BACKGROUND
gnathostomes are unique among vertebrates in utilizing jaws and teeth for food acquisition and processing.
divergent gnathostome lineages exhibit a wide array of dental modifications in association with diverse dietary specializations.
multiple iterations of secondary tooth loss have also evolved in gnathostomes, perhaps most famously in birds, turtles, and several mammalian lineages including baleen whales, pangolins, and anteaters  <cit> .
there are also mammalian species with enamelless teeth, i.e., sloths, armadillos, aardvarks, pygmy and dwarf sperm whales  <cit> .
tooth development is an intricate process that encompasses a complex series of epithelial-mesenchymal interactions involving growth factors, transcription factors, and signal receptors that affect tooth shape, tooth number, and cusp number  <cit> .
several hundred genes are associated with tooth development  <cit> , including members of the hedgehog , fibroblast growth factor  and bone morphogenic protein  families that mediate epithelio-mesenchymal signaling interactions  <cit> .
most or all of the genes that are involved in early tooth development are thought to be pleiotropic and have additional functions outside of tooth formation.
however, there are also genes with putative tooth-specific functions that are expressed later in development by dentin-forming odontoblasts and/or enamel-forming ameloblasts  <cit> .
these genes affect the physical properties of teeth including enamel thickness and structure.
the durability of teeth, in combination with tooth-specific genes that impact the physical structure of dentin and enamel, make teeth a model system for studying the coevolution of morphological change in the fossil record and molecular change in the genome.
previous studies have shown that the genes encoding three enamel matrix proteins , enamelin, amelogenin, and ameloblastin, have become pseudogenized in one or more edentulous/enamelless mammals by frameshift mutations and/or stop codons  <cit>  .
further, the molecular decay of tooth-specific genes in placental mammal lineages has been shown to parallel the morphological degeneration of enamel in the fossil record  <cit> .
the retention of inactivated emp genes in mammalian lineages is not surprising given that all edentulous and enamelless taxa appear to have originated in the cenozoic  <cit> .
by contrast, tooth loss in both birds and turtles occurred in the mesozoic, providing a longer time period for the molecular decay of tooth-specific genes in these lineages.
in birds, the presence of a horny beak and gizzard have presumably compensated for edentulism in food acquisition and processing  <cit> .
edentulism in the ancestry of modern birds occurred in the cretaceous, between  <dig> and  <dig>  ma, and also evolved independently in several lineages of extinct birds that are on the stem to neornithes   <cit> .
even though tooth loss in neornithes occurred in the cretaceous, the chicken  genome retains pseudogenized copies of both amel and enam <cit> .
edentulism in testudines  occurred even earlier, at least as far back as the late jurassic  <cit> .
the oldest turtle is odontochelys semitestacea from the triassic of china   <cit> .
marginal and palatal teeth are both present in this taxon  <cit> .
slightly younger  is proganochelys quenstedti from the upper triassic  <cit> .
marginal teeth are absent in proganochelys, but palatal teeth are present.
phylogenetic analyses suggest that proganochelys, like odontochelys, is a stem testudine  <cit> .
the oldest testudines with unambiguous crown-group affinities, including tooth loss on the palatines, vomer, and pterygoids, are from the late jurassic  <cit> .
to date, remnants of pseudogenized copies of tooth-specific genes in testudines have not been reported.
girondot and sire  <cit>  were unable to amplify fragments of amelogenin in turtles with degenerate pcr primers, and it remains unclear if vestiges of tooth-specific genes are retained in testudine genomes given the antiquity of tooth loss in this clade.
on the other hand, phylogenomic data suggest that testudines have slower rates of nuclear gene evolution than birds and mammals .
the recent release of two testudine genomes, chrysemys picta  and pelodiscus sinensis , provides an opportunity to screen for remnants of tooth-specific genes in turtles.
these taxa, both of which belong to cryptodira , index the basal cladogenic event among crown cryptodires and leave only pleurodira  unrepresented among the three oldest crown-testudine lineages.
cryptodira and pleurodira have traditionally been regarded as reciprocally monophyletic based on morphology  <cit>  and large molecular data sets  <cit> .
an exception is barley et al.’s  <cit>  coalescence analysis, which recovered a basal split between softshells plus the pig-nosed turtle  and pleurodira plus other cryptodira.
however, barley et al.’s  <cit>  concatenation analysis recovered cryptodira and pleurodira.
moreover, the most complete testudine phylogeny with a variety of outgroups  <cit>  supports the monophyly of both cryptodira and pleurodira.
here, we report the results of querying the genomes of chrysemys and pelodiscus for inactivated remnants of three emp genes, enam, amel, and ambn, all of which are hypothesized to have tooth-specific functions.
we also report the results of querying genome sequences of five birds  and three enamelless mammals  for remnants of these emp genes.
the occurrence of pseudogenized remnants of emp genes in highly divergent amniotes, including turtles, birds, and edentulous/enamelless mammals, would provide robust evidence for the hypothesis that the only essential, non-redundant function of amel, ambn, and enam is in enamel formation.
moreover, the search for shared frameshift mutations in emp genes provides an opportunity to date the timing of enamel loss in these clades.
methods
the genomes of chrysemys picta  and pelodiscus sinensis  were queried with blastn using crocodylian mrna sequences for three enamel matrix proteins genes: paleosuchus palpebrosus  amel , caiman crocodilus  ambn , and crocodylus niloticus  enam .
intron-exon boundaries of mrna sequences were determined by blasting crocodylian mrnas against the recently released genome sequence of alligator mississippiensis .
crocodylian sequences were chosen as probes based on recent evidence that provides strong support for a sister group relationship between testudines and archosauria   <cit> .
ambn has  <dig> exons in alligator, all of which include protein-coding regions.
amel includes six exons in alligator, five of which  contain protein-coding regions.
finally, enam includes nine exons in alligator, eight of which  include protein-coding regions.
remnants of emp genes that were recovered from chrysemys were subsequently used to query the pelodiscus genome, and remnants of emp genes from pelodiscus were used to query the chrysemys genome.
five bird genomes  were queried with crocodylian mrnas and virtual pseudogene mrnas for amel  and enam  from gallus, as well as with positive hits from the initial round of screening for the bird genomes.
the ncbi genomes of three enamelless mammals  were queried with gene sequences from mammals that retain enamel-capped teeth .
sequences were aligned with se-al  <cit> .
selection analyses on branches were performed with paml  <dig>   <cit>  to estimate dn/ds values for an assemblage of  <dig> complete or nearly complete mammalian ambn sequences, all of which were derived from ensembl  <dig>  or ncbi .
sequences were aligned with se-al  <cit> .
branch analyses were performed with four branch categories: orycteropus, choloepus, dasypus, and all other branches.
orycteropus, choloepus and dasypus were recognized as separate branches in the paml runs based on the hypothesized independent loss of enamel in these lineages  <cit> .
paml analyses were conducted with codon frequency models  <dig>   <dig>  and  <dig>  we used a composite species tree based on meredith et al.
<cit>  and springer et al.
<cit> .
approximate divergence dates and fossil ages in figure  <dig> are taken from the following sources: amniota and testudines to archosauria  <cit> ; pelodiscus to chrysemys <cit> ; odontochelys <cit> ; neognathae  <cit> ; galloanserae and gallus to meleagris <cit> ; taeniopygia to melopsittacus <cit> ; ichthyornis <cit> ; xenarthra  <cit> ; xenarthra to orycteropus <cit> ; eomaia <cit> .
RESULTS
enam gene
blast searches with crocodylus niloticus  enam  identified almost the entirety of exon  <dig> in chrysemys picta .
remnants of this exon in c. picta were identified on scaffold jh <dig>  which has not yet been mapped onto a chromosome.
the enam sequence for exon  <dig> in chrysemys includes numerous frameshift indels , one of which is a sine insertion that shares homology with members of the cry family of sines in cryptodira  <cit>  .
an additional sine insertion occurs 3’ to the stop codon.
remnants of exon  <dig> in chrysemys comprise ~ <dig> bp after excluding the sine insertions.
chrysemys exon  <dig> retains a “taa” stop codon in the same location as crocodylus.
by contrast with the results of blast searches against the chrysemys genome, blast searches with crocodylus enam resulted in no significant hits to the pelodiscus sinensis genome.
likewise, blast searches with the recovered enam pseudogene segment from chrysemys did not find significant matches in the pelodiscus genome.
numbers correspond to sequence alignment positions in additional files.
additional file  <dig> = af1; additional file  <dig> = af3; additional file  <dig> = af5; additional file  <dig> = af7; additional file  <dig> = af9; additional file  <dig> = af <dig>  cry = cry sine  <cit> .
blast searches with the virtual pseudogene mrna of gallus enam exon  <dig> recovered homologous fragments of this exon in meleagris gallopavo , taeniopygia guttata , anas platyrhynchos , and melopsittacus undulatus .
the 3’ region of m. gallopavo enam was recovered on chromosome  <dig>  whereas the 5’ region was recovered on the z chromosome of this species.
all of the exon  <dig> sequences were characterized by frameshift mutations , including a  <dig> bp deletion near the 3’ end of the coding sequence of exon  <dig> that is shared by all five birds .
enam sequences for orycteropus afer, choloepus hoffmanni and dasypus novemcinctus were reported by meredith et al.
<cit>  and are not duplicated here.
orycteropus enam includes three frameshift mutations  <cit> .
among xenarthrans, choloepus enam includes numerous frameshift mutations whereas dasypus enam includes a single frameshift mutation that is located near the carboxy-terminal end of the coding sequence in exon  <dig>   <cit> .
ambn gene
blast searches with caiman crocodilus  ambn  recovered portions of exons  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  in this order, on chrysemys scaffold jh <dig> .
the ambn exons are located immediately upstream of enam exon  <dig> on scaffold jh <dig>  frameshift mutations occur in exons  <dig>   <dig>  and  <dig> .
ambn sequences were not found in pelodiscus.
among birds, remnants of ambn were discovered in anas , gallus , taeniopygia , and melopsittacus .
frameshift mutations occur in anas , gallus  and taeniopygia  .
a ten-bp frameshift deletion is shared by anas, gallus, and taeniopygia, which together index the common ancestry of neognathae, although this region was not recovered in meleagris and melopsittacus.
there were no frameshifts in melopsittacus exons  <dig> and  <dig>  but a stop codon occurs in the latter exon.
among mammals, we recovered exons 1– <dig>   <dig>  and 10– <dig> in orycteropus afer .
exon  <dig> contains a stop codon and there is a 13-bp frameshift deletion in exon  <dig> .
exon  <dig> has been deleted along with portions of introns  <dig> and  <dig> .
exons  <dig> and  <dig> are 39-bp duplications of exon  <dig> that occur in primates  <cit>  and their absence in orycteropus is not unexpected.
with the exception of elephantulus edwardii, which has two duplications of exon  <dig> as in homo, other afrotherians  are similar to orycteropus in possessing only exon  <dig>  we also recovered sequences for exons 1– <dig> and 10– <dig> in dasypus ambn .
the recovered protein-coding sequence is intact, although the presumed start codon is ten codons downstream of the start codon in the human sequence and occurs in exon  <dig> rather than exon  <dig>  as is also the case for loxodonta africana  .
complete coding sequences for nine exons  and partial sequences for exons  <dig> and  <dig> were recovered from the choloepus genome .
the putative start codon in choloepus is located in exon  <dig> as in dasypus.
there are no frameshift mutations or stop codons in the available choloepus sequence, but intron  <dig> exhibits an inactivating mutation at the acceptor splice site  .
by contrast, all of the donor and accepter splice sites exhibit canonical gt and ag motifs, respectively, in  <dig> mammals with enamel-capped teeth .
selection analyses based on an alignment of  <dig> mammalian ambn sequences  with codon frequency  models  <dig>   <dig>  and  <dig> all suggest elevated dn/ds ratios on the orycteropus , choloepus  and dasypus  branches relative to the background rate  in mammalian taxa with enamel-covered teeth.
amel gene
blast searches with paleosuchus palpebrosus  amel  recovered most of exons  <dig>   <dig>   <dig>  and  <dig> in pelodiscus, with ancestral synteny preserved, on scaffold jh <dig> .
exons  <dig> and  <dig> were identified on scaffold jh <dig> in chrysemys .
chrysemys and pelodiscus both retain the same start codon as in crocodylians.
frameshift mutations are present in sequences of both testudine species , although there are no shared frameshifts in these two exons .
in addition, pelodiscus and chrysemys share exon-flanking sequences that are 3’ to exon  <dig> and 5’ to exon  <dig>  the sequence that flanks exon  <dig> is >  <dig> bp and includes regions that are homologous to intron  <dig> and possibly exon  <dig> of alligator mississippiensis .
intron  <dig> retains the canonical “gt” splice donor site in both testudines .
the homology of exon  <dig> is less certain, but nevertheless of potential importance as there are multiple shared frameshift mutations in chrysemys and pelodiscus .
the sequence that flanks exon  <dig> is >  <dig> bp long and is homologous to a portion of intron  <dig> of a. mississippiensis with the canonical “ag” splice acceptor site in chrysemys  .
in birds, blast searches with gallus amel pseudogene mrna  recovered homologous dna sequences in anas , meleagris , melopsittacus , and taeniopygia  .
all of the birds except for anas share a common start codon with crocodylians.
frameshift mutations occur in all avian taxa, including a one-bp deletion in exon  <dig> that is shared by all five birds .
among mammals, there are two stop codons in exon  <dig> of orycteropus amel .
choloepus amel shows replacement of the methionine initiation codon in exon  <dig> by a threonine codon, an  <dig> bp frameshift deletion in exon  <dig>  and a premature stop codon followed by a frameshift deletion in exon  <dig> .
all of the inactivating mutations in choloepus are corroborated by chromatograms in ncbi’s trace archives.
the sequence for dasypus is intact through exon  <dig> , but a deletion incorporates the 3’ end of intron  <dig>  including the acceptor splice site, and the first  <dig> bp of exon  <dig>  including the stop codon.
orycteropus, choloepus, and dasypus also lack a functional copy of exon  <dig>  either because of stop codons  or a donor splice site mutation  in intron  <dig> that is adjacent to this exon.
however, a functional copy of exon  <dig> is variably present in mammalia  <cit>  and is missing from six other afrotherians  with genome sequences because of stop codons, frameshift mutations, and/or splice site mutations .
discussion
emp genes in turtles
crown testudines comprise  <dig> extant species  <cit> , all of which are toothless and instead have a keratinized beak that in combination with strong jaw muscles allows testudines to tear food and capture prey  <cit> .
the oldest testudine fossil is the late triassic odontochelys semitestacea from china  <cit> .
odontochelys lacked a beak and instead retained both marginal and palatal teeth  <cit> .
proganochelys quenstedti is also known from the late triassic, and is more derived than odontochelys.
proganochelys lacked marginal teeth, but palatal teeth were present.
the morphology of the mandibles suggests that proganochelys possessed a keratinized beak  <cit> .
it remains unclear if palatal teeth, which occur on the vomer, palatine, and pterygoid bones, were lost in the common ancestor of all living turtles or independently in cryptodires and pleurodires owing to alternate phylogenetic hypotheses wherein taxa with palatal teeth such as kayentachelys apix are positioned as stem testudines or as stem cryptodires  <cit> .
in either case palatal teeth were lost no later than the late jurassic in the ancestry of crown testudines.
the retention of pseudogenized copies of three emp genes  in chrysemys is perhaps surprising given the antiquity of tooth loss in turtles and the extended time window for large-scale deletions and/or rearrangements to erase or scramble the genomic instructions for enamel production in this edentulous taxon.
however, remnants of ambn and enam exon  <dig> in chrysemys occur in juxtaposition to each other on the same contig.
kawasaki’s  <cit>  reconstructions of scpp gene order in both stem tetrapods and stem amniotes suggest that ambn is immediately upstream of enam.
thus, it appears that remnants of this original gene order are present in the painted turtle genome.
the protein-coding regions of ambn and enam in chrysemys have been battered by inactivating mutations, including sine insertions in exon  <dig> of enam, but nevertheless retain unambiguous signatures of their heritage.
the only emp gene that was recovered in pelodiscus is amel.
it remains unclear whether ambn and enam were completely deleted from the pelodiscus genome during evolutionary history, are difficult to recognize because of numerous mutations, or are missing from the current assembly of the pelodiscus genome due to incomplete sequencing assembly.
pelodiscus amel is relatively complete and includes exons  <dig>   <dig>   <dig>  and  <dig> in their original syntenic order.
by contrast, we only discovered exons  <dig> and  <dig> of chrysemys amel.
the two exons represent the only overlapping emp fragments that were discovered in both pelodiscus and chrysemys.
frameshift mutations in amel occur in both species, but the only shared frameshift mutations occur in putative exon  <dig>  which has equivocal homology with the same exon in crocodylians.
nevertheless, if these frameshift mutations are genuine then there is molecular evidence for the inactivation of enamel production in the common ancestry of cryptodira.
the genetic and developmental basis of edentulism in turtles involves both tooth-specific and pleiotropic genes.
results presented here demonstrate that three tooth-specific emp genes were pseudogenized in the ancestry of modern cryptodires.
previously, tokita et al.
<cit>  examined patterns of gene expression in the developing lower jaw of pelodiscus sinensis and reported impairment of shh signaling in the oral epithelium along with early-stage arrest of odontoblast development by abrogation of msx <dig> expression in dental mesenchyme.
it remains unclear if changes in gene expression or emp pseudogenization occurred first in the evolutionary history of turtles.
emp genes in birds
amel and enam pseudogenes have previously been reported in gallus <cit> , which belongs to galliformes.
we provide extended evidence for inactivated emp genes in birds and show that there are remnants of amel and enam in four other birds with complete genome sequences: meleagris , anas , melopsittacus , and taeniopygia  .
in addition, we recovered molecular evidence for an inactivated copy of ambn in anas, gallus, taeniopygia, and melopsittacus.
it is noteworthy that there are putative frameshift mutations in ambn, amel, and enam that are shared by representatives of galliformes , anseriformes , and neoaves , which together index the deepest split in neognathae  <cit> .
it will be important to determine if these inactivating mutations are shared with palaeognathae , in which case molecular evidence would be consistent with the loss of enamel in the common ancestor of neornithes rather than independently in neognathae and palaeognathae.
emp genes in mammals
it has previously been shown that enam is a pseudogene in a wide array of edentulous and enamelless mammals  <cit>  .
further, amel and ambn sequences have been reported for baleen whales and in both cases are inactivated in multiple species  <cit> .
however, the functional versus pseudogene status of amel and ambn remains to be investigated in most edentulous and enamelless taxa.
here, we provide evidence of inactivating mutations in amel in orycteropus afer  and choloepus hoffmanni , both of which have enamelless teeth comprised of dentin.
multiple frameshift mutations in choloepus amel are confirmed by trace archives chromatograms .
the two stop codons in orycteropus are based on illumini hi-seq sequencing technology with 44x genome coverage.
previous studies suggest that ambn may play a role in dentin formation/regeneration  <cit> .
however, this hypothesis is contradicted by our finding that there are inactivating mutations in orycteropus and choloepus ambn.
moreover, dn/ds ratios indicate relaxed purifying selection in these taxa.
similarly, mcgowen  <cit>  reported a premature stop codon in exon  <dig>  of monodon monoceros .
narwhals have vestigial teeth, and in the case of males a single enlarged tusk.
both the vestigial teeth and tusk are composed of dentin and cementum, but no enamel  <cit> .
the occurrence of a premature stop codon in monodon ambn, in conjunction with inactivating mutations in two other lineages with enamelless teeth , is consistent with the hypothesis that the only essential, non-redundant role of ambn is in enamel formation.
along these lines, ameloblastin knockout mice do not form an enamel layer, and lack dentin defects  <cit> .
by contrast with choloepus, where enamel loss occurred more than  <dig> million years ago, enamel degeneration in dasypus occurred more recently  <cit> .
dasypus retains a vestigial enamel or enamel-like substance that covers the dentin of some teeth and is quickly worn off.
moreover, there are no frameshifts in enam that are shared by dasypus and other armadillo genera  <cit> .
the single frameshift in dasypus enam precludes a functional, full-length enamelin protein, but the location of the frameshift is close to the 3’ end of the protein-coding sequence in exon  <dig>  and shorter protein products are possible  <cit> .
our finding that ambn is intact in dasypus, and that only the last amino acid is truncated from the coding region of amel, is consistent with the presence of vestigial ‘enamel’ in this species  <cit> .
we also note that a partial ambn sequence  for kogia breviceps  has an intact protein-coding region even though k. breviceps has enamelless teeth and frameshift mutations in the enam gene.
it will be important in future studies to sequence the remaining exons of kogia ambn to determine if the missing exonic regions contain frameshift mutations and/or other inactivating mutations.
CONCLUSIONS
edentulism has evolved independently in multiple lineages of living amniotes including turtles, birds, echidnas, baleen whales, anteaters, and pangolins.
there are also mammals with enamelless teeth including pygmy sperm whale, narwhal, sloths, armadillos, and aardvarks.
in every case these edentulous or enamelless forms have descended from ancestors with enamel-capped teeth.
thus, amniote diversity provides a natural laboratory for testing hypotheses of tooth-specific gene function  <cit> .
moreover, this laboratory includes multiple, replicated experiments.
ambn, amel, and enam have all been postulated to have tooth-specific or even enamel-specific gene functions  <cit> , although pleiotropic functions have been suggested for ambn <cit>  and amel <cit> .
the widespread occurrence of emp pseudogenes in turtles, birds, and several mammalian lineages  provides compelling evidence that the only unique, non-redundant function of these genes in amniotes is in enamel formation: functional copies of these genes have not been retained by natural selection when enamel production was abrogated independently in distantly related lineages.
by contrast, representative sequences from amniotes with enamel-capped teeth retain intact coding sequences for ambn <cit> ; also see additional file  <dig>  amel <cit> , and enam <cit> .
the evolution of tooth loss in multiple amniote lineages also provides a model system for integrating the fossil record, phylogenetics, and genomics.
this system allows for reciprocal hypothesis testing and provides a multifaceted, synthetic view on macroevolutionary transitions in testudines, birds, and edentulous/enamelless mammals  <cit> .
the fossil record and phylogenetics combine to predict the occurrence of molecular fossils of tooth-specific genes in the genomes of edentulous and enamelless amniotes, and molecular fossils have been discovered in all lineages that have been investigated.
we suggest that these striking patterns may prove especially useful for educating the public on the convergence of evidence that clearly documents the evolutionary process over deep time.
competing interests
the authors declare that they have no competing financial interests.
authors’ contributions
jg, rwm, and mss conceived and designed the study.
rwm and mss performed the analyses.
mss wrote the manuscript.
jg and rwm provided comments on the manuscript.
jg and mss prepared figures.
all authors read and approved the final manuscript.
supplementary material
additional file 1
nexus alignment of enam exon  <dig> sequences for two crocodylians, one testudine, and five birds.
the stop codon in crocodylians and chrysemys picta occurs at positions 3788– <dig>  a  <dig> bp deletion that is shared by all five birds occurs near the 3’ end of the coding region at positions 3353– <dig>  additional frameshift mutations are shared by four of five birds including representatives of anseriformes, galliformes, and passeriformes .
cry sine elements  <cit>  in chrysemys occur at positions 994– <dig> and 4782– <dig>  several long strings of ns in chrysemys picta were abridged to shorter strings of ten ns.
click here for file
additional file 2
schematic alignment showing regions of enam exon  <dig> that were identified in chrysemys picta.
green bars below sequence numbers show regions of sequence similarity between c. picta and alligator mississippiensis.
red rectangles = frameshift mutations in c. picta; light blue rectangles = indels within the coding sequence that are in multiples of three base pairs; cry = cry sine insertion; red arrow = position of stop codon in a. mississippiensis.
click here for file
additional file 3
nexus alignment of ambn exon sequences for two crocodylians, one testudine, and one bird.
the start and stop codons in crocodylians occur at positions 127– <dig>  and 1374– <dig> , respectively.
click here for file
additional file 4
schematic alignment showing regions of ambn exons 1– <dig> that were identified in chrysemys picta.
green bars below sequence numbers show regions of sequence similarity between c. picta and alligator mississippiensis.
dark blue rectangles = numbered exons in a. mississippiensis; red rectangles = frameshift mutations in c. picta; light blue rectangles = indels within the coding sequence that are in multiples of three bp; dark blue arrow = position of start codon in a. mississippiensis; red arrow = position of stop codon in a. mississippiensis.
click here for file
additional file 5
nexus alignment of ambn exon sequences forhomo sapiens, orycteropus aferand six other afrotherians, dasypus novemcinctus, and choloepus hoffmanni.
the start and stop codons in homo occur at positions 121– <dig>  and 1468– <dig> , respectively.
the presumed start codon in dasypus occurs at positions 151– <dig>  dasypus shares the same stop codon as homo at positions 1468– <dig>  whereas the presumed stop codon in choloepus is at positions 1477– <dig>
click here for file
additional file 6
nexus alignment of protein-coding ambn sequences for  <dig> mammals that was used in selection analyses with paml  <dig>   <cit> .
click here for file
additional file 7
nexus alignment of amel exon sequences in nexus format for two crocodylians, two testudines, and five birds.
the start and stop codons in crocodylians occur at positions 69– <dig>  and 731– <dig> , respectively.
a putative one-bp frameshift deletion that is shared by all five birds species occurs at position  <dig> in exon  <dig>
click here for file
additional file 8
schematic alignment showing regions of amel exons 1– <dig> that were identified in chrysemys picta and pelodiscus sinensis .
bars below sequence numbers show regions of sequence similarity between alligator mississippiensis and both testudines  or a. mississippiensis and one testudine .
dark blue rectangles = numbered exons in a. mississippiensis; red rectangles = frameshift mutations in c. picta; light blue rectangles = indels within the coding sequence that are in multiples of three bp; dark blue arrow = position of start codon in a. mississippiensis; red arrow = position of stop codon in a. mississippiensis.
click here for file
additional file 9
nexus alignment of alligator mississippiensis amel exon  <dig> , intron  <dig> , and exon  <dig>  with exon  <dig> and flanking sequences from chrysemys picta and pelodiscus sinensis.
click here for file
additional file 10
nexus alignment of amel intron  <dig>  and exon  <dig>  sequences for chrysemys picta and pelodiscus sinensis.
click here for file
additional file 11
nexus alignment of amel sequences  for homo sapiens, orycteropus afer and six other afrotherians , dasypus novemcinctus, and choloepus hoffmanni.
the start and stop codons in homo occur at positions 13– <dig>  and 668– <dig> , respectively.
the mutated stop codon and frameshift mutation in exon  <dig> of choloepus are clear in gnl|ti| <dig> and gnl|ti| <dig>  the stop codon in exon  <dig> of choloepus is confirmed by traces gnl|ti| <dig> and gnl|ti| <dig>  the frameshift mutation in exon  <dig> of choloepus is evident in trace gnl|ti| <dig>  the deletion that includes the 3’ region of intron  <dig> and first  <dig> bp of exon  <dig> in dasypus is evident in gn|ti|  <dig>
click here for file
acknowledgements
this work was supported by nsf .
we thank four anonymous referees for helpful comments on an earlier draft of this manuscript.

 gnathostomes are unique among vertebrates in utilizing jaws and teeth for food acquisition and processing. divergent gnathostome lineages exhibit a wide array of dental modifications in association with diverse dietary specializations. multiple iterations of secondary tooth loss have also evolved in gnathostomes, perhaps most famously in birds, turtles, and several mammalian lineages including baleen whales, pangolins, and anteaters. there are also mammalian species with enamelless teeth, i.e., sloths, armadillos, aardvarks, pygmy and dwarf sperm whales. tooth development is an intricate process that encompasses a complex series of epithelial-mesenchymal interactions involving growth factors, transcription factors, and signal receptors that affect tooth shape, tooth number, and cusp number. several hundred genes are associated with tooth development, including members of the hedgehog, fibroblast growth factor and bone morphogenic protein families that mediate epithelio-mesenchymal signaling interactions. most or all of the genes that are involved in early tooth development are thought to be pleiotropic and have additional functions outside of tooth formation. however, there are also genes with putative tooth-specific functions that are expressed later in development by dentin-forming odontoblasts and/or enamel-forming ameloblasts. these genes affect the physical properties of teeth including enamel thickness and structure. the durability of teeth, in combination with tooth-specific genes that impact the physical structure of dentin and enamel, make teeth a model system for studying the coevolution of morphological change in the fossil record and molecular change in the genome. previous studies have shown that the genes encoding three enamel matrix proteins, enamelin, amelogenin, and ameloblastin, have become pseudogenized in one or more edentulous/enamelless mammals by frameshift mutations and/or stop codons. further, the molecular decay of tooth-specific genes in placental mammal lineages has been shown to parallel the morphological degeneration of enamel in the fossil record. the retention of inactivated emp genes in mammalian lineages is not surprising given that all edentulous and enamelless taxa appear to have originated in the cenozoic. by contrast, tooth loss in both birds and turtles occurred in the mesozoic, providing a longer time period for the molecular decay of tooth-specific genes in these lineages. in birds, the presence of a horny beak and gizzard have presumably compensated for edentulism in food acquisition and processing. edentulism in the ancestry of modern birds occurred in the cretaceous, between and ma, and also evolved independently in several lineages of extinct birds that are on the stem to neornithes. even though tooth loss in neornithes occurred in the cretaceous, the chicken genome retains pseudogenized copies of both amel and enam. edentulism in testudines occurred even earlier, at least as far back as the late jurassic. the oldest turtle is odontochelys semitestacea from the triassic of china. marginal and palatal teeth are both present in this taxon. slightly younger is proganochelys quenstedti from the upper triassic. marginal teeth are absent in proganochelys, but palatal teeth are present. phylogenetic analyses suggest that proganochelys, like odontochelys, is a stem testudine. the oldest testudines with unambiguous crown-group affinities, including tooth loss on the palatines, vomer, and pterygoids, are from the late jurassic. to date, remnants of pseudogenized copies of tooth-specific genes in testudines have not been reported. girondot and sire were unable to amplify fragments of amelogenin in turtles with degenerate pcr primers, and it remains unclear if vestiges of tooth-specific genes are retained in testudine genomes given the antiquity of tooth loss in this clade. on the other hand, phylogenomic data suggest that testudines have slower rates of nuclear gene evolution than birds and mammals. the recent release of two testudine genomes, chrysemys picta and pelodiscus sinensis, provides an opportunity to screen for remnants of tooth-specific genes in turtles. these taxa, both of which belong to cryptodira, index the basal cladogenic event among crown cryptodires and leave only pleurodira unrepresented among the three oldest crown-testudine lineages. cryptodira and pleurodira have traditionally been regarded as reciprocally monophyletic based on morphology and large molecular data sets. an exception is barley et al.’s coalescence analysis, which recovered a basal split between softshells plus the pig-nosed turtle and pleurodira plus other cryptodira. however, barley et al.’s concatenation analysis recovered cryptodira and pleurodira. moreover, the most complete testudine phylogeny with a variety of outgroups supports the monophyly of both cryptodira and pleurodira. here, we report the results of querying the genomes of chrysemys and pelodiscus for inactivated remnants of three emp genes, enam, amel, and ambn, all of which are hypothesized to have tooth-specific functions. we also report the results of querying genome sequences of five birds and three enamelless mammals for remnants of these emp genes. the occurrence of pseudogenized remnants of emp genes in highly divergent amniotes, including turtles, birds, and edentulous/enamelless mammals, would provide robust evidence for the hypothesis that the only essential, non-redundant function of amel, ambn, and enam is in enamel formation. moreover, the search for shared frameshift mutations in emp genes provides an opportunity to date the timing of enamel loss in these clades. methods the genomes of chrysemys picta and pelodiscus sinensis were queried with blastn using crocodylian mrna sequences for three enamel matrix proteins genes: paleosuchus palpebrosus amel, caiman crocodilus ambn, and crocodylus niloticus enam. intron-exon boundaries of mrna sequences were determined by blasting crocodylian mrnas against the recently released genome sequence of alligator mississippiensis. crocodylian sequences were chosen as probes based on recent evidence that provides strong support for a sister group relationship between testudines and archosauria. ambn has exons in alligator, all of which include protein-coding regions. amel includes six exons in alligator, five of which contain protein-coding regions. finally, enam includes nine exons in alligator, eight of which include protein-coding regions. remnants of emp genes that were recovered from chrysemys were subsequently used to query the pelodiscus genome, and remnants of emp genes from pelodiscus were used to query the chrysemys genome. five bird genomes were queried with crocodylian mrnas and virtual pseudogene mrnas for amel and enam from gallus, as well as with positive hits from the initial round of screening for the bird genomes. the ncbi genomes of three enamelless mammals were queried with gene sequences from mammals that retain enamel-capped teeth. sequences were aligned with se-al. selection analyses on branches were performed with paml to estimate dn/ds values for an assemblage of complete or nearly complete mammalian ambn sequences, all of which were derived from ensembl or ncbi. sequences were aligned with se-al. branch analyses were performed with four branch categories: orycteropus, choloepus, dasypus, and all other branches. orycteropus, choloepus and dasypus were recognized as separate branches in the paml runs based on the hypothesized independent loss of enamel in these lineages. paml analyses were conducted with codon frequency models and we used a composite species tree based on meredith et al. and springer et al.. approximate divergence dates and fossil ages in figure  are taken from the following sources: amniota and testudines to archosauria; pelodiscus to chrysemys; odontochelys; neognathae; galloanserae and gallus to meleagris; taeniopygia to melopsittacus; ichthyornis; xenarthra; xenarthra to orycteropus; eomaia.  enam gene blast searches with crocodylus niloticus enam identified almost the entirety of exon in chrysemys picta. remnants of this exon in c. picta were identified on scaffold jh which has not yet been mapped onto a chromosome. the enam sequence for exon in chrysemys includes numerous frameshift indels, one of which is a sine insertion that shares homology with members of the cry family of sines in cryptodira. an additional sine insertion occurs 3’ to the stop codon. remnants of exon in chrysemys comprise ~ bp after excluding the sine insertions. chrysemys exon retains a “taa” stop codon in the same location as crocodylus. by contrast with the results of blast searches against the chrysemys genome, blast searches with crocodylus enam resulted in no significant hits to the pelodiscus sinensis genome. likewise, blast searches with the recovered enam pseudogene segment from chrysemys did not find significant matches in the pelodiscus genome. numbers correspond to sequence alignment positions in additional files. additional file = af1; additional file = af3; additional file = af5; additional file = af7; additional file = af9; additional file = af cry = cry sine. blast searches with the virtual pseudogene mrna of gallus enam exon recovered homologous fragments of this exon in meleagris gallopavo, taeniopygia guttata, anas platyrhynchos, and melopsittacus undulatus. the 3’ region of m. gallopavo enam was recovered on chromosome whereas the 5’ region was recovered on the z chromosome of this species. all of the exon sequences were characterized by frameshift mutations, including a bp deletion near the 3’ end of the coding sequence of exon that is shared by all five birds. enam sequences for orycteropus afer, choloepus hoffmanni and dasypus novemcinctus were reported by meredith et al. and are not duplicated here. orycteropus enam includes three frameshift mutations. among xenarthrans, choloepus enam includes numerous frameshift mutations whereas dasypus enam includes a single frameshift mutation that is located near the carboxy-terminal end of the coding sequence in exon. ambn gene blast searches with caiman crocodilus ambn recovered portions of exons and in this order, on chrysemys scaffold jh. the ambn exons are located immediately upstream of enam exon on scaffold jh frameshift mutations occur in exons and. ambn sequences were not found in pelodiscus. among birds, remnants of ambn were discovered in anas, gallus, taeniopygia, and melopsittacus. frameshift mutations occur in anas, gallus and taeniopygia. a ten-bp frameshift deletion is shared by anas, gallus, and taeniopygia, which together index the common ancestry of neognathae, although this region was not recovered in meleagris and melopsittacus. there were no frameshifts in melopsittacus exons and but a stop codon occurs in the latter exon. among mammals, we recovered exons 1– and 10– in orycteropus afer. exon contains a stop codon and there is a 13-bp frameshift deletion in exon. exon has been deleted along with portions of introns and. exons and are 39-bp duplications of exon that occur in primates and their absence in orycteropus is not unexpected. with the exception of elephantulus edwardii, which has two duplications of exon as in homo, other afrotherians are similar to orycteropus in possessing only exon we also recovered sequences for exons 1– and 10– in dasypus ambn. the recovered protein-coding sequence is intact, although the presumed start codon is ten codons downstream of the start codon in the human sequence and occurs in exon rather than exon as is also the case for loxodonta africana. complete coding sequences for nine exons and partial sequences for exons and were recovered from the choloepus genome. the putative start codon in choloepus is located in exon as in dasypus. there are no frameshift mutations or stop codons in the available choloepus sequence, but intron exhibits an inactivating mutation at the acceptor splice site. by contrast, all of the donor and accepter splice sites exhibit canonical gt and ag motifs, respectively, in mammals with enamel-capped teeth. selection analyses based on an alignment of mammalian ambn sequences with codon frequency models and all suggest elevated dn/ds ratios on the orycteropus, choloepus and dasypus branches relative to the background rate in mammalian taxa with enamel-covered teeth. amel gene blast searches with paleosuchus palpebrosus amel recovered most of exons and in pelodiscus, with ancestral synteny preserved, on scaffold jh. exons and were identified on scaffold jh in chrysemys. chrysemys and pelodiscus both retain the same start codon as in crocodylians. frameshift mutations are present in sequences of both testudine species, although there are no shared frameshifts in these two exons. in addition, pelodiscus and chrysemys share exon-flanking sequences that are 3’ to exon and 5’ to exon the sequence that flanks exon is > bp and includes regions that are homologous to intron and possibly exon of alligator mississippiensis. intron retains the canonical “gt” splice donor site in both testudines. the homology of exon is less certain, but nevertheless of potential importance as there are multiple shared frameshift mutations in chrysemys and pelodiscus. the sequence that flanks exon is > bp long and is homologous to a portion of intron of a. mississippiensis with the canonical “ag” splice acceptor site in chrysemys. in birds, blast searches with gallus amel pseudogene mrna recovered homologous dna sequences in anas, meleagris, melopsittacus, and taeniopygia. all of the birds except for anas share a common start codon with crocodylians. frameshift mutations occur in all avian taxa, including a one-bp deletion in exon that is shared by all five birds. among mammals, there are two stop codons in exon of orycteropus amel. choloepus amel shows replacement of the methionine initiation codon in exon by a threonine codon, an bp frameshift deletion in exon and a premature stop codon followed by a frameshift deletion in exon. all of the inactivating mutations in choloepus are corroborated by chromatograms in ncbi’s trace archives. the sequence for dasypus is intact through exon, but a deletion incorporates the 3’ end of intron including the acceptor splice site, and the first bp of exon including the stop codon. orycteropus, choloepus, and dasypus also lack a functional copy of exon either because of stop codons or a donor splice site mutation in intron that is adjacent to this exon. however, a functional copy of exon is variably present in mammalia and is missing from six other afrotherians with genome sequences because of stop codons, frameshift mutations, and/or splice site mutations. discussion emp genes in turtles crown testudines comprise extant species, all of which are toothless and instead have a keratinized beak that in combination with strong jaw muscles allows testudines to tear food and capture prey. the oldest testudine fossil is the late triassic odontochelys semitestacea from china. odontochelys lacked a beak and instead retained both marginal and palatal teeth. proganochelys quenstedti is also known from the late triassic, and is more derived than odontochelys. proganochelys lacked marginal teeth, but palatal teeth were present. the morphology of the mandibles suggests that proganochelys possessed a keratinized beak. it remains unclear if palatal teeth, which occur on the vomer, palatine, and pterygoid bones, were lost in the common ancestor of all living turtles or independently in cryptodires and pleurodires owing to alternate phylogenetic hypotheses wherein taxa with palatal teeth such as kayentachelys apix are positioned as stem testudines or as stem cryptodires. in either case palatal teeth were lost no later than the late jurassic in the ancestry of crown testudines. the retention of pseudogenized copies of three emp genes in chrysemys is perhaps surprising given the antiquity of tooth loss in turtles and the extended time window for large-scale deletions and/or rearrangements to erase or scramble the genomic instructions for enamel production in this edentulous taxon. however, remnants of ambn and enam exon in chrysemys occur in juxtaposition to each other on the same contig. kawasaki’s reconstructions of scpp gene order in both stem tetrapods and stem amniotes suggest that ambn is immediately upstream of enam. thus, it appears that remnants of this original gene order are present in the painted turtle genome. the protein-coding regions of ambn and enam in chrysemys have been battered by inactivating mutations, including sine insertions in exon of enam, but nevertheless retain unambiguous signatures of their heritage. the only emp gene that was recovered in pelodiscus is amel. it remains unclear whether ambn and enam were completely deleted from the pelodiscus genome during evolutionary history, are difficult to recognize because of numerous mutations, or are missing from the current assembly of the pelodiscus genome due to incomplete sequencing assembly. pelodiscus amel is relatively complete and includes exons and in their original syntenic order. by contrast, we only discovered exons and of chrysemys amel. the two exons represent the only overlapping emp fragments that were discovered in both pelodiscus and chrysemys. frameshift mutations in amel occur in both species, but the only shared frameshift mutations occur in putative exon which has equivocal homology with the same exon in crocodylians. nevertheless, if these frameshift mutations are genuine then there is molecular evidence for the inactivation of enamel production in the common ancestry of cryptodira. the genetic and developmental basis of edentulism in turtles involves both tooth-specific and pleiotropic genes. results presented here demonstrate that three tooth-specific emp genes were pseudogenized in the ancestry of modern cryptodires. previously, tokita et al. examined patterns of gene expression in the developing lower jaw of pelodiscus sinensis and reported impairment of shh signaling in the oral epithelium along with early-stage arrest of odontoblast development by abrogation of msx expression in dental mesenchyme. it remains unclear if changes in gene expression or emp pseudogenization occurred first in the evolutionary history of turtles. emp genes in birds amel and enam pseudogenes have previously been reported in gallus, which belongs to galliformes. we provide extended evidence for inactivated emp genes in birds and show that there are remnants of amel and enam in four other birds with complete genome sequences: meleagris, anas, melopsittacus, and taeniopygia. in addition, we recovered molecular evidence for an inactivated copy of ambn in anas, gallus, taeniopygia, and melopsittacus. it is noteworthy that there are putative frameshift mutations in ambn, amel, and enam that are shared by representatives of galliformes, anseriformes, and neoaves, which together index the deepest split in neognathae. it will be important to determine if these inactivating mutations are shared with palaeognathae, in which case molecular evidence would be consistent with the loss of enamel in the common ancestor of neornithes rather than independently in neognathae and palaeognathae. emp genes in mammals it has previously been shown that enam is a pseudogene in a wide array of edentulous and enamelless mammals. further, amel and ambn sequences have been reported for baleen whales and in both cases are inactivated in multiple species. however, the functional versus pseudogene status of amel and ambn remains to be investigated in most edentulous and enamelless taxa. here, we provide evidence of inactivating mutations in amel in orycteropus afer and choloepus hoffmanni, both of which have enamelless teeth comprised of dentin. multiple frameshift mutations in choloepus amel are confirmed by trace archives chromatograms. the two stop codons in orycteropus are based on illumini hi-seq sequencing technology with 44x genome coverage. previous studies suggest that ambn may play a role in dentin formation/regeneration. however, this hypothesis is contradicted by our finding that there are inactivating mutations in orycteropus and choloepus ambn. moreover, dn/ds ratios indicate relaxed purifying selection in these taxa. similarly, mcgowen reported a premature stop codon in exon of monodon monoceros. narwhals have vestigial teeth, and in the case of males a single enlarged tusk. both the vestigial teeth and tusk are composed of dentin and cementum, but no enamel. the occurrence of a premature stop codon in monodon ambn, in conjunction with inactivating mutations in two other lineages with enamelless teeth, is consistent with the hypothesis that the only essential, non-redundant role of ambn is in enamel formation. along these lines, ameloblastin knockout mice do not form an enamel layer, and lack dentin defects. by contrast with choloepus, where enamel loss occurred more than million years ago, enamel degeneration in dasypus occurred more recently. dasypus retains a vestigial enamel or enamel-like substance that covers the dentin of some teeth and is quickly worn off. moreover, there are no frameshifts in enam that are shared by dasypus and other armadillo genera. the single frameshift in dasypus enam precludes a functional, full-length enamelin protein, but the location of the frameshift is close to the 3’ end of the protein-coding sequence in exon and shorter protein products are possible. our finding that ambn is intact in dasypus, and that only the last amino acid is truncated from the coding region of amel, is consistent with the presence of vestigial ‘enamel’ in this species. we also note that a partial ambn sequence for kogia breviceps has an intact protein-coding region even though k. breviceps has enamelless teeth and frameshift mutations in the enam gene. it will be important in future studies to sequence the remaining exons of kogia ambn to determine if the missing exonic regions contain frameshift mutations and/or other inactivating mutations.  edentulism has evolved independently in multiple lineages of living amniotes including turtles, birds, echidnas, baleen whales, anteaters, and pangolins. there are also mammals with enamelless teeth including pygmy sperm whale, narwhal, sloths, armadillos, and aardvarks. in every case these edentulous or enamelless forms have descended from ancestors with enamel-capped teeth. thus, amniote diversity provides a natural laboratory for testing hypotheses of tooth-specific gene function. moreover, this laboratory includes multiple, replicated experiments. ambn, amel, and enam have all been postulated to have tooth-specific or even enamel-specific gene functions, although pleiotropic functions have been suggested for ambn and amel. the widespread occurrence of emp pseudogenes in turtles, birds, and several mammalian lineages provides compelling evidence that the only unique, non-redundant function of these genes in amniotes is in enamel formation: functional copies of these genes have not been retained by natural selection when enamel production was abrogated independently in distantly related lineages. by contrast, representative sequences from amniotes with enamel-capped teeth retain intact coding sequences for ambn; also see additional file amel, and enam. the evolution of tooth loss in multiple amniote lineages also provides a model system for integrating the fossil record, phylogenetics, and genomics. this system allows for reciprocal hypothesis testing and provides a multifaceted, synthetic view on macroevolutionary transitions in testudines, birds, and edentulous/enamelless mammals. the fossil record and phylogenetics combine to predict the occurrence of molecular fossils of tooth-specific genes in the genomes of edentulous and enamelless amniotes, and molecular fossils have been discovered in all lineages that have been investigated. we suggest that these striking patterns may prove especially useful for educating the public on the convergence of evidence that clearly documents the evolutionary process over deep time. competing interests the authors declare that they have no competing financial interests. authors’ contributions jg, rwm, and mss conceived and designed the study. rwm and mss performed the analyses. mss wrote the manuscript. jg and rwm provided comments on the manuscript. jg and mss prepared figures. all authors read and approved the final manuscript. supplementary material additional file 1 nexus alignment of enam exon sequences for two crocodylians, one testudine, and five birds. the stop codon in crocodylians and chrysemys picta occurs at positions 3788– a bp deletion that is shared by all five birds occurs near the 3’ end of the coding region at positions 3353– additional frameshift mutations are shared by four of five birds including representatives of anseriformes, galliformes, and passeriformes. cry sine elements in chrysemys occur at positions 994– and 4782– several long strings of ns in chrysemys picta were abridged to shorter strings of ten ns. click here for file additional file 2 schematic alignment showing regions of enam exon that were identified in chrysemys picta. green bars below sequence numbers show regions of sequence similarity between c. picta and alligator mississippiensis. red rectangles = frameshift mutations in c. picta; light blue rectangles = indels within the coding sequence that are in multiples of three base pairs; cry = cry sine insertion; red arrow = position of stop codon in a. mississippiensis. click here for file additional file 3 nexus alignment of ambn exon sequences for two crocodylians, one testudine, and one bird. the start and stop codons in crocodylians occur at positions 127– and 1374–, respectively. click here for file additional file 4 schematic alignment showing regions of ambn exons 1– that were identified in chrysemys picta. green bars below sequence numbers show regions of sequence similarity between c. picta and alligator mississippiensis. dark blue rectangles = numbered exons in a. mississippiensis; red rectangles = frameshift mutations in c. picta; light blue rectangles = indels within the coding sequence that are in multiples of three bp; dark blue arrow = position of start codon in a. mississippiensis; red arrow = position of stop codon in a. mississippiensis. click here for file additional file 5 nexus alignment of ambn exon sequences forhomo sapiens, orycteropus aferand six other afrotherians, dasypus novemcinctus, and choloepus hoffmanni. the start and stop codons in homo occur at positions 121– and 1468–, respectively. the presumed start codon in dasypus occurs at positions 151– dasypus shares the same stop codon as homo at positions 1468– whereas the presumed stop codon in choloepus is at positions 1477– click here for file additional file 6 nexus alignment of protein-coding ambn sequences for mammals that was used in selection analyses with paml. click here for file additional file 7 nexus alignment of amel exon sequences in nexus format for two crocodylians, two testudines, and five birds. the start and stop codons in crocodylians occur at positions 69– and 731–, respectively. a putative one-bp frameshift deletion that is shared by all five birds species occurs at position in exon click here for file additional file 8 schematic alignment showing regions of amel exons 1– that were identified in chrysemys picta and pelodiscus sinensis. bars below sequence numbers show regions of sequence similarity between alligator mississippiensis and both testudines or a. mississippiensis and one testudine. dark blue rectangles = numbered exons in a. mississippiensis; red rectangles = frameshift mutations in c. picta; light blue rectangles = indels within the coding sequence that are in multiples of three bp; dark blue arrow = position of start codon in a. mississippiensis; red arrow = position of stop codon in a. mississippiensis. click here for file additional file 9 nexus alignment of alligator mississippiensis amel exon, intron, and exon with exon and flanking sequences from chrysemys picta and pelodiscus sinensis. click here for file additional file 10 nexus alignment of amel intron and exon sequences for chrysemys picta and pelodiscus sinensis. click here for file additional file 11 nexus alignment of amel sequences for homo sapiens, orycteropus afer and six other afrotherians, dasypus novemcinctus, and choloepus hoffmanni. the start and stop codons in homo occur at positions 13– and 668–, respectively. the mutated stop codon and frameshift mutation in exon of choloepus are clear in gnl|ti| and gnl|ti| the stop codon in exon of choloepus is confirmed by traces gnl|ti| and gnl|ti| the frameshift mutation in exon of choloepus is evident in trace gnl|ti| the deletion that includes the 3’ region of intron and first bp of exon in dasypus is evident in gn|ti| click here for file acknowledgements this work was supported by nsf. we thank four anonymous referees for helpful comments on an earlier draft of this manuscript.
the fossil record and phylogenetics combine to predict the occurrence of molecular fossils of tooth-specific genes in the genomes of edentulous amniotes, and in every case these molecular fossils have been discovered. previous studies have documented the molecular decay of tooth-specific genes in edentulous mammals, all of which lost their teeth in the cenozoic, and birds, which lost their teeth in the cretaceous. we queried available genomes of testudines, aves, and enamelless mammals for remnants of three enamel matrix protein genes with putative enamel-specific functions. among enamelless mammals, all three emp genes exhibit inactivating mutations in orycteropus and choloepus. the widespread occurrence of emp pseudogenes in turtles, birds, and edentulous/enamelless mammals also provides compelling evidence that in amniotes, the only unique, non-redundant function of these genes is in enamel formation. by contrast with mammals and birds, tooth loss in turtles occurred in the jurassic, providing an extended time window for tooth gene degradation in this clade. our results highlight the power of combining fossil and genomic evidence to decipher macroevolutionary transitions and characterize the functional range of different loci involved in tooth development. we also show that there are inactivated copies of ambn, amel and enam in representatives of divergent avian lineages including galloanserae, passeriformes, and psittaciformes, and that there are shared frameshift mutations in all three genes that predate the basal split in neognathae. secondary edentulism has evolved on multiple occasions in amniotes including several mammalian lineages, birds, and turtles. the release of the painted turtle and chinese softshell turtle genomes provides an opportunity to recover the decayed remains of tooth-specific genes in testudines.
 secondary edentulism has evolved on multiple occasions in amniotes including several mammalian lineages, birds, and turtles. all edentulous amniote clades have evolved from ancestors with enamel-capped teeth. previous studies have documented the molecular decay of tooth-specific genes in edentulous mammals, all of which lost their teeth in the cenozoic, and birds, which lost their teeth in the cretaceous. by contrast with mammals and birds, tooth loss in turtles occurred in the jurassic, providing an extended time window for tooth gene degradation in this clade. the release of the painted turtle and chinese softshell turtle genomes provides an opportunity to recover the decayed remains of tooth-specific genes in testudines.  we queried available genomes of testudines, aves, and enamelless mammals for remnants of three enamel matrix protein genes with putative enamel-specific functions. remnants of the ambn and enam genes were recovered in chrysemys and retain their original synteny. remnants of amel were recovered in both testudines, although there are no shared frameshifts. we also show that there are inactivated copies of ambn, amel and enam in representatives of divergent avian lineages including galloanserae, passeriformes, and psittaciformes, and that there are shared frameshift mutations in all three genes that predate the basal split in neognathae. among enamelless mammals, all three emp genes exhibit inactivating mutations in orycteropus and choloepus.  our results highlight the power of combining fossil and genomic evidence to decipher macroevolutionary transitions and characterize the functional range of different loci involved in tooth development. the fossil record and phylogenetics combine to predict the occurrence of molecular fossils of tooth-specific genes in the genomes of edentulous amniotes, and in every case these molecular fossils have been discovered. the widespread occurrence of emp pseudogenes in turtles, birds, and edentulous/enamelless mammals also provides compelling evidence that in amniotes, the only unique, non-redundant function of these genes is in enamel formation. ameloblastinamelogeninenamel matrix protein genesenamelinpseudogenestestudines 