UMR 8570, Evolution et Adaptations des Systèmes Ostéomusculaires, Paris, France;
UMR 7622, Biologie Moléculaire et Cellulaire du Développement, Equipe "Phylogénie, Bioinformatique et Génome," Université Pierre et Marie Curie, Paris, France;
Institut Jacques Monod, Equipe "Evolution du Développement des Nématodes," Université Denis Diderot, Paris, France;
UPRESA 8079, Ecologie, Systématique et Evolution, Université Paris-Sud, Orsay, France
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Abstract |
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Introduction |
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All of the above features are specific to vertebrate enamel formation and are not encountered in other mineralized tissues (even in nonvertebrate lineages). Another striking character of most of these enamel proteins is that they have not been found expressed in other mineralizable or nonmineralizable tissues: these proteins are specific to enamel, or, in other words, they have no pleiotropic effects.
We have been interested in the evolutionary origin of the tissues composing the dermal skeleton, and the specificity of the enamel proteins has drawn our attention. Because the enamel nonamelogenin genes are known only from a few mammalian species, they cannot be used to study long-term evolution. In contrast, amelogenin sequences are available in several vertebrate lineages (Girondot and Sire 1998
; Ishiyama et al. 1998
; Toyosawa et al. 1998
).
The present study was undertaken to date the appearance of the amelogenin gene in the vertebrate genome. Amelogenin is currently believed to be an orphan gene without any known homologs. If an amelogenin paralog gene were found, knowledge of the date of duplication of the ancestral gene, or of part of this gene, and its standard deviation would provide a range of possible ages for the acquisition of this protein and of the enamel-like tissue in which it was expressed. Because the amelogenin gene encodes a protein that plays an important role in enamel mineralization and shows no known pleiotropic effect, it is an excellent candidate to independently estimate the date of acquisition of a mineralized tissue in vertebrates. Indeed, this date is currently debated.
On the one hand, according to the fossil record, the earliest vertebrates are known from the early Cambrian of China (during the so-called "Cambrian explosion"), but they did not possess mineralized tissues (Chen, Huang, and Li 1999
; Shu et al. 1999
). Mineralization probably appeared later in vertebrates. Indeed, the earliest presumed vertebrate known to possess a mineralized skeleton was Anatolepis, found in the Upper Cambrian (520 MYA) (Repetski 1978
). Its dermal skeleton was ornamented by small tubercles composed of a tissue identified as a kind of dentin. A mineralized tissue of unknown homology covered it (Smith, Sansom, and Repetski 1996
). Enamel and other tissues of uncertain homologies, possibly bone, dentine, and calcified cartilage, were also described in euconodonts from the Upper Cambrian (Sansom et al. 1992
, but these interpretations have been disputed; see Schultze 1996
). These animals of controversial affinities are now considered craniates by many authors (Aldridge and Donoghue 1998
; Janvier 1996b
). Enamel, or enameloid tissue covering tubercles, was clearly described in Ordovician vertebrates (450 MYA) such as the ostracoderms (Ørvig 1989
). On the other hand, according to molecular data, the Bilateralia were estimated to have had their origin during the Proterozoic, between 1,000 and 830 MYA (Bromham et al. 1998
; Bromham and Hendy 2000
).
Did the mineralized tissues of vertebrates appear in the Cambrian, as the fossil record suggests, or did they arise in the Proterozoic? In the present paper, we have tried to discriminate between these hypotheses using the amelogenin gene. We searched DNA banks for similarities between the amelogenin sequence and other sequences and tested the significance of these similarities, and, using several methods, we were able to date the origin of amelogenin exon 2 in the vertebrate genome.
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Materials and Methods |
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Amelogenin genes are unknown outside vertebrates. However, Slavkin and Diekwisch (1996)
published a partial amino acid sequence of a Pacific hagfish amelogenin to support the immunodetection of amelogenin-like proteins in the oral cavity of this jawless craniate (Hyperotreti sensu Janvier 1996b
). Living hagfish lack mineralized tissues, and this is probably a primitive condition (Janvier 1996a
). Therefore, the presence of amelogenin in hagfish would have indicated that this gene was expressed in nonmineralized tissues in a common ancestor of craniates and vertebrates, a result that does not support our hypothesis of an essential role of amelogenin in enamel mineralization. A phylogenetic analysis of the putative hagfish DNA using mammalian amelogenin genes has demonstrated that this sequence was probably due to contamination by rodent DNA during PCR (Girondot, Delgado, and Laurin 1998
). This finding was confirmed when the recently available amelogenin sequences in the caiman, the snake and Xenopus were added to the analysis (on the MBE website, see Supplementary Material, section 1, EMBL ALIGN_000059). Using the ProtML resampling of estimated log likelihoods (RELL) method (Hasegawa and Kishino 1994
), if the putative hagfish sequence is constrained to be the sister group of amniotes, the resulting tree (ln L = -710.6) is found to be worse than the maximum-likelihood tree (10,000 replicates) in 97.75% of the cases. Furthermore, a parsimony analysis using PAUP 3.1.1 (Swofford 1993
) has indicated that four extra steps are required to locate the putative hagfish sequence outside of amniotes.
Searching for Sequence Similarities
A first BLAST search in GenBank using the various amelogenin exons did not reveal significant similarities to other sequences. Nevertheless, our attention was drawn by the following observation: rat ameloblasts (the epithelial cells that produce amelogenin) show a positive signal when hybridized in situ with an RNA probe of SPARC (secreted protein, acidic and rich in cystein) (Liao et al. 1998
). This protein, also called osteonectin, BM-40, and 43k protein, is a glycoprotein that is involved in mediating cell-matrix interactions but does not serve structural roles (Brekken and Sage 2000)
. Liao et al. (1998)
have suggested that this signal is probably due to the presence of 10 identical bases in the rat sequences of SPARC and amelogenin. Indeed, the alignment of SPARC and amelogenin sequences has revealed that the complete translation of the second exons of both genes (the first exon is not translated) shows similarities. We searched for other SPARC sequences in DNA banks using Nentrez or BLAST tools. A total of 13 sequences were found: eight complete SPARC sequences in deuterostomians (dSPARC), two complete sequences in protostomians a nematode, and drosophila (pSPARC), and partial sequences (i.e., lacking exon 2) of three other deuterostomians. Three other SPARC-related genes were included in the analysis: SC1 in rodents and hevin, its homolog in humans, and QR1, its homolog in the quail. In the following, these three SPARC-related genes will be collectively called SC1 (the first identified). Moreover, in the course of our search, we found 19 zebrafish (Danio rerio) expressed sequence tag (EST) clones showing some similarities to SPARC. After extensive checking, we reconstructed the complete zebrafish SPARC mRNA sequence (on the MBE website, see Supplementary Material, section 2), and this sequence was added to our analysis (on the MBE website, see Supplementary Material, section 3, EMBL Align_000006).
Amelogenin sequences were also sought in the DNA banks using the same procedures, and 20 sequences were found. Only 12 amelogenin sequences were complete (with exon 2) and thus retained for the analysis.
In SPARC, SC1, and amelogenin exon 2 is composed of three regions: first region consists of 12 untranslated nucleotides, the second consists of 48 translated nucleotides constituting the signal peptide, and the third consists of six nucleotides that constitute the beginning of the protein itself. Regions 2 and 3 of dSPARC, SC1, and amelogenin exon 2 (a total of 19 sequences) can be aligned with only one tail of three gaps for the 54 total nucleotides (on the MBE website, see Supplementary Material, section 4, EMBL ALIGN_000005). The signal peptide is highly hydrophobic, and it enables the protein being synthesized (which is hydrophilic) to pass through the rough endoplasmic reticulum membrane. Once localized in the lumen of the endoplasmic reticulum, the protein is transported in the Golgi apparatus, then exocytosed by means of intracellular vesicles.
Testing Similarities
We needed to ensure that the dSPARC, SC1, and amelogenin sequences shared a common ancestor, i.e., that the observed similarity reflected a real homology. Thus, we tested the similarities of dSPARC, SC1, and amelogenin exon 2.
The proportions of identity of the various pairs of sequences cannot be directly compared because these values are not independent. Indeed, we suppose that all amelogenins, on the one hand, and dSPARC and SC1, on the other hand, have a common phylogenetic history. Therefore, the most parsimonious ancestral sequence for the second exon of each gene was calculated separately using MacClade 3.07 (Maddison and Maddison 1992
). Due to ambiguities in the ancestral sequence, position i in a sequence j is fully described by the five frequencies fAij, fGij, fCij, fTij, and fIij, which are, respectively, the probabilities that either an A, a G, a C, a T, or a gap is present at this position for this sequence (on the MBE website, see Supplementary Material, section 5). Then, the mean probability that two identical bases are present in an alignment of l bases is estimated as
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For each pair, the distribution of under the null hypothesis (i.e., that the observed
value is not significantly different from what is expected for random sequences) was established using 1,000 replicates of two random computer-generated sequences sharing the same characteristics as the sequences under analysis (same length, same level of ambiguity, and same base composition). A simple position permutation was also used to generate random sequences. Each pair of computer-generated sequences was aligned to maximize the estimated
value. The corresponding value was used to estimate the distribution of
under H0 (
H0). This test was performed with the three bases of the codons as well as with only bases 1 and 2 in order to disregard synonymous substitutions that occur mainly at the third position in each codon.
Searching for the Origin of Amelogenin Exon 2
BIO-NJ trees (Gascuel 1997
) using either Kimura (1980)
two-parameter or Tajima and Nei (1993)
four-parameter distances were produced with only the translated part of SPARC, SC1, and amelogenin exon 2. Several methods were applied because the sequence (54 nt) was short and we needed to ensure that the result was significant. When these trees were rooted using the two pSPARC sequences, three monophyletic groups were always observed: dSPARC, SC1, and amelogenin. However, the relative positions of these three groups were uncertain.
The covarion method was applied to eliminate most of the substitution saturation (Lopez, Forterre, and Philippe 1999
). Positions with more than one change inferred by parsimony in any of the four monophyletic groups (pSPARC, dSPARC, SC1, and amelogenin) were changed to missing character states ("?") in the sequences of that group (equivalent to the H1 matrix of Lopez, Forterre, and Philippe [1999
]), and the gaps were considered "NEWSTATES" for parsimony analysis. This new alignment was analyzed using parsimony, and the relative bootstraps for the three possible positions of dSPARC, SC1, and amelogenin were established using 100 replicates with a heuristic search using PAUP 3.1.1 (Swofford 1993
). Finally, the three possible relative positions of dSPARC, SC1, and amelogenin were tested using the RELL method (Hasegawa and Kishino 1994
) with default options. The phylogenetic positions of sequences among each monophyletic group (dSPARC, SC1, and amelogenin) were chosen according to the current accepted phylogeny of vertebrates (Kumar and Hedges 1998
; Murphy et al. 2001
).
Dating the Origin of Exon 2
Because of the small number of nucleotides in exon 2, it was not possible to date its origin. In contrast, dating the origin of the dSPARC/SC1 duplication (more than 1,000 nt available) was possible.
The different functions that have been attributed to amelogenin, SPARC, and SC1 proteins raise the possibility of differences in evolutionary rates for these paralogous genes; therefore, a method that could take into account this potential is needed. Rambaut and Bromham (1998)
have proposed a quartet method in which the lineage-specific evolutionary rates are calculated using maximum-likelihood. This method requires that four species be grouped into pairs in a phylogeny and that the two internal dates of divergence be known. The following estimates of divergence dates were used: sauropsids versus synapsids, 310 MYA; lissamphibians versus amniotes, 360 MYA; actinopterygians versus sarcopterygians, 410 MYA (Kumar and Hedges 1998
). This method uses internal references for divergence dates and is more suitable for our study than a method that uses external divergence dates, such as the date of divergence between protostomians and deuterostomians, which is highly controversial (Ayala and Rzhetsky 1998
; Bromham et al. 1998
; Gu 1998
; Bromham and Hendy 2000
). Moreover, for this phase of metazoan evolution, the fossil record is extremely poor (Valentine, Jablonski, and Erwin 1999
).
To estimate the duplication date of SPARC and SC1, all of the quartet combinations involving two dSPARC sequences on one hand and two SC1 sequences on the other were produced (n = 84). For each of these quartets, the substitution rate was calculated using the two-parameter substitution model of Hasegawa, Kishino, and Yano (1985)
. The substitution rate estimated was tested against the five-parameter substitution model. A significant difference implies that the two-parameter substitution model is not sufficient to produce a reliable estimate of the substitution rate because the five-parameter substitution model significantly alters the estimate; none of these 84 tests were rejected at the 5% level. Therefore, minimal and maximal dates at the 95% confidence limit were estimated for each of the 84 quartets using the two-parameter substitution model. These 84 values were not independent of each other because these sequences shared a common phylogenetic history; therefore, all of the results of the estimated duplication dates were used. The molecular-clock method used here allowed a molecule-specific evolutionary rate (SPARC or SC1/QR1/Hevin) (Rambaut and Bromham 1998
). The consistency among the 84 estimates suggests that species-specific evolutionary rates for a given molecule (SPARC or SC1/QR1/Hevin) are not widely divergent.
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Results |
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Date for the Presence of Exon 2 in Amelogenin
The tree of exon 2 shows that the signal peptide it encodes was duplicated from a SPARC sequence to amelogenin before the duplication between dSPARC and SC1. This conclusion was supported by all phylogenetic analyses of these sequences (fig. 3
). This duplication occurred before the separation between actinopterygians and sarcopterygians, because amelogenin exon 2 falls outside the cluster formed by the actinopterygian (D. rerio and Oncorhynchus mykiss) and the sarcopterygian (which includes the tetrapod sequences) SPARC.
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Therefore, we conclude that the date of acquisition of exon 2 for amelogenin probably coincides with the date of amelogenin appearance. This probably means that vertebrate enamel (or its precursor) appeared no later than 630 MYA, even taking into account the standard deviation of the estimate (fig. 4 ).
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Discussion |
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The acquisition of exon 2 in the "pre-amelogenin locus" is slightly anterior to the date of dSPARC and SC1 duplication, which occurred nearly 630 MYA (t2 in fig. 1 ). The date calculated for the duplication of dSPARC and SC1 may be considered a reliable estimate for the latest possible date of exon 2 acquisition in the amelogenin locus.
The lack of mineralized fossils in the Proterozoic may be due to the very small sizes of individuals or to the fragility of the mineralized skeleton (e.g., the scales of Anatolepis are less than 0.1 mm thick). Metazoan fossils have been found in only a few Proterozoic sites so far. We would not be surprised if mineralized fossils were found in late Proterozoic deposits.
The history of the bilateralian phyla is characterized by their sudden appearance in the fossil record within the early Cambrian at the Proterozoic-Phanerozoic transition (approximately 543 MYA), in the so-called "Cambrian explosion" (Morris 1997
; Knoll and Carroll 1999
; Valentine, Jablonski, and Erwin 1999
). Vertebrates were no exception to this rule, and the first early Cambrian fossils indisputably belonging to the vertebrate lineage were recently described from a Chinese locality (Chen, Huang, and Li 1999
; Shu et al. 1999
). One of these fossils may be related to lampreys, and the other is probably a basal vertebrate. The sudden appearance of bilateralian clades during the 10-Myr duration of the Cambrian explosion has long been interpreted as the result of a great and rapid evolutionary radiation (Valentine, Jablonski, and Erwin 1999
). However, this finding has been questioned by several molecular studies.
Using various molecular-clock models with several sets of genes, the origins of many bilateralian clades are consistently estimated to be older than the Cambrian explosion (Fortey, Briggs, and Wills 1997
), and the divergence between protostomians and deuterostomians has been estimated to have occurred between 830 and 1,000 Myr during the Proterozoic (Wray, Levinton, and Shapiro 1996
; Bromham et al. 1998
; Gu 1998
). However, all of these estimates are subject to caution, because most of them use a molecular clock that is supposed to tick regularly through the studied period. This hypothesis has recently been tested, and the same conclusion applies (Bromham and Hendy 2000)
.
However, this hypothesis has recently received support from the fossil record: a putative mollusk-like animal (Fedonkin and Waggoner 1998
), many burrows (Valentine, Jablonski, and Erwin 1999
), and some spiralian embryos (such as annelids or mollusks) (Bengtson 1998
; Xiao, Zhang, and Knoll 1998
) have been described from the late Proterozoic. Therefore, the great increase in abundance of metazoan fossils in the Cambrian explosion could simply be a taphonomic artifact due to either environmental conditions not being conducive to fossilization or the paucity of Bilateralia in the Proterozoic, or their small size, or their lack of mineralization. Whatever the condition it made fossilization much less likely and has led to the incorrect conclusion that Bilateralia diversified in a short period. The hypothesis of a lack of mineralization is supported by the observation of a simultaneous acquisition of mineralization in most animal phyla during the Cambrian (Bengston and Conway Morris 1992
). The few Proterozoic fossils that have been interpreted as belonging to the bilateralian clade lack mineralized tissues. The most conclusive example is that of Kimberella, which was reinterpreted as a mollusk (placophore?) without a mineralized shell (Fedonkin and Waggoner 1998
). Also, some of early Cambrian bilateralian fossils, which are sister groups of clades that subsequently acquired mineralization, are themselves not mineralized. For example, lobopods from the early Cambrian may be the sister group of arthropods, but they possess a nonmineralized cuticle (Min, Kim, and Kim 1998
).
The phylogeny of craniates presented by Janvier (1996a)
suggests that the absence of mineralized tissues in hagfish and lampreys is primitive. If this conclusion is accepted, our results suggest that stem vertebrates (such as Anatolepis or Thelodonts) were already present 630 MYA.
Our study is the first attempt to discriminate between hypotheses of a late, rapid evolutionary radiation of metazoans (supported by a literal interpretation of the fossil record) and an earlier, slow evolutionary radiation (supported by molecular dates of divergence of metazoan taxa) using a molecular date of acquisition of amelogenin, a protein involved in enamel mineralization.
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Acknowledgements |
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Footnotes |
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Keywords: amelogenin
enamel
vertebrates
Cambrian explosion
Address for correspondence and reprints: Marc Girondot, UPRESA 8079, Ecologie, Systématique et Evolution, Université Paris-Sud, Orsay, Bâtiment 362, 91405 Orsay Cedex, France. marc.girondot{at}epc.u-psud.fr
.
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