The Evolution of the Pro-Domain of Bone Morphogenetic Protein 4 (Bmp4) in an Explosively Speciated Lineage of East African Cichlid Fishes

Yohey Terai, Naoko Morikawa and Norihiro Okada

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,Citation pp. 1–104; Greenwood 1984,Citation 1991Citation ), which provide spectacular examples of the explosive adaptive radiation of living vertebrates (Fryer and Iles 1972Citation ; Greenwood 1984Citation ). The fishes exploit almost all resources that are available to freshwater fishes in general (Fryer and Iles 1972Citation ; Greenwood 1984Citation ), and they are extremely diverse, both ecologically and morphologically, despite having evolved during a very short evolutionary period (Meyer et al. 1990Citation ; Johnson et al. 1996Citation ). It has been estimated that the species flocks in Lakes Malawi and Victoria are 700,000 years old (Meyer et al. 1990Citation ) and less than 12,400 years old (Johnson et al. 1996Citation ), 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 1972Citation ; Greenwood 1984Citation ).

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 1997Citation ; Peters and Balling 1999Citation ).

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 1980Citation ; Felsenstein 1985Citation ). 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 1996Citation ). 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.


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Table 1 Average Values of Dn/Ds with Standard Errors Among Species from Lake Tanganyika (T. duboisi), East Africa (A. alluaudi), Lake Victoria (H. sp. brownae), and Lake Malawi (L. caeruleus)

 


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Fig. 1.—a, The positions of exons and introns in Bmp4 in a cichlid fish (Labidochromis caeruleus). A cDNA for cichlid Bmp4 encoded a putative protein that was 78% homologous to Bmp4 from zebrafish. b, Phylogeny of the cichlid species used in this study, showing average values of Dn/Ds within groups. The consensus phylogenetic tree of cichlids is based on molecular data from several sources (Nishida 1991Citation ; Kocher et al. 1993Citation ; Sturmbauer and Meyer 1993Citation ; Mayer, Tichy, and Klein 1998Citation ; Takahashi et al. 2001Citation ). Dn and Ds were calculated by computer using the program package paml: codeml (Yang 1997Citation ). The numbers above the branches are the numbers of nonsynonymous substitutions (right) and the numbers of synonymous substitutions (left) at the branches, as estimated by the parsimony method. We used the tribe name for the Lake Tanganyika species (Poll 1986Citation ), and the names of tribes for species endemic to Lake Tanganyika are shown under the species name. Branches I and II indicate that, on each branch, there was a nonsynonymous substitution without synonymous substitution. c, Sliding-window analysis of estimates of average values of Dn and Ds of Bmp4 from the THMV group. We divided the Bmp4 sequence into each of 99 bp region and calculated the average values of all the pairwise values of value of Dn and Ds from the THMV group. A schematic representation of the structure of Bmp4 is indicated above the sliding window. The pro-domain is indicated by a shaded box. Open columns and filled columns indicate the average Ds and Dn values, respectively

 
To examine the evolutionary rate of amino acid changes in Bmp4, we sequenced the protein-coding region (1,212 bp, from ATG to TAA without intron; fig. 1a ) of this gene from a total of 16 species in the two major lineages in the Great Lakes and from riverine cichlids (fig. 1b ) using the same primer set described above. Then we calculated all the pairwise values of Dn, Ds, and the ratio Dn/Ds using the method mentioned above. The nucleotide sequences are deposited in the GenBank under accession numbers AB084637AB084668.

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 1984Citation , 1991Citation ; Ribbink 1991Citation ; Mayer et al. 1998Citation ). The Lake Tanganyika group represents the old lineages in the Great Lakes species (Nishida 1991Citation ) 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. 1990Citation ; Sturmbauer and Meyer 1992Citation ; Johnson et al. 1996Citation ).

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 1972Citation ; Greenwood 1984Citation ; Meyer et al. 1990Citation ; Sturmbauer and Meyer 1993Citation ; Johnson et al. 1996Citation ). 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 1996Citation ). Transformation of teeth from incisors to molars has been induced experimentally by the inhibition of Bmp4 signaling in the mouse (Tucker, Matthews, and Sharpe 1998Citation ). Bmp4 is a member of the TGFß superfamily (Hogan 1996Citation ), 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 1996Citation ). The proteolytic cleavage occurs at the RX(K/R)R sequence, between the pro-domain and the mature domain (Molloy et al. 1992Citation ; Creemers et al. 1993Citation ). The efficiency of cleavage, as well as the life span of the mature region, is regulated by the pro-domain (Constam and Robertson 1999Citation ).

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 1996Citation ), 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 Back

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 . Back

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Accepted for publication May 6, 2002.