Journal of Molecular Evolution

, Volume 72, Issue 2, pp 127–137 | Cite as

Identification and Characterization of Gene Expression Involved in the Coloration of Cichlid Fish Using Microarray and qRT-PCR Approaches

  • Helen M. Gunter
  • Céline Clabaut
  • Walter Salzburger
  • Axel MeyerEmail author


It has been suggested that speciation on the basis of sexual selection is an important mechanism for the generation of new species for East African cichlids, where male body coloration is one of the major discriminatory factors used by females in mate choice. To gain insight into the molecular basis of cichlid coloration, we studied the Lake Malawi cichlid Pseudotropheus saulosi, comparing transcription in the bright blue skin of males to the yellow skin of females. Our cDNA microarray experiments identified 46 clones that exhibited expression differences between the two sexes, of which five were confirmed to be differentially expressed by relative quantitative real-time PCR (qRT-PCR). This gene list includes a representative from the endosomal-to-Golgi vesicle trafficking pathway, Coatomer protein complex, subunit zeta-1 (Copz-1), which is known to be a critical determinant of pigmentation in humans and zebrafish. With the support of microscopic images of the skin of these specimens, we interpret the transcriptional differences between the blue males and yellow females. Here, we provide insight into the putative functional diversification of genes involved in the coloration of cichlids and by extension, on the evolution of coloration in teleost fish.


Adaptive evolution Cichlid species flocks Sexual selection Copz Collagen 1 alpha 



We thank E. Hespeler, J. Haugg, S. Kuraku, and H.-J. Lee for helpful suggestions on the manuscript and the other members of the Meyer-lab and especially Julia Jones for technical assistance. The reading of the arrays was performed at Altana Pharma, Konstanz, under the supervision of P. Hubert and A. Buhmann. This study was supported by the Zukunftskolleg of the University of Konstanz to H.G., by the Frauenfoerderung (University Konstanz) to C.C., by the Landesstiftung Baden-Württemberg, the Center for Junior Research Fellows (University Konstanz) and the EU (Marie Curie fellowship) to W.S., and grants of the Deutsche Forschungsgemeinschaft to A.M.

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  1. Abzhanov A, Kuo WP, Hartmann C, Grant R, Grant PR, Tabin CJ (2006) The calmodulin pathway and the evolution of elongated beak morphology in Darwin’s finches. Nature 442:563–567PubMedCrossRefGoogle Scholar
  2. Albertson RC, Streelman JT, Kocher TD (2003) Directional selection has shaped the oral jaws of Lake Malawi cichlid fishes. Proc Natl Acad Sci USA 100:5252–5257PubMedCrossRefGoogle Scholar
  3. Allender CJ, Seehausen O, Knight ME, Turner GF, Maclean N (2003) Divergent selection during speciation of Lake Malawi cichlid fish inferred from parallel radiations in nuptial coloration. Proc Natl Acad Sci USA 100:14074–14079PubMedCrossRefGoogle Scholar
  4. Altschul SF, Gish W, Miller W, Myers EW, Lipmann DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  5. Bagnara JT (1976) Colour change. In: Lofts B (ed) Physiology of the Amphibia. Academic Press, New York, NY, pp 1–52Google Scholar
  6. Bagnara JT (1998) Comparative anatomy and physiology of pigment cells in nonmammalian tissues. In: Nordlund JJ (ed) The pigmentary system: physiology and pathophysiology. Oxford University Press, Oxford, pp 9–40Google Scholar
  7. Bagnara JT, Hadley ME (1973) Chromatophores and color change: the comparative physiology of animal pigmentation. Prentice-Hall, Englewood Cliffs, NJGoogle Scholar
  8. Bagnara JT, Stackhouse HL (1961) Purine components of guanophores in amphibians. Anat Rec 139:292Google Scholar
  9. Bagnara JT, Matsumoto J, Ferris W, Frost SK, Turner WAJ, Tchen TT, Taylor JD (1979) Common origins of pigment cells. Science 203:410–415PubMedCrossRefGoogle Scholar
  10. Barlow GW, Rogers W, Cappeto RV (1977) Incompatibility and assortative mating in the Midas cichlid. Behav Ecol Sociobiol 2:49–59CrossRefGoogle Scholar
  11. Barlow GW, Francis RC, Baumgartner JV (1990) Do the colours of parents, companions and self influence assortative mating in the polychromatic Midas cichlid? Anim Behav 46:713–722CrossRefGoogle Scholar
  12. Birbeck MSC (1963) Electron microscopy of melanocytes: the fine structure of hair-bulb premelanophores. Ann N Y Acad Sci 100:540PubMedGoogle Scholar
  13. Braasch I, Salzburger W, Meyer A (2006) Asymmetric evolution in two fish-specifically duplicated receptor tyrosine kinase paralogons involved in teleost colouration. Mol Biol Evol 23:1192–1202PubMedCrossRefGoogle Scholar
  14. Braasch I, Schartl M, Volff J-N (2007) Evolution of pigment synthesis pathways by gene and genome duplication in fish. BMC Evol Biol 7:74PubMedCrossRefGoogle Scholar
  15. Braasch I, Volff J-N, Schartl M (2008) The evolution of teleost pigmentation and the fish-specific genome duplication. J Fish Biol 73:1891–1918CrossRefGoogle Scholar
  16. Braasch I, Liedtke D, Volff J-N, Schartl M (2009a) Pigmentary function and evolution of tyrp1 gene duplicates in fish. Pigment Cell Melanoma Res 22:839–850PubMedCrossRefGoogle Scholar
  17. Braasch I, Brunet F, Volff J-N, Schartl M (2009b) Pigmentation pathway evolution after whole-genome duplication in fish. Genome Biol Evol 1:479–493PubMedCrossRefGoogle Scholar
  18. Braasch I, Volff J-N, Schartl M (2009c) The endothelin system: evolution of vertebrate-specific ligand-receptor interactions by three rounds of genome duplication. Mol Biol Evol 26:783–799PubMedCrossRefGoogle Scholar
  19. Burton D (1979) Sexual dimorphism in the integumentary tissues of the threespine stickleback, Gasterosteus aculeatus from Leiurus. Copeia 3:533–555CrossRefGoogle Scholar
  20. Carelton KL, Spady TC, Streelman JT, Kidd MR, McFarland WN, Loew ER (2008) Visual sensitivities tuned by heterochronic shifts in opsin gene expression. BMC Biol 6:22CrossRefGoogle Scholar
  21. Charles A, Ingram JT (1959) Electron microscope observations of the melanocyte of the human epidermis. J Biophys Biochem Cytol 6:41–44PubMedCrossRefGoogle Scholar
  22. Cosson P, Démollière C, Hennecke S, Duden R, Letourneur F (1996) δ- and ζ-COP, two Coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval. EMBO J 15:1792–1798PubMedGoogle Scholar
  23. Crawford DL, Pierce VA, Segal JA (1999) Evolutionary physiology of closely related taxa: analyses of enzyme expression. Am Zool 32:389–400Google Scholar
  24. Cummings ME, Larkins-Ford J, Reilly CRL, Wong RY, Ramsey M, Hofmann HA (2008) Sexual and social stimuli elicit rapid and contrasting genomic responses. Proc R Soc Lond B 275:393–402CrossRefGoogle Scholar
  25. Drochmans P (1960) Electron microscope studies of epidermal melanocytes, and the fine structure of melanin granules. J Biophys Biochem Cytol 8:165–180PubMedCrossRefGoogle Scholar
  26. Elmer KR, Reggio C, Wirth T, Verheyen E, Salzburger W, Meyer A (2009a) Pleistocene desiccation in East Africa bottlenecked but not extirpate the adaptive radiation of Lake Victoria haplochromine cichlid fishes. Proc Natl Acad Sci USA 106:13404–13409PubMedCrossRefGoogle Scholar
  27. Elmer KR, Lehtonen TK, Meyer A (2009b) Color assortative mating contributes to sympatric divergence of neotropical cichlid fish. Evolution 63:2750–2757PubMedCrossRefGoogle Scholar
  28. Elmer KR, Kusche H, Lehtonen TK, Meyer A (2010) Local variation and parallel evolution: morphological and genetic diversity across a species complex of neotropical crater lake cichlids. Philos Trans R Soc B 365:1763–1782CrossRefGoogle Scholar
  29. Epperlein HH, Claviez M (1982) Changes in the distribution of melanophores and xanthophores in Triturus alpestris embryos during their transition from the uniform to banded pattern. Wilhelm Roux’s Arch Dev Biol 192:5–18CrossRefGoogle Scholar
  30. Fang Y, Brass A, Hoyle DC, Hayes A, Bashein A, Oliver SG, Waddington D, Rattray M (2003) A model-based analysis of microarray experimental error and normalisation. Nucleic Acids Res 31:e96PubMedCrossRefGoogle Scholar
  31. Fryer G, Iles TD (1972) The cichlid fishes of the Great Lakes of Africa. T.H.F. Publications, Inc., Neptune City, NJGoogle Scholar
  32. Fujii R (1993a) Coloration and chromatophores. In: Evans DH (ed) The physiology of fishes. CRC Press, Boca Raton, FL, pp 535–562Google Scholar
  33. Fujii R (1993b) Cytophysiology of fish chromatophores. Int Rev Cytol 143:191–255CrossRefGoogle Scholar
  34. Fujii R (2000) The regulation of motile activity in fish chromatophores. Pigment Cell Res 13:300–319PubMedCrossRefGoogle Scholar
  35. Galis F, Metz JAJ (1998) Why are there so many cichlid species? TREE 13:1–2PubMedGoogle Scholar
  36. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11:4241–4257PubMedGoogle Scholar
  37. Gross JM, Perkins BD, Amsterdam A, Egaña A, Darland T, Matsui JI, Sciascia S, Hopkins N, Dowling JE (2005) Identification of zebrafish insertional mutants with defects in visual system development and function. Genetics 170:245–261PubMedCrossRefGoogle Scholar
  38. Hama T (1963) The relation between the chromatophores and pterin compounds. Ann N Y Acad Sci 100:977–986PubMedGoogle Scholar
  39. Hofmann CM, O’Quin KE, Marshall NJ, Cronin TW, Seehausen O, Carelton KL (2009) The eyes have it: regulatory and structural changes both underlie cichlid visual pigment diversity. PLoS Biol 7:e1000266PubMedCrossRefGoogle Scholar
  40. Kamei-Takeuchi I, Hama T (1971) Structural changes of pterinosome (pteridine pigment granule) during the xanthophore differentiation of Oryzias fish. J Ultrastruct Res 34:452–463PubMedCrossRefGoogle Scholar
  41. Katagiri T, Asakawa S, Minagawa S, Shimizu N, Hirono I, Aoki T (2001) Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia. Anim Genet 32:200–204PubMedCrossRefGoogle Scholar
  42. Knight ME, Turner GF, Rico C, van Oppen MJH, Hewitt GM (1998) Microsatellite paternity analysis on captive Lake Malawi cichlids supports reproductive isolation by direct mate choice. Mol Ecol 7:1605–1610CrossRefGoogle Scholar
  43. Kocher TD (2004) Adaptive evolution and explosive speciation: the cichlid fish model. Nat Rev Genet 5:288–298PubMedCrossRefGoogle Scholar
  44. Kocher TD, Lee WJ, Sobolewska H, Penman D, McAndrew B (1998) A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus). Genetics 148:1225–1232PubMedGoogle Scholar
  45. Lang M, Miyake T, Braasch I, Tinnemore D, Siegel N, Salzburger W, Amemiya CT, Meyer A (2006) A BAC library of the East African haplochromine cichlid fish Astatotilapia burtoni. J Exp Zool B 306:35–44CrossRefGoogle Scholar
  46. Le Guellec D, Morvan-Dubios H, Sire J-Y (2004) Skin development in fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio). Int J Dev Biol 48:217–231PubMedCrossRefGoogle Scholar
  47. Maldonado E, Hernandez F, Lozano C, Castro ME, Navarro RE (2006) The zebrafish mutant vps18 as a model for vesicle-traffic related hypopigmentation diseases. Pigment Cell Res 19:315–326PubMedCrossRefGoogle Scholar
  48. Matsumoto J (1965a) Role of pteridines in the pigmentation of chromatophores in cyprinid fish. Jpn J Zool 14:45–94Google Scholar
  49. Matsumoto J (1965b) Studies on fine structure and cytochemical properties of erythrophores in swordtail, Xiphophorus helleri, with special reference to their pigment granules (Pterinosomes). J Cell Biol 27:493–504PubMedCrossRefGoogle Scholar
  50. Matsumoto J, Kalishima T, Hama T (1960) Relation between the pigmentation and pterin derivatives of chromatophores during development in the normal black and transparent scaled types of goldfish (Carassius auratus). Genetics 45:1177–1189PubMedGoogle Scholar
  51. Mellgren EM, Johnson SL (2002) The evolution of morphological complexity in zebrafish stripes. Trends Genet 18:128–134PubMedCrossRefGoogle Scholar
  52. Meyer A (1993) Phylogenetic relationships and evolutionary processes in East African cichlid fishes. Trends Ecol Evol 8:279–284PubMedCrossRefGoogle Scholar
  53. Meyer A, Kocher TD, Basasibwaki P, Wilson AC (1990) Monophyletic origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA sequences. Nature 347:550–553PubMedCrossRefGoogle Scholar
  54. Michaut L, Flister S, Neeb M, White MP, Certa U, Gehring WJ (2003) Analysis of the eye developmental pathway in Drosophila using DNA microarrays. Proc Natl Acad Sci USA 100:4024–4029PubMedCrossRefGoogle Scholar
  55. Moelleken J, Malsam J, Betts MJ, Movafeghi A, Reckmann I, Meissner I, Hellwig A, Russell RB, Söllner T, Brügger B, Wieland FT (2007) Differential localization of coatomer complex isoforms within the Golgi apparatus. Proc Natl Acad Sci USA 104:4425–4430PubMedCrossRefGoogle Scholar
  56. Obika M (1963) Association of pteridines with amphibian larva pigmentation and their biosynthesis in developing chromatophores. Dev Biol 6:99–112PubMedCrossRefGoogle Scholar
  57. Obika M (1993) Formation of pterinosomes and carotenoid granules in xanthophores of the teleost Oryzias latipes as revealed by the rapid-freezing and freeze-substitution method. Cell Tissue Res 271:81–86CrossRefGoogle Scholar
  58. Odenthal J, Rossnagel K, Haffter P, Kelsh RN, Vogelsang E, Brand M, van Eeden FJM, Furutani-Seiki M, Granato M, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Mullins MC, Nüsslein-Volhard C (1996) Mutations affecting xanthophore pigmentation in the zebrafish, Danio rerio. Development 123:391–398PubMedGoogle Scholar
  59. Parichy DM, Turner JM (2003) Temporal and cellular requirements for Fms signaling during zebrafish adult pigment pattern development. Development 130:817–833PubMedCrossRefGoogle Scholar
  60. Pottinger TG, Pickering AD (1985) Changes in skin structure associated with elevated androgen levels in maturing male brown trout, Salmo trutta L. J Fish Biol 26:745–753CrossRefGoogle Scholar
  61. Renn SCP, Aubin-Horth N, Hofmann HA (2004) Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray. BMC Genomics 5:1–13CrossRefGoogle Scholar
  62. Roberts RB, Ser JR, Kocher TD (2009) Sexual conflict resolved by invasion of a novel sex determiner in Lake Malawi cichlid fishes. Science 326:998–1001PubMedCrossRefGoogle Scholar
  63. Salzburger W, Meyer A (2004) The species flocks of East African cichlid fishes: recent advances in molecular phylogenetics and population genetics. Naturwissenschaften 91:277–290PubMedCrossRefGoogle Scholar
  64. Salzburger W, Mack T, Verheyen E, Meyer A (2005) Out of Tanganyika: genesis, explosive speciation, key-innovations and phylogeography of the haplochromine cichlid fishes. BMC Evol Biol 5:17PubMedCrossRefGoogle Scholar
  65. Salzburger W, Niederstätter H, Brandstätter A, Berger B, Parson W, Snoeks J, Sturmbauer C (2006) Colour-assortative mating among populations of Tropheus moorii, a cichlid fish from Lake Tanganyika, East Africa. Proc R Soc Lond B 273:257–266CrossRefGoogle Scholar
  66. Salzburger W, Braasch I, Meyer A (2007) Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes. BMC Biol 5:51PubMedCrossRefGoogle Scholar
  67. Salzburger W, Renn SCP, Steinke D, Braasch I, Hofmann HA, Meyer A (2008) Annotation of expressed sequence tags for the East African cichlid fish Astatotilapia burtoni and evolutionary analyses of cichlid ORFs. BMC Genomics 9:96PubMedCrossRefGoogle Scholar
  68. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  69. Sanetra M, Henning F, Fukamachi S, Meyer A (2009) A microsatellite-based genetic linkage map of the cichlid fish, Astatotilapia burtoni (Teleostei): a comparison of genetic architectures among rapidly speciating cichlids. Genetics 182:387–397PubMedCrossRefGoogle Scholar
  70. Santini S, Boore JL, Meyer A (2003) Evolutionary conservation of regulatory elements in vertebrate Hox gene clusters. Genome Res 13:1111–1122PubMedCrossRefGoogle Scholar
  71. Sartor MA, Medvedovic M, Aronow BJ (2003) Microarray data normalization: the art and science of overcoming technical variance to maximize the detection of biological variance. In: Blalock EM (ed) A beginner’s guide to microarray. Kluwer, Boston, pp 151–178Google Scholar
  72. Scriver CR (1995) The metabolic and molecular basis of inherited disease. McGraw-Hill, New YorkGoogle Scholar
  73. Seehausen O, van Alphen JJM (1999) Can sympatric speciation by disruptive sexual selection explain rapid evolution of cichlid diversity in Lake Victoria? Ecol Lett 2:262–271CrossRefGoogle Scholar
  74. Seehausen O, van Alphen JJM, Witte F (1997) Cichlid fish diversity threatened by eutrophication that curbs sexual selection. Science 277:1808CrossRefGoogle Scholar
  75. Seehausen O, Mayhew PJ, van Alphen JJM (1999) Evolution of colour patterns in East African cichlid fishes. J Evol Biol 12:514–534CrossRefGoogle Scholar
  76. Seehausen O, Terai Y, Magalhaes IS, Carleton KL, Mrosso HDJ, Miyagi R, van der Sluijs I, Schneider MV, Maan ME, Tachida H, Imai H, Okada N (2008) Speciation through sensory drive in cichlid fish. Nature 455:620–627PubMedCrossRefGoogle Scholar
  77. Segal E, Shapira M, Regev A, Pe’er P, Botstein D, Koller D, Friedman N (2003) Module networks: identifying regulatory modules and their condition-specific regulators from gene expression data. Nat Genet 34:166–176PubMedCrossRefGoogle Scholar
  78. Shapiro SS, Wilk MB (1965) An analysis of variance test for normality (complete samples). Biometrica 52:591–611Google Scholar
  79. Simon P (2003) Q-Gene: processing quantitative real-time RT-PCR data. Bioinformatics 19:1439–1440PubMedCrossRefGoogle Scholar
  80. Smith RJF (1978) Seasonal changes in the histology of the gonads and dorsal skin of the fathead minnow, Pimephales promelas. Can J Zool 56:2103–2109CrossRefGoogle Scholar
  81. Stiassny MLJ, Meyer A (1999) Cichlids of the Rift Lakes. Scientific American Magazine (February) 64–69Google Scholar
  82. Streelmann JT, Albertson C, Kocher TD (2003) Genome mapping of the orange blotch colour pattern in cichlid fishes. Mol Ecol 12:2465–2471CrossRefGoogle Scholar
  83. Sturmbauer C, Meyer A (1992) Genetic divergence, speciation and morphological stasis in a lineage of African cichlid fishes. Nature 358:578–581PubMedCrossRefGoogle Scholar
  84. Taylor JS, Van de Peer Y, Meyer A (2001a) Revisiting a recent test of the ancient fish-specific genome duplication hypothesis. Curr Biol 11:R1005–R1007PubMedCrossRefGoogle Scholar
  85. Taylor J, Van de Peer Y, Braasch I, Meyer A (2001b) Comparative genomics provides evidence for an ancient genome duplication in fish. Philos Trans R Soc Lond B 356:1661–1679CrossRefGoogle Scholar
  86. van Oppen MJH, Turner GF, Rico C, Robinson RL, Deutsch JC, Genner MJ, Hewitt GM (1998) Assortative mating among rock-dwelling cichlid fishes supports high estimates of species richness from Lake Malawi. Mol Ecol 7:991–1001CrossRefGoogle Scholar
  87. Verheyen E, Salzburger W, Snocks J, Meyer A (2003) The origin of the superflock of cichlid fishes from Lake Victoria, East Africa. Science 300:325–329PubMedCrossRefGoogle Scholar
  88. Watanabe M, Kobayashi N, Fujiyama A, Okada N (2003) Construction of a BAC library for Haplochromis chilotes, a cichlid fish from Lake Victoria. Genes Genet Syst 78:103–105PubMedCrossRefGoogle Scholar
  89. Watanabe M, Kobayashi N, Shin-i T, Horiike T, Tateno Y, Kohara Y, Okada N (2004) Extensive analysis of ORF sequences from two different cichlid species in Lake Victoria provides molecular evidence for a recent radiation event of the Victoria species flock: identity of EST sequences between Haplochromis chilotes and Haplochromis sp. “Redtailsheller”. Gene 343:263–269PubMedCrossRefGoogle Scholar
  90. Waters MG, Serafini T, Rothman JE (1991) ‘Coatomer’: a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature 349:248–251PubMedCrossRefGoogle Scholar
  91. Whitehead A, Crawford DL (2005) Variation in tissue-specific gene expression among natural populations. Genome Biol 6:R13PubMedCrossRefGoogle Scholar
  92. Williams H, Brenner S, Venkatesh B (2002a) Characterization of the platelet-derived growth factor receptor alpha and c-kit genes in the pufferfish Fugu rubripes. DNA Seq 13:263–270PubMedGoogle Scholar
  93. Williams H, Brenner S, Venkatesh B (2002b) Identification and analysis of additional copies of the platelet-derived growth factor receptor and colony stimulating factor 1 receptor genes in fugu. Gene 295:255–264PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Helen M. Gunter
    • 1
    • 2
  • Céline Clabaut
    • 1
    • 3
  • Walter Salzburger
    • 1
    • 4
  • Axel Meyer
    • 1
    Email author
  1. 1.Lehrstuhl für Zoologie und Evolutionsbiologie, Department of BiologyUniversity of KonstanzConstanceGermany
  2. 2.ZukunftskollegUniversity of KonstanzConstanceGermany
  3. 3.Department of Organismic and Evolutionary BiologyHarvard UniversityCambridgeUSA
  4. 4.Zoologiches InstitutUniversität BaselBaselSwitzerland

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