# Pseudogenes Document Protracted Parallel Regression of Oral Anatomy in Myrmecophagous Mammals

Christopher A. Emerling, Sophie Teullet, Rémi Allio, John Gatesy, Mark S.

Springer, Frédéric Delsuc

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Christopher A. Emerling, Sophie Teullet, Rémi Allio, John Gatesy, Mark S. Springer, et al.. Pseudogenes Document Protracted Parallel Regression of Oral Anatomy in Myrmecophagous Mammals. 2025. [https://hal-05368790](https://hal-05368790)

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# Pseudogenes Document Protracted Parallel Regression of Oral Anatomy in Myrmecophagous Mammals

Christopher A. Emerling¹,²,*, Sophie Teullet², Rémi Allio²,³, John Gatesy⁴, Mark S. Springer⁵
and Frédéric Delsuc²,∗

¹ Biology Department, Reedley College, Reedley, CA, USA

²ISEM, Univ. Montpellier, CNRS, IRD, Montpellier, France

³CBGP, INRAE, CIRAD, IRD, Montpellier SupAgro, Univ. Montpellier, Montpellier, France

⁴Division of Vertebrate Zoology, American Museum of Natural History, New York, NY, USA

⁵Department of Evolution, Ecology, and Organismal Biology, University of California, Riverside, CA, USA

*Correspondence to christopher.emerling@reedleycollege.edu and frederic.delsuc@umontpellier.fr

## ABSTRACT

Adaptation to ant and/or termite consumption (myrmecophagy) in mammals constitutes a textbook example of convergent evolution, being independently derived in several mammalian lineages. Myrmecophagous species are characterized by striking convergent morphological adaptations such as skull elongation, enlargement of salivary glands, and long claws to dig into ant and termite nests. These evolutionary modifications also include anatomical regression, such as dental simplification or loss, reduction of masticatory muscles, and possessing a reduced set of taste buds. To gain insights into the molecular changes underlying the regression of these morpho-anatomical traits, we investigated the functionality of candidate genes related to dentition, gustation, and mastication in nine convergent myrmecophagous mammalian lineages. We examined these genes in a comparative phylogenetic context, paired with molecular evolutionary analyses, to estimate the relative timing of loss of gene function over the evolutionary history of each convergent lineage. We found that gustatory reduction and pseudogenization of masticatory myosin often were associated with the regression of dental genes. Evidence of pseudogenization events linked to oral anatomy dates to as early as the Cretaceous/Paleogene boundary, and is an ongoing process including examples of incipient gene inactivations. Whereas we found evidence for gene inactivations across all three functional
34 categories occurring during distinct temporal intervals, there was variation in the sets of genes
35 lost and the relative timing of inactivation events. The combined evidence suggests that the
36 convergent evolution of myrmecophagy has occurred as a protracted process with distinct
37 phases of anatomical evolution, over timescales as long as 60 Myr.

38
39
40 INTRODUCTION
41
Convergent evolution is a widespread phenomenon in which distantly-related organisms
42 independently derive highly similar phenotypic traits. Such a pattern-based definition of
43 evolutionary convergence does not make any assumptions on the underlying mechanisms and
44 allows one to test if similar traits evolved as a result of similar selection pressures (Stayton
45 2015). Deriving similar anatomy, physiology, and behavior can indeed result from modifications
46 to identical, unique or partially overlapping sets of genes, depending on the trait and its
47 underlying molecular basis. The frequency with which convergent evolution is demonstrably
48 repeatable at the genetic level, particularly among highly divergent taxa, is a topic of ongoing
49 interest (Blount et al. 2018; Cerca 2023).

50
One prominent example of convergent evolution can be found in myrmecophagous
51 mammals, species that specialize in eating ants and/or termites (termitivory), which constitute
52 more than 90% of their diet. Mammalian myrmecophages are phylogenetically widespread
53 (Redford 1987; Reiss 2001), with some of the most specialized myrmecophages being found
54 among the xenarthran anteaters (Vermilingua), pangolins (Pholidota), the aardvark
55 (Tubulidentata, *Orycteropus afer*), tolypeutine armadillos (Cingulata, Tolypeutinae: three-
56 banded [*Tolypeutes* spp.], giant [*Priodontes maximus*], and naked-tailed [*Cabassous* spp.]
57 armadillos), the aardwolves (Carnivora: *Proteles* spp.), the numbat (Dasyuromorphia,
58 *Myrmecobius fasciatus*), and the short-beaked echidna (Monotremata, *Tachyglossus
59 aculeatus*). Other mammals with at least partially myrmecophagous diets include include fairy
60 (Chlamyphorinae), hairy (Euphractinae) and long-nosed (Dasypodidae) armadillos, as well as
61 some carnivorans, such as the bat-eared fox (Canidae, *Otocyon megalotis*) and sloth bear
62 (Ursidae, *Melursus ursinus*) (Redford 1987; Reiss 2001).

63
As these lineages have independently adapted to consuming social insects, there has
64 been a shift from a more typical ancestral mammalian masticatory apparatus (specialized
65 multicuspid teeth, robust jaws and associated muscles) to a characteristically derived
66 myrmecophagous oral apparatus: dental simplification and/or loss, thinning of the mandible, a
67 reduced or absent zygomatic arch, an elongated tongue that can rapidly probe ant and termite
68 nests, enlarged salivary glands which produce a high volume of viscous saliva to capture prey,
69 and the reorganization and reduction of jaw musculature to accommodate this feeding
70 mechanism (Reiss 2001; Ferreira-Cardoso et al. 2019; Ferreira-Cardoso et al. 2020). These
71 morphological characteristics are exemplified in xenarthran anteaters ("anteaters" throughout
72 text) and pangolins, wherein extreme specialization for myrmecophagy and accompanying
73 anatomical modifications had led systematists to formerly conclude that they constitute a
74 monophyletic group (Edentata) (Glass 1985; Reiss 2001). It was not until molecular
75 phylogenetics was applied to this question towards the turn of the 21st century that it was firmly
76 demonstrated that their shared features were the result of convergent evolution (Murphy et al.
77 2001; Delsuc et al. 2002; Springer et al. 2013).

78 Given that convergent evolution of myrmecophagy can lead to the complete
79 reorganization of the oral apparatus, it raises the question of whether this is detectable at the
80 genomic level. One way to test for convergent molecular evolution is by studying the
81 phenomenon of regressive evolution, or vestigialization, whereby phenotypic traits are reduced
82 (e.g., in size or complexity) or completely lost over evolutionary time (Fong et al. 1995; Lahti et
83 al. 2009; Albalat and Cañestro 2016). While adaptations for myrmecophagy frequently include
84 the enlargement or extension of certain traits (e.g., hypertrophied salivary glands, lengthening of
85 tongue), several others can be classified as evolutionary regressions, including the thinning of
86 the mandible, the weakening of the jaw musculature, and reduction of the dentition. When such
87 regressed traits are linked to non-pleiotropic or marginally pleiotropic genes, these loci can
88 accumulate mutations that disrupt gene expression and/or translation (Albalat and Cañestro
89 2016). The end result is 'gene loss', either in the form of nonfunctional unitary pseudogenes,
90 characterized by frameshift indels, nonsense mutations and other inactivating mutations, or
91 genes that are deleted from the genome. Numerous examples of such gene loss linked to
92 vestigialization have been documented, ranging from the loss of claw keratins in snakes and
93 worm lizards (Emerling 2017; Holthaus et al. 2025), the loss of insect-digesting chitinase and
94 trehalase genes in mammals that have shifted from insectivory to carnivory and herbivory
95 (Emerling et al. 2018; Janiak et al. 2018; Jiao et al. 2019), and the inactivation of melatonin
96 synthesis and receptor genes in mammals that have lost their pineal glands (Huelsmann et al.
97 2019; Emerling et al. 2021; Valente et al. 2021; Yin et al. 2021).

98 Here, we test whether convergent myrmecophagous mammals show signals of
99 convergent molecular regression, by examining candidate genes related to three components of
100 oral anatomy: dentition, gustation, and masticatory muscle contraction. The development and
101 maintenance of vertebrate teeth relies on coordination between a suite of genes associated with
the formation of dentin and overlying enamel, and maintaining their point of contact with the gingiva. Pseudogenizations have been extensively documented for a subset of these genes in edentulous (toothless) and enamelless vertebrates, including turtles (Meredith et al. 2013), birds (Meredith et al. 2014), toads (Shaheen et al. 2021), and baleen whales (Randall et al. 2022). Dental simplification and tooth loss in myrmecophagous mammals is expected to lead to the accumulation of inactivating mutations in these genes, which should vary depending on the degree of dental reduction. Analyses of gustatory cells demonstrate that certain genes are linked to specific taste modalities (Chandrashekar et al. 2006; Yarmolinsky et al. 2009; Chaudhari and Roper 2010; Roper and Chaudhari 2017). Reductions in gustation from the hypothesized ancestral vertebrate complement have been observed in certain species exhibiting dietary specializations (Policarpo et al. 2024), particularly hypercarnivorous mammals and the bamboo-specialist giant and red pandas (Zhao et al. 2010; Jiang et al. 2012), as well as in taxa that have reduced or completely lost taste buds, such as cetaceans (Feng et al. 2014; Kishida et al. 2015) and snakes (Emerling 2017). Given the extreme degree of dietary specialization and modifications to the tongue in myrmecophagous mammals, losses of gustatory genes are expected. Finally, a particular myosin protein (myosin heavy chain 16; MYH16) is expressed exclusively in the jaw closing muscles of multiple vertebrate lineages, and has been associated with species that possess a strong bite force (Hoh 2002; Toniolo et al. 2008; Lee et al. 2019). For myrmecophagous mammals, in which mastication has been greatly reduced or even lost (Ferreira-Cardoso et al. 2020), this gene is a promising candidate for pseudogenization.

Using these candidate genes, we addressed two main questions. First, do these genes show evidence of similar patterns of gene loss in myrmecophagous mammals, linked to the degree of commitment to myrmecophagy? Second, given that these genes are related to the processing of food, despite being expressed in distinctly different tissues (teeth, taste buds, skeletal muscles), is the timing of gene losses in these three functional categories of genes similar in convergently evolved lineages? If so, this may point to a concomitant sequence of regressive morphological evolution of the oral apparatus associated with the convergent evolution of myrmecophagy. We addressed these questions by interrogating genomic data in a diverse collection of myrmecophagous mammals, including placentals (three anteaters, six armadillos, eight pangolins, the aardvark, two aardwolves, the bat-eared fox, the sloth bear), a marsupial (the numbat), and a monotreme (the short-beaked echidna), representing at least nine convergent adaptations to myrmecophagy.
136 RESULTS

137 Overall patterns

138 Each of the examined genes was pseudogenized or deleted in at least some of our focal
139 species (Figure 1). *TAS1R3* was inactivated the least frequently (3/24, 12.5%) with *ACP4*
140 showing evidence of pseudogenization in the vast majority of the focal taxa (21/24, 87.5%). The
141 most anatomically extreme myrmecophagous species, pangolins and anteaters, have the
142 highest proportion of gene losses, with 86.7% (13/15) to 100% of the genes lost in pangolins,
143 and 78.6% (11/14) to 85.7% (12/14) lost in anteaters. The white-bellied (*Phataginus tricuspis*)
144 and black-bellied (*P. tetradactyla*) tree pangolins were the only species to present evidence of
145 inactivation for every examined gene. By contrast, myrmecophagous carnivorous, which present
146 far less anatomical regression in the oral apparatus, ranged from 0 to 6.7% (1/15) gene
147 inactivation. In terms of the timing of gene losses, some appear very recent, including evidently
148 incipient examples (i.e., polymorphic pseudogenes), whereas the earliest pseudogenization
149 estimates pre-date the K/Pg boundary (66 Mya). Some of the non-myrmecophagous outgroup
150 species we examined also possessed pseudogenes for some of the genes we studied (e.g.,
151 sloths [Folivora]), and these are due in part to a combination of shared history of gene loss with
152 myrmecophagous species and in other cases may represent adaptations associated with other
153 dietary niches.

<figure><img src="image_2.png" /></figure>
**FIGURE 1.** Summary of gene functional status in the focal taxa. Evolutionary relationships depicted using a composite phylogeny with branch lengths proportional to time (Dos Reis et al. 2012; Gibb et al. 2016; Hassanin et al. 2021; Foley et al. 2023; Heighton et al. 2023). Pholidota = pangolins; Tubulidentata = aardvark; Cingulata = armadillos; Vermilingua = anteaters. Paintings by Carl Buell except giant armadillo, pygmy anteater and numbat (Michelle S. Fabros).

**Pangolins**
Nearly all of the genes examined in this study are pseudogenes or deleted in pangolins (13/15 to 15/15; Figure 1; Supplementary Figures S2–S6). Most of these genes show strong evidence of loss prior to the last common ancestor (LCA) of pangolins, particularly due to shared inactivating mutations, suggesting early losses in this clade. For example, the masticatory myosin gene (*MYH16*), is pseudogenized in all five examined species (Figure 2), with 12 inactivating mutations shared among them (Supplementary Figure S4). The tooth genes likewise share inactivating mutations (Figure 2; Supplementary Figures S2–S4), but the taste receptor genes are more variable in their patterns of gene loss (Supplementary Figures S4–S6).

The tooth genes we examined have been described for some pangolin species, particularly *Manis pentadactyla* (Meredith et al. 2009; Meredith et al. 2014; Choo et al. 2016; Springer et al. 2016; Springer et al. 2019), but here we expand upon these results by characterizing ten dental genes in three Asian pangolins (*Manis* spp.) and two African pangolins (*Smutsia gigantea*, *Phataginus tricuspis*), representing the two major subclades within Pholidota. All ten are pseudogenes or show evidence of whole gene deletion in all five pangolin species investigated. In almost every case, there is evidence of inactivating mutations shared between Asian and African pangolins. For the enamel genes, *ACP4* has 11 shared mutations, *AMELX* has a single shared splice acceptor mutation (intron 3), *ENAM* has four shared mutations, and both *AMBN* and *MMP20* have three shared mutations. *KLK4* returned no BLAST results for all three *Manis* spp. *KLK5* and *KLK15* flank this gene in other boreoeutherian mammals, and both were found on the RefSeq assembly for *Manis javanica* on chromosome 17, suggesting a whole gene deletion. For the African pangolins, we were only able to recover the first two exons of *KLK4*, with exon 2 presenting two shared premature stop codons. For the dento-gingival junction genes, *AMTN* exons 1–4 have been deleted in all pangolins, and *ODAM* has eight shared mutations. The dentin development gene *DSPP* has been completely deleted in Asian pangolins, confirmed by the retention of homologous sequence upstream and downstream of the CDS, whereas African pangolins retain it but possess 10 shared mutations.
Finally, ODAPH, whose function is correlated with tooth retention, has six mutations shared between all five species (Figure 2).

<figure><img src="image_3.png" /></figure>

**FIGURE 2.** Pseudogene dating estimates for pangolins (Pholidota) and DNA sequence alignments for representative genes. Average dates are mapped on a timetree based on divergence times from Heighton et al. (2023). Also included are relevant fossil taxa, indicated by black circles and daggers (see Discussion). \* indicates minimum date of inactivation based on distribution of inactivating mutations. These genes were not dated using dN/dS due to violated assumptions. Paintings by Carl Buell.
Among the gustatory genes, TAS1R2 is inactivated in all five pangolins we examined, with 10 shared disabling mutations (Figure 2). At the other extreme, TAS1R3 is only inactivated in the two *Phataginus* species with up to nine mutations in *P. tricuspis* and five in *P. tetradactyla*, although none of these are shared between the two species. A premature stop codon is present in the *Smutsia gigantea* TAS1R3, though this is only 14 codons upstream from the ancestral stop codon, making the functional impact uncertain. Likewise, TAS1R1 shows relatively minimal evidence of inactivation, presenting as a pseudogene in both *Phataginus* species and *Smutsia temminckii*. Finally, TAS1R1 in the Sunda (*Manis javanica*), Indian (*M. crassicaudata*) and Philippine (*M. culionensis*) pangolins may likewise be a pseudogene, based on an 8-bp insertion in exon 6. However, this is near the ancestral stop codon and results in an additional five residues translated prior to the next stop codon, rendering its status uncertain. All pangolins have inactivating mutations in *PKD2L1* and although none of these are unambiguously shared among all five species, four mutations are shared between the African pangolins, and Asian pangolins possibly share a splice acceptor mutation in intron 8 relative to the ancestral AG condition (GG in *M. crassicaudata* and *M. javanica*; CG in *M. pentadactyla*).

Given that shared inactivation mutations only provide a minimum date for pseudogenization, we performed numerous pseudogene dating analyses, based on dN/dS calculations (Supplementary Tables S3–S17), to provide estimates of when relaxed selection effectively began on each locus. In most cases, our dN/dS estimates were consistent with assumptions of transitioning from a fully-functional gene to a pseudogene. Specifically, on a transitional (“mixed”) branch, the ω parameter value is expected to be higher than the background (“functional”) branches but less than fully relaxed selection or neutral evolution (“pseudogene”, ω = 1).

Of the 12 genes with mutational evidence suggesting loss on the stem Pholidota branch, nine had ω estimates for the crown Pholidota branches that were not a significantly better fit (p > 0.05) relative to models in which ω was fixed at 1 for crown branches (range ω: 0.66–2.75; mean ω: 1.18; median ω: 1.04). This suggests selection was largely relaxed after inactivation. However, three genes deviated from this pattern for crown Pholidota: *DSPP* (ω = 0.54, p = 0.02); *MYH16* (ω = 0.76, p = 0.009); *TAS1R2* (ω = 0.52, p < 0.00001). One interpretation is that even after becoming a pseudogene in the stem Pholidota lineage, these genes still experienced some selective constraint. Alternatively, rapid evolution of CpG sites or other features of the nucleotide substitution process that are not modelled by codeml might have distorted ω estimates for these genes. When performing pseudogene dating analyses, we assumed the “pseudogene” branch category to be ω = 1 for the former nine genes, and implemented the
direct estimates of $\omega$ for the latter three. For the remaining three genes, which all lack direct evidence of inactivation on the stem Pholidota branch, two showed clear evidence of selective constraint in crown Pholidota, estimating an $\omega$ much lower than one, and statistically distinguishable from models in which the crown $\omega$ was fixed as one (TAS1R1, $\omega$ = 0.29; TAS1R3, $\omega$ = 0.24). However, despite the absence of typical inactivating mutations shared across all pangolins, PKD2L1 had a crown $\omega$ of 1 and a stem $\omega$ that was statistically distinguishable from the “functional” category ($\omega$ = 0.28 stem branch vs 0.17 background; $p$ = 0.037). This suggests loss of the gene, or at least extensive relaxed selection, on the apical end of the stem Pholidota branch, prior to the acquisition of clear disabling mutations in the descendant lineages.

For the 13 genes with evidence of gene loss on the stem Pholidota branch (including PKD2L1), nine have $\omega$ estimates that are consistent with assumptions for a “mixed” or transitional branch, being higher than the “functional” $\omega$ of outgroups and lower than the “pseudogene” $\omega$. The three that violated this assumption had mixed branches wherein $\omega$ was higher than one: AMTN (mixed $\omega$: 1.01, functional $\omega$: 0.49, pseudogene $\omega$: 1.14), DSPP (mixed $\omega$: 1.08, functional $\omega$: 0.75, pseudogene $\omega$: 0.54), and ODAPH (mixed $\omega$: 1.67, functional $\omega$: 0.5, pseudogene $\omega$: 1.47). These all are suggestive of issues with how PAML is modelling these genes, or perhaps a mixed history of positive selection along with purifying and/or relaxed selection on the stem Pholidota branch. Regardless, they were excluded from pseudogene dating estimates due to this assumption violation. For the stem Pholidota $\omega$ estimates, nine of 15 genes were statistically distinguishable from the “functional” category. The exceptions were AMBN, AMTN, DSPP, ODAM, TAS1R1 and TAS1R3, of which the latter two show no evidence of gene loss on the stem Pholidota branch.

We performed pseudogene dating analyses for all genes that were consistent with our assumptions (Figure 2). The inactivation dates here and for other taxa below indicate the average and a range provided by assuming one synonymous substitution rate vs. assuming two (see Meredith et al. 2009) during the transition from functional genes to pseudogenes: KLK4: 77 Mya (76.6–77.4); ENAM: 72.8 Mya (71.8–73.8); MYH16: 63.7 Mya (62–65.3); AMELX: 61.3 Mya (59.6–63); ODAM: 60.8 Mya (59.6–61.3); MMP20: 60.4 Mya (58.7–60.4); TAS1R2: 58.6 Mya (56.9–60.2); AMBN: 54.3 Mya (52.7–55.8); ACP4: 54.1 Mya (52.6–55.7); PKD2L1: 45.8 Mya (45.1–46.5).

303
304
305
306 **Anteaters**

307 Like pangolins, anteaters show a large number of gene losses for our dataset (11/14 to 12/14)
308 (Figure 1; Supplementary Figures S7–S8). The dental genes for xenarthrans have already been
309 reported by Emerling et al. (2023). Among the gustatory genes (Supplementary Figures S7–S8),
310 *TAS1R1* is intact in all three anteaters, *TAS1R3* and *PKD2L1* are variably inactivated, and
311 *TAS1R2* is pseudogenic in all three. For *TAS1R2*, all anteaters share eight inactivating
312 mutations, and their sister group, sloths (Folivora), also have a pseudogenized *TAS1R2*
313 ortholog. The two clades of sloths (*Choloepus* spp., *Bradypus* spp.) share two mutations with
314 anteaters: a 19-bp deletion of the exon 4-intron 4 boundary and an 8-bp deletion in exon 5
315 (Figure 3). *Myrmecophaga tridactyla* presents a premature stop codon in exon 3 of *TAS1R3* in
316 one genome assembly, but this was not reproduced in a second. Furthermore, short-read
317 sequence data suggest that this mutation is polymorphic, as this individual is heterozygous for
318 the stop codon. Only *Cyclopes didactylus* shows evidence of *PKD2L1* pseudogenization,
319 possessing a 2-bp deletion in exon 13, which is polymorphic based on short-read sequence
320 data.

321 *MYH16* is a pseudogene in all three anteaters (Supplementary Figure S8), with three
322 shared disabling mutations in *Tamandua tetradactyla* and *Myrmecophaga tridactyla* (Figure 3).
323 While *Cyclopes didactylus* also has a nonfunctional *MYH16*, possessing 10 disabling mutations,
324 none of these are shared with the other anteaters. Among the sloth outgroups, the two-fingered
325 sloths (*Choloepus* spp.) have an intact *MYH16*, while it is a pseudogene with shared mutations
326 in both of the three-fingered species that we examined (*Bradypus* spp.).

327 Given that the lineage leading to anteaters and armadillos is broken up by cladogenic
328 events (Figure 3), we did not perform ω-based pseudogene dating analyses on xenarthrans,
329 and instead used shared inactivating mutations to provide minimum pseudogenization dates,
330 bracketed by likely maxima. First, we found no evidence of shared inactivating mutations
331 between pilosans (anteaters + sloths) and armadillos, suggesting that the entire complement of
332 genes we examined was functional in the LCA of xenarthrans. After this, assuming the dates
333 from Gibb et al. (2016), *ACP4*, *AMELX*, *ENAM*, *MMP20*, *AMTN*, and *TAS1R2* were all lost on
334 the stem Pilosa (anteaters + sloths) branch (68.1–58.9 Mya). Next, on the stem vermilinguan
335 branch (58.9–38 Mya), *ODAM*, *ODAPH* and *DSPP* were inactivated. On the stem
336 *Myrmecophagidae* (*Myrmecophaga* + *Tamandua*) branch (38–12.8 Mya), *MYH16* and *AMBN*
337 were disabled, and *TAS1R3* shows evidence of incipient pseudogenization in *Myrmecophaga*
338 *tridactyla*. Likewise, along the *Cyclopes didactylus* branch (38–0 Mya), *MYH16* and *AMBN* were
339 inactivated, with evidence of incipient loss of *PKD2L1*.
<figure><img src="image_4.png" /></figure>

**FIGURE 3.** Minimum pseudogenization dates in xenarthrans based on shared inactivating mutations and DNA sequence alignments for representative genes. Gene losses were arbitrarily placed midway on pertinent branches of a timetree, with divergence times derived from Gibb et al. (2016). Exceptions are *PKD2L1* in *Cyclopes didactylus* and *TAS1R3* in *Myrmecophaga tridactyla*, which were placed at the branch tips due to being polymorphic pseudogenes. Also included are relevant fossil taxa, indicated by black circles or bars and daggers. Bars indicate the date range for these fossils (see Discussion). Vermilingua = anteaters; Folivora = sloths; Cingulata = armadillos. Paintings by Carl Buell except *Priodontes maximus* and *Cyclopes didactylus* (Michelle S. Fabros).
374 **Armadillos**

375 Armadillos represent the third clade (Cingulata) within Xenarthra, and possess fewer
376 pseudogenes (6/14–8/14) than their anteater relatives (Figure 1). Much like their dental genes
377 (Emerling et al. 2023), armadillo gustatory genes are variably pseudogenic (Supplementary
378 Figures S7–S8). *TAS1R1* has clear positive evidence of inactivation in only *Tolypeutes*
379 *matacus*, with eight inactivating mutations across the gene, validated by two different genome
380 assemblies. Furthermore, *TAS1R2* and *TAS1R3* appear functionally intact in all six armadillos
381 we examined. By contrast, *PKD2L1* is present as a pseudogene in multiple armadillos including
382 *T. matacus* (three inactivating mutations), *Cabassous unicinctus* (two), *Chlamyphorus truncatus*
383 (two) and *Euphractus sexcinctus* (two). By contrast, *MYH16* is universally inactivated in
384 armadillos (Supplementary Figure S8). We found seven mutations shared among all
385 chlamyphorid armadillos (Figure 3). *Dasypus novemcinctus* also has an *MYH16* pseudogene,
386 though none of its mutations are clearly shared with the five chlamyphorids.

387 Given the absence of shared inactivating mutations across dasypodid and chlamyphorid
388 armadillos, the available evidence suggests that the examined genes were intact in the LCA of
389 Cingulata (Figure 3). After the split between dasypodid and chlamyphorid armadillos (45.3 Mya),
390 shared mutations suggest gene losses occurred repeatedly in parallel. On the stem
391 chlamyphorid branch (45.3–37.2 Mya), *MYH16*, *ACP4*, *AMTN*, and *ODAM* were lost. This was
392 followed by multiple parallel losses of enamel genes (*AMELX*, *ENAM*, *AMBN*) and *PKD2L1*, and
393 a single loss of *TAS1R1*. In dasypodids, between 45.3 Mya and the present, *MYH16*, *AMBN*,
394 *ENAM*, *ACP4*, *AMTN* and *ODAM* were lost. Based on results from Emerling et al. (2023), *ACP4*,
395 *AMTN* and *ODAM* show evidence of loss prior to crown Dasypodidae (12.9 Mya), with *AMBN*
396 and *ENAM* being inactivated more recently. Based on the number of inactivating mutations in
397 *MYH16* (47), it is highly likely this gene was also lost on the stem Dasypodidae branch.
398

399

Aardvark

400 The aardvark's (*Orycteropus afer*) dental pseudogene distribution has been described
401 previously (Meredith et al. 2009; Meredith et al. 2014; Springer et al. 2019), which we confirm
402 here. The aardvark has inactivated orthologs for all of the enamel and dento-gingival junction
403 genes while retaining functional *DSPP* and *ODAPH* (Figure 1). *MYH16* is intact in this species,
404 as are the three *TAS1Rs*, whereas *PKD2L1* possesses a 1-bp deletion in exon 8
405 (Supplementary Figure S9).

406 We performed pseudogene dating analyses based on dN/dS modelling (Figure 4;
407 Supplementary Tables S3–S5, S8, S10, S11, S13), with the estimated dates as follows: *ODAM*
408 51.5 Mya (49.5–53.5), MMP20 28.6 Mya (25.7–31.5), ACP4 26.4 Mya (23.6–29.3), AMBN 23.3
409 Mya (20.6–25.9), AMELX 23 Mya (20.3–25.7), ENAM 17.7 Mya (15.4–20), and PKD2L1 11.6
410 Mya (9.9–13.4). We did not date AMTN because it violated our assumptions (“mixed branch”
411 $\omega$ > 1). dN/dS estimates showed statistically elevated values for five pseudogenes (ACP4,
412 AMTN, MMP20, ODAM, PKD2L1), whereas TAS1R1 was statistically reduced compared to the
413 background (Supplementary Tables S3–S8, S10–17).

<figure><img src="image_5.png" /><figcaption>**FIGURE 4.** Pseudogene dating estimates for aardvark (*Orycteropus afer*). Average dates are mapped on a timetree based on dates from Foley et al. (2023). Also included are relevant fossil taxa, indicated by black bars and daggers. Bars indicate the date range for these fossils (see Discussion). Paintings by Carl Buell.</figcaption></figure>

427

429

430

431

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433

**Carnivorans**

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438

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440

Among the carnivorans we examined, disabling mutations were very rare, and when present they always presented as polymorphic (functional/nonfunctional alleles; Supplementary Figures S2, S5 and S6), demonstrating that these mutations are not fixed in their respective species.

First, we found no evidence of pseudogenes in the bat-eared fox. In the sloth bear (*Melursus ursinus*), we found a single inactivating mutation in *ACP4*, being heterozygous for a splice donor mutation (GT → GG) in intron 3 (Supplementary Figure S2). The two aardwolves only show evidence of incipient pseudogenization in the *TAS1Rs* (Supplementary Figures S5–S6). The
Southern aardwolf (*Proteles cristatus*) genome assembly has an 8-bp deletion in exon 1 of *TAS1R1*, which is supported by short read data. However, one individual (NMB12641) is homozygous for this mutation, whereas another individual (NMB12667) is heterozygous. By contrast, its sister-species, the Eastern aardwolf (*P. septentrionalis*), does not possess this mutation, but instead is heterozygous for a 1-bp insertion in exon 3 of *TAS1R2*, which is absent in *P. cristatus*. We did not perform pseudogene dating estimates, given that disabling mutations were not fixed in these species.

The numbat (*Myrmecobius fasciatus*) similarly had sparse evidence of gene dysfunction, although more than one gene was affected in this species (Supplementary Figures S10–S11). First, a single enamel gene (*ACP4*) presents evidence of pseudogenization (Figure 5), possessing three 1-bp deletions spread across exons 10 and 11. Much of the gene was irretrievable from the genome assembly due to a gap (10,000 N's), though some upstream exons were assembled using short reads from our specimen. However, we were able to validate these mutations using short read data from three specimens: NCBI BioProject PRJNA786364 (Peel et al. 2022), PRJNA512907 (https://www.dnazoo.org/), and our specimen (PRJNA1268346). All three 1-bp deletions were supported by PRJNA786364 and PRJNA512907, and two of the deletions were supported by PRJNA1268346 (assembly gap for the third mutation), suggesting this is a fixed pseudogene in numbats. The remaining two genes have polymorphic mutations. We found inactivating mutations in *TAS1R1*, in contrast to Peel et al. (2022), with one premature stop codon in exon 2 and another in exon 4. Both stop codons are heterozygous in the PRJNA786364 specimen, but absent from PRJNA512907 and PRJNA1268346. Similarly, while *TAS1R2* of the genome assembly lacked evidence for gene inactivation, PRJNA1268346 possesses a single heterozygous 1-bp deletion in exon 6, absent in PRJNA786364 and PRJNA512907. A study of the tongue transcriptome in numbat noted the absence of *TAS1R2* transcripts (Peel et al. 2022), in contrast to *TAS1R1* and *TAS1R3*, pointing to the possibility that regulatory elements may be under relaxed selection, with some numbats beginning to present inactivation mutations.

Given evidence of inactivation of *ACP4* in the numbat, we estimated its timing of pseudogenization (Figure 5), and likewise performed dN/dS ratio analyses on the remaining genes (Supplementary Tables S3–S8, S10–S17). For *ACP4*, we used the sequence constructed from our specimen rather than the genome assembly given that the latter was much more fragmented, estimating inactivation at 13 Mya (11.5–14.4). Among all of the genes, eight
475 had ω values lower on the numbat branch than on the background branches, and of the six with 476 higher ω, only ACP4 (ω: 0.47, functional ω: 0.12, p = 0.008) and TAS1R3 had values 477 statistically distinguishable from the background.

<figure><img src="image_6.png" /></figure>

493 **FIGURE 5.** Pseudogene dating estimate and DNA sequence alignment for *ACP4* in numbat 494 (*Myrmecobius fasciatus*). Average date is mapped on a timetree based on divergence times 495 from Westerman et al. (2016). *TAS1R1* and *TAS1R2* were placed at the branch tip due to being 496 polymorphic pseudogenes. Paintings by Carl Buell except numbat (Michelle S. Fabros).

497

498 **Echidna**

499 For the short-beaked echidna (*Tachyglossus aculeatus*), the patterns of gene loss are most 500 similar to those of pangolins and anteaters (Figure 1; Supplementary Figure S12). All of the 501 enamel and dento-gingival genes are either pseudogenes or deleted, and some of these gene 502 losses are shared with the platypus (*Ornithorhynchus anatinus*). Zhou et al. (2021) reported the 503 absence of *AMTN*, *ODAM*, and *MMP20* in both monotremes, *AMBN* and *ENAM* were reported 504 lost uniquely in echidnas, and *DSPP* and *AMELX* were recorded as present and, implied, 505 functional in both species. Here we elaborate on these results, first by confirming that *AMTN* 506 and *ODAM* were completely deleted in both monotremes, and thereby pointing to loss in their 507 LCA. *AMTN*, along with *AMBN* and *ENAM*, is located between *CSN3*, *MUC7* and *JCHAIN* in 508 other mammals. While the latter three were found in synteny in both monotremes, *AMTN* was
absent. AMBN and ENAM likewise were deleted in echidna, while preserved in the platypus.
ODAM is situated between CSN2 and CSN3 in other mammals, both of which were found in
tandem in monotremes with ODAM missing. Furthermore, MMP20 was completely deleted in
the platypus, whereas exons 9 and 10 were retained in the echidna, with a 13-bp deletion in
exon 9. Like other mammals, MMP20 is positioned between MMP7 and MMP8, both of which
can still be found in synteny within the platypus. We recovered AMELX in both monotremes, but
it is a pseudogene in echidna, with no recoverable exons 1–3 and two inactivating mutations in
exon 4 (Figure 6). The monotreme DSPP is difficult to characterize, and indeed appears to have
a quite different exon structure from that of placental mammals, as evidenced by BLAST and
alignment approaches, as well as the gene structure predicted by NCBI genome annotations.
While potential inactivation mutations were observed, RNA sequencing may provide more clarity
as to whether this gene is functional in these species. We do note that the echidna predicted
mRNA (XM_038769188) possesses a 2-bp deletion that was ‘corrected’ during model
construction, further pointing to potential pseudogenization. However, based on reports of
dentin in the egg tooth of echidnas, this may be a false positive (see Discussion).

To these results we add those for ACP4 and ODAPH. The former is a pseudogene in
both monotremes, with major deletions (exons 2–9) in the echidna, and a stop codon in exon 10
of the platypus. We found no evidence of ODAPH in either monotreme, and both BLAST
searches and synteny analyses across vertebrates suggest this may be a therian (Marsupialia +
Placentalia) innovation. Among the non-dental genes, while the TAS1Rs all appear intact in the
echidna, MYH16 and PKD2L1 are both pseudogenes. The former has at least 22 inactivating
mutations (Figure 6), and the latter likewise has numerous inactivating mutations. All five non-
dental genes appear functional in the platypus.

We performed gene inactivation estimates for five pseudogenes in echidna (Figure 6;
Supplementary Tables S3, S5, S10–S11, S14): PKD2L1 49.9 Mya (48.9–50.9); MYH16 20.3
Mya (18–22.6); AMELX 13.4 Mya (11.6–15.2); ACP4 9.9 Mya (8.4–11.3); MMP20 1.6 Mya (1.3–
1.9). dN/dS estimates were statistically elevated compared to the background for two
pseudogenes (MYH16, PKD2L1), and for all three TAS1Rs, potentially suggestive of relaxed
selection on these gustatory genes (Supplementary Tables S11, S14–S17).

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540

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542
<figure><img src="image_7.png" /></figure>

**FIGURE 6.** Pseudogene dating estimates for short-beaked echidna (*Tachyglossus aculeatus*) and platypus (*Ornithorhynchus anatinus*). Long-beaked echidnas (*Zaglossus* spp.) included for comparison, but not examined in this study. Average dates are mapped on a timetree derived from Dos Reis et al. (2012). \* indicates minimum date of inactivation based on whole gene deletions. Also included are relevant fossil taxa, indicated by black circles or bars and daggers. Bars indicate the date range for the earliest fossils (see Discussion). Paintings by Carl Buell.

DISCUSSION

Here we showed that convergent myrmecophagous mammals have lost numerous genes underlying gustation, mastication and dentition in parallel, but their pseudogenization occured at varying degrees and at different evolutionary timescales. Of the genes examined, there was a spectrum of gene loss, ranging from 100% inactivation (African tree pangolins) to complete functional retention (bat-eared fox) or only incipient pseudogenizations (sloth bear, aardwolves). When gene inactivations did occur, the timing of the first estimate was variable, with some lineages showing pseudogenizations near the K/Pg boundary (66 Mya; anteaters), or perhaps even earlier (>70 Mya; pangolins), and others having their first gene loss estimated at 13 Mya
577 (numbat). However, while clusters of pseudogenization events did seem to occur, large variation
578 in timing occurred within lineages. For example, we estimated *KLK4* was inactivated around 77
579 Mya in a stem pangolin, but *TAS1R3* inactivations occurred near 4.5 Mya in the African tree
580 pangolins. Similarly, we estimated six pseudogene events between 68.1 and 58.9 Mya in the
581 lineage leading to anteaters, while also reporting incipient pseudogenizations in the giant and
582 pygmy anteaters, respectively.

583 It is tempting to hypothesize that there may be common patterns in terms of the timing of
584 gene loss across different lineages. Indeed, among dentition genes, three appear to be
585 commonly inactivated earlier in evolutionary history: *ACP4* (anteaters, aardvark, numbat, sloth
586 bear), *AMTN* (anteaters, chlamyphorid armadillos, echidna) and *ODAM* (anteaters, pangolins,
587 aardvark). The gustatory gene *TAS1R1* was rarely pseudogenized, but when it was, it uniformly
588 occurred late (three-banded armadillo, pangolins). However, there were likewise major contrasts
589 for certain genes. For example, while the taste gene *TAS1R2* appears to have been inactivated
590 very early in the history of pangolins and anteaters, and also being among the first to be
591 pseudogenized in an aardwolf and the numbat, it remains intact in all other species. Likewise,
592 the enamel development gene *MMP20* was inactivated early in several convergent lineages
593 (pangolins, anteaters, aardvark), but it perplexingly appears functional in all of the enamelless
594 chlamyphorid armadillos. *PKD2L1* was the most frequently inactivated gustatory gene, but was
595 lost relatively early (echidna), relatively late (xenarthrans, aardvark), or intermediate in timing
596 (pangolins) during the evolution of myrmecophagous mammals. Similarly, we estimated that the
597 masticatory myosin gene *MYH16* has been lost early in pangolins, chlamyphorid armadillos, and
598 echidna, later in anteaters, and was maintained as functional in aardvark. The explanations for
599 this variation in patterns of gene loss may be manifold, with some possibilities discussed below.

600
601 **Myrmecophagy is associated with some degree of taste loss**
602 One of the more remarkable convergent adaptations of myrmecophagous mammals involves
603 the evolutionary remodelling of the tongue. A typically lengthened, often vermiform tongue,
604 paired with rapid protrusive movements and the production of sticky saliva, allows for the
605 consumption of large quantities of ants and termites during feeding bouts. Modifications are also
606 apparent in the papillae of the dorsum of the tongue. For example, while pangolins are reported
607 to have filiform, fungiform and circumvallate papillae (Prapong et al. 2009), the former two are
608 reported to be poorly-developed (Kubota, Kubota, Nakamura, et al. 1962), with taste buds only
609 being situated in two to three circumvallate papillae (Doran and Allbrook 1973; Prapong et al.
610 2009) or completely absent altogether (Kubota, Kubota, Nakamura, et al. 1962; Abayomi et al.
611 2009). Similarly, in anteaters, fungiform papillae appear to be absent, with taste buds restricted
to a pair of weakly innervated circumvallate papillae (Kubota, Kubota, Fukuda, et al. 1962;
612
613 Casali et al. 2017). Given the reshaping of the tongue into a prey-capturing structure, paired
614 with the tendency towards an extremely specialized diet, we hypothesized that a reduction of
615 taste perception might result from such extreme anatomical and behavioral evolution.

616 We found that there are divergent evolutionary patterns for gustation in these species. At
617 one extreme, some myrmecophagous species retained functional orthologs for the four genes
618 we investigated, suggesting the preservation of the sweet, umami, and sour taste modalities.
619 These included species with a presumably more recent and/or weaker commitment to this
620 dietary habit (sloth bear, bat-eared fox, nine-banded armadillo) but also a species with a
621 relatively long history of myrmecophagy (giant armadillo). At the opposite extreme are the
622 African tree pangolins (Phataginus spp.), both of which appear to have lost all three gustatory
623 pathways (Figure 1), suggesting a reduction in taste only matched so far by cetaceans (whales,
624 dolphins) among mammals (Feng et al. 2014). The remainder of the myrmecophagous taxa are
625 along a spectrum, losing one to three gustatory genes, with some being polymorphic
626 pseudogenes. Beyond the genes we examined, the limited studies that have looked at bitter
627 taste receptor genes (TAS2Rs) suggest that this gene family similarly has contracted in
628 myrmecophagous species (Liu et al. 2016; Seymen et al. 2016; Zhou et al. 2021; Li et al. 2024).

629 Loss of gustatory genes has been linked with major shifts in dietary habits, occurring in
630 mammalian hypercarnivores, bamboo specialists and sanguivores (Zhao et al. 2010; Jiang et al.
631 2012; Zhao et al. 2012; Hu et al. 2017). A narrowing of diet may largely explain the reduction of
632 taste observed in most myrmecophages, but there are other possible explanations. First, gene
633 loss may be related to the modification of the tongue to a prey-capturing organ rather than being
634 used to manipulate food and sample tastants. An analogous situation has been described in a
635 few squamate lineages, including snakes, varanids and teiids, which have modified (forked)
636 tongues specialized for vomeronasal sensation, evidently at the expense of most or all taste
637 buds (Schwenk 1985; Young 1997). Indeed, most snakes have lost their sweet and umami
638 receptor genes, with their bitter taste receptor genes likewise reduced (Emerling 2017). Second,
639 perhaps food consumption in myrmecophages is so rapid that it minimizes the need for
640 gustation. This hypothesis has also been put forth to explain taste reduction in cetaceans and
641 penguins (Feng et al. 2014; Cole et al. 2022). All three hypotheses — dietary specialization,
642 tongue modification for prey capture, and rapid consumption of prey — are certainly not mutually
643 exclusive as explanations for the pattern seen in myrmecophagous mammals, and indeed these
644 diverse factors may have reinforced each other. Yet, the retention of some taste modalities in
645 even some of the most specialized myrmecophagous species (e.g., anteaters, most pangolins)
646 point to the possibility that complete taste gene loss is rarely adaptive. The fact that some of
647 these genes are expressed in extragustatory tissues (Roper and Chaudhari 2017) and have
648 been associated with processes as varied as carbohydrate metabolism (Kochem et al. 2024),
649 cerebrospinal fluid monitoring (Liu et al. 2023), skeletal muscle fitness (Serrano et al. 2024),
650 milk production (Kobayashi et al. 2023), testosterone synthesis (Gong et al. 2024), and
651 intestinal inflammation (Shon et al. 2023) may signify that they are unlikely to be lost with
652 regularity due to pleiotropy. Simultaneously, these patterns may indicate how significant it is that
653 gustation loss occurs at all, and has done so repeatedly, in convergent myrmecophagous
654 mammals.
655

656 **Myrmecophagy is associated with loss of masticatory myosin**
657 Myosin proteins, along with actin, tropomyosin and troponin, drive muscle contraction in
658 mammals. Myosins are heterohexamer proteins that are expressed in muscle cells, with the
659 underlying genes belonging to two families: MYHs (myosin heavy chains) and MYLs (myosin
660 light chains). Most myosin genes are expressed in more than one muscle type (Hoh 2002;
661 Toniolo et al. 2008), whereas the masticatory myosin, encoded by *MYH16*, appears to be
662 exclusively expressed in jaw-closing muscles, including the temporalis, masseters and
663 pterygoideus (Hoh 2002; Hoh et al. 2006; Toniolo et al. 2008; Lee et al. 2019). It has been
664 characterized in mammals such as primates, bats, carnivorans, opossums, dasyurids, and
665 bandicoots, as well as in non-mammalian gnathostomes as distantly-related as crocodylians,
666 turtles and sharks (Hoh 2002; Hoh et al. 2006; Toniolo et al. 2008). This myosin is distinctive in
667 that it seems to allow for a powerful bite for species that possess it, which makes it all the more
668 notable that it is not found in all jawed vertebrates. Specifically, it is absent in distinct lineages of
669 herbivorous mammals, such as kangaroos, ruminants and rabbits. While these species have
670 jaw-closing muscles, their sarcomeres are composed of other myosin classes (Hoh 2002).
671 Given that masticatory myosin provides a powerful bite, shifting from faunivory to dietary habits
672 that rely more on lateral, grinding movements may have rendered *MYH16* superfluous.

673 We expand on these observations, demonstrating that myrmecophagous mammals
674 likewise frequently lose *MYH16*. While faunivorous, species that consume social insects tend to
675 have reduced masticatory muscles and minimal to no chewing capacity (Murray 1981; Redford
676 1987; Smith and Redford 1990; Naples 1999; Ferreira-Cardoso et al. 2020; Thomas et al. 2024;
677 Shoshani et al. 1988; Endo et al. 2017; Endo et al. 2007; Endo et al. 1998). Furthermore, much
678 of the jaw musculature and associated skeletal elements have been rearranged in anteaters,
679 pangolins and echidnas such that they are able to facilitate rapid protrusion of the tongue
680 (Murray 1981; Endo et al. 1998; Endo et al. 2007; Endo et al. 2017; Ferreira-Cardoso et al.
681 2020). Indeed, *MYH16* has been pseudogenized in all three of these clades, in addition to
682 armadillos.

683 Still, some myrmecophagous taxa have retained a functional *MYH16*. The carnivoran
684 myrmecophages retain a functional *MYH16* ortholog, which may reflect more recent adaptations
685 to eating social insects or perhaps phylogenetic constraints associated with an ancestral
686 carnivorous diet. Alternatively, a strong bite force may be retained for behaviors unrelated to
687 diet. The strictly termitivorous aardwolves are territorial and protect their breeding dens from
688 jackals, both of which benefit from jaw muscles that provide a powerful bite (Koehler and
689 Richardson 1990).

690
691 **Contrasting patterns of dental pseudogenization between myrmecophagous lineages**
692 One of the best characterized examples of convergent evolution in the context of mammalian
693 myrmecophagy concerns what is apparently a ubiquitous simplification of the dentition. Different
694 taxa display various combinations of the following: a reduction in tooth number, supernumerary
695 teeth, enamel thinning and loss, simplification of crowns and roots, weak to no occlusion,
696 monophyodonty, intermolar diastemata, and variation in tooth number on the left versus right
697 sides of the jaws (Redford 1987; Koyasu 1993; Stefen and Rensberger 1999; Davit-Béal et al.
698 2009; Cooper 2011; Kupczik and Stynder 2012; Charles et al. 2013; Hayssen et al. 2013; Carter
699 et al. 2016; Pérez-Ramos et al. 2019). Seemingly paradoxically, myrmecophagous mammals
700 range from being completely edentulous (anteaters, pangolins, echidnas) to having the most
701 teeth of any terrestrial mammals (up to 100 in the giant armadillo), suggesting that the genomic
702 elements controlling the formation of teeth are generally under relaxed selection in these
703 species.

704 The association of tooth and enamel loss with dental pseudogenes is well-documented
705 (Deméré et al. 2008; Meredith et al. 2013; Meredith et al. 2014; Choo et al. 2016; Springer et al.
706 2019; Shaheen et al. 2021; Zhou et al. 2021; Randall et al. 2022; Emerling et al. 2023). Indeed,
707 the evidence is so extensive that these species have been considered genomic models that
708 may aid in the discovery of loci implicated in congenital dental diseases in humans (Emerling et
709 al. 2017). In this study, we have examined species at opposite extremes of dental gene loss,
710 particularly the edentulous pangolins, anteaters, and the short-beaked echidna at one end of the
711 spectrum and the myrmecophagous carnivorans and the numbat at the other.
Consistent with patterns seen in other edentulous vertebrates, pangolins, anteaters and the short-beaked echidna present evidence of gene loss in all dental genes we examined (Figure 1), with the possible exception of the dentin matrix gene *DSPP* in the echidna (see below). Species with intermediate levels of dental regression with teeth constituted solely of dentin, including the enamelless aardvark and armadillos, all retain genes necessary for dentin and tooth formation (*DSPP*, *ODAPH*), but lack the dento-gingival genes and most to all of the enamel genes. Myrmecophagous species that retain teeth with enamel crowns, namely myrmecophagous carnivorans and the numbat, largely retain intact dental gene complements, with two exceptions. First, the sloth bear has a pseudogenic allele of *ACP4*, a gene that participates in enamel development and for which inactivation leads to hypoplastic (thin) enamel in humans and mice (Kim et al. 2022; Liang et al. 2022). Likewise, the sole dental pseudogene in the numbat is *ACP4*. Notably, when the pseudogenization pattern of this gene has been compared with the other genes in detailed taxonomic datasets, it appears to be one of the first to become inactivated (Randall et al. 2022; Emerling et al. 2023; Hautier et al. 2023; Randall et al. 2024), which may suggest that dental regression in the sloth bear and numbat is indeed underway.

**Pseudogenes of the oral apparatus and their implications for the convergent evolution of myrmecophagy**

Organisms can, and often do, evolve analogous solutions to the same adaptive problem (Blount et al. 2018; Cerca 2023), sometimes to the point of exhibiting highly similar traits. Despite sharing their most recent common ancestor over 100 Mya (Foley et al. 2023), anteaters and pangolins evolved remarkably similar anatomy and behavior as a result of their independent adaptations for myrmecophagy. Indeed their striking similarities led anatomical systematic studies to almost invariably group them together in a taxon known as Edentata (Glass 1985; Reiss 2001; Springer et al. 2013). Indeed, we found that genes linked to their distinctive oral anatomy were lost in high numbers in both anteaters and pangolins, providing a genomic record for some of these commonalities (Figure 1). It was only with the advent of molecular phylogenetics that it became abundantly clear that these shared traits were actually the result of convergent evolution, with anteaters being the sister group to sloths, and pangolins recognized as the closest living relatives of carnivorans (Murphy et al. 2001; Delsuc et al. 2002).

However, while other convergent myrmecophagous mammals display similar anatomical modifications associated with this specific dietary habit, they are rarely as extreme in their morphology as these two clades. One hypothesis is that this is the result of more recent
adaptive forays into eating ants and termites, in which not enough time has elapsed for natural selection to fine-tune oral anatomy to the extremes found in pangolins and anteaters. For example, the termitivorous aardwolves (*Proteles* spp.) are hyaenids, belonging to a radiation of predominantly carnivorous mammals (Carnivora). The three other hyaenid species are characterized by powerful jaws and teeth, capable of crushing bones using specialized enamel, whereas aardwolves have small, peg-like teeth, which can vary in size and number, and possess thin enamel, with cheek teeth separated by intermolar diastemata (Koehler and Richardson 1990; Stefen and Rensberger 1999; Charles et al. 2013). Aardwolves consume termites with the aid of sticky saliva produced by large submaxillary glands, and possess broad, spatulate tongues that collect termites from soil surfaces. While clearly distinct from other hyaenid species, the retention of enamel-capped teeth, a relatively typical mammalian tongue, and powerful jaw muscles paired with robust zygomatic arches and jaws (Koehler and Richardson 1990), likewise contrasts aardwolves strongly with anteaters and pangolins. Aardwolves diverged from other hyaenids about 10 Mya (Koepfli et al. 2006; Hassanin et al. 2021), while the fossil aardwolf *Proteles transvaalensis*, which had larger teeth than *P. cristatus* and even retained functional carnassials (Hendey 1974), dates to about 2.6–0.8 Mya. Together, these suggest a relatively small temporal window for aardwolves to adapt to a myrmecophagous niche. In support of this hypothesis, we found evidence of only incipient pseudogenization of taste receptor genes in each aardwolf species (Figure 1).

In contrast with aardwolves, the highly specialized pangolins and anteaters show strong evidence of far more ancient forays into myrmecophagy. First, both crown clades originated around 40 million years ago (Gibb et al. 2016; Heighton et al. 2023), and their ancestors likely strongly resembled modern species, being completely edentulous, possessing reorganized jaw muscles and skull elements to facilitate rapid protrusion of a vermiform tongue covered in sticky saliva. Their respective stem lineages mirror each other in the parallel loss of eight dental genes and the sweet gustation gene (Figures 2 & 3). Furthermore, a high concentration of gene losses appears to have taken place soon after the K/Pg boundary (six in stem Pilosa, five in stem Pholidota), suggesting that at least some of these adaptations predated the crown clades by nearly 20 million years, occurring during the radiation of placental mammals in the wake of the end-Cretaceous mass extinction. However, analyzing the timing of gene losses reveals a pattern in which these ancient adaptations may not have been derived all at once, but instead occurred gradually over extensive periods of time.

For anteaters (Vermilingua), we have the benefit of a sister-group (sloths, Folivora), which possesses genomic signatures for some of the regressive evolutionary events in their
shared history (stem Pilosa). Furthermore, this ancestral branch length is relatively short (9.2 Myr; Gibb et al. 2016), allowing for some temporal precision. Five genes underlying enamel development possess inactivating mutations shared by anteaters and sloths, strongly suggesting that enamel was lost on the stem pilosan branch during the Paleocene (Emerling et al. 2023). Furthermore, sweet taste gustation likely was also lost on this ancestral branch (Figure 3). While the fossil record is silent on xenarthran cranial anatomy during this epoch, comparative analyses of chitinase genes (CHIAs) suggest that the earliest pilosans were highly insectivorous (Emerling et al. 2018). This, paired with enamelless teeth and reduced gustation, implies that myrmecophagy was the dietary habit of these ancestors. Following the split of sloths (Folivora) and anteaters (Vermilingua), 21 Myr elapsed until the origin of crown vermilinguans (Gibb et al. 2016). Stem vermilinguans likely completely lost their teeth, as evidenced by shared inactivating mutations in *DSPP*, which encodes a dentin matrix protein, and *ODAPH*, a gene correlated with the retention of teeth (Springer et al. 2016; Emerling et al. 2023). After the split of vermilinguans into pygmy (Cyclopedidae) and other anteaters (Myrmecophagidae), the last remaining dental gene as well as the masticatory myosin gene were inactivated independently in each lineage. Consistent with these genomic data, the earliest fossil myrmecophagid and cyclopedid anteaters, *Protamandua* (18–15.2 Mya) and *Palaeomyrmidon* (5–3 Mya), respectively, were both edentulous and possessed incomplete zygomatic arches (Gaudin and Branham 1998). Finally, in the terminal branches, evidence for additional incipient taste losses are suggested by polymorphic pseudogenes. Our genomic results suggest that adaptations for myrmecophagy in the lineages leading to modern anteaters have been ongoing for more than 60 million years, even continuing to the present day.

Our data on pangolins indicate that the pattern of protracted adaptation in anteaters may not be anomalous. Crown Pholidota dates to 41.3 Mya, diverging from their closest living relatives (Carnivora) 79.5 Mya (Heighton et al. 2023). While we can provide minimum dates for gene losses based on shared mutations, with at least 11 genes lost by 41.3 Mya, pseudogene dating estimates give an additional sense of how long these genes may have evolved neutrally (Figure 2). Our results suggest that two enamel genes were soon after their divergence with carnivorans (77–72.8 Mya), well before the K/Pg boundary. Next, we estimated that five gene losses occurred over a 5.1 Myr window (63.7–58.6 Mya), including dental genes, the masticatory myosin gene, and the sweet taste receptor gene. We dated the inactivation of two additional tooth genes between 54.7–54.1 Mya, with the gustatory PKD2L1 being inactivated 45.8 Mya. The dentin/tooth genes *DSPP* and *ODAPH* could not be dated beyond the LCA of Pholidota, but they affirm the complete absence of teeth in this ancestor. We inferred that most
pseudogenization events substantially predate the earliest fossil stem pangolins, all of which date to ~45 Mya (Figure 2) and were already edentulous (Storch 1978; Storch 1981; Gaudin et al. 2009; Gaudin et al. 2020). Despite this long history of myrmecophagy, subsequent inactivations of gustatory genes (TAS1R1, TAS1R3) occurred in parallel within Pholidota, as recently as 4.8–4.3 Mya. The progressive regression and rearrangement of oral anatomy tied to myrmecophagy is not uniquely suggested by DNA, but rather is further supported by analyses of fossil and extant pangolin crania. From *Eurotamandua* and *Eomanis* (45 Mya), to *Patriomanis* (37.7–33.9 Mya), *Necromanis* (28–14 Mya) and modern pangolins, there is a gradual sequence of reductions and/or losses of masticatory anatomy, including the mandibular coronoid and angular processes, the jugal bone and zygomatic arch (Gaudin et al. 2009; Gaudin et al. 2020).

These data raise questions about why such adaptations might occur over such extended periods, and whether it can help explain why other myrmecophagous taxa have oral anatomy that is not quite as extreme as those of anteaters and pangolins. One possibility is that there are other selection pressures acting on the maintenance of certain genes and their emergent phenotypes. Perhaps the most important of these concerns the maintenance of dietary breadth. Given that a complete commitment to myrmecophagy generally involves the loss and reduction of certain oral traits, corresponding with the inactivation and likely permanent loss of critical underlying genes, it may be extremely difficult to switch to certain alternative dietary niches.

For example, the bat-eared fox consumes large quantities of ants and termites, but frequently consumes other arthropods, and even vertebrate prey and plant material (Redford 1987; Clark Jr. 2005). The sloth bear uses mobile, protrusible lips, and lacks two upper incisors, which facilitate suction of ants and termites (Laurie and Seidensticker 1977), but it also ingests food as diverse as grass, fruits, potatoes, honey and other insects (Laurie and Seidensticker 1977). Long-nosed armadillos (*Dasypus* spp.), which commonly consume ants and termites, have diets that can vary geographically and by species, with other invertebrates, plant material and sometimes vertebrates being included in their diets (Redford 1986; Redford 1987; da Silveira Anacleto 2007; Superina and Abba 2014). Even the aardvark, a mammal highly committed to eating ants and termites, does not do so exclusively. This myrmecophage possesses an elongated tongue (up to 30 cm), enlarged mandibular glands that produce strongly adhesive saliva, weak jaw muscles and simplified, enamelless teeth (Shoshani et al. 1988; Davit-Béal et al. 2009). Fossils with enamelless teeth very similar to the extant aardvark date to 20–16 Mya (Pickford 1974; Lehmann 2009), with our pseudogene dating estimates pushing back enamel loss, and presumably myrmecophagy, to around 28.6–23 Mya (Figure 4). However, the retention of most gustatory genes, masticatory myosin and dentition may point to
848 the need for a more generalized, versatile anatomy in this species. Indeed, the aardvark has a
849 mutualistic relationship with the aardvark cucumber (Cucumis humifructus). This plant fruits
850 underground and the cucumbers are dug out and consumed by aardvarks, which then spread
851 their seeds upon burying their feces (Shoshani et al. 1988). These fruits presumably provide
852 water and essential nutrients that would otherwise be inaccessible if the oral anatomy of
853 aardvarks had regressed to the condition seen in anteaters and pangolins.

854 Competing selection pressures that lead to gradual oral regression may not be solely
855 linked to diet. As a unique example, the short-beaked echidna has perhaps the morphology
856 most comparable to pangolins and anteaters, including an elongated, vermiform tongue, sticky
857 saliva, a highly reduced mandible, reorganized jaw musculature and edentulous jaws (Rismiller
858 and Grutzner 2019). Fossils indicate that edentulism in Monotremata followed a protracted
859 period of dental reduction over >100 Myr (Flannery et al. 2022), with enamel gene loss
860 estimates dating to only 13.4–1.6 Mya (Figure 6). Such a long history of dental regression,
861 culminating in much more recent losses of teeth in the short-beaked echidna may be tied to the
862 fact that monotremes are the only extant oviparous mammals. To aid in their emergence from
863 calcified eggs, monotreme hatchlings use an egg ‘tooth’. A recent study characterized the egg
864 tooth of short-beaked echidnas (Fenelon et al. 2023), and found that this ‘tooth’ lacks enamel
865 but retains dentin. This critical function may have slowed and presumably prevented the loss of
866 certain genomic elements linked to tooth development (Davit-Béal et al. 2009), even though
867 echidnas are edentulous as adults.

868 Answers to how and why myrmecophagous mammals have evolved their specialized
869 anatomy over protracted periods of time may be as diverse as the lineages from which they
870 arose. While oral pseudogenes may reveal part of the picture, further insights will undoubtedly
871 be gained from further exploration of the genomes, transcriptomes, anatomies, and fossils of
872 these iconic examples of convergent evolution.

873
874 **MATERIALS AND METHODS**
875
876 **Candidate genes**
877 The genes selected for this project were based on studies that have demonstrated a strong link
878 between gene function and phenotype. Furthermore, these genes are all known to become
879 pseudogenes in at least some instances. For example, numerous genes are known to
880 participate in tooth development, but some do not degrade into pseudogenes in toothless
881 species, likely due to pleiotropy (Deméré et al. 2008; Springer et al. 2016; Emerling et al. 2023).
882 We placed the tooth genes into one of three categories based on their expression patterns and
functions inferred from association with human congenital diseases, mouse knockout studies, and interspecies comparisons (Meredith et al. 2013; Meredith et al. 2014; Smith et al. 2017; Randall et al. 2022; Emerling et al. 2023): 1) Enamel-development: AMELX (amelogenin), ENAM (enamelin), AMBN (ameloblastin), MMP20 (enamelysin), KLK4 (kallikrein-related peptidase 4), and ACP4 (acid phosphatase 4) (Lagerstrom et al. 1991; Rajpar et al. 2001; Kim et al. 2005; Meredith et al. 2009; Poulter et al. 2014; Seymen et al. 2015; Seymen et al. 2016); 2) Expression during both enamel development and throughout adulthood at the dento-gingival junction (i.e., junctional epithelium): AMTN (amelotin) and ODAM (odontogenic ameloblast-associated) (Nishio et al. 2010; Nakayama et al. 2015; Wazen et al. 2015; Smith et al. 2016; Springer et al. 2019); and 3) Dentin development and tooth retention: DSPP (dentin sialophosphoprotein) and ODAPH (odontogenesis-associated phosphoprotein) (Xiao et al. 2001; Parry et al. 2012; Springer et al. 2016; Emerling et al. 2023). KLK4 is specific to boreoeutherian mammals (Laurasiatheria + Euarchontoglires), so we only searched for orthologs in pertinent species (Kawasaki et al. 2014). Furthermore, we ignored exons 7–9 in AMBN and exon 2 in ODAPH due to these exons not being consistently present across mammals (Toyosawa et al. 2000; Springer et al. 2016). Finally, DSPP possesses a highly variable repetitive region towards the 3' end of exon 4 (McKnight and Fisher 2009; Fisher 2011), making this region difficult to assemble and align with confidence. As such, this region was often excluded from analyses.

While multiple genes are associated with gustation, we examined four genes with clear orthologs that are pseudogenized in at least some vertebrates (Zhao et al. 2010; Jiang et al. 2012; Zhao et al. 2012; Feng et al. 2014; Emerling 2017) and known to be linked to specific taste modalities: TAS1R1 (taste receptor type 1 member 1), TAS1R2 (taste receptor type 1 member 2), TAS1R3 (taste receptor type 1 member 3), and PKD2L1 (polycystic kidney disease 2-like 1). The protein TAS1R1 dimerizes with TAS1R3 to make the umami taste receptor, responding to certain amino acids and nucleotides and leading to the perception of savory or umami flavors. TAS1R2 likewise forms a heterodimer with TAS1R3, which instead detects sweet tastants, especially monosaccharides and disaccharides (Yarmolinsky et al. 2009; Roper and Chaudhari 2017). Although the specific function of PKD2L1 is unclear, this gene is expressed in type III cells, which facilitate the perception of sour tastants, making it a strong genetic marker for this taste modality (Tu et al. 2018; Liman and Kinnamon 2021).

Finally, we examined the jaw myosin gene, MYH16 (myosin heavy chain 16). While vertebrate jaw muscles can express several types of myosin proteins, MYH16, also known as the masticatory myosin, appears to be uniquely expressed in jaw-closing muscles. While the
916 gene itself has not been examined in much detail, outside of being a pseudogene in humans
917 (Stedman et al. 2004), the protein has been lost in multiple mammalian species (Hoh 2002; Lee
918 et al. 2019).

919
920 **Genomic dataset**
921 We used a mixture of publicly available genomes and newly generated data to compile gene
922 sequences from the focal myrmecophagous species, as well as from related 'outgroup' species
923 for comparison. Most sequences were taken from whole genome assemblies on NCBI's RefSeq
924 (Reference Sequence) and WGS (whole genome shotgun) databases (Supplementary Table
925 S1). Gene sequences for the following species were extracted from whole genome assemblies
926 generated by the ConvergeAnt project (Allio 2021; Bioproject ID: PRJNA1238091): the pale-
927 throated three-fingered sloth, *Bradypus tridactylus* (108x); giant anteater, *Myrmecophaga
928 tridactyla* (119x); southern tamandua, *Tamandua tetradactyla* (118x); pygmy anteater, *Cyclopes
929 didactylus* (91x); giant armadillo, *Priodontes maximus* (71x); pink fairy armadillo, *Chlamyphorus
930 truncatus* (92x; PRJNA1238091); six-banded armadillo, *Euphractus sexcinctus* (71x); and giant
931 pangolin, *Smutsia gigantea* (90x; now published, GCA_032199065.1 [Heighton et al. 2023]).

932 Details on the hybrid assembly procedure and genome annotation steps can be found in Allio et
933 al. (2021). The Southern tamandua (*Tamandua tetradactyla*), giant anteater (*Myrmecophaga
934 tridactyla*), and bat-eared fox (*O. megalotis*) hybrid genome assemblies were later upgraded to
935 chromosome-length by the DNA Zoo using complementary Hi-C data (Dudchenko et al. 2017;
936 Dudchenko et al. 2018) and re-annotated using the same strategy. Similarly, we used the DNA
937 Zoo annotations of the Hi-C genome assembly for the white-bellied tree pangolin (*Phataginus
938 tricuspis*). We also constructed a genomic library for numbat (*Myrmecobius fasciatus*;
939 PRJNA1268346) using a kit from SeqOnce Biosciences. The average insert size for the numbat
940 library was ~410 bp. Paired-end sequencing (150 bp) was performed on a NovaSeq platform at
941 the University of California San Francisco and generated 300 Gb of sequence, which
942 corresponds to ~88X coverage assuming a genome size of 3.42 Gb (Peel et al. 2022, doi:
943 10.46471/gigabyte.47). Finally, we used short read sequences downloaded from NCBI's SRA
944 (Sequence Read Archive) database for the Eastern aardwolf (*Proteles septentrionalis*;
945 SRX9615643; Allio et al. 2021) and the sloth bear (*Melursus ursinus*; ERX1025771; Kumar et
946 al. 2017).

947
948
949
950 **Gene assembly**

951 A critical goal of this study is to find inactivated genes that may have been evolving neutrally as 952 pseudogenes for perhaps tens of millions of years. This is a challenging task that requires a 953 diversity of approaches to extract ancient pseudogenes from both assembled genomes and, in 954 some cases, short read data. Conventional bioinformatic pipelines typically have limitations in 955 their capabilities to discern ancient, highly degraded pseudogenes, score certain inactivating 956 mutations or validate whole gene deletions. By using a diversity of approaches, including 957 BLAST searches at various thresholds and reference sequences, synteny, and read mapping to 958 target loci sequences, we were able to mine highly divergent inactivated gene loci.

959 To assemble these genes, we first obtained reference mRNA sequences from NCBI 960 (GenBank) for the targeted genes in our study, typically for *Homo sapiens*. When this was not 961 possible, we used alternative curated mammal mRNA sequences or gene models derived from 962 the NCBI eukaryotic genome annotation pipeline (Thibaud-Nissen et al. 2016). We imported the 963 full mRNA sequence into Geneious Prime (v2019.2.3; Kearse et al. 2012) and used the 964 annotations provided by GenBank to identify exon/intron boundaries and coding sequence 965 structure. Since we did not evaluate non-coding DNA in this study, we did not analyze the 966 structure of untranslated regions. As such, exon numbering throughout this study is based on 967 coding exons only.

968 Once we obtained the coding regions of the reference mRNA sequences, we used these 969 to obtain sequences derived from whole genome assemblies. We began by obtaining 970 sequences from NCBI's RefSeq and WGS databases by performing similarity searches with 971 BLAST (discontiguous megablast) using the mRNA reference sequence against target 972 assemblies. We obtained the hits with the highest E-values that likewise spanned the largest 973 portions of the reference mRNA sequence, especially those found towards the 5' and 3' ends. 974 We then downloaded the contig or scaffold regions encompassing the entire coding sequence 975 (CDS) and some flanking sequence (100+ bp) and imported them into Geneious. Next, we 976 performed automated sequence alignments using MUSCLE (ver 3.8.425; Edgar 2004) within 977 Geneious, aligning the reference mRNA against the contig or scaffold to identify coding exons. 978 Some genome assemblies also had exon annotations for comparison. In cases where 979 sequences had long strings of unknown bases (e.g., >50 Ns), all but ~10 Ns were deleted to 980 facilitate better sequence alignments. If after aligning the sequence the orthology appeared 981 dubious, we performed reciprocal BLAST (megablast) searches against NCBI's core nucleotide 982 database to confirm gene identity. This was validated a second time using gene tree analyses
983 (see below). After we obtained a whole gene sequence closely-related to a focal taxon, we then
984 used this in subsequent BLAST searches for a subset of representative species..

985 After extracting whole gene sequences from representative taxa from NCBI, these data
986 were used to mine targeted gene sequences locally from new genome assemblies. Once these
987 assemblies were imported into Geneious, we used a combination of BLAST searches and short-
988 read mapping to pull out gene sequences. BLAST searches followed the same basic
989 parameters as above in NCBI, but we also set BLAST results to return an additional 100-bp
990 flanking on each hit. For mapping, we used Minimap2 v2.24 (Li 2018) via the long-read spliced
991 alignment mode (with default parameters) as implemented in Geneious Prime v2022.1.1. This
992 splice-aware alignment enables the mapping of CDS, cDNA, or mRNA sequences against
993 reference genomic sequences containing introns by dividing the query protein-coding sequence
994 to its different mapping locations. The genomic regions that included both exons and
995 intercalating introns were then extracted for downstream analyses.

996 We used short read data derived from whole genome sequencing to assemble genes for
997 the sloth bear (Melursus ursinus; NCBI's SRA), a numbat (Myrmecobius fasciatus) specimen,
998 and the Eastern aardwolf (Proteles septentrionalis). For the sloth bear and numbat, we first
999 imported whole genome sequencing reads into Geneious, and obtained reference gene
1000 sequences from closely-related species (ursids and dasyurids, respectively) using BLAST
1001 methods on NCBI (WGS, RefSeq). We mapped the paired-end short reads to their respective
1002 reference sequences using the Geneious Prime mapper, with settings at Medium-Low
1003 Sensitivity/Fast with no fine-tuning (fast / read mapping), no sequence trimming and mapping
1004 multiple best matches randomly. Each mapping result was then examined by eye, and in cases
1005 of abundant erroneous mappings (i.e., nonhomologous sequences), we remapped those same
1006 reads at Low Sensitivity/Fastest up to five iterations, in which reads were mapped to the
1007 consensus from the previous iteration. Again, we examined each mapping alignment by eye and
1008 manually adjusted as needed.

1009 For the Eastern aardwolf (Proteles septentrionalis), paired-end short reads from SRA
1010 were mapped to orthologs from the Southern aardwolf (P. cristatus) using bowtie2 v2.3.4.3
1011 (Langmead and Salzberg 2012), with default parameters. Mapped reads were extracted from
1012 the bam mapping files and converted to fastq files with SAMtools v1.10 (Danecek et al. 2021).
1013 These reads were then imported into Geneious Prime and remapped against reference gene
1014 sequences for visualization.
1015
1016
1017 Sequence alignments and gene functionality analyses

1018 While obtaining orthologs for a single gene, we performed successive DNA sequence
1019 alignments with MUSCLE in Geneious, starting by aligning two closely related taxa, then
1020 aligning these two to a third, those three to a fourth, and so on. By doing this, we were able to
1021 anchor the alignment using highly similar sequences and then progressively add more divergent
1022 ones, thereby minimizing alignment errors. In addition, this allowed us to examine each
1023 alignment by eye to search for errors, a common issue when aligning divergent pseudogene
1024 and intron sequences. Each alignment generally only included orthologs from species belonging
1025 to a specific clade, to maximize our ability to determine homology.

1026 Next, we examined the gene structure for every ortholog to determine gene
1027 completeness. When data appeared to be missing, based on exon annotations from the
1028 reference mRNA, we executed additional BLAST searches with more relaxed parameters
1029 and/or alternative reference sequences. In some cases, there was evidence of partial or whole
1030 gene deletion, which required us to examine additional sequence data upstream or downstream
1031 from our original BLAST searches. In some cases where genes appeared to be deleted, we did
1032 synteny analyses to validate their absence. This involved two strategies used in tandem. First,
1033 we would examine the vertebrate-wide synteny comparisons provided in Genomicus v110.01
1034 (Nguyen et al. 2022) to determine which genes should be flanking the putatively deleted gene.
1035 In most cases, we then obtained NCBI RefSeq sequences from closely-related species that
1036 contained the gene of interest along with annotated genes upstream and downstream of the
1037 focal gene, comparing them to the Genomicus synteny maps. We then searched genome
1038 assemblies (NCBI RefSeq, WGS) for these flanking genes in the species appearing to lack the
1039 gene, and then downloaded and aligned these multigenic sequences to the related comparison
1040 species. If the resulting alignment showed a major gap where the focal gene should be, not
1041 explainable by an assembly gap indicated by N's, we considered this a whole gene deletion.

1042 Once our gene alignments were complete (Supplementary Dataset S1), we searched for
1043 evidence of the following categories of inactivating mutations: start codon mutations, frameshift
1044 insertions and deletions, premature stop codons, splice site mutations, alterations to the
1045 ancestral stop codon, exon-intron boundary deletions, and whole exon deletions. To search for
1046 premature stop codons and mutations in the ancestral start and stop codons, we generated
1047 alignments encompassing the coding sequences only. To do so, we removed all introns, deleted
1048 all frameshift insertions, and inserted Ns to restore the correct reading frame due to frameshift
1049 deletions.
1050 When we found evidence of at least one disabling mutation in a species, we typically 1051 attempted to rule out sequencing or assembly errors by using data from at least one of four 1052 different sources. First, if the sister taxon shared the mutation, we considered this validation that 1053 the mutation was inherited from a common ancestor (see below). Second, in some cases, 1054 alternative genome assemblies, sometimes from separate individuals, were available either in 1055 NCBI's WGS database or in our local datasets to validate the mutation. Third, for some NCBI- 1056 derived sequences, we BLASTed (megablast) the relevant regions against short reads in 1057 available experiments in SRA, downloaded them and mapped them in Geneious. Then we 1058 examined the short reads mapped to the mutation by eye. Finally, for others (Cyclopes 1059 didactylus, Proteles cristatus, Myrmecobius fasciatus), we mapped short reads using de novo 1060 sequence data on the gene sequence with bowtie2 or the Geneious mapper, as described 1061 above. Two individuals were used for P. cristatus (NMB12641 and NMB12667), one for C. 1062 didactylus (M2300) and three for M. fasciatus (NCBI PRJNA512907, PRJNA786364, 1063 PRJNA1268346). Together, these methods enabled us to confirm that an inactivating mutation 1064 was not the result of a sequencing or assembly error, and in a few cases determine whether a 1065 particular mutation was heterozygous or homozygous.

1066 In addition to characterizing inactivating mutations in individual species, we checked 1067 whether any were shared among two or more closely-related species. Such shared mutations 1068 are suggestive of loss of gene function in a common ancestor, thereby providing a minimum 1069 pseudogenization date. The only shared mutations were found in xenarthrans and pangolins, so 1070 we referred to the phylogenies of Gibb et al. (2016) for the former and Heighton et al. (2023) for 1071 the latter. Most of the gene sequences we collected for pangolins were from five species only 1072 (Smutsia gigantea, Phataginus tricuspis, Manis pentadactyla, M. javanica, and M. 1073 crassicaudata), because genome assemblies for S. temminckii, P. tetradactyla and M. 1074 culionensis were not available until we were far into our analyses. These new assemblies were 1075 rendered largely redundant by our initial sample of five species due to the presence of shared 1076 inactivating mutations in all five, Asian pangolins only (Manis spp.) or African pangolins only 1077 (Phataginus spp., Smutsia spp.), representing the deepest splits in Pholidota. For the subset of 1078 candidate genes that did not show evidence of loss on these early branches, we obtained, 1079 aligned and examined the orthologs for the remaining three pangolin species. 1080 1081 1082 1083
1084 **Selection analyses based on dN/dS and gene inactivation dating**

1085 Given the phylogenetic distribution of gene losses during the xenarthran radiation (see Results
1086 below), we were able to provide minimum date estimates of gene inactivation based on patterns
1087 of shared mutations in this clade. However, for other myrmecophagous taxa, there are long
1088 branches on which genes were lost as autapomorphies, which do not permit any resolution of
1089 the relative timing of inactivation events in different genes. As such, we estimated the timing of
1090 pseudogenization in the remaining species that possess fixed (i.e., not polymorphic) inactivating
1091 mutations (pangolins, the aardvark, short-beaked echidna, numbat) by employing the dN/dS-
1092 based method of Meredith et al. (2009).

1093 To begin with, for each gene, we aligned functional orthologs from outgroup taxa
1094 (Supplementary Table S2) and modeled their evolution over a fixed phylogeny (Supplementary
1095 Dataset S2, Supplementary Tables S3-S17), to estimate the expected background ω value
1096 when the gene is under functional constraint (“functional” branches). These orthologs were
1097 provided from annotated mRNAs and predicted mRNA models from GenBank, the latter of
1098 which are derived from NCBI’s Eukaryotic Genome Annotation Pipeline
1099 (https://www.ncbi.nlm.nih.gov/genome/annotation_euk/process/). Our taxonomic sampling for
1100 outgroup taxa was broadly representative of major lineages of mammals, with the following
1101 clades being represented, when possible, for every dN/dS analysis (Supplementary Table S2):
1102 Marsupialia: Didelphimorphia, Microbiotheria, Dasyuromorphia, Diprotodontia; Afrotheria:
1103 Afrosoricida, Macroscelidea, Sirenia, Proboscidea; Euarchontoglires: Scandentia, Dermoptera,
1104 Primates, Lagomorpha, Rodentia; Laurasiatheria: Eulipotyphla, Chiroptera, Perissodactyla,
1105 Carnivora, Cetartiodactyla. In some cases, the targeted genes were not available from some
1106 outgroup taxa, perhaps due to genome assembly gaps, being a heavily degraded pseudogene
1107 or the entire gene having been deleted. The Meredith et al. (2009) method assumes that
1108 outgroup orthologs are functional, so we also examined the annotations of predicted mRNA
1109 models for information suggestive of pseudogenization. At NCBI, a gene’s product is given a
1110 “LOW QUALITY PROTEIN” designation when frameshift indels and/or premature stop codons
1111 are ‘corrected’ during the construction of the gene model. Any such gene models were removed
1112 from the dN/dS analysis dataset. The final datasets had as few as nine outgroup species (KLK4)
1113 and as many as 18 (median of 17; Supplementary Table S2).

1114 These outgroup sequences were imported into Geneious, where we removed any
1115 portions of the gene models outside of the coding sequence annotation, and then performed
1116 translation alignments (MUSCLE), which we then examined by eye. We manually removed stop
1117 codons, taxon-specific predicted exons and insertions, and any codons with questionable
1118 homology (e.g., straddling a deletion with ambiguous homology). Next, we performed a
1119 maximum likelihood phylogenetic analysis using RAxML (Stamatakis 2014; v8.2.11;
1120 GTR+GAMMA model, Rapid hill-climbing) to validate orthology of the genes by comparing
1121 topology and branch length to what is predicted by published species trees (Supplementary
1122 Figure S1).

1123 We then analyzed the outgroup taxa for each candidate gene using PAML v4.8 codeml
1124 analyses (Yang 2007) to determine the best fit codon frequency model. For placental mammal
1125 relationships, we implemented the topology from Fig. 4a in Foley et al. (2023), and for marsupial
1126 relationships, we referred to Mitchell et al. (2014). We then performed one ratio model analyses
1127 using the 1/61 each, F1X4 and F3X4 codon frequency models and determined the best fit using
1128 the Akaike information criterion. The best fitting model was then implemented for subsequent
1129 taxon-specific pseudogene dating analyses (Supplementary Table S3–S17).

1130 To estimate gene inactivation dates for specific taxa, we ran separate analyses for
1131 pangolins, the aardvark, the short-beaked echidna and the numbat, respectively
1132 (Supplementary Dataset S2). In each case, coding sequences of focal taxa were aligned to the
1133 corresponding outgroup alignment (MUSCLE, Translation align). Each resulting alignment was
1134 examined by eye, and we manually removed stop codons, partial codons resulting from
1135 frameshift deletions, lineage-specific insertions and any codons with ambiguous homology. We
1136 then implemented branch models in codeml of PAML to estimate the ω values for the branches
1137 on which pseudogenization occurred (“mixed” branches) and, when relevant (i.e., most pangolin
1138 analyses), branches on which selection is expected to be completely relaxed (“pseudogene”
1139 branches). We then entered the relevant ω categories into the calculations provided by
1140 Meredith et al. (2009), utilizing divergence times provided by different studies.

1141 For pangolins, we usually only included four species in the analyses: *Smutsia gigantea*,
1142 *Phataginus tricuspis*, *Manis pentadactyla* and *M. javanica* or *M. crassicaudata*. Since these taxa
1143 represent the deepest divergences within Pholidota, they are probably sufficient for generating
1144 dN/dS estimates for genes lost on the stem Pholidota branch. Given the lack of shared
1145 inactivating mutations in *TAS1R1* and *TAS1R3*, we included all eight pangolin species in those
1146 analyses. We implemented the topology from Fig. 2 of Heighton et al. (2023), and set the
1147 following branch categories: the “functional” background branches received one ω, the “mixed”
1148 branch(es), on which gene inactivation likely occurred, received its own ω, and then the
1149 branches that post-dated the pseudogenization event (“pseudogene”) received their own ω.
1150 Though the “pseudogene” branches are expected to have a ω of 1 (relaxed selection), we ran a
1151 second model in which the “pseudogene” branches were fixed at 1. We compared the two
models with a likelihood ratio test, and if there was no statistically significant difference between the models, we assumed the “pseudogene” ω was 1 when performing pseudogene dating calculations. By contrast, if there was a significant difference in models, we used the estimated ω for the calculations. Divergence times used for these calculations were likewise derived from Fig. 2 of Heighton et al. (2023).

For the aardvark, short-beaked echidna, and numbat dN/dS models, the branch leading to the focal taxon always represented the “mixed” branch, and all other branches were “functional” branches. No branches were designated as “pseudogene” branches, so the pseudogene ω was assumed to be 1. For the aardvark, we assumed that its lineage split from other afroinsectiphilian mammals 66.3 Myr ago (Foley et al. 2023, neutral 241 species average). For the short-beaked echidna, we assumed it diverged from the platypus (Ornithorhynchus anatinus) 56.4 Mya and monotremes from therian mammals at 184.7 Mya (Dos Reis et al. 2012). Since the platypus shows evidence for relevant gene losses (Zhou et al. 2021), we obtained orthologs by interrogating genome assemblies for this species using the methods described above and included these sequences in dN/dS analyses. Finally, for the numbat we assumed a divergence from Dasyuridae 36.5 Mya (Westerman et al. 2016).

For each taxon set (pangolins, aardvark, echidna, numbat), we performed dN/dS estimates for cases in which there was no evidence of gene loss on the branch of interest, to compare patterns of dN/dS estimates in pseudogenes versus putatively functional orthologs as controls. We also compared each of these to models in which the focal branch ω was fixed as part of the background. This allowed us to test whether the dN/dS was statistically distinguishable from the background, as determined by a likelihood ratio test.

## 1175 ACKNOWLEDGEMENTS

This is dedicated to Averie Jeanne-Lorraine 玉蘭. We thank Lionel Hautier for insightful discussions and comments on the manuscript, and two anonymous reviewers for their constructive feedback. This work has been supported by grants from the European Research Council (ConvergeAnt project: ERC-2015-CoG-683257; FD) and Investissements d'Avenir of the Agence Nationale de la Recherche (CEBA: ANR-10-LABX-25-01; CEMEB: ANR-10-LABX-0004; FD); a National Science Foundation Postdoctoral Research Fellowship in Biology (award no. 1523943; CAE); a National Science Foundation Postdoctoral Fellow Research Opportunities in Europe award (CAE); the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. PCOFUND-
1185 GA-2013-609102, through the PRESTIGE programme coordinated by Campus France (CAE).

1186 This is contribution ISEM 2025-XXX of the Institut des Sciences de l'Evolution de Montpellier.

1187

1188 **Data availability statement**

1189 Supporting data are available from zenodo.org (https://doi.org/10.5281/zenodo.14895250).

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