The Evolution of Enamel Microstructure: How Important Is Amelogenin?

Transcription

1 Journal of Mammalian Evolution, Vol. 7, No. 1, 2000 The Evolution of Enamel Microstructure: How Important Is Amelogenin? Ajay Kishore Mathur 1 and P. David Polly 1,2,3 The shape, size, and orientation of enamel prisms have heretofore been thought to be controlled solely by the shape of the Tomes process. It is known, however, that amelogenin proteins play an important role in enamel deposition and maturation and it is possible that they contribute independently to enamel structure. Using a phylogenetic framework, we clarify the role of amelogenin proteins in the formation of enamel microstructure. We found a negative association between evolutionary changes in amelogenin protein sequences and enamel complexity: amelogenin evolution slows as enamel complexity increases. This is probably because selective constraints on amelogenin increase as enamel complexity increases. Monotremes, which have lost their adult dentition, have particularly high rates of amelogenin evolution while rodents, which have very complex enamel, have very low rates. There is a positive correlation between the number of different amelogenin proteins in a given species and the complexity of its enamel microstructure. An increased number of amelogenins may be necessary for the formation of multiple enamel types in the same tooth. Alternative splicing of amelogenin exons, which allows multiple protein products to be produced from the same gene, may be a key innovation in the diversification of enamel microstructure. KEY WORDS: enamel microstructure: amelogenin; alternative splicing; amelogenesis; Monotremata. INTRODUCTION The evolution of enamel microstructure structural patterns found within enamel has become an increasingly important topic over the past decade because of its functional and phylogenetic significance. In spite of advances in our knowledge of both the comparative structure of enamel and its molecular developmental biology, the mechanisms controlling microstructural patterns and their evolution are still only poorly understood. The dominant hypothesis is that microstructural patterns, which are due to the orientations of hydroxyapatite (HAP) crystallite bundles within enamel, are determined by the 1 Molecular and Cellular Biology Section, Biomedical Science Division, Queen Mary & Westfield College, London E1 4NS United Kingdom. 2 Department of Palaeontology, The Natural History Museum, London, United Kingdom. 3 To whom correspondence should be addressed / 00/ $18.00/ Plenum Publishing Corporation

2 24 Mathur and Polly shape of the enamel-producing ameloblast cells at the time of deposition (Boyde, 1967; Lester and Koenigswald, 1989). While ameloblast shape is undoubtedly a factor, it may not by itself explain the full range of observed structures or their distribution among nonmammalian vertebrates (Sander, 1997). Enamel proteins, particularly amelogenins, may also play an important role in the formation of microstructural patterns and their evolution (Deutsch, 1989). When enamel matrix is first laid down, it is 80 90% protein and fluid by volume and only 10 20% mineral; at maturation, however, enamel is 80 90% mineral by volume (Robinson et al., 1998). Amelogenins, which constitute some 90% of the original protein volume, are differentially removed during maturation and are known to aid in the growth and orientation of HAP crystallites (Diekwisch et al., 1993). It follows that evolutionary changes in amelogenin proteins may result in correlated evolutionary changes in microstructure that are independent of evolutionary changes in the shape of ameloblasts. Specifically, complex microstructures, such as those found in many mammals, may require a range of amelogenin proteins to produce a variety of HAP crystallite sizes and orientations, while the more uniform microstructure of nonmammalian enamel may require fewer and simpler proteins. In this paper, we test this hypothesis by looking for patterns of coevolution between amelogenin proteins and enamel microstructure across a range of mammalian and nonmammalian vertebrates. We compare amelogenin sequence evolution with enamel structure complexity in ten species five eutherian mammals, one marsupial, two monotremes, one archosaur, and one lissamphibian (Fig. 1). If amelogenin is a factor in determining the complexity of enamel structure, then we expect the rate of sequence change along branches of the phylogenetic tree to be correlated with changes in enamel complexity along those same branches. In other words, increased enamel complexity is expected to be associated with an increased rate of amelogenin evolution, while decreased complexity is expected to be associated with a lower rate. A Comparative Overview of Enamel Structure In most vertebrates, enamel covers the surface of the teeth and is the substance which comes into contact with objects put into the mouth. It is composed largely of hydroxyapatite (HAP) crystallites whose size, orientation, and packing form microstructural patterns. One of the most fundamental differences in enamel structure is the presence or absence of prisms. Wood and Stern (1997) defined enamel prisms based on the presence of a prism sheath, elaborating that the sheath is a cylindrical or semi-cylindrical surface of discontinuity in enamel (p. 71). thus, prisms are areas within the enamel where HAP crystallites are grouped in parallel bundles bounded, at least in part, by interprismatic crystallites oriented at a sharp angle to those in the prism. Following Boyde (1976), it is accepted that each prism was associated with a single ameloblast, that the prism s boundary and crosssectional shape are determined by the shape of the ameloblast s Tomes process (which determines the initial orientation of HAP crystallites), and that the orientation of the prism relative to the enamel dentine junction (EDJ) and to other prisms is the result of intercellular movement of ameloblasts during the mineralization process (see review by Carlson, 1990). When present, prisms arise from the EDJ and extend toward the surface of the tooth. Prisms themselves are classified by their cross-sectional shape and packing (Boyde

3 Amelogenins and the Evolution of Enamel Microstructure 25 Fig. 1. A phylogenetic tree of the ten vertebrate species considered in this study. Branch lengths are proportional to paleontological estimates of the time of last common ancestry. Both paleontological estimates and molecular clock estimates of the time of last common ancestry are noted for each node. 1 Benton (1990); 2 Kumar and Hedges (1998); 3 Retief et al. (1993); 4 McKenna and Bell (1997); 5 Webb (1998). and Martin, 1984). The three-dimensional arrangement and packing of prisms can vary within a single tooth, between teeth, and also between species (Koenigswald and Clemens, 1992). Some species possess only one type of enamel, whereas others have multiple types. There is a strong phylogenetic component to the distribution of enamel structure.

4 26 Mathur and Polly The enamel of most nonmammalian vertebrates is prismless, simply composed of semiparallel HAP crystallites that radiate outward from the EDJ (Sander, 1997). Structural organization in nonmammals is often apparent, but crystallite orientation does not form sharp prism boundaries as it does in mammals (Wood et al., 1999). Most mammals, recognized here as the clade stemming from the last common ancestor of morganucodontids and extant monotremes, marsupials, and placentals, have prismatic enamel. Plesiomorphic prismatic enamel had small prisms with arc-shaped sheaths, widely separated by interprismatic matrix. In many taxa, there was a thick aprismatic outer layer of enamel (Wood and Stern, 1997). It is not clear whether prisms had a single phylogenetic origin or have evolved several times in parallel, but it is certain that they have been lost in several clades (see reviews by Carlson, 1990; Wood and Stern, 1997; Wood et al., 1999). In mammals, the angle of prisms relative to the enamel surface or the EDJ is used to classify enamel into several types, including radial, tangential, Hunter Schreger, and irregular enamel (von Koenigswald and Clemens, 1992). These types are distinguished from each other by the angle of their long axis relative to one another and to the EDJ and whether an individual prism maintains the same angle throughout its length from the EDJ toward the enamel surface (Clemens, 1997). Most mammalian teeth are covered with more than one enamel type; marsupials often have at least two types (Stern et al., 1989) and placentals often have three or more (Koenigswald, 1997a,b). In more complex enamel, several types of enamel occur in successive layers between the EDJ and enamel surface (the schmeltzmuster ). Rodents, particularly arvicolines, have the most complex distribution of enamel types across single teeth and the dentition of any vertebrates yet studied (von Koenigswald, 1982). At the level of the dentition, there are differences in the complexity of enamel in different areas of teeth in different mammalian species (von Koenigswald and Clemens, 1992; Koenigswald, 1997b). For example, the organization of enamel in the incisors of rats differs from that of the molars. Extant mammals have a remarkable variety of enamel structure. Marsupials are known to have several types of prisms and prism-packing patterns and some have Hunter Schreger bands. Very complex enamel is often found in placental mammals, especially carnivores, ungulates, and proboscideans (Stefan, 1997; Rensberger and von Koenigswald, 1980), with the most complex yet known occurring in rodents (Koenigswald, 1997a). Monotremes, which do not have functional teeth as adults, have very simple enamel that has lost much of its prismatic structure, although it still has some incremental lines and radial features (Lester and Boyde, 1986; Woood and Stern, 1997). Amelogenin Structure and Function Amelogenin proteins play an important role in the maturation of enamel, guiding the formation, growth, and orientation fo HAP crystallites. Enamel is first deposited by ameloblast cells as an enamel matrix secretion that is 80 90% protein matrix and 10 20% mineral by volume (Moss-Salentijn et al., 1997). During matrix deposition the shape of ameloblasts changes, becoming more elongate and developing projections on the secretory surface known as Tomes processes. The shape of the Tomes process contributes to the initial orientation of the matrix proteins and, thus, ultimately to the orientation of HAP crystallites and enamel prisms (Lester and Koenigswald, 1989; Boyde, 1967; Moss, 1969). Enamel deposition begins at the level of the EDJ and proceeds outward toward the

5 Amelogenins and the Evolution of Enamel Microstructure 27 future crown surface. It is thought that individual prisms correspond to the path taken by a single ameloblast during amelogenesis (Boyde, 1967) and that decussation is mediated by intercellular movements in the ameloblast layer (Nishikawa, 1992). There is an initimate association between amelogenins and mineralization. They make up 90% of the initial enamel matrix and regulate the mineralization process. They bind apatite and can inhibit hydroxyapatite formation in vitro (Fincham et al., 1992) and inhibition of amelogenin translation is known to interfere with both crystal growth and orientation (Diekwisch et al., 1993). The proteins shield the surfaces of the growing HAP crystal from adventitious molecules, which might otherwise hinder or distort growth (Robinson et al., 1998). The selective disappearance of amelogenins before and during enamel maturation suggests that they regulate the size and growth of the crystals (Gibson et al., 1998; Girondot and Sire, 1998; Robinson et al., 1998). Amelogenins also mediate the inhibition of lateral crystallite growth at the enamel surface, preventing premature mineralization and sealing off the enamel surface (Brookes et al., 1995). Because of their role in crystal growth, it is plausible that amelogenins are direct contributors to enamel structural patterns, supplementing and elaborating the initial patterns laid down by the ameloblasts during the secretory phase. As elaborated below, it is likely that the number of amelogenin proteins present in the enamel matrix at deposition is related to the diversity of size and orientation of HAP crystallites in the mature enamel. Amelogenin protein sequences vary among mammals, and in many eutherians there are several variants present within a single developing tooth. For example, in the pig, six amelogenin isoforms have been identified which are, respectively, 190, 173, 157, 56, 41, and 40 amino acids in length (Hu et al., 1996b; Gibson et al., 1991b). The 173-residue amelogenin, which has a mass of 25-kDa is the main protein secreted by ameloblasts, but it is only found in the most superficial layer of developing enamel. It is cleaved by a proteinase enzyme, close to its carboxy-terminus. The 23-kDa protein resulting from this splitting then undergoes a series of apparently discrete cleavages. This produces a diverse family of amelogenins. A 20-kDa amelogenin protein (being the most abundant amelogenin species in the greater thickness of the enamel) may be responsible for supporting the immature crystals (Brookes et al., 1995). Once elongation is complete, the supporting protein matrix is largely withdrawn, allowing crystallites to mature and become selfsupporting through close packing. In the mouse, seven amelogenin mrnas have been isolated, which code for peptides that are 194, 180, 156, 141, 59, and 44 amino acids in length (Simmer et al., 1994). In the rat, five amelogenin proteins have been isolated (Robinson et al., 1998). The size of amelogenin proteins varies between and, sometimes, within species. Cows produce two proteins, one from the X chromosome, consisting of 213 amino acids, and a second from the Y, consisting of 192 amino acids; likewise, humans have X and Y variants of 191 and 192 amino acids, respectively. Opossums have amelogenins of 202 and 57 amino acids (Hu et al., 1996a). The amelogenin gene and/ or cdnas have been cloned from a number of species, including mouse (Snead et al., 1985), cow (Shimokawa et al., 1987; Gibson et al., 1991a, 1992), human (Salido et al., 1992), rat (Bonass et al., 1994a,b; Li et al., 1995), opossum (Hu et al., 1996a), pig (Hu et al., 1996b), as well as the toad, caiman, platypus, and echidna (Toyosawa et al., 1998). The DNA base sequence itself is relatively constant and in general, the protein-coding regions are highly conserved. Where they exist, the various forms of amelogenin protein seem to be produced by alternative splicing of the

6 28 Mathur and Polly initial gene product (Bonass et al., 1994b; Gibson et al., 1991a). In at least some mammals (e.g., human, cow, mouse, and rat), the amelogenin gene appears to consist of seven exons, but the mature mrna of just six (Gibson et al., 1991a,b; Salido et al., 1992; Bonass et al., 1994a). Amelogenin gene exons can either be spliced in numerous ways to produce mature mrnas from the DNA, each composed of one or more exons, or can be formed by the deletion of part of an exon. For example, a leucine-rich amelogenin peptide has been identified in the developing enamel of the cow (Gibson et al., 1991b), mouse (Lau et al., 1992), and rat (Bonass et al., 1994b) and represents a major product of amelogenin gene expression. This peptide is produced by the colinear splicing of exons 1, 2, 3, and 5. The 3 end of exon 5, in this case, instead of being spliced to the 5 end of exon 6, recognizes an alternative splice site near the 3 end of exon 6 (Gibson et al., 1991a). The mineral-binding properties of amelogenins appear to depend on the overall conformation of the intact molecule (Brookes et al., 1995). Amelogenins with radically different sizes and strutures may thus play different roles in mineralization and HAP crystallite growth. This idea is supported by the topographic localization of certain amelogenin products close to the surface of the developing enamel (Gibson et al., 1991b). Evolutionary changes in the structure of amelogenin gene may, therefore, be responsible, in part, for the diverse microstructure of enamel. Changing protein structure and the evolutionary multiplication of the number of amelogenins may be correlated with the differing levels of structural complexity seen in the enamel of various vertebrates. Structural changes to the protein brought about by mutation may therefore have differing effects on enamel maturation, which would be expressed in changes in the complexity of the enamel as a whole. To test this, we examined the correlation between the evolution of amelogenin protein sequences and the evolution of enamel microstructure. MATERIALS AND METHODS Ten species, all of which are represented by sequenced amelogenin proteins (Toyosawa et al., 1998), were considered in this study. These include one amphibian (Xenopus laevis), one archosaur reptile (Paleosuchus palpebrosus), two monotremes (Ornithorhynchus anatinus and Tachyglossus aculeatus), one marsupial (Monodelphis domestica), and five placental mammals (Homo sapiens, Mus musculus, Rattus norvegicus, Sus scrofa, and Bos taurus). Amelogenin protein sequences were identified using published literature and downloaded from GenBank. Two sequences (one each from the X and Y chromosomes) from Homo and Bos were used. Two alternatively spliced protein sequences were available for Sus and three for Rattus. Xenopus has two amelogenin genes, both of which were used in this study. These aligned sequences are shown in the Appendix; all GenBank accession numbers are listed by Toyosawa et al. (1998). Protein sequences were aligned using Clustal X (Thompson et al., 1994) with weights set at 10.0 for gap openings and 0.05 for gap extensions. Substitutions were weighted with the Blosum 30 protein weight matrix. Because the goal of the study was to document structural changes in the protein rather than to reconstruct phylogenetic relationships, gaps were not excluded from the final analysis. A neighbor-joining tree (Saitou and Nei, 1987) was constructed from the aligned protein sequences. The N J method, which does not assume constancy of rate, was used because a major goal of this study was to document changes in the rate of evolution of protein sequences. Branch lengths

7 Amelogenins and the Evolution of Enamel Microstructure 29 from the resulting tree were used as measures of amelogenin evolution. The numbers reported are scaled from 1.0 to 0.0 and represent percentage sequence change (e.g., 1.0 represents 100% sequence change and 0.5 represents 50% change). Protein sequence changes were mapped onto a phylogenetic tree of the 10 species (Benton, 1990; Gauthier et al., 1988). Branch lengths in millions of year were also calculated. Most of the paleontological dates come from Benton (1990), although dates for the divergence of Sus and Bos come from Webb (1998); Mus and Rattus come from McKenna and Bell (1997). Molecular clock divergence times all came from Kumar and Hedges (1998) except for the divergence of the two monotremes from one another and from other vertebrates whose estimates were taken from Retief et al. (1993). All node ages are reported in Fig. 1. There is no precise consensus about times of divergence either among paleontologists or among molecular workers; however, we believe that the results we present here are robust against the uncertainties. Rates of protein evolution were calculated by dividing the percentage change along each branch by the length of the branch in millions of years. The resulting rates are thus in units of percentage sequence change per million years. Multiple rates were calculated for those species with more than one available amelogenin sequence (Xenopus, Homo, Sus, Bos, and Mus). Because of disagreements in the time of last common ancestry, rates were calculated twice, once with the paleontological estimates and once with the molecular clock estimates. It should be noted, that there is an important discrepancy between the molecular clock and the paleontological estimates of the divergence time of marsupials relative to other mammals. Kumar and Hedges (1998) dates put marsupials outside of monotremes, while more established phylogenies place marsupials as the sister group of eutherians (e.g., McKenna and Bell, 1997; Novacek, 1992; Benton, 1990). This means that nodes 3 and 5 (Fig. 1 and subsequent) are reversed if the molecular dates are correct. We resolved this discrepancy in two ways: we did not include the branch between nodes 3 and 5 in our correlations (see below) and we collapsed those nodes (assigning it a mean age of million years before present) when calculating the rates of protein evolution from molecular clock dates. Data on the enamel microstructure of each of the ten species were collected from published literature as follows: Xenopus (Poole, 1966), Paleosuchus (Poole, 1966, data from Gavialis used as proxy), Monodelphis (Paulson and McGlumphy, 1983; Paulson and Larkin, 1981, supplemented with data on Didelphis from Stern et al., 1989), Tachyglossus (Griffiths, 1978), Ornithorhynchus (Lester and Boyde, 1986; Lester et al., 1987), Homo (Schroeder, 1991), Sus (Boyde, 1967; Nishikawa, 1992), Bos (Pfretzschner, 1994, supplemented with data on Ovis from Grine et al., 1986), Mus (Lyngstadaas et al., 1998), and Rattus (Warshawsky et al., 1991). Data on evolutionary transitions came from Wood and Stern (1997), Sander (1997), and Carlson (1990). A rank order variable for enamel complexity (EC) was assigned to each species based on whether enamel was prismatic or prismless, whether single or multiple prism types were present, whether single or multiple enamel types were present, whether there was prism decussation, and whether there was variation within the dentition. These range from 0 (the least complex) to 7 (the most complex). A second rank order variable for change in enamel complexity (CEC) was assigned to each branch in the tree. When there was an increase in complexity along a branch, it was given either a +1 (for small changes) or +2 (for multiple changes). If there was a decrease in complexity, a score of 1 was assigned. Branches on which there

8 30 Mathur and Polly was no change were given a 0 (zero). Our purpose was not to develop a comprehensive quantitative classification of vertebrate enamel complexity, but rather to provide a coarse descriptor of the variety of enamel complexity among those species with known amelogenin sequences, in order to arrive at a crude estimate of the change in enamel structure complexity along branches of the phylogenetic tree connecting those species. It is those rank-order changes that we compare to changes in the rate of amelogenin evolution along the same branches. Qualitative conclusions about the coevolution of amelogenin proteins and enamel microstructure were statistically tested using correlation analyses. The rate of protein evolution along each branch was tested for association with the enamel complexity change variable (CEC). Because the quantification of enamel complexity was necessarily arbitrary, the nonparametric Spearman s rank correlation test was used. The same nonparametric test was used to assess the relationship between enamel complexity (EC) and the number of amelogenin proteins in each of the ten species. RESULTS AND DISCUSSION A neighbor-joining tree depicting the relationships and percent divergences of the 16 amelogenin proteins considered in this study is shown in Fig. 2. The branch lengths (in percentage sequence divergence) are reported when they were 0.01% or larger. For the most part, the relationships among the proteins mirrors the phylogeny of the animals Fig. 2. A neighbor-joining tree depicting relationships of sixteen amelogenin proteins from ten species. Branch lengths are drawn proportional to percentage sequence divergence along them and each branch with a length greater than 0.01 is labeled.

9 Amelogenins and the Evolution of Enamel Microstructure 31 Fig. 3. The phylogenetic tree from Fig. 1 with percentage amelogenin sequence divergences mapped onto it. More than one distance is reported on branches for species with more than one version of the protein. from which they were taken. One exception is that the monotreme amelogenins cluster closer to eutherian amelogenins than does the marsupial protein. Another exception is that all of the rodent amelogenins cluster together with the Mus protein in the midst of the Rattus ones. This is not surprising as three Rattus amelogenins are alternative splicings of the same underlying amelogenin gene. In those species with two amelogenin genes (Xenopus, Homo, Sus, and Bos), the two associated proteins cluster together. This implies that amelogenin gene duplications happened independently in each of these taxa. Amelogenin sequence divergences are mapped onto a phylogenetic tree in Fig. 3. Where multiple proteins are found in the same species, a corresponding number of distances are reported. As would be expected, protein divergence is greater among species whose last common ancestor is more ancient and less among those whose last common ancestor is more recent. The greatest sequence divergences are found between Xenopus and the other vertebrates the last common ancestor with Xenopus was approximately 360 mya. The smallest divergences are between Mus and Rattus, which shared a common ancestor less than 40 mya. Protein divergence rates are reported in Table I. These were calculated using both the paleontological and molecular clock divergence dates from Fig. 1. Surprisingly, some of the lowest rates of amelogenin sequence divergence are found within placental mammals the percentage divergence per million years is between nodes 5 and 6 (when calibrated using the molecular clock dates), while the rate of sequence divergence is almost 200 to 300 times as fast within the monotremes ( between node 4 and

10 32 Mathur and Polly Table I. Amelogenin and Enamel Microstructure Evolution a Tree branch PPC MY-M PC-M MY-P PC-P CEC Xenopus I r Node Xenopus II r Node Node 2 r Paleosuchus Node 2 r Node Node 3 r Node Node 4 r Tachyglossus Node 4 r Ornithorhynchus Node 5 r Monodelphis Node 5 r Node Node 6 r Homo X Node 6 r Homo Y Node 6 r Node Node 7 r Sus I Node 7 r Sus II Node 7 r Bos X Node 7 r Bos Y Node 6 r Node Node 8 r Mus Node 8 r Rattus I Node 8 r Rattus II Node 8 r Rattus III a Percentage amelogenin protein change (PPC), branch length in millions of years using molecular clock calibrations (MY-M), rate of protein sequence change using molecular age estimates (PC-M), branch length in millions of years using paleontological calibrations (MY-P), rate of protein change using paleontological estimates (PC-P), and the rank value of change in enamel complexity (CEC) for each branch in the phylogenetic tree of the ten species considered in this study. Tachyglossus, calculated with molecular dates or calculated with paleontological dates). We return to this below. Data on the complexity of enamel microstructures and their evolutionary changes are summarized in Fig. 4 and Table II. The nonmammals (Xenopus and Paleosuchus) have the simplest enamel, while the rodents (Mus and Rattus) have the most complex. There were several major evolutionary changes in enamel microstructure. True enamel evolved from enameloid between nodes 1 and 2 some 300 to 350 mya. Prismatic enamel evolved in mammals along the branch between nodes 2 and 3 sometime in the late Triassic (a little more than 200 mya). In living monotremes, the adult dentition has been lost and in Tachyglossus the deciduous dentition has been lost as well. Ornithorhynchus has vestigial deciduous teeth with rudimentarily prismatic enamel (Lester and Boyde, 1986; Lester et al., 1987). In spite of this reduction and loss, both species still produce amelogenin proteins, although they are considerably reduced in length compared to those found in other vertebrates (Appendix). Within mammals, a number of changes occurred, including the evolution of prism decussation, multiple prism types, and multiple enamel types. Within rodents, less than 40 mya, there was a dramatic increase in complexity of enamel. There is a much greater range of enamel patterning; decussation occurs to a much greater extent throughout the dentition, and there is also a much greater variety of enamel structure within the dentition in both rats and mice (Koenigswald, 1982). The variety of enamel structures are probably related to function, although the degree to which this would have

11 Amelogenins and the Evolution of Enamel Microstructure 33 Fig. 4. Evolution of enamel microstructure in tetrapod vertebrates. Table II describes the distribution of enamel microstructures among the ten species considered in this study and reports the enamel complexity rank (EC) assigned to each based on that distribution. Major evolutionary changes in enamel microstructure are shown here mapped onto the phylogenetic tree. Ranks for the direction and magnitude of those changes are shown in parentheses. See text for details. an effect on the development of enamel complexity is unclear. It has been argued that complex enamel microstructures always occur in animals that have developed complex occlusion that involves shearing (Clemens, 1997). The distribution of enamel microstructure does not have a simple relationship with diet. Paleosuchus is a carnivore it eats crustaceans, terrestrial invertebrates, such as coleopterans, crabs, molluscs, and shrimps (Magnusson et al., 1987) while Xenopus eats decomposed organic material, larvae, and insects (Deuchar, 1975). Yet, both have prismless enamel and simple, conical teeth. Humans, who have quite complex enamel, are omnivorous while Bos and Sus, which have similar enamel, are herbivores. Rodents, which can be herbivores, granivores, or omnivores, have the most complex enamel structures. The evolution of enamel microstructures, however, does have a strong relationship with change in amelogenin structure (Fig. 5). Interestingly, the relationship between protein change and complexity is negative: as enamel becomes more complex, amelogenin evolution slows, but it speeds up in clades where enamel becomes less complex. Compare the rates in Table I with the changes documented in Fig. 4 and Table II. In murine rodents (node 8 and above), rates of amelogenin evolution have been low but enamel complexity is at its highest. Amelogenin evolved more quickly in the lineage leading to Paleosuchus (from node 2) than in rodents. It appears that amelogenin protein evolution has been more rapid in lineages with simple, prismless enamel than in lineages where it is more complex. This is best illustrated in the monotreme clade (node 3 to Tachyglossus

12 34 Mathur and Polly Table II. Enamel Structures, Complexity, and Amelogenins in Ten Vertebrates a Xenopus Paleosuchus Tachyglossus Ornithorhynchus Monodelphis Homo Sus Bos Mus Rattus Enamel present? Y Y N Y Y Y Y Y Y Y Prismatic? N N N Y Y Y Y Y Y Y Decussation? N N N N N Y Y N Y Y No. of prism types No. of enamel types Variation in dentition? N N N N N N N N Y Y Complexity rank b No. of amelogenins a N, no; Y, yes. b See text and Fig. 6 for details. and Ornithorhynchus). Extant monotremes have reduced or lost the adult dentition, but the rate of amelogenin evolution increased sometime during the evolution of that clade. This suggests that the rate of amelogenin change is inversely related to its function in amelogenesis in monotremes. Because adult dentition has become vestigial, the functional constraint on protein expression has been removed, thus allowing an increase in the rate of evolution of the gene in extant monotremes. Statistically, the inverse correlation between the rate of protein change and change in enamel complexity is significant. When paleontological dates are used to calculate rates, Spearman s rho is (P 0.03) and when molecular dates are used, it is (P 0.02). The exceptionally high rates of protein divergence in monotremes largely account for this, but even without them there is still a lesser trend toward decreased protein rate with increased enamel complexity (Fig. 5). This finding is evidence that enamel complexity is explained not only by ameloblast activity, but also by changes in amelogenin proteins themselves. This is even more evident in the positive correlation between the number of amelogenin protein variants in a species and the overall complexity of its enamel (Fig. 6). The correlation between the number of amelogenins and the enamel complexity (EC) values assigned in Table II is statistically significant. Spearman s rho for this association is (P < 0.01). As far as is known, rodents have the most amelogenin products (more than five) and the most complex enamel structures. Nonmammals, with the exception of Xenopus, have only one or two amelogenins and have relatively less complex enamel structures. Thus, the formation of more complex enamel structures (multiple enamel types in complex layers distributed across the entire dentition) may require several amelogenin proteins with different structures and different sizes. This fits well with previous observations that amelogenins in species with multiple isoforms are differentially distributed through enamel layers (some being found near the EDJ, some at the surface, and some intermediate) and that different isoforms have different roles in supporting, orienting, and terminating HAP crystallite growth (Brookes et al., 1995).

13 Amelogenins and the Evolution of Enamel Microstructure 35 Fig. 5. The relationship between change in enamel complexity (CEC) and rate of evolution for all the branches in the phylogeny of the ten species considered in this study. (a) graph showing rates calculated using paleontological age estimates for divergence times. (b) graph showing rates calculated using molecular clock estimates for divergence times. In both cases, the correlation is statistically significant according to Spearman s rank correlation test. In both cases evolutionary changes within the monotremes (the upper and right-most data point in both graphs) contribute substantially to the correlation. In monotremes, enamel microstructures are greatly simplified or lost while the rate of amelogenin evolution increases substantially.

14 36 Mathur and Polly Fig. 6. The relationship between enamel microstructure complexity (EC) and the number of amelogenin proteins present within a single individual of a species. See text for discussion. It is interesting that species with the greatest number of products also have the slowest evolving protein structures. This seems counterintuitive Rattus and Mus might well have the fastest evolving proteins since they seem to have the most rapidly evolving enamel. However, functional constraint may play a significant role: rodents have continuously growing enamel in their incisors (and sometimes in their molars) that wear down throughout life. It may be that selection on their enamel microstructure is more intense and is reflected by a decrease in the rate of amelogenin evolution. Certainly it is clear that enamel microstructure is important in resisting abrasion and wear in function regions of the tooth (Maas, 1993; Fortelius, 1985; Rensberger and Koenigswald, 1980). Presuming that amelogenins do contribute to the formation of enamel microstructure, then functional selection on tooth macrostructure should have a dampening effect on the rate of amelogenin protein evolution. It is unlikely that random mutations have positive effects and it is generally thought that the rate of mutation in a functionally important gene will have a slower rate of mutation compared to one that does not have the same functional importance (Ohta and Gillespie, 1996). This interpretation of amelogenin evolution is further bolstered by the increased rate in monotremes where functional selection on enamel microstructure has almost certainly decreased or disappeared. CONCLUSIONS The formation of enamel structures during amelogenesis have heretofore been explained primarily in terms of the shape of the Tomes processes of ameloblast cells

15 Amelogenins and the Evolution of Enamel Microstructure 37 (e.g., Boyde, 1967; Lester and Koenigswald, 1989). The formation of prisms, the pattern of their packing, and their course from EDJ to tooth surface are in large part determined by the shape of the secretory surface and intercellular movements within the ameloblast sheet (Carlson, 1990; Nishikawa, 1992). However, enamel microstructures (prisms, prism outlines, decussation patterns, etc.) are all emergent phenomena resulting from the size and orientation of HAP crystallites. Tomes processes are demonstrably associated with the initial orientation of these crystallites, but the majority of crystallite growth is mediated by amelogenin proteins. This postdepositional maturation is significant, enamel changes from only 10 to almost 90% mineral content by volume, and leaves ample opportunity for additional factors to help determine the final pattern of enamel microstructure. It is known that inhibiting transcription of amelogenin interferes with maturation by reducing HAP crystal sizes and by altering their orientations (Diekwisch et al., 1993). This suggests that amelogenins themselves may be partly responsible for enamel microstructural patterns. While ameloblasts lay down the initial enamel matrix and orient the protocrystallites, amelogenins may be responsible for the variety of mature crystallite sizes and the sharp discontinuities found in complex mammalian enamel. Using a phylogenetic framework, our study supports this interpretation. There is a significant correlation between evolutionary changes in amelogenin protein sequence and enamel complexity. Surprisingly, this is a negative correlation: amelogenin evolution slows as enamel complexity increases. We explain this in terms of functional constraint. Complex enamel is associated with complex occlusal and masticatory patterns, which place strong selective pressure on enamel structure and, therefore, amelogenins. This may slow the rate of evolution in the proteins by increasing selection against new variants. In clades where that selection is lifted (such as monotremes, which have lost their adult dentition), the rate of amelogenin evolution has increased. We also found a positive correlation between the number of different amelogenin proteins in a species and the complexity of enamel microstructure: the more complex the enamel, the more amelogenin variants are present. This makes intuitive sense because amelogenins are differentially removed during enamel maturation. If there are several amelogenins, each with a different size and structure, the pattern of removal can be more complex than if there is only one amelogenin. An increased number of amelogenins may allow the formation of multiple enamel types in the same tooth. If so, this suggests that the evolution of multiple amelogenin products, which seems to have happened both by gene duplication and alternative splicing of exons, may be a key innovation allowing the rapid diversification of enamel structure and, therefore, tooth structure and function. ACKNOWLEDGMENTS Jessica Theodor and Wighart von Koenigswald provided bibliographical references. Ms. S. Banerjee helped throughout the project. This paper was greatly improved by thorough reviews from C. B. Wood, J. Jernvall, and an anonymous reviewer. The Drapers Company and Dental Old Londoners Club provided financial assistance to AKM. This work was supported by grant number GR8/ from the Natural Environment Research Council (NERC).

16 38 Mathur and Polly APPENDIX. Amino Acid Alignment of Amelogenin Sequences Analyzed Homo Y MGTWILFACLVGAAFAMPLPPHPGHPGYIN Homo X MGTWILFACLLGAAFAMPLPPHPGHPGYIN Rattus 1 QASLSFRQKVTEHTLKNHQEMGTWILFACLLGAAFAMPLPPHPGSPGYIN Rattus MGTWILFACLLGAAFAMPLPPHPGSPGYIN Rattus MGTWILFACLLGAAFAMPLPPHPGSPGYIN Mus MGTWILFACLLGAAFAMPLPPHPGSPGYIN Bos Y MGTWILFACLLGAAYSMPLPPHPGHPGYIN Bos X MGTWILFACLLGAAFSMPLPPHPGHPGYIN Sus MGTWIFFACLLGASLAMPLPPHPGHPGYIN Sus MGTWILFACLLGAAFSMPLPPHPGHPGYIN Tachyglossus Ornithorhynchus Monodelphis IPLPPHPGHPGYIN Paleosuchus MEGWMLITCLLGATFAIPLPPHPHHPGYVN Xenopus MRPLVMLTALIGAAFSLPLPPQPQHPGYVN Xenopus MRPWLMLTALIGVAFSVPLPPHPQHPGYVN Homo Y FSYEVLTPLKWYQSMIRPPYSSYGYEPMGGWLHHQIIPVVSQQHPLTHTL Homo X FSYEVLTPLKWYQS IRPPYPSYGYEPMGGWLHHQIIPVLSQQHPPTHTL Rattus 1 LSYEVLTPLKWYQSMIRQP Rattus 2 LSYEVLTPLKWYQSMIRQPHPPS HTL Rattus 3 LSYEVLTPLKWYQSMIRQPHPSYGYEPMGGWLHHQIIPVLSQQHPPSHTL Mus LSYEVLTPLKWYQSMIRQPYPSYGYEPMGGWLHHQIIPVLSQQHPPSHTL Bos Y FSYEVLTPLKWYQNMLRYPYPSYGYEPVGGWLHHQIIPVVSQQSPQNHAL Bos X FSYEVLTPLKWYQSMIRHPYPSYGYEPMGGWLHHQIIPVVSQQTPQNHAL Sus 1 FSYEVLTPLKWYQNMIRHPYTSYGYEPMGGWLHHQIIPVVSQQTPQSHAL Sus 2 FSYEVLTPLKWYQNMIRHPYTSYGYEPMGGWLHHQIIPVVSQQTPQSHAL Tachyglossus LHHQIIPVLSQHQTPTHAL Ornithorhynchus LHHQIIPVLSQQQTPTHAL Monodelphis FSYEVLTPLKWYQSMMRHEYPSYGYEPMGGWLHHQIIPVLSQQHSPSHSL Paleosuchus FSYEVLTPLKWYQSLMRQPYSSYGYEPMGGWLHQPMLP IAQQHPPIQTL Xenopus 1 FSYEILSPIKWYQSMMKNQYPNYGYEPVSGWLQSPMIP VPPMMQQQQL Xenopus 2 FSYEILSPLKWYQSMMTHQYPNYGYEPVSGWLQNPIIP APPMMPQQQ Homo Y QSHHHIPVVPAQQPR VRQQALMPVPGQQSMTPTQHHQPNLPLPAQQPFQ Homo X QPHHHIPVVPAQQPV IPQQPMMPVPGQHSMTPIQHHQPNLPPPAQQPYQ Rattus Rattus 2 QPHHHLPVVPAQQPV APQQPMMPVPGHHSMTPTQHHQPNIPPSAQQPFQ Rattus 3 QPHHHLPVVPAQQPV APQQPMMPVPGHHSMTPTQHHQPNIPPSAQQPFQ Mus QPHHHLPVVPAQQPV APQQPMMPVPGHHSMTPTQHHQPNIPPSAQQPFQ Bos Y QPHHHNPMVPAQQPV VPQQPMMPVPGQHSMTPIQHHQPNLPLPAQQSFQ Bos X QPHHHIPMVPAQQPV VPQQPMMPVPGQHSMTPTQHHQPNLPLPAQQPFQ Sus 1 QPHHHIPMVPAQQPG IPQQPMMPLPGQHSMTPTQHHQPNLPLPAQQPFQ Sus 2 QPHHHIPMVPAQQPG IPQQPMMPLPGQHSMTPTQHHQPNLPLPAQQPFQ Tachyglossus QSHHHIPVMATQQPT QPPQPMMPMPGQHSVTPTQHHQSNLPQPGQQPFQ Ornithorhynchus QPHHHIPVMAAQQPM QPQQPMMPMPGQPSVTPTQHHQSNLPQPAQQPFQ Monodelphis PPQHHIPIMAAQQPA PPQQPVMPVPGQHPMAPTQHHQPNLPQPGQQPYQ Paleosuchus TPHHQIPFLS PQHPLMQMPGPHQMMPIPQQQPSLQMPVQEPVQ Xenopus 1 PSQNAVPKLPSHHPLLIPQQPLVPVPVHHPLIPLTPQHTHQLKPIYLFNS Xenopus QNAVPKLPPHHPLLIPQHPLVPVPVHHPVFPLIPQHTHQLKPTYLSNP Homo Y PQPVQ PQPHQPMQ PQPPV Homo X PQPVQ PQPHQPMQ PQPPV Rattus Rattus 2 QPFQPQAIP PQSHQPMQ PQSPL Rattus 3 QPFQPQAIP PQSHQPMQ PQSPL Mus QPFQPQAIP PQSHQPMQ PQSPL

17 Amelogenins and the Evolution of Enamel Microstructure 39 APPENDIX. Continued Bos Y PQPIQ PQPHQPLQ PQPPV Bos X PQSIQ PQPHQPLQPHQPLQ PMQPMQPLQPLQPLQPQPPV Sus PQPVQ PQPHQPLQ PQSPM Sus PQPVQ PQPHQPLQ PQSPM Tachyglossus PQFPQ KPTHRPIQ PQAPV Ornithorhynchus PQVPQ QPPHQPIQ PQAPA Monodelphis PQPAQQPQPHQPIQPIQPIQPIQPMQPMQPMQPMQPMQPMQPQTPV Paleosuchus PQAGEHPSPPVQPQQPGHPNPP MQPQLPGSPH Xenopus DGQYPTNTQLLE PSKPD Xenopus DGQYPTNTQ PD Homo Y QPMQPLLPQPPLPPMFPLRPLPPILPDLHLEAWPATDKTKQEEVD Homo X HPMQPLPPQPPLPPMFPMQPLPPMLPDLTLEAWPSTDKTKREEVD Rattus PLSPILPELPLEAWPATDKTKREEVAFSPMK Rattus 2 HPMQPLAPQPPLPPLFSMQPLSPILPELPLEAWPATDKTKREEVAFSPMK Rattus 3 HPMQPLAPQPPLPPLFSMQPLSPILPELPLEAWPATDKTKREEVAFSPMK Mus HPMQPLAPQPPLPPLFSMQPLSPILPELPLEAWPATDKTKREEVD Bos Y HPIQRLPPQPPLPPIFPMQPLPPVLPDLPLEAWPATDKTKREEVD Bos X HPIQPLPPQPPLPPIFPMQPLPPMLPDLPLEAWPATDKTKREEVD Sus 1 HPIQPLLPQPPLPPMFSMQSL- - - LPDLPLEAWPATDKTKREEVD Sus 2 HPIQPLLPQPPLPPMFSMQSL- - - LPDLPLEAWPATDKTKREEVD Tachyglossus HPMPP MPQPQLPPMFPLQPLPPLLPDLPLEPWPAS Ornithorhynchus HPMPP MPQPPLPPMFPMQPLPPLLPDLPLEQWPAT Monodelphis HAVRPLPPQPPLPPMFPMQPMSP MLPDMEAWPATDKTKREEVD Paleosuchus PPMRPQQPGIPNPPMYPMQPLPPLLPDMPLEPWRPMDKTKQEEID Xenopus 1 HE SQNGQPTFPLHPLPPLVEERPQEPWQEAGNDKQEELD Xenopus 2 N QNGKPIFPLQPMPPLVEDRPQEPWQAAGNTKQEELD Homo Y Homo X Homo Y Homo X Rattus 1 WYQGTARHPLNMETTTEK Rattus 2 WYQGTARHPLNMETTTEK Rattus 3 WYQGTARHPLNMETTTEK Mus Bos Y Bos X Sus Sus Tachyglossus Ornithorhynchus Monodelphis Paleosuchus Xenopus Xenopus LITERATURE CITED Benton, M. J. (1990). Phylogeny and the major tetrapod groups: Morphological data and divergence dates. J. Mol. Evol. 30: Bonass, W. A., Robinson, P. A., Kirkham, J., Shore, R. C., and Robinson, C. (1994a). Molecular cloning and DNA sequence of rat amelogenin and a comparative analysis of mammalian amelogenin protein sequence divergence. Biochem. Biophys. Res. Commun. 198: Bonass, W. A., Kirkham, J., Brookes, S. J., Shore, R. C., and Robinson, C. (1994b). Isolation and characterization of an alternatively-spliced rat amelogenin cdna: LRAP a highly conserved, functional alternatively spliced amelogenin? Biochem. Biophys. Acta 1219: Boyde, A. (1967). The development of enamel structure. Proc. Roy. Soc. Med. 60:

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