The genetic basis of biodiversity: genomic studies of cichlid fishes

Transcription

1 The genetic basis of biodiversity: genomic studies of cichlid fishes Thomas D. Kocher, R. Craig Albertson, Karen L. Carleton, and J. Todd Streelman Hubbard Center for Genome Studies University of New Hampshire, Durham, NH 03824, USA Summary. Genomic techniques allow new approaches to long-standing questions in evolutionary biology. In particular, the development of linkage maps will accelerate the identification of genes underlying adaptive traits. We discuss the application of these technologies to understanding the radiation of cichlid fishes in Lake Malawi, East Africa. We have developed a linkage map for Oreochromis niloticus and are using it to identify the genetic factors controlling adaptive differences in jaw morphology among closely related Malawi cichlids. Cloning of visual pigment genes is leading to a new understanding of the visual capabilities of these fishes. We argue that a combination of linkage mapping, transcriptome profiling and analysis of candidate genes is likely to be the most efficient approach to identifying the genetic basis of adaptive evolution. Key words. Teleost, Morphology, Opsin, Linkage map The diversity of African cichlids The 23,600 described species of teleost fishes make up more than half of the living vertebrates (Helfman et al. 1997). These fishes have evolved numerous specializations adapting them to diverse habitats and unique trophic niches. Perhaps the most successful among them are the Perciformes - the largest order of vertebrates with more the 9,300 species. Among the Perciformes, the family Cichlidae is particularly diverse. In the Great Lakes of East Africa, this family has undergone a spectacular radiation to produce more that 1500 species. The earliest radiation began in Lake Tanganyika about 8 MY ago. Additional radiations in the last 1 MY produced several hundred species in Lake Victoria, and more than 500 species in Lake Malawi These are the highest rates of speciation known in vertebrates.

2 The radiation of cichlids in Lake Malawi can be attributed to three major selective forces (Danley and Kocher, 2001). The primary radiation occurred as species evolved particular habitat preferences (e.g. rock vs. sand). A secondary radiation occurred as species evolved specialized jaw morphologies to feed on different prey. The third, continuing radiation is occurring in response to sexual selection on male color patterns. Because all of these species have evolved in a short period of time, Lake Malawi cichlids are a particularly useful system in which to study the genetic mechanisms by which morphology and behavior have evolved. In this paper we describe our studies to identify genes important in trophic adaptation of these fishes. We also describe patterns of expression of opsin genes, which are important components of the visual communication system acted upon by sexual selection. Genetic basis of evolutionary change Genomics has the potential to transform evolutionary biology. For most of the past century an impressive body of population genetic theory has been applied to the study of random or neutral markers. While this has led to many insights into evolutionary mechanisms, there is a disjunct between these studies and analyses of natural selection. Genomic approaches now allow us to identify and study the genes underlying adaptive evolution. It is our belief that knowledge of the genetic basis of phenotypic differences will enlighten evolutionary theory. Our goal is to identify the genetic basis of Evolutionarily Significant Phenotypes (ESPs). Specifically, we hope to identify the DNA sequence differences which are responsible for these phenotypes, and to place these sequence differences in a phylogenetic and ecological context. General strategy for identifying ESPs Genomics is not a single technique, but a suite of high-throughput approaches aimed at identifying functional differences in the sequences of genomes. The task is akin to finding a needle in a haystack. Which of 35,000 genes, or 10 9 base pairs, is responsible for a particular phenotypic difference?

3 Figure 1 shows three general approaches to winnowing functionally significant differences from the universe of genetic differences between two organisms (Streelman and Kocher, 2000). To date we have used primarily genetic linkage studies (genome scans) to identify chromosomal segments responsible for Genome Scan differences in phenotypes. We have also studied candidate genes (e.g. opsins) which we expect to show significant Candidates differences among species. Surveys of gene expression (transciptome scans) can also be used to identify genetic differences among individuals with Transcriptome scan different phenotypes. Ideally more than one approach will be used, because the intersection of genes identified by each method will identify a smaller set of genes potentially affecting the trait. Figure 1. Approaches to identifying genes underlying particular phenotypic traits. Genome studies in teleost fishes Several teleosts are the subject of major genomics efforts. Detailed linkage maps of the zebrafish (Danio rerio) have been developed and more than 50,000 ESTs sequenced (Kelly et al. 2000). A first draft sequence of the pufferfish (Fugu rubripes) genome is expected in late Low-resolution maps of medaka (Naruse et al ), tilapia (Kocher et al. 1998) and rainbow trout (Sakamoto 2000) have been reported. Extensive databases of ESTs are available for Japanese flounder (Nam et al. 2000) and channel catfish (Ju et al. 2000). Gene order is probably highly conserved among fish species. Humans and fish share ~200 blocks of genes (Barbazuk et al. 2000), even though they last shared a common ancestor X00MY ago. Levels of conserved synteny among teleosts are likely much higher because of the relatively slow rate of chromosomal evolution in fishes (ref). Comparative maps will provide useful links between QTL identified in cichlid fishes and the sequences of other model organisms.

4 Tilapia genomics Tilapia are a group of species of cichlid fish (genus Oreochromis, Sarotherodon and Tilapia) native to tropical Africa. World production of these food fishes is >500,000 tons, increasing at >10% per year. Tilapia are closely related to the cichlids of East Africa, so that genomic resources developed in tilapia are easily applied to studies of Lake Malawi species. Our first genetic linkage map of the tilapia O. niloticus linked 162 microsatellite and AFLP markers (Kocher et al. 1998). Our second generation map is based on an F 2 cross between O. niloticus and O. aureus, and contains over 500 microsatellite markers (Kocher et al. in prep). Linkage analysis has identified markers linked to sex, color and growth in this cross (Figure 2) Series Position Figure 2. QTL for growth on linkage group 4. Arrows indicate position of microsatellite markers.

5 Genetic analysis of jaw morphology Lake Malawi cichlids feed on a variety of items including attached algae, benthic invertebrates, zooplankton and other fishes. Individual species have evolved specialized jaw morphologies to optimize their feeding on particular prey types. Because inter-specific, and even inter-generic hybridization is easily performed among Lake Malawi cichlids, crosses can be used to identify genes contributing to quantitative traits. We have focussed on a cross between Labeotropheus fuelleborni (an algal scraper) and Metriaclima zebra (a generalized planktivore; Figure 3). Figure 3. External morphology of Metriaclima zebra (left) and Labeotropheus fuelleborni (right). We have used the methods of geometric morphometrics to quantify the differences in jaw shape between these species (Albertson and Kocher 2001). We first identified landmarks which could be compared among specimens and which captured the developmental units and functional relationships of each element. We then used the technique of thin plate splines to quantify the shape differences between species (Figure 4). The shape variables identified by this method can be reduced in a principal components analysis, but also visualized via deformation grids which describe the exact regions of change. A comparison of the variance in the F 1 and F 2 generations can be used to estimate the number of genetic factors controlling the shape of each jaw element. Our preliminary estimates for each of several elements are shown in Figure 5. Current efforts are directed at identifying molecular markers for each of these factors.

6 Figure 4. Geometric morphometric analysis of the lower jaw. Position of landmarks is shown in the first panel, and scatter of individual animals from the mean form is show in the second panel. Below are representations of the mean form (middle panels) and the deformations needed to produce the form of M. zebra (left) and L. fuelleborni (right).

7 Figure 5. Estimates of the number of genetic factors controlling the shape of each skeletal element in the cross between M. zebra and L. fuelleborni.

8 Mate choice and the visual system One of the most striking features of the Lake Malawi species flock is the diversity of bright male mating colors. These colors are thought to have evolved by sexual selection. We wondered whether differences in the visual system among species might create sensory biases and lead to the evolution of different colors among species. As a first step we determined the absorption spectra for isolated retinal cone cells from two species. Dimidiochromis compressiceps is a sandy shore predator with visual sensitivities similar to humans (Figure 3a). Red and green opsin pigments are found in double cones, and a blue-sensitive pigment is present in the single cones. In contrast, the rocky-shore algal grazer Metriclima zebra has three pigments that are blue-shifted ~50nm (Figure 3b). The shortest wavelength pigment is sensitive to UV radiation, which may explain the use of UV-reflective color elements in this species. Normalized pigment absorption for D compressiceps Wavelength (nm) Normalized pigment absorption for M. zebra Wavelength (nm) Figure 6. Absorption spectra for visual pigments of two species of Lake Malawi cichlid.

9 To discover how these differences in visual sensitivity arise we cloned and sequenced the opsin genes from each species (Carleton and Kocher, 2001). Lake Malawi cichlids have five classes of opsin gene, but individual species typically express only three genes simultaneously (Table 1). Future studies will survey patterns of opsin gene expression among species and develop behavioral assays to assess the importance of these visual system differences to mating preferences. Table 1. Opsin gene expression in adult cichlids Species Gene Class SWS-1 SWS-2a SWS-2b RH2 LWS (UV) (Blue) (Blue/green) Green Red D. compressiceps M. zebra O. niloticus Conclusions As the cost of high-throughput methodologies continues to drop, genomic tools increasingly will be available to study model systems in evolutionary biology. An integrated approach incorporating genomic tools into studies of morphology and behavior is already yielding results in the Lake Malawi system. Identification of genes underlying evolutionarily significant phenotypes will provide new insight into the evolutionary process. The theoretical engine of population genetics will be applied not to random or neutral markers, but to the genes actually undergoing selection. This will allow the design of experiments to assess selection pressures on relevant genotypes, to survey frequencies of relevant alleles among populations, and the study of character states in a phylogenetic context. A whole new era of evolutionary biology is upon us. References Albertson RC, Kocher TD (2001) Assessing morphological differences in an adaptive trait: a landmark-based morphometric approach to jaw morphology in Lake Malawi cichlid fish. Journal of Experimental Zoology 289: Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E, Bedell JA, McPherson JD, Johnson SL (2000) The syntenic relationship of the zebrafish and human genomes. Genome Research 10: Carleton, KL, Kocher TD (2001) Cone opsin genes of African cichlid fishes: tuning spectral sensitivity by differential gene expression. Molecular Biology and Evolution 18(8):

10 Danley PD, Kocher TD (2001) Speciation in rapidly diverging systems: lessons from Lake Malawi. Molecular Ecology 10: Helfman GS, Collette BB, Facey DE (1997) The diversity of fishes. Blackwell Science, Malden, Massachusetts Ju Z, Karsi A, Kocabas A, Patterson A, Li P, Cao D, Dunham R, Liu Z (2000) Transcriptome analysis of channel catfish (Ictalurus punctatus): genes and expression profile from the brain. Gene 261: Kelly PD, Chu F, Woods IG, Ngo-Hazelett P, Cardozo T, Huang H, Kimm F, Liao L, Yan YL, Zhou Y, Johnson SL, Abagyan R, Schier AF, Postlethwait JH, Talbot WS (2000) Genetic linkage mapping of zebrafish genes and ESTs. Genome Research 10: Kocher TD, Lee W-J, Sobolewska H, Penman D, McAndrew B (1998) A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus). Genetics 148: Nam BH, Yamamoto E, Hirono I, Aoki T (2000) A survey of expressed genes in the leukocytes of Japanese flounder, Paralichthys olivaceus, infected with Hirame rhabdovirus. Dev Comp Immunol 24:13-24 Naruse K, Fukamachi S, Mitani H, Kondo M, Matsuoka T, Kondo S, Hanamura N, Morita Y, Hasegawa K, Nishigaki R, Shimada A, Wada H, Kusakabe T, Suzuki N, Kinoshita M, Kanamori A, Terado T, Kimura H, Nonaka M, Shima A (2000) A detailed linkage map of medaka, Oryzias latipes: comparative genomics and genome evolution. Genetics 154: Sakamoto T, Danzmann RG, Gharbi K, Howard P, Ozaki A, Khoo SK, Woram RA, Okamoto N, Ferguson MM, Holm LE, Guyomard R, Hoyheim B (2000) A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates. Genetics 155: Streelman JT, Kocher TD (2000) From phenotype to genotype. Evolution and Development 2: Acknowledgements Woo-Jai Lee, Jeff Markert and Bo-Young Lee contributed to the development of the tilapia genome map. We thank the UNH Agricultural Experiment Station, the USDA-NRI Competitive Grants Program, the National Science Foundation and Genomar AS for support.