Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama, Japan
Lakes Victoria, Malawi, and Tanganyika in the East African Rift Valley harbor approximately 200, 400, and 170 endemic species of cichlid fishes, respectively (Fryer and Iles 1972,
pp. 1104; Greenwood 1984,
1991
), which provide spectacular examples of the explosive adaptive radiation of living vertebrates (Fryer and Iles 1972
; Greenwood 1984
). The fishes exploit almost all resources that are available to freshwater fishes in general (Fryer and Iles 1972
; Greenwood 1984
), and they are extremely diverse, both ecologically and morphologically, despite having evolved during a very short evolutionary period (Meyer et al. 1990
; Johnson et al. 1996
). It has been estimated that the species flocks in Lakes Malawi and Victoria are 700,000 years old (Meyer et al. 1990
) and less than 12,400 years old (Johnson et al. 1996
), respectively, and that the species in these lakes have speciated within such very short periods of time. In general, members of a species are defined by their reproductive physiology, sexual behavior, and morphology. Therefore, it is reasonable to postulate that morphogenetic genes responsible for the morphological diversity of cichlid species must have changed at an accelerated rate in parallel with the morphogenetical diversification of species. In an attempt to identify the possible correlation between changes in morphology and changes in genes, we focused on genes responsible for the morphogenesis of the cichlid jaw and dentition as possible candidates. The structure of the cichlid jaw and dentition, i.e., the trophic morphology, vary more considerably among closely related cichlid species in an explosively speciated lineage of East Africa than in species inhabiting rivers (Fryer and Iles 1972
; Greenwood 1984
).
In this study we postulated that if the morphogenetic genes have changed with morphological changes of cichlids, amino acid substitutions should have occurred more frequently in the Great Lakes lineage than in the ancestral riverine lineage. But if the genes are unrelated to morphological changes, the frequency of the amino acid substitutions should be similar. We compared the evolutionary rate of amino acid substitutions between the Great Lakes lineage and the riverine lineage to identify such genes.
Studies on the morphogenesis of the jaw and dentition in model animals, such as the mouse and the zebrafish, should provide opportunities to study morphogenetic genes in other species. As a first step toward the understanding of the molecular basis of morphological diversity in cichlids, we cloned and characterized the cichlid homologs of Dlx1, Dlx2, Pax9, Otx1, Otx2, Bmp2, and Bmp4 genes, all of which were reported to play roles in the morphogenesis of the jaw and dentition in the mouse (Kuratani, Matsuo, and Aizawa 1997
; Peters and Balling 1999
).
We amplified partial sequences of cichlid homologs of Dlx1, Dlx2, Pax9, Otx1, Otx2, Bmp2, and Bmp4 genes by the polymerase chain reaction (PCR) using degenerate primers (Dlx1: Dlx1F [5'-GTTYAAGAARCTGATGAAGCA-3'] and Dlx1R [5'-CATSAGYTGWGGCTGCTGCAT-3']; Dlx2: Dlx2F [5'-CAAAGTTCAAGAAGWTGTGGA-3'] and Dlx2R [5'-CASGGGTAGTTTSYCAGAAA-3']; Pax9: Pax9F [5'-ATCYGRCCTTGTGACATCAG-3'] and Pax9R [5'-GGGTCCCTTCTGCTTGTAAGT-3']; Otx1 and Otx2: OtxF [5'-ATGATGTCRTAYCTCAARCA-3'] and OtxR [5'-CTGRTCTTTGTARTCCAARCA-3']; BMP2 and Bmp4: BmpF [5'-GTRGGSTGGAATGACAGGAT-3'] and BmpR [5'-CTCCACWACCATGTCCTGRTA-3']) and genomic DNA from Labidochromis caeruleus as a template. Total RNA was prepared from the whole body of an adult specimen of L. caeruleus and Haplochromis sp. brownae. With reference to the partial sequences, we synthesized nested primers for Bmp4, Bmp2, and Pax9 (Bmp4: Bmp4FS1 [5'-CCCTTTCCTCTGGCAGATCATCT-3'], Bmp4FS2 [5'-AGACACTGGTGAACTCTGTGAACAAC-3'], Bmp4RS1 [5'-CATGTTCATCTAGGTAGAGCATGGAGA-3'], and Bmp4RS2 [5'-TGTCTGAACAATGGCGTGGTT-3']; Bmp2: BMP2FS1 [5'-GCTACCATGCCTTTTATTGCCAT-3'], BMP2FS2 [5'-AGCAGACCACCTCAATTCTACTAACCA-3'], BMP2RS1 [5'-TGAGTTGACAAGTGTCTGCACAATG-3'], and BMP2RS2 [5'-TGGTTAGTAGAATTGAGGTGGTCTGCTA-3']; and Pax9: Pax9FS1 [5'-GTGTCAGCAAGATCCTGGCTCGCTACA-3'], Pax9FS2 [5'-CAGCAAGATCCTGGCTCGCTACAA-3'], Pax9RS1 [5'-TGTAAGTCCGTATGTGTTTGACAACG-3'], and Pax9RS2 [5'-ATGGAGCCGGTCTCGTTGTAG-3']) for 3' or 5' rapid amplification of cDNA ends (RACE). We isolated the Otx1 and Otx2 genes by genomic PCR and full-length cDNA of Pax9, Bmp2, and Bmp4 from the DNA or the RNA of L. caeruleus by 3'RACE and 5'RACE, respectively.
To estimate the evolutionary rate of amino acid substitutions among four closely related species, we determined the sequences of Pax9 (608 bp), Otx1 (477 bp), Otx2 (737 bp), Bmp2 (697 bp), and Bmp4 (1,212 bp) from Tropheus duboisi belonging to the Tropheini tribe in Lake Tanganyika, Astatoreochromis alluaudi from East African riverine, Haplochromis sp. brownae from Lake Victoria, and L. caeruleus from Lake Malawi. We performed PCR to amplify the homologous sequences using appropriate primers (Pax9: Pax9u1 [5'-CTTCGGTGAAGTGAACCAACT-3'] and Pax9d1 [5'-CAAAAGTTGGTTATATGTGGTGCAAT-3']; Otx1: Otx1u1 [5'-CCCAAGAAGAAGTCCTCTCCT-3'] and Otx1d1 [5'-CGGCAGTCTGGTCTTTGTAGTC-3']; Otx2: Otx2u1 [5'-GGATCGATTATGATGTCGTAACTCAA-3'] and Otx2d1 [5'-GCAATCGGCATTGAAGTTCAG-3']; Bmp2: BMP2u1 [5'-TGGTACTGTTGCTCGCTCAG-3'] and BMP2d1 [5'-ACGAGTCCTGGTCCTGGTGTAG-3']; and Bmp4: Bmp4u1 [5'-CCTTTAGTGGGAACATTTATACAAGAG-3'] and Bmp4d1 [5'-CAGTCCAAGCCCACACTTTAGT-3']) and genomic DNA or cDNA from T. duboisi, A. alluaudi, L. caeruleus, and Haplochromis sp. brownae.
We calculated all the pairwise values of nonsynonymous substitutions per nonsynonymous sites (Dn) and synonymous substitutions per synonymous sites (Ds) for these genes (table 1
). The ratio Dn/Ds provides an estimate of the evolutionary rate of amino acid substitutions as well as standard errors (SE) by bootstrap resampling (Miyata and Yasunaga 1980
; Felsenstein 1985
). Then, we calculated the average values of Dn/Ds among the four species and compared the average values of Dn/Ds among these genes (table 1 ). Because we found that there was no amino acid substitution in Otx1, Otx2, and Pax9 among the four species, we compared the values of Bmp2 and Bmp4. These two morphogenetic proteins are members of the TGFß superfamily (Hogan 1996
). The average values of Dn/Ds for Bmp4 were higher than those for Bmp2 (table 1
). In this study, therefore, we focused on Bmp4 and determined exon-intron structures of the gene by interexon PCR (fig. 1a
). Bmp4 includes four exons (fig. 1a
) and encodes a protein of 403 amino acids that is 78% homologous to zebrafish Bmp4.
|
|
Referring to the presently accepted phylogeny of cichlid species (see legend to fig. 1
), we divided the fishes into three groups; riverine group, Lake Tanganyika group, and THMV group. The riverine group includes a small number of species and can serve as an outgroup for the other groups (Greenwood 1984
, 1991
; Ribbink 1991
; Mayer et al. 1998
). The Lake Tanganyika group represents the old lineages in the Great Lakes species (Nishida 1991
) and includes a large number of species (
100). The THMV group includes the Tropheini and Haplochromini tribe in Lake Tanganyika as well as East Africa riverine Haplochromine, and Lake Victoria and Lake Malawi flocks, and is designated the THMV (Tropheini, Haplochromine, Lake Malawi and Lake Victoria flock) group. Species in this third group appear to have been subjected to rapid speciation very recently (Meyer et al. 1990
; Sturmbauer and Meyer 1992
; Johnson et al. 1996
).
We calculated the average values of Dn/Ds for Bmp4 for each of the three groups and compared them. We postulated that if the gene for Bmp4 has changed with morphological changes, amino acid substitutions should have occurred at an accelerated rate and the average values of Dn/Ds for the Great Lakes groups should be higher than those for the riverine group because of the high morphological diversity in the Great Lakes groups. But if the gene was unrelated to morphological changes, the values should be similar.
Figure 1b
shows the phylogeny of the cichlid species used in this study and the average values of Dn/Ds within each group. The average values of Dn/Ds for Bmp4 from the riverine group, the Lake Tanganyika group, and the THMV group were estimated to be 0.072 ± 0.037, 0.041 ± 0.029, and 0.359 ± 0.167, respectively (fig. 1b
). The average value for the THMV group is nine times higher than that for the Lake Tanganyika group and five times higher than that for the riverine group (fig. 1b
). The confidence intervals of the two estimates for the THMV group and the other groups do not overlap, indicating the robust nature of the estimations of these values. The higher average value of Dn/Ds within the THMV group suggests that the amino acids in Bmp4 changed at an accelerated rate in this group. The THMV group is characterized by high morphological diversity, a vast number of species (>700), and explosive speciation (Fryer and Iles 1972
; Greenwood 1984
; Meyer et al. 1990
; Sturmbauer and Meyer 1993
; Johnson et al. 1996
). Thus, there is an apparent correlation between the morphological diversity of this lineage and the high rate of change of Bmp4. Although the morphological diversity of Lake Tanganyika species is also high, we did not detect the high value of Dn/Ds from the Lake Tanganyika group (fig. 1b
). Bmp4 may affect some particular morphological change among THMV species.
Bmp4 is a signaling molecule that regulates many aspects of morphogenesis (Hogan 1996
). Transformation of teeth from incisors to molars has been induced experimentally by the inhibition of Bmp4 signaling in the mouse (Tucker, Matthews, and Sharpe 1998
). Bmp4 is a member of the TGFß superfamily (Hogan 1996
), and like all members of TGFß superfamily, Bmp4 is synthesized as a large precursor, which is processed and proteolytically cleaved to yield a carboxy-terminal mature protein (Hogan 1996
). The proteolytic cleavage occurs at the RX(K/R)R sequence, between the pro-domain and the mature domain (Molloy et al. 1992
; Creemers et al. 1993
). The efficiency of cleavage, as well as the life span of the mature region, is regulated by the pro-domain (Constam and Robertson 1999
).
To identify the region of Bmp4 that changed at an accelerated rate in the THMV group, we performed a sliding-window analysis of the average estimates of Dn and Ds for Bmp4 for each of the several species from the THMV group and then we calculated average values, as shown in figure 1c .
Our analysis showed that the variations in amino acids were restricted to the pro-domain of Bmp4. No variations were found in the signal peptide and the mature domain. Thus, it is likely that the observed accelerated changes in amino acids have not affected the function of Bmp4 itself; rather, they have affected the posttranslational regulation of the mature protein in the THMV group. The amino acid sequence of the pro-domain might regulate morphology in a manner that is somehow related to the adaptation of cichlids in lacustrine environments.
In theory, when the ratio of Dn/Ds < 1, the gene has evolved under negative selection. The average ratio of Dn/Ds for Bmp4 among the THMV group was 0.359 ± 0.167 < 1, and this result indicates that Bmp4 might have evolved under negative selection and that the amino acids might have been changed by the relaxation of negative selection in parallel with the morphological changes of cichlids in the THMV group. Alternatively, it is possible that the amino acid substitution in the pro-domain of Bmp4 might have evolved under positive selection during a certain evolutionary period when the morphological change occurred (for example, in fig. 1b branches I and II; on each branch, there was a nonsynonymous substitution without synonymous substitution). After the period, the synonymous substitutions in the pro-domain of Bmp4 might have accumulated, and the average ratio of Dn/Ds for Bmp4 might have become less than 1.
The function of a gene is regulated at many stages, for example, transcription, mRNA processing, transportation from the nucleus to the cytoplasm, translation and posttranslation. Because the Bmp4 signaling pathway includes many morphogenetic genes (Hogan 1996
), the number of regulatory steps in this pathway must be much greater than the number of such genes. The pro-domain of Bmp4 might be a regulatory region that regulates one of the steps in this signaling pathway. Because it is possible that a small change in the pro-domain of Bmp4 might induce larger changes in a cascade of the Bmp4 signaling pathway, by the accumulation of substitutions in genes in each regulatory step to change the morphology and to create the diversity of cichlids, it will be interesting to analyze the regulatory region of other morphogenetic genes included in the Bmp4 signaling pathway.
Evolution was studied initially in terms of changes in morphology, and the correlation between changes in morphology and changes in genes remains to be clarified. The analysis of morphogenetic genes in the East African cichlids will provide an opportunity for studies on the evolution of morphology at the molecular level. Various genes that affect the morphogenesis of the jaw and dentition have been identified by genetic analysis of model animals, and studies of orthologous loci in cichlids should help us to understand some details of the morphological diversity in cichlids.
Acknowledgements
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Footnotes
Naruya Saitou, Reviewing Editor
Keywords: cichlid
adaptive radiation
morphogenetic gene
Bmp4
Address for correspondence and reprints: Norihiro Okada, Graduate school of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. E-mail: nokada{at}bio.titech.ac.jp
.
References
Constam D. B., E. J. Robertson, 1999 Regulation of bone morphogenetic protein activity by pro-domains and proprotein convertases J. Cell Biol 144:139-149
Creemers J. W., P. J. Kormelink, A. J. Roebroek, K. Nakayama, W. J. Van de Ven, 1993 Proprotein processing activity and cleavage site selectivity of the Kex2-like endoprotease PACE4 FEBS Lett 336:65-69[ISI][Medline]
Felsenstein J., 1985 Confidence limits on phylogenies: an approach using the bootstrap Evolution 39:783-791[ISI]
Fryer G., T. D. Iles, 1972 The cichlid fishes of the great lakes of Africa Oliver and Boyd, Edinburgh
Greenwood P. H., 1984 African cichlids and evolutionary theories Pp. 141154 in A. A. Echelle and I. Kornfield, eds. Evolution of fish species flock. University of Maine at Orono Press, Orono
. 1991 Speciation Pp. 86102 in M. H. A. Keenleyside, ed. Cichlid fishes: behavior, ecology and evolution. Chapman and Hall, London
Hogan B. L., 1996 Bone morphogenetic proteins: multifunctional regulators of vertebrate development Genes Dev 10:1580-1594[ISI][Medline]
Johnson T. C., C. A. Scholz, M. R. Talbot, K. Kelts, R. D. Ricketts, G. Ngobi, K. K. Beuning, I. Ssemmanda, J. W. McGill, 1996 Late Pleistocene desiccation of Lake Victoria and rapid evolution of cichlid fishes Science 273:1091-1093[Abstract]
Kocher T. D., J. A. Conroy, K. R. McKaye, J. R. Stauffer, 1993 Similar morphologies of cichlid fish in Lakes Tanganyika and Malawi are due to convergence Mol. Phylogenet. Evol 2:158-165[Medline]
Kuratani S., I. Matsuo, S. Aizawa, 1997 Developmental patterning and evolution of the mammalian viscerocranium: genetic insights into comparative morphology Dev. Dyn 209:139-155[ISI][Medline]
Mayer W. E., H. Tichy, J. Klein, 1998 Phylogeny of African cichlid fishes as revealed by molecular markers Heredity 80:702-714[ISI][Medline]
Meyer A., T. D. Kocher, P. Basasibwaki, A. C. Wilson, 1990 Monophyletic origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA sequences Nature 347:550-553[ISI][Medline]
Miyata T., T. J. Yasunaga, 1980 Molecular evolution of mRNA: a method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application J. Mol. Evol 16:23-36[ISI][Medline]
Molloy S. S., P. A. Bresnahan, S. H. Leppla, K. R. Klimpel, G. Thomas, 1992 Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen J. Biol. Chem 267:16396-16402
Nishida M., 1991 Lake Tanganyika as an evolutionary reservoir of old lineages of East African cichlid fishes: inference from allozyme data Experientia 47:974-979[ISI]
Peters H., R. Balling, 1999 Teeth: where and how to make them Trends Genet 15:59-65[ISI][Medline]
Poll M., 1986 Classification des Cichlidae du lac Tanganyika: tribus, genres et especes Ac. Roy. de Belg. (2E serie) XLV:1-163
Ribbink A. J., 1991 Distribution and ecology of the cichlids of the African Great Lakes Pp. 3659 in M. H. A. Keenleyside, ed. Cichlid fishes: behavior, ecology and evolution. Chapman and Hall, London
Sturmbauer C., A. Meyer, 1992 Genetic divergence, speciation and morphological status in a lineage of African cichlid fishes Nature 358:578-581[ISI][Medline]
. 1993 Mitochondrial phylogeny of the endemic mouthbrooding lineages from Lake Tanganyika in East Africa Mol. Bio. Evol 10:751-768[Abstract]
Takahashi K., Y. Terai, M. Nishida, N. Okada, 2001 Phylogenetic relationships and ancient incomplete lineage sorting among cichlid fishes in Lake Tanganyika as revealed by analysis of the insertion of retroposons Mol. Biol. Evol 18:2057-2066
Tucker A. S., K. L. Matthews, P. T. Sharpe, 1998 Transformation of tooth type induced by inhibition of BMP signaling Science 282:1136-1138
Yang Z., 1997 PAML: a program package for phylogenetic analysis by maximum likelihood Comput. Appl. Biosci 13:555-556[Medline]