Total evidence: molecules, morphology, and the phylogenetics of cichlid fishes

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.     

Positive Darwinian Selection Drives the Evolution of the Morphology-Related Gene, EPCAM, in Particularly Species-Rich Lineages of African Cichlid Fishes

The study of genetic evolution within the context of adaptive radiations offers insights to genes and selection pressures that result in rapid morphological change. Cichlid fishes are very species-rich and variable in coloration, behavior, and morphology, and so provide a classical model system for studying the genetics of adaptive radiation. In this study, we researched the evolution of the epithelial cell adhesion molecule (EPCAM), a candidate gene for the adaptive evolution of morphology broadly, and skin development specifically, in fishes. We compared EPCAM gene sequences from a rapidly speciating African cichlid lineage (the haplochromines), a species-poor African lineage (Nile tilapia Oreochromis niloticus), and a very young adaptive radiation in the Neotropics (sympatric crater lake Midas cichlids, Amphilophus sp.). Our results, based on a hierarchy of evolutionary analyses of nucleotide substitution, demonstrate that there are different selection pressures on the EPCAM gene among the cichlid lineages. Several waves of positive natural selection were identified not only on the terminal branches, but also on ancestral branches. Interestingly, significant positive or directional selection was found in the haplochromine cichlids only but not the comparatively species-poor tilapia lineage. We hypothesize that the strong signal of selection in the ancestral African cichlid lineage coincided with the transition from riverine to lacustrine habitat. The two neotropical species for which we collected new sequence data were invariant in the EPCAM locus. Our results suggest that functional changes promoted by positive Darwinian selection are widespread in the EPCAM gene during African cichlid evolution.     

Temporal Patterns of Diversification across Global Cichlid Biodiversity (Acanthomorpha: Cichlidae)

The contrasting distribution of species diversity across the major lineages of cichlids makes them an ideal group for investigating macroevolutionary processes. In this study, we investigate whether different rates of diversification may explain the disparity in species richness across cichlid lineages globally. We present the most taxonomically robust time-calibrated hypothesis of cichlid evolutionary relationships to date. We then utilize this temporal framework to investigate whether both species-rich and depauperate lineages are associated with rapid shifts in diversification rates and if exceptional species richness can be explained by clade age alone. A single significant rapid rate shift increase is detected within the evolutionary history of the African subfamily Pseudocrenilabrinae, which includes the haplochromins of the East African Great Lakes. Several lineages from the subfamilies Pseudocrenilabrinae (Australotilapiini, Oreochromini) and Cichlinae (Heroini) exhibit exceptional species richness given their clade age, a net rate of diversification, and relative rates of extinction, indicating that clade age alone is not a sufficient explanation for their increased diversity. Our results indicate that the Neotropical Cichlinae includes lineages that have not experienced a significant rapid burst in diversification when compared to certain African lineages (rift lake). Neotropical cichlids have remained comparatively understudied with regard to macroevolutionary patterns relative to African lineages, and our results indicate that of Neotropical lineages, the tribe Heroini may have an elevated rate of diversification in contrast to other Neotropical cichlids. These findings provide insight into our understanding of the diversification patterns across taxonomically disparate lineages in this diverse clade of freshwater fishes and one of the most species-rich families of vertebrates.     

Using SINEs to probe ancient explosive speciation: "hidden" radiation of African cichlids?

Cichlid fishes of the east African Great Lakes represent a paradigm of adaptive radiation. We conducted a phylogenetic analysis of cichlids including pan-African and west African species by using insertion patterns of short interspersed elements (SINEs) at orthologous loci. The monophyly of the east African cichlids was consistently supported by seven independent insertions of SINE sequences that are uniquely shared by these species. In addition, data from four other loci indicated that the genera Tilapia (pan-African) and Steatocranus (west African) are the closest relatives to east African cichlids. However, relationships among Tilapia, Steatocranus, and the east African clade were ambiguous because of incongruencies among topologies suggested by insertion patterns of SINEs at six other loci. One plausible explanation for this phenomenon is incomplete lineage sorting of alleles containing or missing a SINE insertion at these loci during ancestral speciation. Such incomplete sorting may have taken place earlier than 14 MYA, followed by random and stochastic fixation of the alleles in subsequent lineages. These observations prompted us to consider the possibility that cichlid speciation occurred at an accelerated rate during this period when the African Great Lakes did not exist. The SINE method could be useful for detecting ancient exclusive speciation events that tend to remain hidden during conventional sequence analyses because of accumulated point mutations.     

Speciation via introgressive hybridization in East African cichlids?

Speciation caused by introgressive hybridization occurs frequently in plants but its importance remains controversial in animal evolution. Here we report a case of introgressive hybridization between two ancient and genetically distinct species of Lake Tanganyika cichlids that led to the formation of a new species. Neolamprologus marunguensis contains mtDNA haplotypes from both parental species varying on average by 12.4% in the first section of the control region and by 5.2% in a segment of the cytochrome b gene. All individuals have almost identical DNA sequences in the flanking regions of the single-copy nuclear DNA locus TmoM27, and show a mosaic of alleles derived from both parental lineages in six microsatellite loci. Hence, our finding displays another mode of speciation in cichlid fishes. The increase of genetic and phenotypic diversity due to hybridization may contribute to the uniquely rapid pace of speciation in cichlids.     

Molecular phylogeny and speciation patterns in host-specific monogeneans (Cichlidogyrus,Dactylogyridae) parasitizing cichlid fishes (Cichliformes,Cichlidae) in Lake Tanganyika

Cichlidogyrus (including Scutogyrus) is the most speciose dactylogyridean monogenean genus known from African and Levantine cichlid fishes (Cichlidae). While its taxonomy is well established, little is known about the phylogenetic relationships and evolutionary history of this ectoparasite, especially from hosts belonging to one of the most impressive vertebrate radiations, the cichlid fishes from the East African Great Lakes and surrounding hydrological systems. Phylogenetic inference based on DNA sequences of the nuclear 18S, internal transcribed spacer 1 and 28S rDNA genes revealed that Cichlidogyrus parasitizing mainly West African cichlid tribes is paraphyletic with respect to species parasitizing hosts belonging to the East African cichlid radiation, which constitute a well-supported monophylum. Members of Cichlidogyrus from tylochromine and oreochromine hosts that colonised Lake Tanganyika only recently, cluster with their non-Lake Tanganyika relatives, indicating that they colonised Lake Tanganyika with their current host species, and did not jump over from any of the many cichlid species already present in the lake. The diversification of Cichlidogyrus in Lake Tanganyika seems to be driven by failure to diverge in old lineages of cichlids, cospeciation in more recently evolved ones, and host switching followed by parasite duplication at the level of the various host tribes. Evaluation of host specificity and structural evolution of haptoral and reproductive organs in Lake Tanganyika Cichlidogyrus revealed that strict specialist species together with larval hook size represent the ancestral state of haptor configuration, suggesting that members of Cichlidogyrus in this system evolved from a very simple form to a more complex one similarly to their West African congeners. Generalist species among Cichlidogyrus with a sclerotized vagina parasitizing ancient Lake Tanganyika lineages seem to have developed a different hook configuration, most probably to ensure successful colonisation of new, phylogenetically unrelated hosts.     

A targeted next‐generation sequencing toolkit for exon‐based cichlid phylogenomics

Cichlid fishes (family Cichlidae) are models for evolutionary and ecological research. Massively parallel sequencing approaches have been successfully applied to study relatively recent diversification in groups of African and Neotropical cichlids, but such technologies have yet to be used for addressing larger‐scale phylogenetic questions of cichlid evolution. Here, we describe a process for identifying putative single‐copy exons from five African cichlid genomes and sequence the targeted exons for a range of divergent (>tens of millions of years) taxa with probes designed from a single reference species (Oreochromis niloticus, Nile tilapia). Targeted sequencing of 923 exons across 10 cichlid species that represent the family's major lineages and geographic distribution resulted in a complete taxon matrix of 564 exons (649 549 bp), representing 559 genes. Maximum likelihood and Bayesian analyses in both species tree and concatenation frameworks yielded the same fully resolved and highly supported topology, which matched the expected backbone phylogeny of the major cichlid lineages. This work adds to the body of evidence that it is possible to use a relatively divergent reference genome for exon target design and successful capture across a broad phylogenetic range of species. Furthermore, our results show that the use of a third‐party laboratory coupled with accessible bioinformatics tools makes such phylogenomics projects feasible for research groups that lack direct access to genomic facilities. We expect that these resources will be used in further cichlid evolution studies and hope the protocols and identified targets will also be useful for phylogenetic studies of a wider range of organisms.     

Mitochondrial DNA Phylogeny of the Family Cichlidae: Monophyly and Fast Molecular Evolution of the Neotropical Assemblage

A mitochondrial DNA (mtDNA) phylogeny of cichlid fish is presented for the most taxonomically inclusive data set compiled to date (64 taxa). 16S rDNA data establish with confidence relationships among major lineages of cichlids, with a general pattern congruent with previous morphological studies and less inclusive molecular phylogenies based on nuclear genes. Cichlids from Madagascar and India are the most basal groups of the family Cichlidae and sister to African–Neotropical cichlids. The cichlid phylogeny suggests drift-vicariance events, consistent with the fragmentation of Gondwana, to explain current biogeographic distributions. Important phylogenetic findings include the placement of the controversial genus Heterochromis basal among African cichlids, the South American genus Retroculus as the most basal taxon of the Neotropical cichlid assemblage, and the close relationship of the Neotropical genera Cichla with Astronotus rather than with the crenicichlines. Based on a large number of South American genera, the Neotropical cichlids are defined as a monophyletic assemblage and shown to harbor significantly higher levels of genetic variation than their African counterparts. Relative rate tests suggest that Neotropical cichlids have experienced accelerated rates of molecular evolution. But these high evolutionary rates were significantly higher among geophagine cichlids. Received: 18 September 1998 / Accepted: 16 December 1998     

Mitochondrial genome primers for Lake Malawi cichlids

Resolving the evolutionary history of rapidly diversifying lineages like the Lake Malawi Cichlid Flock demands powerful phylogenetic tools. Although this clade of over 500 species of fish likely diversified in less than two million years, the availability of extensive sequence data sets, such as complete mitochondrial genomes, could help resolve evolutionary patterns in this group. Using a large number of newly developed primers, we generated whole mitochondrial genome sequences for 14 Lake Malawi cichlids. We compared sequence divergence across protein‐coding regions of the mitochondrial genome and also compared divergence in the mitochondrial loci to divergence at two nuclear protein‐coding loci, Mitfb and Dlx2. Despite the widespread sharing of haplotypes of identical sequences at individual loci, the combined use of all protein‐coding mitochondrial loci provided a bifurcating phylogenetic hypothesis for the exemplars of major lineages within the Lake Malawi cichlid radiation. The primers presented here could have substantial utility for evolutionary analyses of mitochondrial evolution and hybridization within this diverse clade.     

Molecular and fossil evidence place the origin of cichlid fishes long after Gondwanan rifting

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.     

Modelling of essential fish habitat based on remote sensing,spatial analysis and GIS

Lake 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.     

Phylogeny of the Lake Tanganyika cichlid species flock and its relationship to the Central and East African haplochromine cichlid fish faunas

Lake Tanganyika, the oldest of the East African Great Lakes, harbors the ecologically, morphologically, and behaviorally most complex of all assemblages of cichlid fishes, consisting of about 200 described species. The evolutionary old age of the cichlid assemblage, its extreme degree of morphological differentiation, the lack of species with intermediate morphologies, and the rapidity of lineage formation have made evolutionary reconstruction difficult. The number and origin of seeding lineages, particularly the possible contribution of riverine haplochromine cichlids to endemic lacustrine lineages, remains unclear. Our phylogenetic analyses, based on mitochondrial DNA sequences of three gene segments of 49 species (25% of all described species, up to 2,400 bp each), yield robust phylogenies that provide new insights into the Lake Tanganyika adaptive radiation as well as into the origin of the Central- and East-African haplochromine faunas. Our data suggest that eight ancient African lineages may have seeded the Tanganyikan cichlid radiation. One of these seeding lineages, probably comprising substrate spawning Lamprologus-like species, diversified into six lineages that evolved mouthbrooding during the initial stage of the radiation. All analyzed haplochromines from surrounding rivers and lakes seem to have evolved within the radiating Tanganyikan lineages. Thus, our findings contradict the current hypothesis that ancestral riverine haplochromines colonized Lake Tanganyika to give rise to at least part of its spectacular endemic cichlid species assemblage. Instead, the early phases of the Tanganyikan radiation affected Central and East African rivers and lakes. The haplochromines may have evolved in the Tanganyikan basin before the lake became a hydrologically and ecologically closed system and then secondarily colonized surrounding rivers. Apparently, therefore, the current diversity of Central and East African haplochromines represents a relatively young and polyphyletic fauna that evolved from or in parallel to lineages now endemic to Lake Tanganyika.     

Molecular phylogeny and biogeography of the Malagasy and South Asian cichlids (Teleostei: Perciformes: Cichlidae)

Phylogenetic relationships of the Malagasy and South Asian cichlids are investigated using nucleotide characters from two mitochondrial genes, a 544 bp region of the large ribosomal subunit (16S) and a 649 bp region of cytochrome c oxidase subunit I (COI). This is the first molecular analysis to include a thorough taxonomic sampling of all Malagasy-South Asian genera, and a near complete taxonomic inventory of all valid species. Parsimony analysis of the combined data set (1193 aligned nucleotide positions) results in a single, completely resolved phylogenetic hypothesis. Results of this analysis, and that based on more comprehensive taxonomic sampling across Cichlidae for 16S alone (554 bp for 73 taxa), indicate that the Malagasy cichlids are paraphyletic, whereas the Malagasy and South Asian cichlids comprise a monophyletic group. In both analyses, the African and Neotropical assemblages are monophyletic. The Malagasy-South Asian cichlids are not recovered as plesiomorphic members of the family in either analysis. Two major clades are recovered within the Malagasy-South Asian assemblage and given subfamilial rank, Etroplinae, comprising Paretroplus (Madagascar) and Etroplus (southern India and Sri Lanka), and Ptychochrominae, comprising Ptychochromis, Ptychochromoides, and Oxylapia, all endemic Malagasy genera. Placement of the endemic Malagasy genus Paratilapia is equivocal depending on the gene fragment(s) analyzed. Inter- and intrageneric relationships within Ptychochrominae and Etroplinae are presented and discussed. The hypothesis of relationships for Cichlidae based on nucleotide characters from 16S alone, arguably the most comprehensive and broadly sampled data set across all major geographic assemblages to date, is congruent with prevailing hypotheses regarding the sequence of Gondwanan fragmentation and a vicariance scenario to explain the current distribution of cichlid fishes.     

Phylogeny and biogeography of cichlid fishes (Teleostei: Perciformes: Cichlidae)

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. © The Willi Hennig Society 2004.     

African cichlid fish: a model system in adaptive radiation research

The African cichlid fish radiations are the most diverse extant animal radiations and provide a unique system to test predictions of speciation and adaptive radiation theory. The past few years have seen major advances in the phylogenetics, evolutionary biogeography and ecology of cichlid fish. Most of this work has concentrated on the most diverse radiations. Unfortunately, a large number of small radiations and 'non-radiations' have been overlooked, potentially limiting the contribution of the cichlid system to our understanding of speciation and adaptive radiation. I have reviewed the literature to identify 33 intralacustrine radiations and 76 failed radiations. For as many as possible I collected information on lake size, age and phylogenetic relationships. I use these data to address two questions: (i) whether the rate of speciation and the resulting species richness are related to temporal and spatial variation in ecological opportunity and (ii) whether the likelihood of undergoing adaptive radiation is similar for different African cichlid lineages. The former is a key prediction of the ecological theory of adaptive radiation that has been presumed true but remains untested for cichlid radiations. The second is based on the hypothesis that the propensity of cichlids to radiate is due to a key evolutionary innovation shared by all African cichlids. The evidence suggests that speciation rate declines through time as niches get filled up during adaptive radiation: young radiations and early stages of old radiations are characterized by high rates of speciation, whereas at least 0.5 Myr into a radiation speciation becomes a lot less frequent. The number of species in cichlid radiations increases with lake size, supporting the prediction that species diversity increases with habitat heterogeneity, but also with opportunity for isolation by distance. Finally, the data suggest that the propensity to radiate within lakes is a derived property that evolved during the evolutionary history of some African cichlids, and the appearance of which does not coincide with the appearance of proposed key innovations in morphology and life history.     

Genetic population structure indicates sympatric speciation of Lake Malawi pelagic cichlids.

Allopatric processes of speciation have routinely been presented to explain the extraordinary radiation of the East African Great Lakes cichlid fish species flocks. The 21 or more species of pelagic cichlids within the Lake Malawi flock appear to have lake-wide distributions that challenge such a concept. Data from six microsatellite DNA loci indicate single, panmictic populations across the lake of three Diplotaxodon species. Levels of variability at these loci suggest that populations have been large and stable. Mitochondrial DNA sequence data (872 bp of control region + 981 bp of the NADH-2) from 90 species, representing all major clades within the Lake Malawi flock, indicate reciprocal monophyly of the pelagic clade. We suggest that these data support a hypothesis that speciation in sympatry is more plausible (and widespread) within the cichlid species flocks than previously thought.     

Positive selection and gene conversion in SPP120, a fertilization-related gene, during the East African cichlid fish radiation

The ability to infer historical natural selection from sequence data aides in finding genes that might be important in adaptation and the formation of new species. As the fastest evolving and largest known vertebrate radiation, the cichlid fish of the African Great Lakes exhibit a wide range of recent morphological diversification. We used DNA databases, mostly of expressed sequence tags, to find candidate orthologous coding sequences from 2 tribes of cichlids and, using an automated procedure, scanned these sequence pairs for high dN/dS, the signal of positive selection and protein adaptation. The results included vertebrate genes commonly found to be under selection (e.g., major histocompatibility complex [MHC] loci) as well as genes known to be important specifically in the cichlid radiation (e.g., long-wave-sensitive opsins). Further investigation focused on a gene encoding a fertilization-related protein, SPP120, which was previously known only from cichlids. Using maximum likelihood analysis on novel SPP120 cDNA sequences from a range of African cichlids, we demonstrate the influence of positive selection in a specific subregion of the protein. We also show that SPP120 is a tandemly arranged, multicopy gene evolving with occasional interlocus gene conversion. A search of the Medaka genome database also revealed a tandem arrangement of multiple SPP120 copies and evolutionary rate differences between Medaka gene subregions mirroring those found for cichlids. Combined, these results suggest that SPP120 has been under repeated diversifying selection for over 100 Myr.     

Evolutionary history of Lake Tanganyika's scale-eating cichlid fishes

Although Lake Tanganyika is not the most species-rich of the Great East African Lakes it comprises by far the greatest diversity of cichlid fishes in terms of morphology, ecology, and breeding styles. Our study focuses on the Tanganyikan cichlid tribe Perissodini, which exhibits one of the most peculiar feeding strategies found in cichlids-scale-eating. Their evolutionary history was reconstructed from 1416 bp DNA sequence of two mitochondrial genes (ND2 and partial control region) and from 612 AFLP markers. We confirm the inclusion of the zooplanktivorous genus Haplotaxodon in the tribe Perissodini, and species status of Haplotaxodon trifasciatus. Within the Perissodini, the major lineages emerged within a short period roughly 1.5-2 MYA, which makes their radiation slightly younger than that of other Tanganyikan cichlid tribes. Most scale-eaters evolved in deep-water habitat, perhaps associated with the previously documented radiations of other deep-water dwelling cichlid lineages, and colonized the shallow habitat only recently.     

A novel primer set for multilocus phylogenetic inference in East African cichlid fishes

The cichlid fishes in the East African Great Lakes are a prime model system for the study of adaptive radiation. Therefore, the availability of an elaborate phylogenetic framework is an important prerequisite. Previous phylogenetic hypotheses on East African cichlids are mainly based on mitochondrial and/or fragment‐based markers, and, to date, no taxon‐rich phylogeny exists that is based on multilocus DNA sequence data. Here, we present the design of an extensive new primer set (24 nuclear makers) for East African cichlids that will be used for multilocus phylogenetic analyses in the future. The primers are designed to work for both Sanger sequencing and next‐generation sequencing with the 454 technology. As a proof of principle, we validate these primers in a phylogenetically representative set of 16 cichlid species from Lake Tanganyika and main river systems in the area and provide a basic evaluation of the markers with respect to marker length and diversity indices.