Understanding Explosive Diversification Through Cichlid Fish Genomics

Reviews

Understanding explosive diversification through cichlid fish genomics

Walter Salzburger Abstract | Owing to their taxonomic, phenotypic, ecological and behavioural diversity and propensity for explosive diversification, the assemblages of cichlid fish in the East African Great Lakes Victoria, Malawi and Tanganyika are important role models in evolutionary biology. With the release of five reference genomes and many additional genomic resources, as well as the establishment of functional genomic tools, the cichlid system has fully entered the genomic era. The in-depth​ genomic exploration of the East African cichlid fauna — in combination with the examination of their ecology , morphology and behaviour — permits novel insights into the way organisms diversify.

Model organisms Why is species richness so unequally distributed across and morphological diversity as a consequence of adap- Non-​human species studied in the tree of life? Why did some organismal lineages diver- tation to novel or hitherto underutilized ecological detail in the context of a sify into new forms in a seemingly explosive manner, niches14,15 — combine the advantages of laboratory and particular research question whereas others have lingered phenotypically unvaried natural model species in the context of the genesis of with the motivation to be able to make more general over millions of years? These questions have puzzled biodiversity. Therefore, iconic examples of adaptive radi- statements about the generations of biologists ever since Darwin and Wallace ation, such as Darwin’s finches on the Galapagos archi- functioning of organisms. jointly introduced their theory of evolution by natural pelago, anole lizards on the islands of the Caribbean, selection1. 160 years of scholarly study later, there is a rea- threespine stickleback fish in post-​glacial rivers and Clades sonable understanding of how and under which circum- lakes, and cichlid fish in East Africa (Box 1), have long Branches on an evolutionary 2–7 tree, consisting of a common stances new species can originate . However, the causal been recognized as essential models to study organis- 12,16–18 ancestor and all its factors that determine species richness and the rate mal diversification . Scientific interest in many of descendants (a clade is, hence, at which new species form remain largely elusive8,9. these radiations can be traced back to the 19th cen- equivalent to a monophyletic Particularly in light of the global biodiversity crisis that tury, such that, for a long time, these groups have been group). our planet is currently facing10, it is no longer of purely intensely investigated with respect to their evolution, academic interest to know how novel species form and, ecology, ontogenetic development and behaviour. The consequently, how biodiversity arises. close relatedness of the species emerging from adaptive Unravelling how variation at the genomic level is radiations facilitates genetic and genomic investiga- interlinked with phenotypic evolution is key to under- tions12, for example, on the basis of hybrid crosses or standing organismal diversification2,11–13. To this end, divergence mapping. Moreover, representatives of these we must understand how organisms evolve, how they adaptive radiations were among the first vertebrates to function and how they interact with other organisms and have their genomes sequenced19–23. the environment. The problem is that many widely used The species flocks of cichlid fish in the East African model organisms provide limited insights into the under- Great Lakes Victoria, Malawi and Tanganyika repre- pinnings of rapid — by way of comparison — organismal sent the most species-​rich and phenotypically diverse diversification; many traditional laboratory-based​ model adaptive radiations in vertebrates and are characterized organisms tell us little about how organisms adapt, by exceptionally fast diversification rates18,24,25 (Box 1; Zoological Institute, behave and diversify in the wild, while model species in Fig. 1). To put cichlid radiations into a temporal context, Department of Environmental ecology and evolution often lack tractability in the labo- during the evolutionary time span of our own species, Sciences, University of Basel, Basel, Switzerland. ratory and fundamental data on genomics and develop- starting with the split between chimpanzees and humans e-mail:​ walter.salzburger@ ment. Importantly, most established model organisms do some 5–7 million years ago, approximately 2,000 spe- unibas.ch not belong to extensively diversifying clades. cies of cichlid fish evolved in East Africa, the geographic https://doi.org/10.1038/ Instances of adaptive radiation — that is, the rapid region where the chimpanzee–human split initially s41576-018-0043-9 (sometimes ‘explosive’) origin of taxonomic, ecological occurred. Within the time span that it took for 14 species

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Box 1 | What is a cichlid? The family Cichlidae (Bonaparte, 1835) is one of 536 families in fish117. Cichlidae, together with only two species of convict blennies in the family Pholidichthyidae, make up the percomorph order Cichliformes in the series Ovalentaria118,119. Etymologically, the family name descends from the Greek word κίχλη (‘kichle’) for thrushes and some marine wrasses120,121. The oldest known written record of a cichlid is a hieroglyph in ancient Egypt specifically assigned to the Nile tilapia (Oreochromis niloticus)122. The first cichlid artefacts also appeared in ancient Egypt, displaying the mouthbrooding tilapia as a symbol of rebirth123. Another tilapia, Sarotherodon galilaeus, embodies the biblical St. Peter’s fish in the Sea of Galilee. Cichlids inhabit fresh and brackish waters in — from East to West — southern India and Sri Lanka, Madagascar, the Middle East, Africa, the West Indies and the Americas (South and Central America, Mexico and Texas). Wherever they occur, cichlids show a strong tendency to form species flocks with high levels of endemism (the exclusive occurrence of a species or higher-level​ taxon in a confined geographic area)31,124–126. Species flocks are conceptually related to the phenomenon of adaptive radiation because most, if not all, species flocks are the outcome of such radiations35,85,127. Cichlids show substantial diversity across different levels of biological organization: Taxonomic diversity. Approximately 1,700 cichlid species in more than 200 genera are recognized to date117,121, but many more species have yet to be described. The total number of cichlid species has been estimated in the range of 3,000–4,000, suggesting that roughly every tenth teleost fish species is a cichlid. More than half of this estimated taxonomic diversity is allocated to the cichlid faunas of the African Great Lakes Tanganyika (250 species), Malawi (800–1,000 species) and Victoria (700 species, including its surrounding water bodies)25,39,85,128. Morphological diversity. Morphologically, cichlids are characterized by a single nostril on each side of their snout rather than two as in most other fish; their lateral line is interrupted; their dorsal and anal fins consist of spines (7–25 and 3–15, respectively) and soft rays (3–15 and 4–30, respectively); and they possess a characteristic pharyngeal jaw apparatus, a second set of jaws in the pharynx that is functionally decoupled from the oral jaw apparatus, for processing food117,121. Although none of these characteristics is unique to cichlids, their combination is. Cichlids vary widely in body shape — from rounded, as exemplified by the freshwater angelfish (Pterophyllum scalare) or the discus fish (Symphysodon discus), to elongated, as in Crenicichla percna — as well as in body size — from less than 3 cm and 0.1 g in, for example, Telmatochromis bifrenatus to >80 cm and >3 kg in Boulengerochromis microlepis, both from Lake Tanganyika. Above all, cichlids differ in ecologically relevant traits, such as the mouth apparatus and head shape17,61. Ecological diversity. Cichlids occupy a wide range of feeding niches, ranging from eating algae, sponges, invertebrates or other fish to mollusc crushing or scale eating, and habitat types, ranging from small streams or rivers to lakes and, within lakes, from the shallow to the deep benthic and limnetic zones. Cichlids occur on rocky, sandy, weedy and muddy substrates, yet others are found in caves or use empty snail shells as shelter17,18. As in other cases of adaptive radiation, there is a strong correlation between phenotype and environment in cichlids17,61. Colour diversity. Cichlids are highly diverse with respect to body coloration and pigmentation, hence their German name ‘Buntbarsche’ (colourful perches). Coloration in cichlids has been implicated in sexual selection via female choice with respect to male nuptial coloration129 but also with mimicry17,130 and camouflage17. Not least because of their conspicuous coloration, cichlids are popular with aquarists. Behavioural diversity. Cichlids show a very broad behavioural repertoire. Many cichlid species are territorial, others form large schools. All cichlids show some kind of brood-​care behaviour, ranging from substrate spawning to maternal, paternal or biparental mouthbrooding17,18,120.

of Darwin’s finches to evolve on the Galapagos archipel- Explosive diversification in cichlids ago22, about 1,000 cichlid species evolved in Lake Malawi The adaptive radiations of cichlid fish in Lakes Victoria, alone26–28. In addition, since the last ice age, which is Malawi and Tanganyika (Fig. 1) differ from all other cases when sticklebacks began to diverge into replicate species of adaptive radiation in vertebrates — including those of pairs in the Northern hemisphere20, hundreds of cichlid cichlids elsewhere — by their unparalleled degree 29,30 24,31 Divergence mapping species evolved in Lake Victoria . of phenotypic and taxonomic diversity in sympatry . Genetic-marker-based​ search In this Review, I discuss how the examination of That cichlids are unusual had been realized already for genomic regions exhibiting recently available genome-​wide sequence data of East over the course of the earliest biological explorations exceptionally strong African cichlids has deepened our understanding of the of the African Great Lakes in the late 19th century32,33. differentiation between different biological entities, phenomena of adaptive radiation and explosive diversifi- Ever since, researchers repeatedly came to the con- such as populations or species. cation in general and in cichlids in particular. I start with clusion that the current understanding of speciation a discussion of the explosive nature of species formation was insufficient to explain the plethora of cichlid spe- Species flocks in East African cichlids and the resultant difficulties in cies in East Africa17,34,35. With specific reference to the Unusually taxon-rich​ (ref.34) assemblages of closely related delineating species. Then, I focus on the challenges that cichlids, Woltereck in 1931 introduced the term species that coexist in the emerge in the reconstruction of the evolutionary history ‘Artexplosion’ for outbursts of endemic diversity on same area, such as an island, a of rapidly diversifying clades at the interface between islands and in some ancient lakes. More widely known lake or a section of a river. population genetics and phylogenetics. Finally, I sum- as explosive speciation, this term refers to a substantial marize what we have learned about the genomes of East increase in speciation rate in a clade relative to a com- Lateral line A sensory system along the African cichlids thus far and discuss which features in parable group, irrespective of ecological and morpho- body in aquatic vertebrates their genomes are potentially linked to their propensity logical differentiation, and is thus distinct from the that consists of sensory cells. to diversify explosively. ­phenomenon of adaptive radiation36.

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Sympatry The process of species formation applied to cichlids and often fails on the basis that repro- The existence of two or more Generally speaking, speciation is the formation of a new ductive isolation is usually incomplete between sister species in the same geographic species that is distinct from all other species; it is com- taxa. In fact, many East African cichlid species are inter- area such that they encounter monly defined as the build-​up of reproductive isolation crossable51,52, even when belonging to distinct phylo­ each other frequently. between an ancestral species and the newly formed spe- genetic lineages and being derived from different adaptive 2 53,54 Reproductive isolation cies . Most of the earlier work on this topic has focused radiations , and cichlids do interbreed in the wild as Any property that prevents (or on the role of geographical separation in the origin of spe- evidenced by occasionally observed hybrid specimens55 reduces the probability of) cies (reviewed in ref.2). More recently, the field has shifted as well as molecular analyses demonstrating substantial members of one species towards a more process-​oriented approach, emphasizing levels of gene flow between species41,56,57. Delineating breeding successfully with 5,6 members of another species. the importance of ecology in speciation — through cichlid species by means of genetic markers is problem- divergent natural selection in distinct environments — atic too. DNA barcoding, a widely used method for iden- Gene flow and establishing that speciation is possible, perhaps even tifying species on the basis of the mitochondrial COX1 The movement or exchange of common, in light of some levels of gene flow between the gene58, performs poorly when applied to cichlids59. This genes into or through a 37,38 population by interbreeding or diversifying lineages . Both of these features, ecologi- is not surprising, given the high levels of DNA sequence by migration and subsequent cal speciation and gene flow, are particularly common in similarity in cichlids (for example, the average genome-​ interbreeding. adaptive radiations, in the course of which new species wide sequence divergence between Lake Malawi cichlids typically form in the absence of geographical barriers14,15. is only 0.1–0.25%)27 as well as mitochondrial haplotype Speciation continuum Speciation is often (but not always) a gradual process sharing between species42,43. Defining species according The bandwidth of variation phylogenetic species concept between diversifying that has a clearly defined starting point — a single spe- to the is equally problem- populations ranging from cies — and is completed when at least one new species atic, as there is no a priori level of genetic distinctive- virtually no variation (panmixia) (if the ancestral species continues to exist) or a mini- ness above which two sister taxa should be considered through partially discontinuous mum of two species (if the ancestral species becomes different species and because reciprocal monophyly of variation (incipient barriers to gene flow) to strongly extinct) has emerged. In between, along the so-​called sister taxa does not help in deciding whether these are discontinuous variation speciation continuum, it is impossible to conclusively populations of the same species or different species. (complete reproductive determine whether there is one or more than one spe- Grouping individuals (or lineages thereof) into species isolation). cies. Any consideration of the process of speciation is according to their shared ecology — as suggested in the therefore inextricably interwoven with the questions of ecological species concept — is difficult in cichlids, as there Tribes The taxonomic rank between what a species is and how it can be distinguished from is substantial niche and resource overlap and, hence, little the genus and the family level. other, closely related ones. competitive exclusion (whereby two species cannot stably coexist in the same ecological niche) between some spe- Sister taxa Species delineation in cichlids cies17,60,61. In practice, to facilitate the expedient naming Reciprocally closest relatives of one another. The delineation of species is not an easy undertaking in of distinct taxonomic units in cichlids, species are seen cichlids. Although species belonging to different lineages as clusters of individuals that are morphologically and Ring species (that is, tribes or genera) in the longer persisting cichlid ecologically similar and distinct from other such clusters Two reproductively isolated radiations of Lakes Tanganyika and Malawi are pheno­ (that is, the vernacular species concept)62. populations are connected typically and ecologically clearly distinct from one another Taken together, there is no straightforward way of through a geographic ring of (Fig. 1) populations that interbreed; no , the status of species within such lineages as well species delineation in cichlids. Phenotypically distinct morphological character can as within younger species flocks is often unclear. This is, yet closely related cichlids within species flocks may per- be used, except arbitrarily, to in part, due to the sheer number of species, which makes haps best be characterized as multispecies, that is, sets divide the ring into discrete it difficult for taxonomists to keep track of taxonomic of closely related species that co-​occur and that occa- taxonomic units. units and to sort species according to diagnostic charac- sionally exchange genes63. This is exemplified by two 39,40 Biological species concept teristics . However, the main issues in deciding what a closely related Pundamilia species from Lake Victoria, A concept positing that species species is in cichlids emerge from their close relatedness Pundamilia nyererei and Pundamilia pundamilia, which, are groups of actually or and the fact that radiations are still ongoing: morpholog- when co-​occurring, are genetically more similar to one potentially interbreeding ically distinct sister taxa are typically of very recent ori- another than two geographically separated (allopatric) natural populations that are 4,30,41,42 4,41 reproductively isolated from gin (in the range of a few hundred to a few thousand populations of the same species . Importantly, what other such groups. years), and, in many cases, it is not clear whether they have makes species delineation so difficult in cichlid spe- reached the end of the speciation continuum. In other cies flocks — namely, that there are many species, that Haplotype cases, geographically isolated sister taxa are connected species are very young and that speciation is often ‘caught A stretch of DNA on a single 43–45 molecule (chromosome, plastid through intermediate forms , much as in the case of in the act’ — is exactly what makes cichlids such a useful or mitochondrion) that is ring species, rendering it impossible to define clear bound- model for the study of the diversification process. Note that inherited as a single unit. aries between them. By contrast, closely related cichlids of to account for the situation in which, in many cases, speci- uncertain taxonomic status often mate assortatively with ation is not yet complete, one should use the more general Phylogenetic species respect to their own source population46–48, that is, indi- term ‘diversification’ instead of ‘speciation’ in the context concept A concept positing that species viduals mate more frequently with members of their own of the adaptive radiations of East African cichlids, as the are ‘tips’ on a phylogeny, that population than expected under a random pattern, or even latter designation requires completion of the process. is, the smallest set of occur in sympatry in parts of their distribution ranges43,44 organisms that share an (Fig. 2), suggesting that these sister taxa are valid species. Reconstructing cichlid evolution ancestor and can be distinguished from other such The classic species concepts provide little guidance The availability of accurate, comprehensively sampled sets. for species delineation in cichlids. The most widely and time-​calibrated phylogenetic hypotheses is crucial used definition in biology for the category species, the to understanding the progression of adaptive radiations biological species concept49,50, is not very practical when and explosive diversification64, as well as to interpret

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Haplochromis sp. Haplochromis sp. Pundamilia sp. Paralabidochromis Haplochromis sauvagei aeneocolor Nile tilapia (Oreochromis niloticus)

Haplochromis Haplochromis Yssichromis Haplochromis sp. Pundamilia nyererei paropius thereuterion piceatus ‘Victoria Nile’

Lake Victoria region | ~700 species

Lake Tanganyika | 250 species SouthSouth Sudan

Democratic UgandaUganda Republic Lake Kioga Altolamprologus sp. Ctenochromis of the Congo ‘Sumbu shell’ Neolamprologus brichardi horei Lake Albert

Lake Edward Lake Kivu Kenya Lamprologus Neolamprologus Petrochromis sp. Rwanda Lake Victoria signatus longior ‘Texas’ Burundi

Lakeake TTaanganynganyikikaa

Lepidiolamprologus Julidochromis Astatotilapia burtoni elongatus transcriptus Lake Rukwa

Tanzania Lake Mweru

Lake Bangweulu Asprotilapia Greenwoodochromis Tropheus moorii leptura christyi Lake Malawi Mozambique Zambia MalawiM i

Eretmodus cyanostictus Boulengerochromis microlepis

Lake Malawi | 800-1,000 species

Cheilochromis Dimidiochromis Exochochromis Nimbochromis Placidochromis sp. Maylandia zebra euchilus compressiceps anagenys livingstonii

Astatotilapia Labeotropheus Melanochromis Pseudotropheus Lichnochromis Rhamphochromis esox calliptera fuelleborni auratus johannii acuticeps

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◀ Fig. 1 | The species flocks of cichlid fish in East Africa. The lakes and rivers in the area entire evolutionary history of a group, making the con- harbour diverse assemblages of endemic cichlids, of which the species flocks in Lakes cept of a clade and the quest for bifurcating branching Tanganyika and Malawi and in the Lake Victoria region are, by far, the most species-rich.​ diagrams questionable. Dotted boxes highlight the five species that were selected for the initial round of The analysis of genome-​wide markers neverthe- genome sequencing19. Images courtesy of W. Gessl, University of Graz, Austria. less provides novel insight regarding evolutionary relationships in cichlids, not least because incomplete general patterns of these phenomena (Box 2). At the lineage sorting and (introgressive) hybridization can same time, the close relatedness of species within adap- now be looked at on a genome-​wide scale and across tive radiations, occasional gene flow between species and many taxa26,27,76. By and large, the phylogeny of the East the pace at which new species form pose considerable African cichlids reflects the dynamic geological his- challenges to the phylogenetic reconstruction of rapidly tory of the area (Fig. 4). Lake Tanganyika, the oldest and diversifying clades22,27,64–66 (Fig. 3), even if genome-​wide geologically most stable of the African Great Lakes31, is data are available. home to the phenotypically most diverse cichlid spe- cies flock17. Taxonomically, the Tanganyikan cichlids The first cichlid genomes have been grouped into 14 tribes, which differ sub- The initial round of genome sequencing in cichlids stantially in species number (ranging from 1 species in, involved a set of five phylogenetically representative for example, Boulengerochromini to about 100 species African species19: the Nile tilapia (Oreochromis nilot- in Lamprologini)61,77. Some tribes — even if endemic icus), as a member of a less species-​rich yet geographi- to Lake Tanganyika today — must have evolved else- cally widespread sister lineage to the cichlid radiations where, that is, before the formation of the present lake in the East African Great Lakes; the Princess of Burundi some 9–12 million years ago (notably, the Bathybatini, (Neolamprologus brichardi), from the most species-​rich Boulengerochromini and Trematocarini), whereas the tribe within Lake Tanganyika, the Lamprologini; and three origin of other tribes is compatible with a scenario of Monophyly 26,78,79 The condition of forming a members of the tribe Haplochromini, namely, Burton’s in situ evolution . Although the respective mono- monophyletic group (that is, a cichlid (Astatotilapia burtoni) from Lake Tanganyika phyly of these tribes is usually well supported in phylo­ clade). and nearby rivers, the Zebra mbuna (Metriaclima zebra) genetic analyses using genome-​wide data26,78–80, there from the mbuna clade of Lake Malawi and P. nyererei from is strong evidence for past gene flow between some of Ecological species concept (Figs. 1,4) 26,78,79 (Fig. 4) A concept positing that species Lake Victoria . To facilitate genome annotations, them . 19 are sets of organisms that are the study of Brawand et al. also established reference The radiations in Lakes Malawi and Victoria involve adapted to a particular set of transcriptomes for these five species. In the meantime, only one of the African cichlid tribes, the Haplochromini resources, that is, to the same many more genomes of East African cichlids have been (note that in all three African Great Lakes, a few mem- ecological niche. sequenced at low coverage with short-​read (Illumina) bers of the Oreochromini are found, which did not radi- 27,41,57,67,68 Incomplete lineage sorting sequencing approaches . The assemblies of two ate). In the early stages of the cichlid adaptive radiation The imperfect segregation of a of the reference genomes, the Nile tilapia and the Zebra in Lake Malawi, three major clades emerged in closely gene into all evolutionary mbuna, have been much improved with long-​read timed lineage-splitting​ events: a pelagic clade formed by lineages, that is, a gene fails to (PacBio) sequencing69,70, and additional transcriptomes Diplotaxodon and Rhamphochromis; a clade including coalesce within the duration of a species. from more species and more tissues have been gener- shallow and deep benthic species as well as the utaka ated71–75. These advances have confirmed that the genomes lineage; and the mbuna clade. The presumed ancestor Introgression of rapidly diversifying cichlids are frequently subjected to of the radiation, the generalist species Astatotilapia Also referred to as incomplete lineage sorting and introgression (Fig. 3c,d), result- calliptera, continued to exist in rivers and lakes in the introgressive hybridization. The ing in mosaic genomes that consist of small segments with area (including Lake Malawi)27 (Fig. 4); this species is, transfer of genetic material 12,19,27,57 (Fig. 3g) from one species into another different evolutionary histories . hence, phylogenetically nested within the Lake Malawi 27,81 via repeated and asymmetrical cichlid radiation . The inspection of genome-​wide backcrossing with one of the The phylogeny of East African cichlids data further revealed multiple events of gene flow parental lineages after a Incongruence between gene trees and between gene trees within and between the main cichlid clades in Lake hybridization event. and the species tree is particularly pronounced in East Malawi27. The current age estimates for the onset of the 26,27 Mosaic genomes African cichlids. For example, analysis of the initially Lake Malawi cichlid radiation are compatible with a Genomes heterogeneous in sequenced cichlid reference genomes revealed that more time-calibrated​ paleoecological record82, which revealed ancestry, emerging from than 40% of all single nucleotide polymorphisms (SNPs) that the lake transitioned into its current state as a more introgression, incomplete support topologies among the three representatives of the or less closed system with deepwater habitats approxi- lineage sorting or lateral gene transfer. tribe Haplochromini that are in conflict with the species mately 800,000 years ago, making cichlid diversification tree19. Phylogenetic analyses of thousands of segments in into deepwater habitats possible only afterwards28. Gene trees the genomes of 5 closely related Neolamprologus species The situation in Lake Victoria differs somewhat given Actual evolutionary from Lake Tanganyika showed that all of the 15 possible that the cichlid fauna of the lake is part of a geograph- relationships between the versions of a gene as present in topologies connecting the 5 species received support from ically more extended species assemblage, the so-​called different taxa. at least a few dozen segments in the genome, whereby Lake Victoria region super-​flock, which includes the one topology stood out as being supported by about half adaptive radiations of cichlids in Lakes Victoria, Edward, Species tree of all segments57. In addition, the analysis of 2,543 non-​ Albert and Kivu, among others42,76 (Fig. 1). The onset of Actual evolutionary overlapping windows, each containing 8,000 SNPs, across the diversification of the super-flock​ has been estimated relationships between a set of 26,42,76,83 species. The species tree the genomes of 73 Lake Malawi cichlid species produced at 100,000–200,000 years ago , and most, if not all, 27 reflects the ‘true’ evolutionary 2,542 different topologies . Obviously, in such situations, species within Lake Victoria must have evolved within history of a clade. a single tree-​like phylogeny can no longer capture the the past 15,000 years following its refill after complete

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Tropheus sp. ‘black’

Tropheus brichardi

Tropheus sp. ‘lunatus’

Tropheus duboisi

Tropheus annectens Tropheus sp. ‘maculatus’ Tropheus sp. ‘Mpimbwe’

Tropheus sp. ‘red’

Tropheus moorii Fig. 2 | The Tropheus species complex from Lake Tanganyika. At Lake Tanganyika, basically every stretch of rocky shoreline is inhabited by its own colour morph of Tropheus, with more or less sharp boundaries of gene flow between adjacent populations44,135. Six nominal species are currently described in this genus, but a few more await formal description136. Up to four different taxonomic units (formally described species, undescribed species and local colour morphs) occur in sympatry at certain locations, suggesting that these are valid species. Distribution ranges are according to refs45,136. Drawings courtesy of J. Himes, University of Basel, Switzerland.

desiccation in the late Pleistocene29,30. The extremely groups of animals31, which is corroborated by the scaling young age of the roughly 700 species in this super-flock​ of the number of endemic species in a lake with such a thus makes classic phylogenetic analyses difficult. The size, relative stability and depth31,84,87. Still, the number most thorough analysis so far on the basis of restriction-​ of endemic cichlid species in Lakes Victoria, Malawi site associated DNA (RAD) sequencing suggests that not and Tanganyika is at least an order of magnitude higher all radiations in the lakes of the Lake Victoria region are than the number of endemic species in any other fam- reciprocally monophyletic and that hybridization played ily resident in these lakes (except, perhaps, ostracods a key part in triggering these radiations76. in Lake Tanganyika)31, and none of approximately 20 non-​cichlid fish families that also occur in each of these Genomic basis of cichlid diversification lakes has brought forth more than a handful of endemic The phenomenon of explosive diversification in species17,88. These findings suggest that there is some- cichlids has long been implicated with the particu- thing special about cichlids — some kind of ‘cichlidness’ lar environment in which cichlid evolution has taken — that permits these fish to diversify explosively. That place, as well as with intrinsic features of the cich- cichlids in general feature an intrinsic propensity for Ecological opportunity lids themselves17,35,84–86. Without a doubt, the ample diversification is substantiated by numerous examples of The availability of ecologically ecological opportunity accessible resources that may provided by the African Great smaller-​scale (in comparison to the ones in the African be exploited by an Lakes is conducive for diversification through adaptive Great Lakes) adaptive radiations in rivers and lakes evolutionary lineage. radiation, not only in cichlids but also across different throughout their distribution range84,89–93, of which the

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Box 2 | General patterns in (cichlid) adaptive radiations element expansions were detected in cichlids. Many more cichlid genomes have been inspected since, call- Adaptive radiations in different groups of organisms are thought to follow the same ing for a revisit to the question of what the genomic general patterns, which include an early burst of phenotypic divergence, a taxonomic underpinnings of adaptive radiation and explosive overshooting and a progression in stages (reviewed in ref.15). In the adaptive radiations diversification in cichlids are. of cichlids in East Africa, early bursts of divergence are a likely scenario (given the generally young age of the species flocks) but are difficult to prove in the absence of independent sources of information, such as fossils in chronological resolution. Sources of genetic variation The phenomenon of overshooting due to decreasing speciation and/or increasing There is a general consensus that divergent natural extinction rates along the course of an adaptive radiation might explain why there are selection, partly in combination with sexual selection, more cichlid species in the lake of intermediate age, Lake Malawi, than in the younger has played a key part in the diversification of cich- (Lake Victoria) and the older (Lake Tanganyika) lakes30 (Fig. 1). However, differences in lids18,19,31,84,97. Heritable phenotypic variation in fitness-​ (estimated) species numbers between the African Great Lakes also reflect the different related traits is a prerequisite for selection to operate. taxonomic strategies that have been deployed when assigning taxa into species, and This variation can arise from mutations at the level of 17,31 overall morphological disparity is greatest in Lake Tanganyika . Similar to the early-​ single nucleotides, of genes or of chromosomes and via burst scenario, it is difficult to test the overshooting effect on the basis of phylogenomic the reshuffling of existing genetic material during mei- reconstructions alone. The so-called​ stage model of adaptive radiation posits that diversification initially occurs with respect to macrohabitat adaptations and then with osis (recombination), making these sources of variation respect to microhabitat specializations (trophic adaptations), after which disruptive at the molecular level prime targets in the quest for the sexual selection based upon mating traits becomes the dominant mode of genomic basis of explosive diversification in cichlids. speciation15,131,132. More recent work has cast doubts on the validity of the stage model in this chronological order, suggesting instead that trophic traits diversified earlier Mutation rate. Interestingly, the nucleotide mutation than traits implicated in macrohabitat adaptation97,133. One of the most fascinating rate is not very high in East African cichlids. Quite to aspects of adaptive radiation is the frequent occurrence of convergent forms12. the contrary, the nucleotide mutation rate has been esti- This phenomenon is particularly pronounced within cichlids, where convergent mated — using trio sequencing — at 3.5 × 10−9 per bp per 75,134 61 evolution is prevalent and occurs not only between but also within species flocks. generation (Table 1), which is 3–4-fold lower than the rate in humans27. Therefore, the exceptionally high spe- ciation rates in East African cichlids cannot be linked to ones in small volcanic crater lakes in Africa and Central an elevated mutation rate. America are the most widely perceived3,67,94–96. Early on, the extraordinary diversity of cichlids has spurred spec- Gene duplications. The rate of lineage-​specific gene ulations that particular genomic features might underlie duplications, by contrast, has been found to be 4.5–6-fold their propensity to diversify35. Examining this ‘genomic higher in the common ancestor of the explosively diver- substrate’ for diversification has become one of the main sifying East African cichlid lineages than in other fish; research targets within the cichlid genome project19. it is highest in the common ancestor of the most species-​ rich lineage in cichlids, the Haplochromini19, suggesting Comparative cichlid genomics a link between gene duplication and diversification. The in-depth​ comparative analysis of the initial cichlid genome sequence data19 identified several distinctive Transposable elements. Transposable element insertions features in the genomes of the radiating East African provide another source of genetic variation, and there cichlids that could potentially — individually or jointly is evidence from the cichlid genomes that the insertion — be responsible for explosive diversification in this of transposable elements near genes has altered their group, thereby confirming earlier assumptions. First, expression19. This is exemplified by a case study showing the cichlid genomes turned out to be genetically more that the insertion of a short interspersed nuclear element diverse than expected in light of the very recent ori- (SINE) upstream of a previously unknown colour gene, gin of the species flocks, which was attributed mainly fhl2b, in the ancestor of the Haplochromini lineage led to the accumulation of standing genetic variation to a gain of expression in iridophores (a specific type before the radiations (as opposed to new mutations). of pigment cells), which in turn has been linked to the Second, the four genomes of the explosively diversify- origin of a new pigmentation trait in this group71. ing cichlid lineages from Lakes Victoria, Malawi and Tanganyika showed an increased rate of gene duplica- Chromosome numbers. At the level of entire chromo- tions compared with the Nile tilapia and other teleosts, somes, there is little variation among the East African and about one-​fifth of the duplicated genes showed cichlids, with chromosome numbers ranging from evidence of neo-​functionalization, the gain of a new 2n = 40 to 2n = 46 according to karyotyping; most species function in one of the gene copies. Third, the same have 2n = 44 (refs98,99) (Table 1). Differences in chromo- four cichlid genomes are characterized by a greater some number, thus, do not seem to have an important dynamic in gene regulatory processes than observed role in the origin or maintenance of cichlid species. The Trio sequencing in other fish species, as evidenced by increased rates contribution of smaller-​scale chromosomal rearrange- Whole-genome​ sequencing of of regulatory element evolution and novel and func- ments (such as inversions) to cichlid diversification is two parents and one of their tionally diverse microRNAs. Fourth, these genomes less clear to date, as its examination is currently ham- offspring, allowing accurate exhibit accelerated coding sequence evolution, as evi- pered by the insufficient quality of genome assemblies phasing (and the determination of the nucleotide mutation rate denced by elevated rates of non-synonymous​ to synon- based on short-​read sequence data, and too few genome if sequence coverage is high ymous (dN/dS) substitutions compared with the rates assemblies based on long-​read data in combination with enough). in the Nile tilapia. Finally, three waves of transposable genetic mapping are currently available.

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a Hard polytomy b Soft polytomy c Gene duplication or loss

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◀ Fig. 3 | Phylogenetic inference in rapidly diversifying clades. A number of factors changes in the form of lowstands of the lake level that contribute to discordance among individual gene trees and between gene trees and the lead to increased water turbidity and/or bring together | species tree, resulting in mosaic genomes. a In the event of an adaptive radiation, populations in dense contact27,28,44,82, incomplete line- particularly if novel species emerge through explosive diversification, the species tree is age sorting and, at least in some cases, hybridization at not a priori expected to be strictly bifurcating (neither are the underlying gene trees). This is because an ancestor may give rise to more than two daughter species at a given the onset of adaptive radiation can explain why cichlid time and continue to persist as its own species. In this case, polytomies in the species tree genomes are genetically diverse and why much of the and in gene trees are ‘hard’ and reflect true multifurcations. b | In the course of an adaptive variation is shared between cichlid species (Fig. 3). For radiation, new species form rapidly (in some cases explosively). As a consequence, there is example, within Lake Malawi, more than 80% of the often insufficient time for the accumulation of branch-specific​ diagnostic nucleotide heterozygous sites are shared between species27, and it substitutions between consecutive speciation events, leading to non-bifurcating​ gene has been estimated that more than half of the SNPs that trees (‘soft’ polytomies) or gene trees with very short internal branches64,137; in many such are polymorphic in Lake Malawi are also polymorphic cases, more mutations accumulate after rather than during speciation events, resulting in cichlids outside this lake101. Taken together, it seems 138 | in bottom-heavy​ phylogenies . c The duplication of genes and the subsequent that, in East African cichlids, the reshuffling of existing (stochastic) loss (marked with x) of one of the gene copies — a phenomenon that is genetic variation has played a major part in creating the known to occur in adaptive radiations139 — can also lead to phylogenetic incongruence. d | When speciation events are closely timed, which is prototypical of both adaptive raw genetic material for selection to act upon. radiation and explosive diversification, ancestral genetic variation can stochastically persist as polymorphisms between speciation events. This phenomenon — called Multifarious selection incomplete lineage sorting — causes discordance between individual gene trees as a Comparative genomic analysis in East African cichlids function of when in the past the respective genes coalesce; incomplete lineage sorting suggests that selection is multifarious during explosive also causes discordance between the gene trees affected by incomplete lineage sorting diversification and targets many loci across the genome, and the species tree. e | Closely related species that coexist in the same area — a defining as evident from the multiple genomic regions with ele- feature of species flocks, adaptive radiations and instances of explosive diversification — vated levels of divergence — so-called​ outlier regions — 12,22,140 are prone to the exchange of genetic material via introgression . Introgression (or between sister taxa19,67,102,103, which is in turn a probable introgressive hybridization) leads to incongruence between gene trees in such a way that consequence of syndrome selection12. For example, in a a larger proportion of gene trees is equivalent to the species genealogy , while a smaller fraction of gene trees reveals the donor lineage of the genetic material that comparison between two A. calliptera ecomorphs that introgressed57,141. f | In some instances, hybridization may lead to the formation of a new are at the verge of speciation in a small crater lake near 67 species7, in which case about half of the gene trees place the hybrid species together with Lake Malawi, Malinsky et al. identified 55 genomic one of the parental lineages, whereas the other half of the gene trees reveals the second regions that were characterized by high divergence (in parental lineage. For this reason, the species tree becomes reticulate. g | As a result of both FST and DXY) relative to the genome-​wide back- rapid divergence (parts a and b), duplication or deletion of genes (part c), incomplete ground. Interestingly, these ‘islands of speciation’ are not lineage sorting (part d) and introgressive hybridization (part e), chromosomes become a randomly distributed across the genome; approximately mosaic of segments with different evolutionary histories (as shown by local phylogenies), half of these islands cluster on only five chromosomes67, whereby the size of each segment is primarily determined by the number of that is, they form archipelagos104. Note that the fairly large recombination events that have occurred. Depending on the number of generations number of outlier regions is somewhat contradictory to since divergence, population sizes and the recombination rate, segments might become excessively small and plentiful (for example, for the cichlids of the Lake Victoria region, theoretical models, which predict that rapid diversifica- it has been estimated that such ancestry blocks are in the size of 500–1,000 bp only).76 tion is most probable if the number of underlying loci is small15 and that genomic regions of high divergence are not necessarily indicative of divergent selection105. Recombination. Besides mutational change, recombina- Gene candidates emerging from divergence map- tion is the other major factor that can lead to an increase ping — that is, genes showing elevated levels of diver- in genetic variation. The effect of recombination on gence between diversifying populations or more rapid genetic variation is expected to be especially strong when coding sequence evolution — include genes with chromosomes from genetically more distinct parents are functions in morphogenesis19,67, cytoskeleton develop- involved, as is the case when two species hybridize. In ment67, the sensory system (including the visual opsin this way, hybridization can instantaneously boost genetic genes)4,19,27,67,76,102,103, oxygen transport27,103, hormone variation100. In East African cichlids, hybridization has signalling27,67, protein translation27,67, pigmentation19 been identified as an important factor for establishing and the immune system27,102. To date, very few of these Syndrome selection and maintaining genetic variation in species flocks27,57,76. gene candidates have been studied in sufficient detail to Selection on a combination of For example, it has been shown that explosive diversifi- allow their assignment to discrete functions and to vary- traits (for example, body and cation in cichlids of the Lake Victoria region was pre- ing phenotypes in cichlids, and a direct link between any mouth morphology and coloration in cichlids) in a given dated by a hybridization event involving two distantly of these genes and explosive diversification is currently environment. related riverine lineages, creating a hybrid swarm, lacking. Future work should thus focus on the identifi- whereby fixed differences in the parental lineages recom- cation and in-​depth functional characterization of the F ST bined to form many new combinations of alleles in the genes involved in diversification in cichlids, making use Known as the fixation index, FST 76 is a measure of differentiation emerging species . of divergence mapping and laboratory crosses. between two populations due Obvious signatures of introgression were found in to genetic structure. the cichlid assemblages of all three large lakes26,27,41,57,79 Conclusions and outlook (Fig. 4). Based on the analysis of genome-​wide data, it The first wave of genomic exploration of the exception- D XY has further been suggested that there is occasional gene ally diverse cichlid species flocks of the East African Average number of differences between two individuals exchange between the cichlid faunas in rivers and the Great Lakes Victoria, Malawi and Tanganyika has identi- 26,101 randomly sampled from two African Great Lakes . Recurrent introgression events, fied gene duplication, accelerated coding sequence evo- populations. potentially triggered by large-​scale environmental lution, transposable element insertion and regulatory

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Tylochromini Oreochromini Trematocarini ? Bathybatini Boulengerochromini

Lamprologini Lake Ta

Limnochromini

Cyphotilapiini nganyika Perissodini Benthochromini ? Cyprichromini Ectodini Eretmodini CSA lineage ? Tropheini A. burtoni Astatotilapia sp.

Rhamphochromis Diplotaxodon Lake Malawi Shallow and deep benthic, ‘utaka’

A. calliptera

‘Mbuna’

Astatotilapia sp. Thoracochromis sp. Lake Vi ctoria r egion

Haplochromini Astatotilapia sp.

9 8 7 6 5 4 3 2 1 MYA Fig. 4 | Phylogenetic relationships among the cichlid faunas in the East Africa Great Lakes Victoria, Malawi and Tanganyika. Phylogenetic hypotheses are based on data in refs26,27,76,78,80. Lacustrine clades are indicated in blue and scaled according to species numbers. The arrows highlight gene exchange via (introgressive) hybridization (according to refs26,27,41,55–58,76,79). Dotted lines indicate more recent intruders from more ancestral lineages (Oreochromini and Tylochromini). The grey box indicates the members of the most species-rich​ cichlid tribe, Haplochromini. A. burtoni, Astatotilapia burtoni; A. calliptera, Astatotilapia calliptera; CSA , Congolese/South African; MYA , million years ago

evolution, but not increased nucleotide mutation rate, as variation27,57,76, which can be tied directly to novel pheno­ candidate genomic features underlying explosive diver- types via transgressive segregation106,107. Occasional sification19,27. The reshuffling of existing allelic variation gene exchange between evolutionary lineages in cichlids via hybridization at the onset or in the form of intro- is possibly facilitated by the relative overall stability of gression in the course of adaptive radiation has further their genomes in terms of genome size and the number been identified as an important factor boosting genetic of chromosomes as well as comparably low nucleotide

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Table 1 | The genomic features of East African cichlids apart stably or whether other mechanisms, be they genetic or ecological ones, are involved. Clearly, more Characteristic Value or range Refs experimental work — ideally under (semi-)natural con- 19 Genome size (in Gb) 0.92Astbur−1.01Orenil ditions — is needed to disentangle the relative roles of 69 Anchored sequence 868,591,263Orenil signal and mate choice evolution, genetic incompatibil- information (in bp) ities, habitat preferences and/or spatiotemporal isolation 98,99 in cichlid diversification, which in turn would expand Number of 2n = 40Astbur−46Erecya (44Orenil; 44Mayzeb) chromosomes our knowledge of the nature of cichlid species. Similarly, 69 more experimental work is needed to clarify the relative Number of genes 38,412Orenil and pseudogenes contribution of phenotypic plasticity to cichlid evolu- 108,109 Number of protein-​ 29,249 69 tion and the potential role that the high turnover Orenil 110–113 coding genes rates in sex determination systems might have .

69 Ultimately, it will of course be important to know Percentage of repeat 37.6%Orenil elements more about the genes underlying explosive diversifi- • DNA transposons: 13% cation in cichlids, as well as their exact functions. The • LINE: 8% • SINE: 0.7% possibility of generating and rearing artificial hybrids for • LTR: 3.2% genetic mapping of particular traits51,52,113 in combination • Low complexity (satellite and simple repeats): 2.7% with divergence mapping in natural populations67,102,114 • Rolling-circle​ (Helitrons): 0.3% and the prospect of CRISPR–Cas9 gene editing in cich- • Unknown: 9.7% lids115 promise exciting new insights into this topic, −9 27 Nucleotide mutation 3.5 × 10 per bp per generationAstcal,Alnstu,Letlet (95% CI: especially when these strategies are combined. Overall, −9 −9 rate 1.6 × 10 to 4.6 × 10 ) only a small fraction of the East African cichlid fauna 113 Mean recombination 2.3 cM/MbPunpun, Pspred has been subjected to genome (re-)sequencing to date. rate Many more genomes from many more species and in a 19,27 Heterozygosity • 1/365Neobri−1/1,800Astcal (natural populations) much better quality would be needed to fully understand • 1/976Astbur−1/4,365Orenil (inbred strains) the genomic underpinnings of adaptive radiation and Effective population 50,000–130,000 breeding individuals 27 explosive diversification in cichlids. The cichlid system

size (Ne) offers an ideal comparative framework in this context, as Alnstu, Aulonocara stuartgranti; Astbur, Astatotilapia burtoni; Astcal, Astatotilapia calliptera; excessively proliferating lineages can be compared with Erecya, Eretomdus cyanostictus; Letlet, Lethrinops lethrinus; LINE, long interspersed nuclear their non-radiating​ sister lineages living outside the lakes elements; LTR , long terminal repeats; Mayzeb, Maylandia zebra; Neobri, Neolamprologus brichardi; Orenil, Oreochromis niloticus; Punpun, Pundamilia pundamilia; Pspred, Pundamilia as well as with comparatively species-​poor lineages that sp. ‘red head’; SINE, short interspersed nuclear elements. aNote that some of the reference diversified side by side to species-​rich ones in the same genomes were generated from inbred individuals, which necessarily results in lower levels of lake and that descend from the same common ances- heterozygosity. tor (Fig. 4). Importantly, such considerations make sense only in an integrative framework, that is, when similar mutation rates (Table 1), allowing species to hybridize efforts, such as with genome sequencing, are undertaken across fairly large phylogenetic distances (see refs53,54). to scrutinize the morphology, ecology, physiology and It is less clear whether hybridization has a causal role in behaviour of the East African cichlids, rather than with the generation of taxonomic and phenotypic diversity a sole focus on ecomorphological traits in the study of throughout the course of cichlid adaptive radiations cichlid divergence. These empirical efforts should be or whether hybridization is rather a by-​product of the accompanied by the development of new theory tai- proliferation and subsequent coexistence of numerous lored to the phenomenon of explosive diversification recently diverged taxa that are reproductively isolated and the establishment of novel analytical pipelines to primarily based on pre-mating​ isolation mechanisms. handle these data. Finally, more emphasis should be An important question is then what maintains the devoted to better incorporate information provided by taxonomic and phenotypic diversity in the cichlid the fossil and paleoecological record buried in the lakes’ species flocks of the African Great Lakes in light of sediments116, including the examination of ancient DNA. (occasional) gene flow between lineages and whether assortative mating alone is sufficient to keep species Published online 15 August 2018

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