bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Multiple evolutionary origins and losses of tooth

2 complexity in squamates

3

4 Fabien Lafuma*a, Ian J. Corfe*a, Julien Clavelb,c, Nicolas Di-Poï*a

5

6 aDevelopmental Biology Program, Institute of Biotechnology, University of Helsinki, FIN-

7 00014 Helsinki, Finland

8 bDepartment of Life Sciences, The Natural History Museum, London SW7 5DB, United

9 Kingdom

10 cLaboratoire d’Écologie des Hydrosystèmes Naturels et Anthropisés (LEHNA), Université

11 Claude Bernard Lyon 1 – UMR CNRS 5023, ENTPE, F-69622 Villeurbanne, France

12

13 *Mail: [email protected]; [email protected]; [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

14 Teeth act as tools for acquiring and processing food and so hold a prominent role in

15 vertebrate evolution1,2. In mammals, dental-dietary adaptations rely on tooth shape and

16 complexity variations controlled by cusp number and pattern – the main features of the

17 tooth surface3,4. Complexity increase through cusp addition has dominated the

18 diversification of many mammal groups3,5-9. However, studies of Mammalia alone don’t

19 allow identification of patterns of tooth complexity conserved throughout vertebrate

20 evolution. Here, we use morphometric and phylogenetic comparative methods across

21 fossil and extant squamates (“lizards” and snakes) to show they also repeatedly evolved

22 increasingly complex teeth, but with more flexibility than mammals. Since the Late

23 Jurassic, six major squamate groups independently evolved multiple-cusped teeth from a

24 single-cusped common ancestor. Unlike mammals10,11, reversals to lower cusp numbers

25 were frequent in squamates, with varied multiple-cusped morphologies in several groups

26 resulting in heterogenous evolutionary rates. Squamate tooth complexity evolved in

27 correlation with dietary change – increased plant consumption typically followed tooth

28 complexity increases, and the major increases in speciation rate in squamate evolutionary

29 history are associated with such changes. The evolution of complex teeth played a critical

30 role in vertebrate evolution outside Mammalia, with squamates exemplifying a more

31 labile system of dental- dietary evolution.

32 As organs directly interacting with the environment, teeth are central to the acquisition and

33 processing of food, determine the achievable dietary range of vertebrates1, and their shapes are

34 subject to intense natural selective pressures8,12. Simple conical to bladed teeth generally

35 identify faunivorous vertebrates, while higher dental complexity – typically a result of more

36 numerous cusps – enables the reduction of fibrous plant tissue and is crucial to the feeding

37 apparatus in many herbivores4,8,13. Evidence of such dental-dietary adaptations dates back to the

38 first herbivorous tetrapods in the Palaeozoic, about 300 million years ago (Ma)13. Plant bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

39 consumers with highly complex teeth have subsequently emerged repeatedly within early

40 synapsids13, crocodyliforms14, dinosaurs15, and stem- and crown-mammals6,7,9,16. Since the

41 earliest tetrapods had simple, single-cusped teeth8, such examples highlight repeated,

42 independent increases of phenotypic complexity throughout evolution17. Many such linked

43 increases in tooth complexity and plant consumption have been hypothesised to be key to

44 adaptive radiations6,9, though such links have rarely been formally tested. It is also unclear

45 whether the known differences in tooth development between tetrapod clades might result in

46 differences in the evolutionary patterns of convergent functional adaptations18,19.

47 To understand the repeated origin of dental-dietary adaptations and their role in vertebrate

48 evolution, we investigated tooth complexity evolution in squamates, the largest tetrapod

49 radiation. Squamata is recognized for including species bearing complex multicuspid teeth

50 within heterodont dentitions20, and squamate ecology spans a broad range of past and present

51 niches. Squamates express dental marker genes broadly conserved across vertebrates18, with

52 varying patterns of localization and expression compared to mammals, and structures at least

53 partially homologous to mammalian enamel knots (non-proliferative signalling centres of

54 ectodermal cells) determine tooth shape in some squamate clades19,21,22. In mammals – the most

55 commonly studied dental system – novel morphologies arise from developmental changes in

56 tooth morphogenesis23. Epithelial signalling centres – the enamel knots – control tooth crown

57 morphogenesis24, including cusp number and position and ultimately tooth complexity, by

58 expressing genes of widely conserved signalling pathways18,25. Experimental data show most

59 changes in these pathways result in tooth complexity reduction, or complete loss of teeth25, yet

60 increasing tooth complexity largely dominates the evolutionary history of mammals6-9,16. To

61 determine whether similar patterns of tooth complexity underlie all tetrapod evolution or are

62 the specific results of mammalian dental development and history, we used morphometric and

63 phylogenetic comparative methods with squamate tooth and diet data. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

64 We analysed cusp number and diet data for 545 squamate species spanning all living and extinct

65 diversity and identified species with multicuspid teeth in 29 of 100 recognized squamate

66 families (Figure 1a & Extended Data Figure 1). Within extant “lizards”, we found multicuspid

67 species in almost 56% of families (24/43). While lacking entirely in mostly predatory clades

68 (dibamids, geckos, snakes), multicuspidness dominates Iguania and Lacertoidea, the two most

69 prominent groups of plant-eating squamates. A Kruskal–Wallis test and post-hoc pairwise

70 Wilcoxon–Mann–Whitney tests show squamate dietary guilds differ statistically in tooth

71 complexity, with the proportion of multicuspid species and cusp number successively

72 increasing along a plant consumption gradient, from carnivores to insectivores, omnivores, and

73 herbivores (p-value < 0.001; Fig. 1b, Extended Data Table 1). We quantified tooth outline shape

74 in a subset of taxa spanning all major multicuspid groups with two-dimensional semi-

75 landmarks, which showed the teeth of herbivores are more protruding with a wider top cusp

76 angle (Fig. 1c). A regularized phylogenetic multivariate analysis of variance (MANOVA) on

77 principal component scores confirm statistically significant differences between diets overall

78 (p-value = 0.001; Fig.1c) with negligible phylogenetic signal in the model’s residuals (Pagel’s

79  = 0.03). Herbivore teeth differ from both the insectivorous and omnivorous morphospace

80 regions (Fig.1c, Extended Data Table 2), similarly to observations from mammals and

81 saurians4,20. Furthermore, we find support for shifts in the rate of evolution of tooth shape

82 outline independent of cusp number among the 75 species examined (log Bayes Factor = 319),

83 with particularly high rates characterising Iguanidae (Extended Data Figure 2).

84 Using Maximum-Likelihood reconstructions of ancestral character states26 across our squamate

85 phylogeny (Fig. 2, Extended Data Figure 3 and 4, Supplementary Table 1 and 2), we found

86 dental-dietary adaptations to plant consumption repeatedly evolved, arising from the

87 convergent evolution of multicuspidness. Since the Late Jurassic, six major clades and 18

88 isolated lineages independently evolved multicuspid teeth from a unicuspid ancestral bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

89 morphology, mostly through single-cusp addition events. Similar numbers – 10 clades, 13

90 isolated lineages – show independent origins of plant consumption from carnivorous or

91 insectivorous ancestors (Fig. 2, Supplementary Table 3). Across the tree, most lineages and

92 terminal taxa are unicuspid insectivores retaining the reconstructed ancestral squamate

93 condition. However, of 102 lineages showing cusp number or plant consumption increases, 42

94 (41%) of increases are along the same phylogenetic path than an increase in the other character

95 (see Methods).

96 Squamate dental evolution was however labile and included repeated reversals towards lower

97 tooth complexity. Both complexity and diet changed similarly through much of squamate

98 evolution, though there were more changes in diet than complexity (115 vs. 92 lineages), and

99 reversals in diet were more common than for complexity (56% vs. 44%) (Fig. 3, Extended Data

100 Figure 3). Such flexibility is reflected in the reconstructed transition rates underlying our models

101 of evolution for tooth complexity and diet, where higher relative rates characterise decreases in

102 cusp number and plant consumption compared to increases (Extended Data Figure 4,

103 Supplementary Table 1 and 2). Moreover, 38% of inferred complexity decreases were due to

104 the simultaneous loss of two cusps or more, while multiple-cusp addition events were half

105 (20%) as frequent. We identify two lineages (genera Gallotia and Phrynosoma) in which

106 multicuspid teeth re-evolved following earlier loss (Fig. 2, Extended Data Figure 3). Most often,

107 reversals to lesser cusp numbers followed a decrease in plant consumption (52% of paired

108 events; Supplementary Table 3), likely resulting from the relaxation of selective pressures for

109 plant consumption.

110 Furthermore, we find the observed dental-dietary patterns derive from the correlated evolution

111 of tooth complexity and plant-based diets under highly variable rates of phenotypic evolution.

112 Our results show strong support for a correlated model of the evolution of multicuspidness and

113 plant consumption, which assumes transition rates in one trait directly depend on character state bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

114 in the other trait (log Bayes Factor = 21, Supplementary Table 4). Additionally, a model with

115 heterogenous character transition rates throughout the tree better fits the macroevolutionary

116 pattern of each trait than a constant model, with the highest rates observed resulting in a

117 relatively balanced mixture of tooth complexity and diet character states for the clades

118 concerned (e.g., Lacertidae) (Figs. 2, 4a, 4b, Supplementary Table 5).

119 These evolutionary increases in tooth complexity and plant consumption appear to have

120 contributed to the diversification of Squamata. Using models with variable rates of

121 diversification implemented in a Bayesian framework (through a reversible jump Markov Chain

122 Monte Carlo algorithm) and allowing for the inclusion of fossil taxa, we identified multiple

123 events of increased speciation (13 events in the studied tree with up to eight-fold magnitude for

124 the focal group versus its outgroup; Fig. 4c, Extended Data Table 3). Five speciation increases

125 coincide exactly with increases in tooth complexity or plant consumption, and three more are

126 just one node away from such increases. The equivalent results for decreases are two and one

127 lineage respectively (Extended Data Table 3, Supplementary Table 6). We further tested this

128 apparent association between diversification shifts and transitions of cusp number and diets

129 using a “hidden state” trait-dependent model of speciation and extinction. Results from this

130 model suggest each trait (tooth complexity and diet) contributed considerably to the group’s

131 diversification – with rates of speciation and extinction increasing with transitions to

132 multicuspidness or plant-based diets – despite the influence of unobserved factors beyond our

133 study (Extended Data Table 4 and Figure 5). Combining these results, we propose plant

134 consumption and tooth complexity changes – principally increases – were critical innovations

135 for squamate evolution.

136 The evolution of tooth complexity in Squamata encompasses multiple independent radiations

137 defined by increasingly complex teeth. This mirrors patterns of mammalian diversification, in

138 which stem-mammals show repeated independent evolutions of multicuspid teeth through the bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

139 Palaeozoic and Mesozoic7,9,13, the key adaptations of tribosphenic or pseudo-tribosphenic

140 molars separately originated in the Jurassic27, and quadritubercular molars with a hypocone

141 appeared multiple times in the Cenozoic3,5,6. It differs from the mammalian pattern, however,

142 in that the most recent common ancestor (MRCA) of Mammalia was multicuspid7, whereas we

143 reconstruct the MRCA of Squamata as unicuspid and infer at least 24 independent acquisitions

144 of multicuspidness in squamate lineages. Squamate tooth evolution was also not mainly

145 unidirectional as in mammals, with numerous lineages losing tooth complexity, including

146 reversals to the ancestral unicuspid condition. Moreover, tooth complexity at times

147 subsequently re-emerged within lineages that previously underwent such reversals, in

148 opposition to Dollo’s law28. Despite the lack of a similar large-scale phylogenetic assessment,

149 studies suggest relatively few mammalian lineages experienced reversals towards reduced tooth

150 complexity (including complete tooth loss)10,11,29, and even fewer re-evolved cusps once lost30.

151 We confirm here across the whole of Squamata the link noted previously between plant-eating

152 squamates and a specialized, typically more complex dentition20, similar to those hypothesized

153 or discovered for early tetrapods13, crocodyliforms14, and mammals4. The generality of these

154 findings suggests similar ecological and dietary selective pressures for complex dental

155 phenotypes operate across all tetrapods. We find strong support for correlated evolution of

156 multicuspidness and plant consumption, both of which promoted increased diversification of

157 several major squamate groups (e.g., Pleurodonta, Polyglyphanodontia), and propose

158 environmental factors such as the floral turnovers of the Cretaceous Terrestrial Revolution

159 (KTR – 125–80 Ma)31,32 and Cenozoic33 are the most plausible drivers of increasing plant

160 consumption in squamate evolution34. During the KTR, squamate speciation locally peaked, net

161 turnover was highest until the Late Miocene, and extinction was overall highest. All three

162 metrics drop prior to and across the Cretaceous-Paleogene (K-Pg) boundary, suggesting the K-

163 Pg extinction event had less of an effect on squamate diversification than the KTR. These KTR bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

164 diversification shifts coincide with the majority of the only period where reductions in both

165 tooth complexity and plant consumption outnumber increases, suggesting a rapidly shifting set

166 of available dietary niches as previously proposed for mammals of the same time35. Reductions

167 of squamate cusp number most often followed plant consumption reductions, suggesting

168 relaxed selective pressures on diet enabled the loss of tooth complexity, as with mammals11,29.

169 However, selective pressures on squamate teeth may not be as intense as for mammals. Most

170 plant-eating squamates still consume insects36, suggesting that, unlike in Mammalia, no hyper-

171 specialist ratchet operated37,38.

172 The patterns of squamate dental complexity evolution we observe offer a valuable counterpoint

173 to the mammalian picture, exemplifying dental-dietary adaptations that responded to similar

174 selective pressures, while resulting in more labile dental complexity throughout evolution.

175 Despite vertebrates sharing a basic tooth gene-network18, mammal teeth are more integrated

176 structures, less prone, through intense selective pressures, to loss of complexity, though also

177 capable of accumulating significantly more variance and reaching farther phenotypic extremes

178 over time39. Since such finely tuned dental morphologies and precise occlusion have a critical

179 role in ensuring mammals meet their high energy needs8, endothermy may limit the possibilities

180 of mammalian dental simplification compared to ectothermic squamates. Several dental

181 developmental differences to mammals can be suggested to explain why squamates didn’t fall

182 into a developmental complexity trap40, but instead evolved complex teeth highly liable to

183 developmental instability and simplification41. These include simpler, less compartmentalised

184 expression of dental development genes during tooth formation19,22, a less complex

185 morphological starting point than mammal teeth7, and potentially simpler and/or looser gene

186 regulatory networks18. We propose these characteristics of squamates explain both the

187 evolutionary lability of their dental complexity and diet, and the near-complete absence of

188 mammal-like teeth in over 250 million years of squamate history42. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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a Cusps Polyglyphanodontia Sc P inc 1 2 3 >3 olyglyphanodontidae oid a ea ide Gymnophthalmidae rto ce e.g. La Dibamia

†

Rhineuridae †

T T Bipedidae Amphisbaenidaerogonophidae eiidae Gekkota Blan usiidae

†

Eolacertidae Piramicephalosauridae † a Slavoiidae i Scincidae r idae Car u Xantusiidae G a Dolichosauridae † Gerrhosauridae e s Cordylidae k a Lacertidae k s Paramacellodidaeonidae † o o Aigialosaur Dibamidae t Dibamia M † a Gekk Mosasauridae † Phyllodactylidae idae idae † Sphaerodactylidae Madtsoiidae Eublephar Scincoidea Leptotyphlopidae Pygopodidae † Carphodactylidae Typhlopidae Diplodactylidae Anomalepididae Eichstaettisauridae † † Polyglyphanodontia † achtleri Pachyophidae † Megachirella w Aniliidae Sphenodon punctatus † Gephyrosaurus bridensis Uropeltidae Lacertoidea Sophineta cracoviensis † 50 My Iguanidae Cylindrophiidae Tropidur idae Anomochilidae Crotaphytidae Mosasauria † Xenopeltidae Hoplocercidae Loxocemidae Polychrotidae Liolaemidae Pythonidae Boidae Leiosaur Serpentes Opluridae Bolyeridae Corytophanidaeidae Leiocephalidae Dactyloidae AcrochordidaePareidae Phr iperidae Tem V ynosomatidae Anguimorpha Agamidae Chamaeleonidae ujiniidae † Pr Elapidae Anguidae Diploglossidae

Heloder Homalopsidae icidae iscagamidae

Natr idae

anidae Lamprophiidae Colubridae

matidae Iguania ar

V

† Lanthanotidae

Shinisauridae Necrosauridae † Xenosaur

0 20 40 60 80 100 Relative species proportion (%) b c 4 100 Carnivores InsectivoresOmnivores Herbivores

80 2

60

0

40 Principal Component 2 (8.08%) Carnivores Relative species proportion (%) Insectivores 20 -2 Omnivores Herbivores

-4 -2 0 2 4 6 8 0 Principal Component 1 (80.82%) bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

283 Fig.1 | The diversity of squamate dental morphologies correlates with a gradient of plant

284 consumption. a, Relative proportions (%) of tooth complexity levels in all known squamate

285 suborders/super-families (left) and 76 families (right) based on cusp number data for 545 living

286 and extinct species (including the most ancient known squamate Megachirella wachtleri), two

287 rhynchocephalians, and the stem-lepidosaurian Sophineta cracoviensis, with example teeth for

288 each complexity level redrawn from microCT-scan data (not to scale). b, Relative proportions

289 (%) of tooth complexity levels in 545 squamates sorted by diet. c, Discrete Cosine Transform

290 analysis of multicuspid tooth labial view profiles from 75 extant and fossil squamate species,

291 with 95% confidence ellipses for insectivorous, omnivorous, and herbivorous species.

292 Theoretical tooth profiles at the extreme positive and negative values of each axis reconstructed

293 from the first 21 harmonic coefficients. Scalebar = 50 million years (My). † = extinct taxon.

294 Silhouettes: the authors, Phylopic, and public domain (see Methods for license information). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Number of cusps 1 2 3 >3

Diet S cin s.l. co Insectivorous idea ide eiio a Omniv T Ng ea Ge id rr orous to h orous r os ce Pg a a ur L id Herbiv ae

Dibamia K . .l s 1 e a id t r e c 2 G a 3 L e k J k o t a

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4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

295 Fig.2 | Multiple independent acquisitions of multicuspid teeth and plant consumption are

296 found across the squamate phylogeny. Known and Maximum-Likelihood ancestral state

297 reconstructions of the number of cusps (branch colours) and diet (node pie charts and branch

298 tip small circles) in squamates. Pie charts indicate the most ancient nodes with >50% combined

299 relative likelihood for omnivorous and herbivorous diets; also shown are the first nodes with

300 >50% relative likelihood for herbivory within already omnivorous clades. Branch tip circles

301 indicate omnivorous/herbivorous species. Six major clades showing independent originations

302 of multicuspid teeth – 1: Gerrhosauridae. 2: Teiioidea + Polyglyphanodontia (informally

303 Teiioidea sensu lato). 3: total group Lacertidae (informally Lacertidae sensu lato). 4:

304 Chamaeleonidae. 5: non-Uromastycinae agamids (informally Agamidae sensu stricto). 6: total

305 group Pleurodonta. P: Permian. Tr: Triassic. J: Jurassic. K: Cretaceous. Pg: Paleogene. Ng:

306 Neogene. Scalebar = 10 million years. Silhouettes: the authors, Phylopic, and public domain

307 (see Methods for license information). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Cusp number decrease Cusp number increase 30

20 KTR Lineages 10

0 Jurassic Cretaceous Paleogene Neogene Q 0

10

20 KTR Lineages 30 Plant consumption decrease Plant consumption increase bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

308 Fig.3 | Dynamics of squamate tooth complexity and plant consumption evolution over the

309 last 165 million years. Lineages showing increasing (n = 61) or decreasing (n = 31) tooth

310 complexity (top) and increasing (n = 51) or decreasing (n = 64) plant consumption (bottom) per

311 ten million year-time bins. KTR: Cretaceous Terrestrial Revolution (125–80 Ma). Q:

312 Quaternary. Decreases in both cusp number and plant consumption proportion first outnumber

313 increases during the Cretaceous Terrestrial Revolution (KTR), while the Cretaceous–Paleogene

314 boundary (in red) shows the change towards the Cenozoic pattern of approximately twice as

315 many cusp increases as decreases, and similar numbers of plant consumption increase and

316 decrease from the Paleogene on. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b log10(scalar) Unicuspid log10(scalar) Predator Multicuspid Plant consumer Sci 2 Sci nco nco 2 ide ide a Ng a a Ng a de 1 de oi oi 1 rt rt ce Pg ce Pg a a L 0 L 0 Dibamia Dibamia K K 1 1

G G 3 e 3 3 e 2 k 2 k J k J k o o a a i t i t r a r a u u Rhynchocephalia Rhynchocephalia

a a s Tr s Tr a a

s s

o o

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6 6 5 5

4 4

Mosasauria ntes c Serpe Speciation rate shift Lac ert 8.0x oid ea a ph G or m D ui 4.0x ng A 2.0x 1.0x

-1 Speciation rate (My ) H 0.34 F 3 B S 0.15 c in c o 4 E id 0.041 C e a

0.0025 ia n a 2 u 5 g I K K–Pg KTR 0.08 Dibamia 66/I 1 J 0.06

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k Rhynchocephalia Extinction/ L 0.04 k o

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a 0.02 A Net turnover rate

Speciation/ 250 200 150 100 50 0 M 0.00 P Tr J K Pg Ng d Time before present (m.y.) bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

317 Fig.4 | Dental-dietary rates of phenotypic evolution and squamate macroevolution. Log-

318 transformed averaged rate scalars of the character transition rates of tooth complexity (a) and

319 diet (b) across squamates. Positive values (i.e., rate scalar > 1) indicate increased relative

320 transition rates. c, Rates of squamate speciation for one maximum shift credibility configuration

321 (MSC) out of ten similar independent replicates. 13 rate shifts (A–M) present in at least five

322 MSC replicates are indicated proportionally to their magnitude compared to the background

323 rate. d, Mean rates of squamate speciation, extinction, and net turnover through time (in My-1).

324 1: Gerrhosauridae. 2: Teiioidea + Polyglyphanodontia (informally Teiioidea sensu lato). 3: total

325 group Lacertidae (informally Lacertidae sensu lato). 4: Chamaeleonidae. 5: non-Uromastycinae

326 agamids (informally Agamidae sensu stricto). 6: total group Pleurodonta. P: Permian. Tr:

327 Triassic. J: Jurassic. K: Cretaceous. Pg: Paleogene. Ng: Neogene. Q: Quaternary. KTR:

328 Cretaceous Terrestrial Revolution (125–80 Ma). K–Pg: Cretaceous–Paleogene extinction event

329 (66 Ma). Scalebars = 50 million years. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

330 Methods

331 Phylogenies

332 We gathered our own observations and reports from the literature on cusp number and diet for

333 548 species (429 extant species and 119 fossil species equally distributed between Mesozoic and

334 Cenozoic). The data include all major squamate groups plus squamate stem taxa – including

335 the oldest known squamate Megachirella wachtleri, two rhynchocephalians (the extant

336 Sphenodon punctatus and the fossil Gephyrosaurus bridensis), and a stem-lepidosaurian

337 (Sophineta cracoviensis). To provide a phylogenetic framework for our analyses, we assembled

338 an informal super-tree43 for the 548 taxa. For topology we followed the total evidence phylogeny

339 of Simões et al.44 – the first work to find agreement between morphological and molecular

340 evidence regarding early squamate evolution. The same source provided time calibrations for

341 Sophineta cracoviensis, fossil and extant Rhynchocephalia, stem squamates and crown

342 squamate groups. Using additional sources, we gathered complementary information on the

343 stem and crown of Gekkota45-47, Dibamia47, Scincoidea46,47, Lacertoidea46-52 including

344 Polyglyphanodontia46,53, Mosasauria46, Serpentes46,47, Anguimorpha46,47, and Iguania46,47,54-56.

345 To avoid over-sampling Liolaemidae, we randomly selected species according to relative

346 abundance of dietary categories within the group34 and of liolaemids among squamates. In the

347 absence of time-calibrated phylogenetic information, we used temporal ranges from the

348 Paleobiology Database (http://www.paleobiodb.org) and checked accuracy by comparison with

349 cited sources. Each squamate group stated above was grafted onto the backbone of the Simões

350 et al.44 phylogeny according to its proposed calibrations. Node calibrations falling within the

351 95% highest posterior density for the corresponding node in the Simões et al.44 phylogeny were

352 kept unchanged. Where a calibration fell beyond that range, the calibration of Simões et al.44

353 was preferred. For taxa and nodes not included in Simões et al.44 and with phylogenetic data bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

354 lacking time-calibration, we used the code of Mitchell et al.57,58 to generate calibrations based

355 on last appearance dates and estimated rates of speciation, extinction, and preservation. The

356 method – derived from Bapst59 – allows the stochastic estimation of node age based on the

357 inferred probability of sampling a fossil and probability density of unobserved evolutionary

358 history, though nodes are sampled downwards towards the root rather than upwards from it.

359 We used preliminary BAMM 2.657 runs not including the taxa concerned to generate estimates

360 of speciation and extinction rates and selected a preservation rate of 0.01 (see below). Our tree

361 includes 27 unresolved nodes, denoting phylogenetic uncertainty. For methods requiring a fully

362 dichotomous tree, we used the function multi2di in ape 5.360 for R 3.6.161 to generate a random

363 dichotomous topology. Because of the sensitivity of BAMM to zero-length branches, we then

364 used the method of Mitchell et al58. to generate non-zero branch lengths in randomly resolved

365 polytomies with fossil taxa. We used the same randomly resolved and calibrated tree in all

366 analyses requiring a dichotomous tree. We referred to the August 12th, 2019 version of the

367 Reptile Database (http://www.reptile-database.org) and the Paleobiology Database

368 (http://www.paleobiodb.org) for taxonomical reference of extant and fossil species

369 (respectively).

370 Dietary data

371 We followed Meiri62 and Pineda-Munoz & Alroy63 for dietary classification. Accordingly,

372 when quantitative dietary data were available, we classified species based on the main feeding

373 resource in adults (i.e., >50% of total diet in volume63). Species consuming >50% plant material

374 were classified as herbivores. We followed Meiri62 and Cooper & Vitt64 in defining omnivorous

375 diets as including between 10 and 50% of plants, to account for accidental plant consumption

376 by some predators. Among predators, carnivores are defined as feeding mostly on vertebrates.

377 Predators consuming primarily arthropods and molluscs are “insectivores.” We could find no

378 published dietary hypothesis for 64 out of 119 fossil species, which we assigned to the most bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

379 plausible of our diet categories based on tooth complexity and the diets of closely related taxa

380 (see Supplementary Data File 1).

381 Geometric morphometrics

382 Specimens of 75 species were selected to represent all major groups of squamates with multiple-

383 cusped teeth and based on the quality of the material available. We extracted two-dimensional

384 outlines for geometric morphometric analyses from 52 X-ray computed microtomography scans

385 (microCT-scans), 12 photographs, 10 anatomical drawings of specimens, and one scanning

386 electron microscopy (SEM) image. Sources included the literature, the Digital Morphology

387 (DigiMorph) library, four new photographs, and four new microCT-scans (see below and

388 Supplementary Data File 1).

389 To analyse morphological variation of tooth shapes, we collected two-dimensional open

390 outlines of a left upper posterior maxillary multicuspid tooth in labial view with ImageJ 1.47v65.

391 We chose whenever possible the tooth with the most numerous cusps in the quadrant. If no left

392 maxillary tooth was sampled or suitable for tracing an outline, we referred to the right quadrant

393 or lower jaws and mirrored the outline adequately to retain the same orientation. We used the

394 EqualSpace function from PollyMorphometrics 10.166 for Mathematica 1067 to normalize teeth

395 outlines as sets of 200 equally spaced points based on Bézier splines functions.

396 We used Momocs 1.3.068 for R61 to perform geometric morphometric analyses of tooth outlines.

397 We first applied a Bookstein alignment69 and, for each outline, computed by Discrete Cosine

398 Transform (DCT) the first 21 harmonic amplitudes70. Harmonic coefficients were then

399 processed by Principal Component Analysis (PCA)71. We limited graphical representation of

400 the PCA to its first two axes, accounting for 89% of all morphological variation. To determine

401 the significance of our dietary grouping, we fitted a phylogenetic multivariate linear model

402 using penalized likelihood (PL)72,73 on all PC scores using mvMORPH 1.1.174 for R61. Because

403 we sampled only two carnivorous species, we added these to our insectivorous sample (n = 51, bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

404 so making a “predatory” group, to avoid spurious conclusions arising from groups with

405 extremely low sample sizes. Model fit was performed using Pagel’s 75 to jointly estimate the

406 phylogenetic signal in model residuals. We then used a one-way phylogenetic PL-MANOVA

407 to evaluate overall differences between dietary groups. To test between-group differences, we

408 used general linear hypothesis testing through contrast coding. We fitted a model for which

409 each group was explicitly estimated to test compound contrasts.

410 Ancestral character state reconstructions

411 We reconstructed the evolution of cusp number and diet using Maximum-Likelihood (ML)

412 ancestral character state reconstruction under a time-reversible continuous Markov transition

413 model26,76 as implemented in phytools 0.6-9977. We retrieved marginal ancestral states at the

414 nodes of the tree with the re-rooting algorithm from the same package78 and generated a model

415 of character evolution by averaging three character transition matrices (all transitions allowed

416 with either all rates different, symmetrical rates, or equal rates) according to their respective fit

417 through Akaike-weights model averaging79 (see Supplementary Table I). Finally, we used

418 stochastic character mapping (1,000 simulations) to compute the most likely character states at

419 each node based on the model-averaged transition matrix80. In contrast with tooth complexity

420 ancestral states reconstructions, extant data allow the formulation of informed hypotheses on

421 possible dietary transitions in squamates. Insects are an important food resource for the

422 juveniles of many squamate species, and several extant species of plant consumers show an

423 ontogenetic dietary shift from insectivorous juveniles to omnivorous or herbivorous

424 adults36,64,81,82. Moreover, extant data show that predatory squamates may rely on plant material

425 depending on environmental conditions34,64,83-85. Therefore, it has been hypothesized that

426 squamate plant consumption originated in predatory animals, which evolved increasingly more

427 plant-based diet through time under selective pressure34,64. We thus chose to test a specific

428 hypothesis of dietary transitions against naïve models and base our reconstructions on the best- bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

429 performing model. We compared the respective fit of three default models (all transitions

430 allowed) to three variants of our hypothesis of dietary transitions (limiting transitions to

431 carnivore-insectivore, insectivore-omnivore, and omnivore-herbivore, with three transition rate

432 regimes) and selected the model with highest relative fit (i.e., the custom model with all rates

433 different) to retrieve ancestral states at the nodes (see Supplementary Table 1). Based on these

434 ancestral reconstructions, we gathered a list of changes in cusp number and plant consumption.

435 Subsequently, we identified pairs of increases or decreases in both traits belonging to the same

436 phylogenetic path (the unique succession of branches connecting a descendent lineage to one

437 of its ancestors) and noted whether each initiated by a change in cusp number, plant

438 consumption, or whether both changes happened on the same branch.

439 Tests of correlated evolution

440 We used BayesTraits 3.0.2 (www.evolution.rdg.ac.uk) to run Markov Chain Monte Carlo

441 (MCMC) models of evolution of tooth complexity and diet with independent or correlated (i.e.

442 assuming rates of transition in one trait depend on the character state of the other) rates of

443 character transition. To improve rate estimations with our discrete dataset, in each run we scaled

444 our tree to obtain an average branch length of 0.1 (i.e., scaling factor = 5.017e-3) as

445 recommended by the software manual. Due to method limitations, we transformed our discrete

446 tooth complexity and diet characters into binary traits (teeth bearing one cusp vs two cusps or

447 more, and carnivores and insectivores (predators) vs omnivores and herbivores (plant

448 consumers), respectively). Each model ran for 110,000,000 iterations with default rate priors,

449 and we discarded the first 10,000,000 iterations as burn-in. We sampled parameters every 10,000

450 iterations and checked each chain for convergence and large effective sample size (using CODA

451 0.19-386 for R61). We used a steppingstone sampler87 to retrieve the marginal likelihood of each

452 model (250 stones, each run for 10,000 iterations), which we compared with a log Bayes Factor

453 to provide a measure of relative support of each model88. Analyses of the following bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

454 combinations of different binarization of the dataset yielded similar results, i.e., correlated

455 evolution of cusp number and diet: one or two cusps vs three cusps or more combined with

456 carnivores and insectivores vs omnivores and herbivores, and one to three cusps vs more than

457 three cusps combined with herbivores vs other diets. As expected, a correlated model was

458 weakly or not supported for other combinations (Supplementary Table 4).

459 Rates of phenotypic evolution

460 We estimated the evolutionary rate of tooth shape change through the variable rates model of

461 BayesTraits 3.0.289,90. In this approach, a reversible-jump Markov Chain Monte Carlo

462 (rjMCMC) algorithm is used to detect shifts in rates of continuous trait evolution – modelled

463 by a Brownian motion (BM) process – across the branches of a phylogenetic tree. This is

464 achieved by estimating the location of the shifts in rates (the product of a homogeneous

465 background rate with a set of rate scalars) by using two different proposal mechanisms (one

466 updating one branch at a time and one updating complete subclades). We used the default

467 gamma priors on rate scalar parameters. Support for rate heterogeneity was then further

468 confirmed by comparing the fit of the variable rates model against a null single-rate Brownian

469 model. Here, we ran a variable rates model and a homogeneous Brownian model on the scores

470 of the first 12 pPC axes from our phylogenetic PCA of tooth outlines, accounting for over 99%

471 of the total variance. Because PC axes can be correlated in a phylogenetic context, we used the

472 phylogenetic PC scores to remove evolutionary correlations91,92. We ran a phylogenetic

473 principal component analysis (pPCA)92 on the first 21 harmonics obtained by DCT using

474 phytools 0.6-9977 for R61. As for the original PCA, we found the two first pPC axes accounted

475 for the largest part of all morphological variation (83% of cumulative variance). All parameters

476 used were the same as for the correlated evolution tests (see above): 110,000,000 iterations, 10%

477 burn in, default priors, rescaling factor = 5.017e-3, sampling every 10,000 iterations,

478 convergence and sample size checks, stepping stone sampler with 250 stones run 10,000 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

479 iterations. Finally, we plotted our species tree (using phytools 0.6-9977, ggtree 1.8.193, and viridis

480 0.5.194 for R61) with branches scaled by the averaged rate scalars across posterior samples

481 (returned by the Variable Rates Post Processor;

482 http://www.evolution.reading.ac.uk/VarRatesWebPP/), thus indicating the relative deviation

483 from the background rate of change.

484 Likewise, we used a variable rates model approach on discrete data to detect heterogeneity in

485 character transition rates for tooth complexity and diet. The variable rates model operates on

486 discrete data by breaking the assumption of a single character transition rate matrix defined for

487 the entire tree, which it achieves by re-scaling this transition matrix in different parts of the tree

488 using an rjMCMC algorithm. As for continuous data, the process generates a posterior

489 distribution of scalars for each branch, and comparison with a null MCMC model with a

490 constant transition matrix allows evaluation of support for heterogeneity in the strength of

491 character transition rates. We ran the variable rates and null models similarly to tooth shape

492 data (see above), using binarized tooth complexity and dietary data to avoid over-

493 parameterization. The Variable Rates Post Processor returned the averaged branch rate scalars

494 used to plot the tree according to local deviations from the background transition matrix. The

495 large variances returned by the post-processor for some rate scalars, however, denote a

496 relatively complex model to fit and warrant adequate caution in interpreting absolute rate scalar

497 values, though relative rate differences should be fully representational. For clarity, we coloured

498 each branch according to a common log-transformed scale. We again tested alternative

499 binarizations of diet and tooth complexity and found support for a variable rates model for the

500 alternative binarization of diet and one other binarization of tooth complexity (one or two cusps

501 vs three cusps or more) (Supplementary Table 5). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

502 Models of diversification

503 We fitted different trait-dependent models of speciation and extinction (BiSSE, HiSSE) and

504 associated trait-independent null models with hisse 1.9.595 for R61, for which we compared

505 relative fit using Akaike weights79. In addition, we used an rjMCMC algorithm with BAMM

506 2.657 and BAMMtools 2.1.6 for R61 to model rates of speciation and extinction independently

507 from trait evolution on a random resolution of our super-tree (see above). This is currently the

508 only available method allowing branch-specific estimation of diversification rates on non-

509 ultrametric trees (i.e., including fossil taxa) by using a fossilized birth-death process57. We ran

510 ten independent replicates for 20,000,000 generations using priors generated by the

511 setBAMMpriors function of BAMMtools, a preservation rate prior of 0.01 (to reflect the

512 sampling biases affecting the squamate fossil record96), and a global sampling fraction of 0.048

513 accounting for our sampling relative to the total diversity of living and extinct squamates

514 referenced in both the Reptile Database (http://www.reptile-database.org) and the Paleobiology

515 Database (http://www.paleobiodb.org). We set a 10% burn-in and checked convergence and

516 effective sample size with CODA 0.19-386. Because we encountered many equiprobable

517 configurations, for each run we computed the maximum shift credibility (MSC) configuration

518 and extracted speciation and extinction rates for clades defined by each node immediately above

519 a shift, plus mean rates outside these clades (background rate). We then calculated a mean shift

520 magnitude for each clade using the ratio of its mean speciation rate over the mean background

521 rate97. To control for the influence of aquatic taxa during the KTR, we repeated analyses on a

522 tree devoid of Cretaceous aquatic taxa (ten mosasaurs and three snakes) and found no changes

523 to our results.

524 Statistics

525 We performed all univariate non-parametric tests using rcompanion 2.3.25

526 (https://www.rcompanion.org) and the base stat package in R 3.6.161. All effect sizes98 and their bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

527 95% confidence intervals were computed by bootstrap over 10,000 iterations. Sample size for

528 all tests is n = 548. A Kruskal–Wallis H test99 on tooth complexity levels among squamate

529 dietary categories showed a statistically significant effect of diet on the level of tooth

530 complexity (χ² = 144.27, df = 3, p-value = 4.5e-31, ² = 0.26 [0.20, 0.34]). Post-hoc pairwise two-

531 sided Wilcoxon–Mann–Whitney tests100,101 showed statistically significant differences between

532 all dietary categories (see Extended Data Table 1 for full reporting).

533 We used mvMORPH 1.1.174 for R61 to perform regularised phylogenetic one-way multivariate

534 analyses of variance (MANOVA) and multivariate general linear hypothesis tests in a penalized

535 likelihood framework72,73. For each test, we assessed significance over 10,000 permutations of

536 the Pillai trace102 obtained through regularised estimates72,73. A regularised phylogenetic

537 MANOVA on the principal component scores of 75 tooth outlines showed statistically

538 significant differences in 2D tooth shape between diets (V = 1.04, p-value = 0.001). We then

539 used general linear hypothesis tests to evaluate simple and compound contrasts between groups,

540 of which all but one were statistically significantly different (see Extended Data Table 2 for full

541 reporting).

542 Two-sided Wilcoxon–Mann–Whitney tests100,101 on macroevolutionary rates inferred using the

543 best-performing trait-dependent model of speciation and extinction (see Extended Data Table

544 4 and Figure 5) show multiple-cusped taxa have both statistically significantly higher speciation

545 and extinction rates than taxa with single-cusped teeth. Likewise, plant-consuming (i.e.,

546 omnivorous and herbivorous) taxa have both statistically significantly higher speciation and

547 extinction rates than mainly predatory taxa (i.e., carnivores and insectivores) (see Extended

548 Data Figure 5 for full reporting).

549 Photographs and X-ray computed microtomography

550 Photographs of ten specimens were captured at the Museum für Naturkunde (Berlin, Germany).

551 New microCT-scan data was generated for 24 specimens using a Skyscan 1272 microCT bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

552 (Bruker) at the University of Helsinki (Finland), a Skyscan 1172 microCT (Bruker) at the

553 University of Eastern Finland (Kuopio, Finland), and a Phoenix nanotom CT (GE) at the

554 Museum für Naturkunde (Berlin, Germany). Three-dimensional surface renderings were

555 generated using Amira 5.5.0103.

556 Specimen collection

557 Specialised retailers provided specimens of five species (see Supplementary Data File 1). The

558 Laboratory Animal Center (LAC) of the University of Helsinki and/or the National Animal

559 Experiment Board (ELLA) in Finland approved all reptile captive breeding (license numbers

560 ESLH‐2007‐07445/ym‐23 and ESAVI/7484/04.10.07/2016).

561 Art credits

562 Figure 1: the authors after Darren Naish (used with permission), Phylopic courtesy of Michael

563 Keesey, David Orr, Ian Reid, Alex Slavenko, and Steven Traver, and public domain. Figure 2:

564 the authors after Dick Culbert (CC-BY 2.0), Scott Robert Ladd (CC-BY 3.0), and Darren Naish

565 (used with permission), Phylopic courtesy of Michael Croggie, Michael Keesey, Alex

566 Slavenko, and Jack Meyer Wood. See https://www.phylopic.org for additional license

567 information.

568 Data availability statement

569 All datasets generated and analysed during the current study (tip-state dataset, polytomous and

570 dichotomous versions of our phylogeny, 2D outlines; see Fig. 1-4 and Extended Data Figure 1-

571 5) are available as Supplementary Information. CT-scan data are available through NDP, upon

572 reasonable request. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

573 Method references

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709 Acknowledgements

710 We thank Ilpo Hanski and Martti Hildén (Luonnontieteellinen keskusmuseo, Helsinki, Finland)

711 for specimen loans, Johannes Müller (Museum für Naturkunde, Berlin, Germany) for specimen

712 loans and access to collections and CT-scanning facilities, Jessie Maisano (University of Texas,

713 Austin, TX) for sharing data from the DigiMorph database, Arto Koistinen (University of

714 Kuopio, Finland) and Heikki Suhonen (University of Helsinki, Finland) for access to CT-

715 scanning facilities, Arto Koistinen, Simone Macrì, Kristin Mahlow, and Filipe Oliveira da Silva

716 for acquiring morphological data, as well as Jukka Jernvall, Mikael Fortelius, and the Helsinki

717 Evo-Devo community for discussions. We thank Vincent Bonhomme, David Caetano, Andrew bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

718 Meade, and Johnathan Mitchell for their help in implementing Momocs, HiSSE models,

719 BayesTraits, and BAMM 2.6 respectively. We also thank Robert Espinoza for precisions on

720 liolaemid diets. This work was supported by funds from the Integrative Life Science doctoral

721 program (ILS; to FL), the Center for International Mobility scholarship program (CIMO; to

722 FL), the University of Helsinki (to NDP), the Institute of Biotechnology (to NDP), Biocentrum

723 Helsinki (to NDP), and the Academy of Finland (to NDP).

724 Authors contributions

725 FL, IJC, and NDP designed the experimental approach. FL and NDP collected the specimens

726 for microCT-scanning. FL character-coded species from the literature and specimen data. FL

727 collected tooth outline semi-landmark data. FL performed the research. FL analysed the data,

728 with contribution from JC, IJC and NDP. FL made the figures. FL produced the first draft, and

729 FL, JC, and IJC wrote the paper, to which all authors contributed in the form of discussion and

730 critical comments. All authors approved the final version of the manuscript.

731 Competing interest declaration

732 The authors declare no conflict of interests.

733 Additional information

734 Correspondence and requests for materials should be addressed to FL, IJC, and NDP.

735 Supplementary information is available for this paper. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Hemisphaeriodon gerrardii

ti

Tiliqua scincoides

Tiliqua nigrolutea

Dicrodon guttulatum Aspidoscelis uniparens yi

airii

PholidoscelisHolcosus e bridgesii escens Teius suquiensis Kentrop

Aspidoscelis tigris T Gymnophthalmus speciosus eius oculatus K Pholidobolus montium Ameiva bifrontata Scriptosaura catimbau entrop Ameiv Teius teyou Colobodactylus tauna ylepis sulcata Colobosaura modesta Ameivula nativo ylepis laevis yx altamazonica CalyptommatusNothobachia nicterus ablephara

rach Callopistes flavipunctatus Bachia bicolor yx pelviceps rach Egernia striolata Egernia cunninghami a ameiva Egernia stokesii Cyclodomorphus branchialis Tiliqua rugosa Liopholis whitii Corucia zebrata T Callopistes bicuspidatus T Chioninia coctei Eutropis multifasciata Eutropis carinata Dasia vyneri asciatus xsul Phoboscincus bocour Leiolopisma telf Eugongylus ruf Callopistes maculatus Mochlus sundevalli Coeranoscincus reticulatus Silvascincus murra nia gillespieae T Eulamprus quoyii ropidophorus misaminius Scincella lateralis upinambis quadrilineatus Sphenomorphus solomonis Crocodilurus amazonicus T Salvator merianae Chalcides chalcides Chalcides ocellatus eylinia currori Salv Chalcides sepsoides Tupinambis teguixin Chalcides viridanus ermis Eger F Melanoseps occidentalis Dracaena guianensis Scelotes arenicolus ator rufescens Callopistes rionegrensis Brachyseps splendidus Plestiodon f yi Plestiodon gilberti Plestiodon chinensis Scincus scincus Eumeces schneideri Pyramicephalosaurus cherminicus Brachymeles gracilis Sineoamphisbaena he Acontias cregoi Acontias aurantiacus Rhineura floridanaDracaena colombiana Acontias meleagrisyphlosaurus v Acontias lineatus Bipes canaliculatus T Typhlosaurus braini T Bipes biporus rogonophis wiegmanni Diplometopon zarudnyi P Dyticonastis rensbergeri Blanus strauchi eneteius aquilonius Dicothodon bajaensis T P Gilmoreteius chulsanensis chingisaurus multiv Xantusia vigilis olyglyphanodon sternbergi Lov Xantusia bezyi Xantusia henshawi GeocalamusCynisca acutus leucura Blanus thomaskelleri Amphisbaena fuliginosa Gobinatus arenosus Xantusia extorris eridgea ionidesii Cherminsaurus kozlowskii Amphisbaena kingii Blanus gracilis Globaura venusta Xantusia riversiana Leposternon microcephalum Spathorh Slavoia dare Xantusia downsi yma smithii Rhineura hatcherii xatabularis yma gaigeae AmphisbaenaAmphisbaena ridleyi alba

Amphisbaena bakeri ynchus fossorium Pyrenasaurus evansae Lepidoph Amphisbaena caeca Lepidoph Lepidophyma flavimaculatum

agus vskii natus T Cricosaura typica

epe xantusia borealis Gerrhosaurus nigrolineatus Psammodromus algirus Gerrhosaurus skoogi Axonoscincus sabatieri xisaurus tepexii Matobosaurus validus xantusia allisonialaeo Cordylosaurus subtessellatus P Broadleysaurus major Zonosaurus or

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Rena dulcis Gobekko cretacicus Letheobia caeca Dinilysia patagonica Xerotyphlops vermicularis Eichstaettisaurus Norellius schroederi nyctisaurops Huehuecuetzpalli mixtecus Indotyphlops braminus Sphenodon punctatus Liotyphlops albirostris Marmoretta oxoniensis Haasiophis terrasanctus Megachirella wachtleri Gephyrosaurus bridensis Eupodophis descouensi Sophineta cracoviensis Ctenosaura quinquecarinata Anilius scytale Ctenosaura clarki Tropidophis haetianus Ctenosaura similis Uropeltis woodmasoni Ctenosaura pectinata Cylindrophis ruffus Ctenosaura acanthura Ctenosaura hemilopha Cylindrophis melanotus Conolophus subcristatus Anomochilus leonardi Amblyrhynchus cristatus Xenopeltis unicolor Iguana iguana Loxocemus bicolor Iguana delicatissima Malayopython reticulatus Pumilia novaceki Aspidites melanocephalus Sauromalus ater Python regius Cyclura cornuta ython fischeri Armandisaurus explorator Python bivittatus Palaeop Python molurus Dipsosaurus dorsalis Brachylophus f T Calabaria reinhardtii ropidurus torquatusasciatus Tropidurus etheridgei Ungaliophis continentalis Candoia carinata Plica plica Eryx colubrinus Plica umbra Eryx jaculus Microlophus peruvianus Uranoscodon superciliosus Boa constrictor tulanus Stenocercus guentheri Crotaph Corallus hor Crotaph ytus oligocenicus Chilabothrus angulifer Crotaph ytus bicinctores ytus collaris Chilabothrus inornatus Gambelia sila Chilabothrus monensis Gambelia wislizenii Chilabothrus subflavus Chilabothrus fordii Aciprion formosum Gambelia corona ysogaster Morunasaurus annularis Chilabothrus gracilis Enyalioides laticeps Hoplocercus spinosus Chilabothrus chr Chilabothrus strigilatus Chilabothrus exsul Polychrus marmoratus Chilabothrus striatus Liolaemus darwinii Casarea dussumieri Liolaemus olongasta Liolaemus albiceps AcrochordusAplopeltura granulatus boa Liolaemus uspallatensis Pareas hamptoni Liolaemus telsen Liolaemus fitzingerii CaususDaboia rhombeatus russelii Liolaemus melanops Bitis arietanseae Liolaemus cuyanus Bitis gabonica Liolaemus scapularis Azemiops f Liolaemus gravenhorstii Liolaemus tenuis Azemiops kharini Ph Babibasiliscus alxi Ctenoblepharymaturus palluma Bothrops asper Tropidolaemus wagleri Diplolaemus bibronii Pristidactylus casuhatiensis Fordonia leucobalia Leiosaurus catamarcensis ys adspersa Homalopsis buccataphyriacus Pristidactylus torquatus Geiseltaliellus Geiseltaliellus longicaudus maarius Urostrophus vautieri Acanthophis antarcticus Oplurus cyclurus Hydrophis gracilis Chalarodon madagascariensis Pseudechis por Geiseltaliellus pradiguensis HydrophisNaja platurusnigricollisNaja naja Corytophanes percarinatus Cor Duberria lutrix ytophanes cristatus Laemanctus longipes Basiliscus plumifrons Prosymna meleagris Basiliscus basiliscus Basiliscus vittatus Psammophis sibilans Boaedon fuliginosus ma pulchrum Magnuviator Temujinia ovimonsensis ellisoni Atractaspis boulengeri neri Saichangur Igua minuta Leiocephalus barahonensis Atractaspis irregularis Polrussia mongoliensis Zapsosaurus sceliphros Leiocephalus schreibersii Amblyodipsas unicolor Gobider Jeddaherdan aleadonta Isodontosaurus gracilis Leiocephalus carinatus Aparallactus modestus EstesiaAiolosaurus mongoliensis oriens Leiocephalus melanochlorus Polemon gabonensis Anolis garmani Afronatrix anoscopusNatrix natrix Anolis sagrei a vel da Anolis chamaeleonides Iguanognathus wer Anolis porcus OpisthotropisNerodia latouchii sipedon Anolis allisoni tensi vidsoni Gueragama sulamericana Anolis carolinensis yas aestiv Anolis equestris Heterodon platirhinos va Anolis baracoae Diadophis punctatus Arrhyton taeniatum Phr Philodr Phrynosoma ditmarsi Saniwa sp. Phr ynosoma hernandesi Phalotris mer Phrynosoma modestum Cypressaurus h Helicops angulatus Phrynosomaynosoma taurus orbiculare Phimophis guerini Phr Phrynosoma coronatum Eirenis rothii Phr ynosoma braconnieri Sibynophis collaris Phrynosoma platyrhinos Phr ynosoma solare Lycodon aulicus Exostinus lancensis Phr Eirenis decemlineatus Ctenomastax par Phrynosoma asio Necrosaurus eucarinatus ynosoma mcallii Scaphiodontophis annulatusDasypeltis scabra Cophosaurus te Priscagama gobiensis ynosoma cornutum Mimeosaurus crassus Phrynosomimus asper Uma scoparia Odaxosaurus piger ypsodontus Sceloporus variabilis P Coluber constrictor Barbature Uta stansburianaetrosaurus mear Coronella austriaca Trimorphodon biscutatus neensis

antherophisLampropeltis obsoletus getula P

xanus aceus Calotes emma x morrisoni Calotes mystaceus Calotes v Varanus griseus Bronchocela jubata Gonocephalus chamaeleontinus Varanus niloticus Glyptosaurus sylvestris Coryphoph Japalura polygonata nsi Lanthanotus bor ator Draco quinquef Draco volans Draco fimbriatus

Varanus albigularis aranus gouldii Agama Agamaagama hispida aranus acanthurusaranus semotus Phr Varanus oliv V Phrynocephalus mystaceus V V P ersicolor Eurheloderma gallicum P Varanus varius Helodermoides tuberculatus Diporiphora winneckei Varanus exanthematicus Moloch horridus Hypsilurus boydii ogona vitticeps Ph ogona barbata ynocephalus arabicus Hydrosaurus pustulatus Hydrosaurus amboinensis Leiolepis ree Ophisaurus sp. Leiolepis belliana Varanus dumerilii Leiolepis triploida ylax subcristatus

aranus komodoensis Uromastyx aegyptia Varanus rudicollis Saara hardwickii ysignathus cocincinus Chamaeleo calyptratus Peltosaurus granulosus Chamaeleo zeylanicus Chamaeleo chamaeleon Chamaeleo lae Calumma brevicorne Bradypodion pumilum V Varanus salv Trioceros melleri

aranus palawanensis ernalis asciatus V

yurus Xenosaurus grandis

Shinisaurus crocodilurus Xenosaurus platycepsHeloderma horridum Anniella pulchra

vesii Anguis fragilis Pseudopus ahnikoviensis er pardalis Heloderma suspectum Celestus crusculus

vigatus

Elgaria multicarinata oweni Trioceros

Furcif Pseudopus apodus

Diploglossus lessonae Brookesia brygooi

rioceros jacksonii Gerrhonotus inf

T Celestus enneagrammus

Rieppeleon kerstenii

Brookesia superciliaris

Rieppeleon brach

Rhampholeon spectrum bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

736 Extended Data Figure 1 | Time-calibrated squamate phylogeny. Informal super-tree

737 including 545 extant and extinct squamates species and three outgroup species (see Methods).

738 Scalebar = 50 million years. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Ctenosaura quinquecarinata Conolophus subcristatus Iguana iguana Dipsosaurus dorsalis Brachylophus fasciatus Tropidurus torquatus Plica plica Microlophus peruvianus Uranoscodon superciliosus Stenocercus guentheri Morunasaurus annularis Enyalioides laticeps Hoplocercus spinosus Aciprion formosum † Polychrus marmoratus Gambelia wislizenii Diplolaemus bibronii Pristidactylus torquatus Urostrophus vautieri Oplurus cyclurus Chalarodon madagascariensis Phymaturus palluma Anolis sagrei Anolis porcus Anolis allisoni Anolis carolinensis Anolis equestris Leiocephalus barahonensis Leiocephalus schreibersii Basiliscus basiliscus Cophosaurus texanus Uma scoparia Phrynosoma platyrhinos Petrosaurus mearnsi Uta stansburiana Saichangurvel davidsoni † Igua minuta † Bronchocela jubata Gonocephalus chamaeleontinus Coryphophylax subcristatus Calotes emma Draco quinquefasciatus Agama hispida Pogona vitticeps Pogona barbata Physignathus cocincinus Leiolepis triploida Bradypodion pumilum Brookesia brygooi Lacerta bilineata Takydromus sexlineatus Zootoca vivipara Latastia longicaudata Ichnotropis capensis Gallotia galloti Gallotia stehlini Psammodromus algirus Diplometopon zarudnyi Trogonophis wiegmanni Tupinambis teguixin Callopistes maculatus Colobosaura modesta Pholidobolus montium Kentropyx altamazonica Aspidoscelis tigris Teius suquiensis Gilmoreteius chulsanensis † Peneteius aquilonius † Pyramicephalosaurus cherminicus † Gerrhosaurus nigrolineatus Gerrhosaurus skoogi Zonosaurus ornatus Xantusia riversiana Liopholis whitii Eulamprus quoyii Scalar Cusp number Diet 2 Carnivorous 0.0 0.5 1.0 3 Insectivorous >3 Omnivorous Herbivorous bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

739 Extended Data Figure 2 | Rates of 2D tooth shape evolution among 75 squamate species

740 with multicuspid teeth. Branch lengths are transformed by the mean of the respective posterior

741 distribution of scalars generated under a variable rates model, reflecting changes in the rate of

742 shape evolution. † = extinct taxon. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Cusp number increase a Sc inc Cusp number decrease oid ea Ng Cusp number ea id 1 rto ce Pg a 2 L 3 >3 Dibamia K 1

3 G 2 e k J k o t a a i r

u

a

s Tr Rhynchocephalia a

s

o

M P

6

5

4

b Plant consumption increase Sc inc Plant consumption decrease oid ea Ng Diet ea id Carnivorous rto ce Pg a Insectivorous L Omnivorous Herbivorous Dibamia K 1

3 G 3 2 e k J k o t a a i r

u

a

s Tr Rhynchocephalia a

s

o

M P

6

5

4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

743 Extended Data Figure 3 | Squamate dental and dietary evolution. Known and Maximum-

744 Likelihood ancestral state reconstructions of tooth complexity (a) and diet (b) in squamates. Pie

745 charts indicate the relative likelihood of each character state at the corresponding node. Branch

746 tip circles indicate character state at tips. Coloured branches indicate an increase or a decrease

747 in tooth complexity/plant consumption. 1: Gerrhosauridae. 2: Teiioidea + Polyglyphanodontia

748 (informally Teiioidea sensu lato). 3: total group Lacertidae (informally Lacertidae sensu lato).

749 4: Chamaeleonidae. 5: non-Uromastycinae agamids (informally Agamidae sensu stricto). 6:

750 total group Pleurodonta. P: Permian. Tr: Triassic. J: Jurassic. K: Cretaceous. Pg: Paleogene.

751 Ng: Neogene. Scalebar = 50 million years. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b

>3 cusps Omnivorous

3 cusps 1 cusp Insectivorous Carnivorous

2 cusps Herbivorous

Diet Carnivorous Cusp number Insectivorous 1 Omnivorous 2 Herbivorous 3 >3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

752 Extended Data Figure 4 | Transition models used for ancestral state reconstructions.

753 Relative character transition rates for tooth complexity (a) and diet (b). Arrow widths are scaled

754 by the log-transformed rates of transition. The orientation of arrows denotes the direction of

755 character transitions. Note: the transition from two-cusped to three-cusped teeth is represented

756 with a dotted line, due to its relative rate being negligible compared to all other transition rates. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b

W = 18552, p = 1.59e-21, e² = 0.17, CI95% [0.11, 0.23] W = 18553, p = 1.60e-21, e² = 0.17, CI95% [0.11, 0.23]

0.04 8e -11

-1 0.03 -1 e 6 -11

0.02 4e -11 Extinction rate (My ) Speciation rate (My )

0.01 2e -11

Unicuspid Multicuspid Unicuspid Multicuspid (n = 333) (n = 215) (n = 333) (n = 215) HiSSE state HiSSE state c d

W = 12776, p = 5.64e-18, e² = 0.14, CI95% [0.08, 0.20] W = 8646, p = 1.17e-29, e² = 0.23, CI95% [0.18, 0.30]

0.04

7.5e-11

-1 0.03 -1

5.0e-11

0.02 Extinction rate (My ) Speciation rate (My )

2.5e-11 0.01

Predator Plant consumer Predator Plant consumer (n = 425) (n = 123) (n = 425) (n = 123) HiSSE state HiSSE state bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

757 Extended Data Figure 5 | Squamate macroevolutionary rates among different levels of

758 tooth complexity and diet Speciation (a–c) and extinction rates (b–d) per tooth complexity or

759 diet character state for the best supported model of trait-dependent speciation and extinction.

760 Violin plots indicate the density of data points. Boxes include 50% of the data points, with the

761 black line and dot indicating the median and mean, respectively. Whiskers incorporate the

762 whole range of the data. All pairs are statistically significantly different (Wilcoxon–Mann–

763 Whitney test); see panels for their respective W statistic and effect size (²), including 2.5th and

th 764 97.5 confidence interval percentiles (CI95%) in brackets. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

765 Extended Data Table 1 | Tooth complexity of squamates analysed by dietary category

Group 1 Group 2 Sample size W p-value

Carnivores Herbivores 154 490 4.9e-21

Carnivores Insectivores 425 12458 1.8e-08

Carnivores Omnivores 197 1583 7.3e-21

Herbivores Insectivores 351 10188 8.2e-13

Herbivores Omnivores 123 2330 4.6e-04

Insectivores Omnivores 394 7543 4.5e-10 766 1 767 Two-sided pairwise Wilcoxon–Mann–Whitney tests on the tooth complexity level of 548

768 species grouped by diet (carnivores, n = 114; insectivores, n = 311; omnivores, n = 83; herbivores,

769 n= 40), with W statistic and Bonferroni-corrected p values. Significant differences (p < 0.05)

770 are in bold. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

771 Extended Data Table 2 | Pairwise comparisons of dietary categories in 2D tooth

772 morphospace

Group 1 Group 2 Sample size V p-value

Herbivores Omnivores 22 0.63 3.0e-04

Herbivores Predators 64 0.76 1.0e-04

Omnivores Predators 64 0.29 0.80

Herbivores + Predators 75 0.64 2.0e-04 Omnivores

Omnivores + Herbivores 75 0.73 1.0e-04 Predators 773 1 774 Multivariate general linear hypothesis testing through phylogenetic penalized likelihood

775 MANOVA of PC scores for the multicuspid teeth of 75 extant and fossil squamate species

776 grouped by diet (predators, n = 53 (including insectivores, n = 51, and carnivores, n = 2);

777 omnivores, n = 11; herbivores, n = 11). We assessed statistical significance over 10,000

778 permutations of the Pillai trace (V). Significant differences (p < 0.05) are in bold. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

779 Extended Data Table 3 | Squamate clades showing rate shifts in trait-independent models

780 of speciation and extinction.

Mean Mean Cusp Plant Standard Ratio to mean Standard Ratio to mean Node Replicates speciation extinction number consumption deviation outgroup rate deviation outgroup rate rate (My-1) rate (My-1) transition transition 555 (A) 9/10 0.0523 0.0159 4.40 0.0092 0.0030 0.65 - - 679 (B) 6/10 0.1296 0.0659 2.59 0.0128 0.0083 1.36 - I → O 691 1/10 0.0511 0.0207 1.01 0.0122 0.0049 1.82 - - 697 (C) 9/10 0.0875 0.0489 1.72 0.0961 0.0572 11.06 2 → >3 - 705 1/10 0.0523 0.0212 1.03 0.0004 0.0002 0.05 - - 763 2/10 0.1918 0.0899 3.69 0.1133 0.0553 11.00 2 → 1 - 782 (D) 10/10 0.1439 0.0482 2.86 0.0308 0.0247 3.37 - I → O 802 (E) 10/10 0.0596 0.0203 1.17 0.0557 0.0189 6.39 - - 824 (F) 7/10 0.0624 0.0296 1.26 0.0021 0.0014 0.21 - - 826 1/10 0.0778 0.0315 1.54 0.0060 0.0024 0.49 - - 846 (G) 10/10 0.3912 0.0966 7.79 0.0433 0.0456 4.69 - - 914 (H) 10/10 0.1460 0.0231 2.92 0.0143 0.0111 1.55 - - 996 (I) 5/10 0.0797 0.0417 1.71 0.0100 0.0052 1.15 1 → 3 I → O 1003 (J) 5/10 0.0802 0.0420 1.71 0.0056 0.0030 0.61 - - 1010 (K) 10/10 0.1586 0.0154 3.16 0.0159 0.0153 1.72 3 → 1 - 1055 (L) 10/10 0.3224 0.0939 6.43 0.0268 0.0181 2.89 - - 1084 (M) 6/10 0.2555 0.1330 5.15 0.0272 0.0198 3.04 3 → >3 - 1085 4/10 0.2934 0.1555 5.91 0.0347 0.0260 3.63 - - 781 1 782 Mean rates of speciation and extinction for the 18 clades defined by a rate shift in 10 maximum

783 shift credibility configuration (MSC) independent replicates, including the 13 clades with rate

784 shifts in at least five replicates (as indicated in Fig. 4c), and their ratio to the corresponding

785 mean rate for the outgroup. Shift location is given by the number of the node immediately above

786 it. Letters in brackets in the “Node” column denote clade labels in Fig. 4c. Column “Replicates”

787 indicates the number of occurrences of a given shift among the ten MSC replicates. Column

788 “Cusp number transition” indicates changes in tooth complexity inferred at a given node, if any.

789 Column “Plant consumption transition” indicates changes in plant matter proportion in the diet

790 inferred at a given node, if any (I: insectivorous, O: omnivorous). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042796; this version posted April 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

791 Extended Data Table 4 | Tests of trait-dependent models of squamate speciation and

792 extinction.

Model Hidden Character- Number of Number of Dual AICc (tooth AICc AICc AICc states dependent diversification transition transitions complexity) (tooth (diet) (diet) rates rates complexity)

HiSSE complete ARD Yes Yes 4 12 Yes 5231.30 0.004 5301.83 0.068

HiSSE complete ER Yes Yes 4 1 Yes 5275.11 < 0.001 5381.02 < 0.001

HiSSE node dual ARD Yes Yes 4 8 No 5221.05 0.595 5296.89 0.802

HiSSE no dual 3 rates Yes Yes 4 3 No 5247.47 < 0.001 5306.42 0.007

HiSSE no dual ER Yes Yes 4 1 No 5271.91 < 0.001 5370.23 < 0.001

BiSSE No Yes 2 2 - 5285.85 < 0.001 5362.89 < 0.001

CID-4 3 rates Yes No 4 3 No 5221.96 0.378 5300.65 0.123

CID-4 ER Yes No 4 1 No 5244.80 < 0.001 5353.53 < 0.001

CID-2 complete ARD Yes No 2 12 Yes 5245.97 < 0.001 5362.36 < 0.001

CID-2 complete ER Yes No 2 1 Yes 5270.88 < 0.001 5377.14 < 0.001

CID-2 no dual ARD Yes No 2 8 No 5239.53 < 0.001 5345.35 < 0.001

CID-2 no dual 3 rates Yes No 2 3 No 5227.58 0.023 5310.76 < 0.001

CID-2 no dual ER Yes No 2 1 No 5260.71 < 0.001 5364.75 < 0.001 793

794 Description of model parameters and relative goodness of fit. For both tooth complexity and

795 plant consumption, a “hidden state” speciation and extinction model (HiSSE) is best supported

796 to account for squamate macroevolutionary patterns. Corrected Akaike Information Criterion

797 (AICc) values and AICc model weights (AICc) for the binary tooth complexity dataset and

798 the binary diet dataset. The model with the highest AICc weight is highlighted in bold. HiSSE:

799 hidden state speciation and extinction model; BiSSE: binary state speciation and extinction

800 model; CID: character-independent (null) model; “complete”: all character state transitions

801 possible, including simultaneous transitions in both the observed and hidden trait; “no dual”:

802 simultaneous transitions in both the observed and hidden trait are excluded; ARD: all rates

803 different; ER: equal rates.