA—suggest that Lake Tanganyika harbors several ancient lineages that did not undergo rampant speciation (e.g., Bathybatini, Trematocarini). We find strong support for the monophyly of the most species-rich Tanganyikan group, the Lamprologini, and we propose a new taxonomic group that we term the C-lineage. The Haplochromini and Tropheini both have an 11-bp deletion in the intron of RAG1, strongly supporting the monophyly of this clade and its derived position. Mapping the phylogenetically informative positions revealed that, for certain branches, there are six times fewer apomorphies in RAG1. However, the consistency index of these positions is higher compared to the mitochondrial ND2 gene. Nuclear data therefore provide, on a per–base pair basis, less but more reliable phylogenetic information. Even if in our case RAG1 has not provided as much phylogenetic information as we expected, we suggest that this marker might be useful in the resolution of the phylogeny of older groups.

title: Evolution of NADH dehydrogenase subunit 2 in east African cichlid fish.
abstract: The complete sequence (1047 bp) of the mitochondrially encoded ND2 gene was obtained from 31 species of cichlid fishes to investigate the evolutionary history of the species flocks of the East African lakes. The observed pattern of nucleotide substitution in this sequence is typical of mitochondrial genes, showing a high transition bias and rapid mutational saturation, especially at codon positions where base frequencies are unequal. The base composition of the third position of codons is heterogeneous among species, suggesting frequent shifts in the pattern of substitution. Phylogenetic analysis of the sequences shows that the mtDNA variation in Lake Malawi cichlids is nested monophyletically within the range of variation shown by Tanganyikan cichlids. The closest Tanganyikan relatives of the Malawi flock are members of the tribe Tropheini. Classifications based on morphology are generally supported by the mtDNA data, with some significant exceptions in the Tropheini and Lamprologini. Because of an apparently rapid radiation of the Tanganyikan lineages, it is difficult to assess the basal topology of the Tanganyikan radiation at this time. Divergences among tribes are consistent with an intralacustrine radiation within the past 10 million years.

title: Molecular and fossil evidence place the origin of cichlid fishes long after Gondwanan rifting
abstract: Cichlid fishes are a key model system in the study of adaptive radiation, speciation and evolutionary developmental biology. More than 1600 cichlid species inhabit freshwater and marginal marine environments across several southern landmasses. This distributional pattern, combined with parallels between cichlid phylogeny and sequences of Mesozoic continental rifting, has led to the widely accepted hypothesis that cichlids are an ancient group whose major biogeographic patterns arose from Gondwanan vicariance. Although the Early Cretaceous (ca 135 Ma) divergence of living cichlids demanded by the vicariance model now represents a key calibration for teleost molecular clocks, this putative split pre-dates the oldest cichlid fossils by nearly 90 Myr. Here, we provide independent palaeontological and relaxed-molecular-clock estimates for the time of cichlid origin that collectively reject the antiquity of the group required by the Gondwanan vicariance scenario. The distribution of cichlid fossil horizons, the age of stratigraphically consistent outgroup lineages to cichlids and relaxed-clock analysis of a DNA sequence dataset consisting of 10 nuclear genes all deliver overlapping estimates for crown cichlid origin centred on the Palaeocene (ca 65–57 Ma), substantially post-dating the tectonic fragmentation of Gondwana. Our results provide a revised macroevolutionary time scale for cichlids, imply a role for dispersal in generating the observed geographical distribution of this important model clade and add to a growing debate that questions the dominance of the vicariance paradigm of historical biogeography.

title: The Lake Tanganyika cichlid species assemblage: recent advances in molecular phylogenetics
abstract: Tanganyika is not the most species-rich of the Great East African Lakes, but comprises the greatest diversity of cichlid fishes in terms of morphology, ecology, and breeding styles. The lake contains a polyphyletic assemblage of cichlid lineages, which evolved from several ancient species that colonized the emerging lake some 9–12 million years ago. Based on morphological characteristics, the Tanganyikan cichlids have been classified into 12, or, more recently, 16 tribes, which are largely supported by molecular data. The radiations of East African cichlids are believed to be driven by complex interactions between extrinsic factors, such as climatic changes and geological processes, and intrinsic biological characteristics of the involved organisms. Diversification within different lineages occurred simultaneously in response to drastic habitat changes such as the establishment of lacustrine deep-water conditions 5–6 MYA and subsequent major lake-level fluctuations. This seems particularly true for the mouthbrooding lineages whereas the substrate breeders underwent a more gradual process of diversification. This review presents an account of the taxonomy and phylogeny of the Lake Tanganyika cichlid species assemblage, its relationship to the African cichlid fauna, key factors leading to the astonishing diversity and discusses recently proposed alternative age estimates for the Lake Tanganyika cichlid species assemblage.

title: Age of cichlids: new dates for ancient lake fish radiations.
abstract: 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.

title: Bayesian Phylogenetic Estimation of Clade Ages Supports Trans‐Atlantic Dispersal of Cichlid Fishes
abstract: Divergence‐time estimation based on molecular phylogenies and the fossil record has provided insights into fundamental questions of evolutionary biology. In Bayesian node dating, phylogenies are commonly time calibrated through the specification of calibration densities on nodes representing clades with known fossil occurrences. Unfortunately, the optimal shape of these calibration densities is usually unknown and they are therefore often chosen arbitrarily, which directly impacts the reliability of the resulting age estimates. As possible solutions to this problem, two nonexclusive alternative approaches have recently been developed, the “fossilized birth‐death” (FBD) model and “total‐evidence dating.” While these approaches have been shown to perform well under certain conditions, they require including all (or a random subset) of the fossils of each clade in the analysis, rather than just relying on the oldest fossils of clades. In addition, both approaches assume that fossil records of different clades in the phylogeny are all the product of the same underlying fossil sampling rate, even though this rate has been shown to differ strongly between higher level taxa. We here develop a flexible new approach to Bayesian age estimation that combines advantages of node dating and the FBD model. In our new approach, calibration densities are defined on the basis of first fossil occurrences and sampling rate estimates that can be specified separately for all clades. We verify our approach with a large number of simulated data sets, and compare its performance to that of the FBD model. We find that our approach produces reliable age estimates that are robust to model violation, on par with the FBD model. By applying our approach to a large data set including sequence data from over 1000 species of teleost fishes as well as 147 carefully selected fossil constraints, we recover a timeline of teleost diversification that is incompatible with previously assumed vicariant divergences of freshwater fishes. Our results instead provide strong evidence for transoceanic dispersal of cichlids and other groups of teleost fishes.

title: Salt-tolerance of fresh-water fish groups in relation to zoogeographical problems
abstract: The distribution of fresh-water fishes like that of other groups, has been widely utilized by zoogeographers, but with widely divergent acumen and success. At one extreme are those non-ichthyologists who have uncritically utilized for evidence certain groups whose distribution happens to support whatever theory they may be espousing. At the other extreme is the work of careful ichthyologists like DE BEAUFORT (1913) and REGAN (1922) whose thorough knowledge of the groups with which they are working demands the most careful consideration of their conclusions. However, no zoogeographer who utilizes the evidence of diverse groups can be familiar at first hand with all of them, and the difficulty facing such workers is that of seeking out the really dependable evidence in those groups he does not know well. Aside from the difficulty of selecting dependable authorities or systematic works, the zoogeographer desiring to use the evidence of fresh-water fishes has another troublesome matter to contend with. This is the differing tolerance of salt-water exhibited by different groups of fresh-water fishes. As one example, and one which has frequently troubled zoogeographers, we may mention the Galaxiidae, fresh-water fishes of Southern South America, Africa, Australia, and New Zealand, whose distribution has been held by some to be evidence for continental drift or southern intercontinental land-bridges. Ichthyologists now know that these fishes are, as a group, salt-tolerant and possibly either anadromous or catadromous, and that they are not really strong evidence for continental connections simply because it seems possible that they may cross ocean barriers.

title: Repeated colonization and hybridization in Lake Malawi cichlids
abstract: Through adaptive radiation, ancestral species rapidly diversify into multiple species with different ecological adaptations. The haplochromine cichlid fishes of the East African Great Lakes are considered classic examples of adaptive radiation, but our understanding of the evolutionary origins of these radiations has been limited by inadequate taxonomic and genomic sampling . Perhaps the largest of these radiations is from Lake Malawi, estimated to contain between 500 and 800 endemic species. Surprisingly, its monophyly — the origin from a single ancestral species — has never been critically tested. This is because river populations which could have seeded the radiation, with one very limited exception , have never been included in phylogenetic reconstructions. Moreover, phylogenies have relied heavily on mitochondrial DNA (mtDNA), which can be a misleading phylogenetic marker for species capable of hybridization  because its non-recombining nature means that transfer to other species can occur via asymmetric introgression and ‘allele surfing' [6]. Here, we used broad taxonomic sampling and nuclear DNA markers with wide genomic coverage and find that the Lake Malawi radiation is not monophyletic, but instead contains genetic material from divergent riverine ancestors indicating multiple invasions and hybridization.

title: The Role of Alternative Splicing and Differential Gene Expression in Cichlid Adaptive Radiation
abstract: Species diverge eco-morphologically through the continuous action of natural selection on functionally important structures, producing alternative adaptive morphologies. In cichlid fishes, the oral and pharyngeal jaws are such key structures. Adaptive variation in jaw morphology contributes to trophic specialization, which is hypothesized to fuel their rapid speciation in the East African Great Lakes. Much is known about the genes involved in cichlid jaw and craniofacial development. However, it is still unclear what salient sources of variation gave rise to trophic-niche specialization, facilitating adaptive radiation. Here, we explore two sources of transcriptional variation that may underlie species-specific disparities in jaw morphology. Using whole transcriptome RNA-sequencing, we analyze differences in gene expression and alternative splicing, at the end of postlarval development, in fully functional jaws of six species of cichlids from the Lake Tanganyika tribe Tropheini. Our data reveal a surprisingly high degree of alternative splicing events compared with gene expression differences among species and trophic types. This suggests that differential trophic adaptation of the jaw apparatus may have been shaped by transcriptional rewiring of splicing as well as gene expression variation during the rapid radiation of the Tropheini. Specifically, genes undergoing splicing across most species were found to be enriched for pharyngeal jaw gene ontology terms. Overall, jaw transcriptional patterns at postlarval developmental stage were highly dynamic and species-specific. In conclusion, this work indicates that shifts in alternative splicing could have played a more important role in cichlid adaptive radiation, and possibly adaptive radiation in general, than currently recognized.

title: Ecological opportunity and sexual selection together predict adaptive radiation
abstract: A fundamental challenge to our understanding of biodiversity is to explain why some groups of species undergo adaptive radiations, diversifying extensively into many and varied species, whereas others do not. Both extrinsic environmental factors (for example, resource availability, climate) and intrinsic lineage-specific traits (for example, behavioural or morphological traits, genetic architecture) influence diversification, but few studies have addressed how such factors interact. Radiations of cichlid fishes in the African Great Lakes provide some of the most dramatic cases of species diversification. However, most cichlid lineages in African lakes have not undergone adaptive radiations. Here we compile data on cichlid colonization and diversification in 46 African lakes, along with lake environmental features and information about the traits of colonizing cichlid lineages, to investigate why adaptive radiation does and does not occur. We find that extrinsic environmental factors related to ecological opportunity and intrinsic lineage-specific traits related to sexual selection both strongly influence whether cichlids radiate. Cichlids are more likely to radiate in deep lakes, in regions with more incident solar radiation and in lakes where there has been more time for diversification. Weak or negative associations between diversification and lake surface area indicate that cichlid speciation is not constrained by area, in contrast to diversification in many terrestrial taxa. Among the suite of intrinsic traits that we investigate, sexual dichromatism, a surrogate for the intensity of sexual selection, is consistently positively associated with diversification. Thus, for cichlids, it is the coincidence between ecological opportunity and sexual selection that best predicts whether adaptive radiation will occur. These findings suggest that adaptive radiation is predictable, but only when species traits and environmental factors are jointly considered.

title: Evolutionary Strategies and Morphological Innovations: Cichlid Pharyngeal Jaws
abstract: Liem, Karel F. (Museum of Comparative Zoology, Harvard University, Cambridge, Mass. 02138) 1974. Evolutionary strategies and morphological innovations: Cichlid pharyngeal jaws.  percoid fish family Cichlidae possesses a phenomenal ability to colonize lakes and to diversify to an extent unmatched by any other vertebrate family in the presence of predator pressure and strong competition. The invading cichlids successfully occupy contiguous and occasionally overlapping adaptive zones and specialize progressively into diversified subzones, ramifying prodigiously and covering a breadth of total adaptation that would have been entirely unpredictable if we were aware only of the rudiments of the evolutionary process. This evolutionary avalanche can be attributed to the cooccurrence of a wide range of prospective adaptive zones in the lacustrine environment, and the presence of a unique morphological key innovation of maximum versatility. The new adaptive complex has been revealed in this study by electromyographic analysis synchronized with cineradiography of the cichlid pharyngeal jaw apparatus. The morphological novelty characterizing the family Cichlidae involves the development of: a synarthrosis between the lower pharyngeal jaws, a strategic shift of insertion of the two fourth levator externi muscles, and synovial joints between upper pharyngeal jaws and basicranium. This specialized, highly integrated key innovation enables the cichlids not only to transport (deglutination) but also to prepare food, freeing the premaxillary and mandibular jaws to evolve numerous specializations dealing with the collection of dramatically diverse foods. The functional integration of the innovation is so basic and its potential adaptive versatility so rich that it is maintained throughout the adaptive radiation even though numerous nondisruptive evolutionary changes do take place, providing prodigious opportunities for explosive evolution during the exploitation of rich resources of food in the lacustrine environment. The conversion of the preexisting elements into a new and significantly improved cichlid adaptive complex of high selective value may have evolved by rapid steps under influence of strong selection pressure acting on the minor reconstruction of the -genotype which is involved in evolutionary changes of the pertinent ontogenetic mechanisms. Such relatively simple evolutionary processes are probably the cause for the general phenomenon that only slight reconstructions of existing structures are necessary for successful and rapid adaptation to drastic shifts of adaptive zones.