Stepanova and Bauer BMC Ecol Evo (2021) 21:86 BMC Ecology and Evolution https://doi.org/10.1186/s12862-021-01821-w

RESEARCH Open Access Phylogenetic history infuences convergence for a specialized ecology: comparative skull morphology of African burrowing skinks (Squamata; Scincidae) Natasha Stepanova1,2* and Aaron M. Bauer1

Abstract Background: Skulls serve many functions and as a result, are subject to many diferent evolutionary pressures. In squamates, many fossorial species occupy a unique region of skull morphospace, showing convergence across families, due to modifcations related to head-frst burrowing. As diferent substrates have variable physical properties, particular skull shapes may ofer selective advantages in certain substrates. Despite this, studies of variation within burrowers have been limited and are typically focused on a single origin of fossoriality. We focused on seven skink genera (Acontias, Typhlosaurus, Scelotes, Sepsina, Feylinia, Typhlacontias, and Mochlus; 39 sp.) from southern Africa, encompassing at least three independent evolutions of semi-fossoriality/fossoriality. We used microCT scans and geometric morphometrics to test how cranial and mandibular shape were infuenced by phylogenetic history, size, and ecology. We also qualitatively described the skulls of four species to look at variation across phylogenetic and functional levels, and assess the degree of convergence. Results: We found a strong efect of phylogenetic history on cranial and mandibular shape, with size and substrate playing secondary roles. There was a clear gradient in morphospace from less specialized to more specialized bur- rowers and burrowers in sand were signifcantly diferent from those in other substrates. We also created an anatomi- cal atlas for four species with each element described in isolation. Every bone showed some variation in shape and relative scaling of features, with the skull roofng bones, septomaxilla, vomer, and palatine showing the most variation. We showed how broad-scale convergence in traits related to fossoriality can be the result of diferent anatomical changes. Conclusions: Our study used geometric morphometrics and comparative anatomy to examine how skull morphol- ogy changes for a highly specialized and demanding lifestyle. Although there was broad convergence in both shape and qualitative traits, phylogenetic history played a large role and much of this convergence was produced by difer- ent anatomical changes, implying diferent developmental pathways or lineage-specifc constraints. Even within a single family, adaptation for a specialized ecology does not follow a singular deterministic path. Keywords: Squamates, Computed tomography, Macroevolution, Anatomy, Convergent evolution, Fossorial, Cranium

Background

*Correspondence: [email protected] Biologists have long been interested in the patterns 2 Present Address: Department of Ecology and Evolutionary Biology, and processes that underlie morphological diversity. University of Michigan, Ann Arbor, MI, USA Two major approaches in studying morphology are Full list of author information is available at the end of the article

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morphometrics and comparative anatomy. Morphomet- width can impact burrowing performance between two rics uses measurements or landmarks to quantify mor- closely related species [30] and that skull variation within phological variation, and has been used to study topics the genus Leposternon is correlated with biogeographical including ecomorphology [1, 2], modularity [2, 3], and variables related to soil type [31]. On the other hand, a convergent evolution [4, 5]. Comparative anatomy is study on Caribbean amphisbaenians found no relation- often more time-intensive but ofers a detailed view of ship between skull shape and location (a proxy for soil structures that can provide further insight into systemat- type), and instead found a tight link with phylogenetic ics [6], ontogeny [7], biomechanics/function [8, 9], and history and skull shape [18]. Together, these results indi- adaptation [10, 11]. Increasingly, studies incorporate both cate that although variation refecting specifc microhabi- methods [7, 8, 12, 13], which together provide a deeper tats can exist, this might be overshadowed by variation understanding of evolution. For instance, geometric mor- related to phylogenetic history in more distantly related phometrics can identify general convergence in shape, species. Outside of amphisbaenians, gymnophthalmid whereas anatomical study can investigate whether that lizards and acontine skinks have also shown a relation- convergence is the result of the same anatomical changes, ship between head shape and microhabitat [23, 32]. Tese implying similar developmental pathways, or diferent studies indicate the existence of skull specialization for changes that may point to historical contingency. Tis diferent substrates, but do not test it across independent imperfect convergence may imply variation in genetic derivations of fossoriality. pathways, functional trade-ofs, many-to-one mapping, Scincidae (i.e., skinks) is one of the most speciose squa- diferent ancestral states, or other lineage-specifc con- mate families with more than 1700 species worldwide straints [4]. Here, we employ both approaches to study [33]. Te family is also morphologically and ecologically how skull morphology has changed for a specialized eco- diverse, occurring in a range of habitats on every conti- logical lifestyle and examine convergence across multiple nent apart from Antarctica. Whereas the typical skink levels. has four well-developed limbs, limb reduction and loss Te skull serves many functions including protection are common, with numerous independent events of limb of the brain and sensory organs, manipulation and break- reduction estimated in the group [34, 35], including mul- down of food, attachment for masticatory and axial mus- tiple times within some genera [36, 37]. Limb reduction cles, and in some cases, locomotion or defense [14]. As a is typically correlated with a fossorial lifestyle and accom- result, the skull is subject to many diferent evolutionary panied by a suite of other adaptations [38, 39]. Despite pressures, including ones that confict with one another this diversity, cranial descriptions of the group are few, (e.g. burrowing performance and bite force; [15, 16]). In with only ~ 250 species (~ 16% of the family) having oste- squamates (lizards and snakes), the skull has been found ological descriptions for complete skulls and/or individ- to vary with phylogenetic history [17, 18], allometry [19, ual disarticulated bones [11]. 20], diet [21, 22], and habitat use [1, 2, 20, 23]. Burrowing We focused on skinks from southern Africa because squamates, in particular, occupy unique regions of mor- of the repeated evolution of burrowing forms in this phospace and show convergence across families [2, 6, 24, region. Other regions like southeast Asia and Aus- 25]. In addition to having elongate bodies and reduced or tralia also show diversity in fossorial skinks, although no limbs, they possess many skull modifcations related it tends to be limited to a single lineage [38, 40]. We to head-frst burrowing [26]. Common modifcations looked at the genera Acontias, Typhlosaurus, Scelotes, include closure of the braincase, increased compactness Sepsina, Feylinia, Typhlacontias, and Mochlus (Fig. 1), of the skull roof, loss or reduction of various structures, which encompass three subfamilies and at least three and an overall streamlining of the skull [10, 24, 27, 28]. independent derivations of semi-fossoriality/fossorial- Despite this unique anatomy, studies of variation ity [41]. Acontias and Typhlosaurus are in the subfamily within burrowing squamates have been relatively limited. Acontinae, which is the sister group to all other skinks Soil properties, especially those related to hardness and and contains only limbless species [36, 41]. Scelotes, penetrability, should impact an organism’s ability to bur- Sepsina, Feylinia, and Typhlacontias are in the sub- row through it and thus diferent head shapes may ofer family Scincinae. Te southern African scincines are advantages in particular substrates. Within squamates, all semi-fossorial and fossorial, and show great varia- most work on this topic has focused on amphisbaeni- tion in specialization for burrowing and limb reduction ans, the largest and most specialized group of fossorial [36]. Mochlus retains all four limbs and is within a clade squamates. Early work identifed three specialized head of lygosomine skinks that includes semi-fossorial and shapes, in addition to the more generalized head shape, non-fossorial genera [42]. Previous anatomical studies which are typically associated with diferent substrates have looked at cranial osteology in Acontias [10, 43– [29]. More recent work has revealed that changes in skull 47], Typhlosaurus [10, 43], Scelotes [48], and Feylinia Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 3 of 53

Fig. 1 A Multi-locus squamate phylogeny [39] trimmed to show species in this study. Species names are colored by substrate. The data matrix shows number of limbs, number of digits, jugal condition, and condition of the upper temporal fenestrae, with boxes color-coded to indicate character state. Apposition refers to the closure of the upper temporal fenestrae through close contact of the squamosal with the parietal. Photos: B Acontias litoralis, C Typhlacontias brevipes, and D Sepsina angolensis. Credit to Johan Marais (B and C) and Luis M. P. Ceríaco (D)

[10, 49, 50]. By focusing on African skinks, where there examining each bone in detail, to create an anatomi- have been multiple independent origins of fossoriality, cal atlas and investigate variation across phylogenetic we can tease apart the interplay of phylogenetic history, and functional levels. We looked at variation within a body size, and ecology on skull morphology and better single genus (Typhlacontias brevipes and Typhlacon- understand the evolution of a specialized ecology. tias gracilis) and compared them with a less special- We used micro-computed tomography (CT) scans ized burrower in the same subfamily (Sepsina alberti) to untangle the effects of the evolutionary history and and with a more distantly related species that shares a ecological adaptation on skull morphology in seven more similar lifestyle (Acontias occidentalis). None of genera of African skinks. First, using geometric mor- these species have been the subject of a detailed ana- phometrics and phylogenetic comparative methods, tomical study before and two of the genera (Typhlac- we tested how cranial and mandibular shape varied ontias and Sepsina) have no such studies for the genus with phylogenetic history, size, substrate, and degree [11]. We also used these descriptions to assess whether of limb reduction, which is often used as a proxy for fossorial adaptations show perfect convergence (simi- the degree of burrowing specialization [51]. Next, we lar overall traits due to the same changes to anatomy, described the skull anatomy of four African skinks, implying similar developmental or genetic shifts). Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 4 of 53

Results and short frontals. Te PC2 axis is related to the length We CT scanned 39 species from seven genera and seg- of the braincase: species with negative scores have a rela- mented the cranium and lower jaw of each specimen. tively long braincase and species with positive scores Image stacks, models, and metadata are available on have shorter braincases. Te increase in length can be Morphosource (https://​www.​morph​osour​ce.​org/​proje​ the result of a longer sphenoid, longer basipterygoid pro- cts/​00000​C482). For our morphometrics, we used 21 cesses, or a combination of both features. fxed landmarks on the cranium and 13 on the mandi- Te multivariate regression found a signifcant relation- ble (Fig. 2). Because not all species were represented on ship between cranial shape and centroid size (p = 0.0034). the phylogeny, we ran analyses on three datasets for the In general, smaller skinks have relatively longer brain- cranium and mandible each. Te frst included all species cases, less distance between the quadrate and braincase, and was analyzed with non-phylogenetic methods. Te and narrow rectangular parietals. Larger skinks have second excluded Mochlus sundevallii (the only species relatively short braincases, greater distance between in its genus included in study) to run pairwise compari- the quadrate and braincase, and square or hourglass- sons between genera and subfamilies. Te third included shaped parietals (Fig. 3A). When comparing patterns of only the species in the phylogeny and was analyzed with allometry between genera (Additional fle 1: Table S2), both non-phylogenetic and phylogenetic methods. For Acontias and Scelotes both signifcantly difered in slope our phylogenetic analyses, we used a tree trimmed from vector length from Typhlacontias and Typhlosaurus, a multi-locus squamate phylogeny [41]. We mapped and Sepsina difered in slope vector length from Typhlo- burrowing-related traits on the tree to show their dis- saurus (Fig. 3B). Te allometric patterns for Acontinae tribution in our sampling (Fig. 1). After, we provide ana- and Scincinae signifcantly difered both in slope vec- tomical descriptions for the four species we examined in tor length (p = 0.0011) and slope vector orientation greater detail. Details on scanning, segmentation, and (p = 0.0082; Fig. 3C). In the reduced dataset, both the analyses can be found in “Methods” section. multivariate and phylogenetic regressions found a sig- nifcant relationship between cranial shape and centroid Cranial morphometrics size (Additional fle 1: Table S4). Tere were signifcant Te frst two principal components PC1 and PC2 interactions between centroid size and genus, subfam- explained 42% and 13% of the cranial shape variation ily, digits, and substrate (Additional fle 1: Tables S3, S4); respectively (Additional fle 1: Table S1). Species with a therefore, pairwise comparisons were run both with and negative PC1 score possess taller crania with relatively without centroid size as a covariate to account for this. short snouts and long frontals, and species with a positive Tere was a signifcant relationship between cra- score have depressed skulls with relatively long snouts nial shape and both genus (p < 0.001) and subfamily

Fig. 2 Location of landmarks on the cranium and right mandible of Acontias occidentalis (CAS 196430) in A dorsal; B lateral; C ventral; D labial; and E lingual views. We placed 21 fxed landmarks on the cranium and 13 on the mandible Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 5 of 53

Fig. 3 The relationship between size and shape in African burrowing skinks. A The largest skull Acontias plumbeus (MCZ R-18358, top row, skull length 31 mm) and the smallest skull Typhlacontias rohani (MCZ R-190458, bottom row, skull length 5.13 mm) in dorsal, lateral, ventral, and labial = = views. Multivariate regression between centroid size and cranial shape (Regression score). Ordinary least squares regression lines are displayed for each B genus, excluding Mochlus which only had one species; and C subfamily except for Lygosominae, which only had one species. Multivariate regression between centroid size and mandibular shape (Regression score). Ordinary least squares regression lines are displayed for each D genus; and E subfamily

(p < 0.001) in the full dataset. Acontinae and Scincinae longer diferent. Te PCA supported these results, as retained signifcantly diferent cranial shapes after size genera formed discrete clusters in morphospace except correction (p < 0.001). Every genus was signifcantly for Scelotes and Sepsina (Fig. 4A). Te ANCOVA for the diferent from every other genus prior to size correc- reduced dataset also showed a signifcant relationship tion except for Scelotes and Sepsina (Additional fle 1: between cranial shape and both genus and subfamily; Table S5). Most results remained unchanged after size however, the PGLS did not (Additional fle 1: Table S4). correction except Acontias and Typhlosaurus were no Te K-statistic’s generalization for multivariate data suggested that less phylogenetic signal was present than Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 6 of 53

Fig. 4 PCA plots showing the relationship between cranial shape and A genus and B substrate; and mandibular shape and C genus and D substrate. Lines connect each species point to the group mean. For the genus PCAs, the pictured crania, from top to bottom and left to right, are Mochlus sundevallii (MCZ R-28682), A. plumbeus, Scelotes gronovii (CAS 173306), Typhlacontias brevipes (CAS 224004), and Typhlosaurus vermis (CAS 196406). The pictured jaws are A. plumbeus, T. vermis, Scelotes sexlineatus (CAS 206813), and T. brevipes. For the substrate plots, the specimen closest to the group mean is shown. The crania are Scelotes arenicola (MCZ R-21292), Acontias gracilicauda (CAS 147466), Scelotes kasneri (CAS 207016), and Acontias kgalagadi (CAS 125809). The jaws are A. gracilicauda, Acontias litoralis (CAS 206810), Sepsina alberti (CAS 263923), and Sepsina copei (CAS 263921)

expected under a Brownian motion (BM) model of trait the reduced dataset (Additional fle 1: Table S4). In the evolution for cranial shape (K = 0.57592, p < 0.001). ANCOVA (Additional fle 1: Table S7), prior to size cor- We found a signifcant relationship between cranial rection, sand burrowers were signifcantly diferent from shape and both limbs (p < 0.001) and digits (p < 0.001) the other three substrates and skinks in leaf litter were in the full dataset. Tis result was driven by a large sig- diferent from those in soil. When corrected for size, nifcant diference between limbless skinks and the other sand burrowers were signifcantly diferent from those in limb states (p < 0.001). Skinks with two limbs and four leaf litter and sandy soil, but not soil. In the PGLS (Addi- limbs did not difer from each other. Although both limbs tional fle 1: Table S8), prior to size correction, sand was and digits showed a signifcant relationship with cranial signifcantly diferent from sandy soil and soil. After size shape in the ANCOVA for the reduced dataset, these correction, only sand and sandy soil were signifcantly relationships ceased to be signifcant when corrected for diferent. phylogeny (Additional fle 1: Table S4). A signifcant relationship was found between cra- Mandibular morphometrics nial shape and substrate in the full dataset (p < 0.001; Te frst two principal components PC1 and PC2 Fig. 4B). Before size correction, all but two pairs, sandy explained 58% and 13% of the mandibular shape vari- soil with soil and with leaf litter, showed a signifcant dif- ation respectively in the full dataset (Additional fle 1: ference (Additional fle 1: Table S6). After size correc- Table S9). Te PC1 axis describes the dentary. Species tion, the only signifcant diferences left were between with a negative score have a longer dentary, with the sand with leaf litter and with sandy soil (Additional fle 1: most posterior point along the lateral side of the mandi- Table S6). Te ANCOVA and PGLS both found a signif- ble. Tey also have a relatively short tooth row. Species cant relationship between cranial shape and substrate in with a positive score have a shorter dentary, with the Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 7 of 53

most posterior point along the ventral side of the mandi- were signifcantly diferent from species with either two ble and a long tooth row extending to the coronoid. Te or four limbs (p < 0.001), but species with two limbs were PC2 axis describes both the coronoid process of the den- not diferent from species with four limbs. Although both tary and the coronoid. Species with a negative score have limbs and digits showed a signifcant relationship with a very short coronoid and species with a positive score mandibular shape in the ANCOVA for the reduced data- have a tall coronoid and coronoid process. set, these relationships were not signifcant when cor- Te multivariate regression for the full dataset showed rected for phylogeny (Additional fle 1: Table S12). a signifcant relationship between mandibular shape Tere was a signifcant relationship between man- and centroid size (p = 0.0167). Smaller skinks have rela- dibular shape and substrate in the full dataset (p < 0.001; tively narrow jaws and larger skinks have thicker, more Fig. 4D). Skinks in sand were signifcantly diferent robust jaws (Fig. 3A). When comparing patterns of from skinks in leaf litter, sandy soil, and soil before and allometry between genera, there were signifcant pair- after size correction (Additional fle 1: Table S14). Te wise diferences between Acontias with Typhlacontias ANCOVA and PGLS both found a signifcant relation- and Typhlosaurus, Feylinia with Typhlosaurus, Scelotes ship between mandibular shape and substrate in the with Typhlacontias and Typhlosaurus, and Sepsina with reduced dataset (Additional fle 1: Table S12). In the Typhlosaurus (Fig. 3D; Additional fle 1: Table S10). Te ANCOVA (Additional fle 1: Table S15), sand burrowers patterns of allometry between Acontinae and Scincinae had signifcantly diferent mandibles from those in leaf did not difer in slope vector length (p = 0.2739), but they litter, sandy soil, and soil. After size correction, sand bur- were signifcantly diferent in slope vector orientation rowers only varied from those in leaf litter and sandy soil. (p = 0.039; Fig. 3E). In the reduced dataset, both the mul- In the PGLS (Additional fle 1: Table S16), prior to size tivariate and phylogenetic regressions showed a signif- correction, burrowers in sand were signifcantly diferent cant relationship between cranial shape and centroid size from those in sandy soil and soil. After size correction, (Additional fle 1: Table S12). Tere were also signifcant they were diferent only from those in sandy soil. interactions between centroid size and all other variables (Additional fle 1: Tables S11, S12). Anatomical descriptions We found a signifcant relationship between man- Below, we provide anatomical descriptions for the skulls dibular shape and both genus (p < 0.001) and subfamily of four species—T. brevipes (CAS 224004), T. gracilis (p < 0.001) in the full dataset. Acontinae and Scincinae (MCZ R-18023), S. alberti (CAS 263923), and A. occiden- had signifcantly diferent mandibular shapes even after talis (CAS 196430)—based on examination of a single size correction (p < 0.001). Prior to size correction, there adult specimen. We begin with a general overview of the were many signifcant pairwise diferences between gen- skull before moving on to descriptions of each individual era (Additional fle 1: Table S13) including Acontias and bone. all genera except Feylinia, Feylinia from Scelotes and Typhlosaurus, Scelotes from Typhlacontias and Typhlo- General skull morphology saurus, Sepsina from Typhlacontias and Typhlosaurus, Te skull length (from the most anterior point of the and Typhlacontias from Typhlosaurus. After size cor- premaxilla to the most posterior point of the braincase rection, the remaining diferences were: Acontias from on the ventral side) and width (from the lateral extent Scelotes and Sepsina, Feylinia from Scelotes and Sepsina, of one quadrate to the other) respectively are 8.33 mm Scelotes from Typhlacontias and Typhlosaurus, Sepsina (8.4% SVL) and 4.26 mm for T. brevipes, 6.03 mm (7.6% from Typhlosaurus, and Typhlacontias from Typhlosau- SVL) and 2.93 mm for T. gracilis, 9.06 mm (11.2% SVL) rus. Te PCA supported these relationships, with many and 4.5 mm for S. alberti, and 13.51 mm (7.5% SVL) and genera occupying unique regions of morphospace with 6.25 mm for A. occidentalis. Te skull is similarly propor- low overlap (Fig. 4C). Te ANCOVA, but not the PGLS, tioned with the length twice or more the width. Te skull for the reduced dataset supported a signifcant relation- of T. brevipes is triangular and wedge-shaped and the ship between mandibular shape and both genus and skull of T. gracilis forms a rounded wedge (Fig. 5A). Te subfamily (Additional fle 1: Table S12). Te K-statistic’s lateral view of S. alberti is similar to T. gracilis although generalization for multivariate data suggested that less with a longer snout (Fig. 6A). Te skull of A. occidentalis phylogenetic signal was present than expected under a has a more robust, rectangular snout (Fig. 6A). In dorsal BM evolutionary model of trait evolution for mandibular view, the snout is more rounded in S. alberti and more shape (K = 0.62051, p-value < 0.001). pointed in the other three (Figs. 5B and 6B). Te palate In the full dataset, we found a signifcant relationship shows closure in all species with the most closure in A. between mandibular shape and the number of limbs occidentalis and T. brevipes, and the least in S. alberti (p < 0.001) and digits (p < 0.001). Species with no limbs (Figs. 5C and 6C). Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 8 of 53

Fig. 5 Articulated skulls of T. brevipes (left) and T. gracilis (right). Views are: A lateral; B dorsal; C ventral; D anterior; E posterior. c, choanae; en, external nares; fov, fenestra ovalis; iptv, interpterygoid vacuity; orb, orbit; ovna, opening for the vomeronasal apparatus; ptfen, posttemporal fenestra; sof, suborbital fenestra. Scale bars 1 mm =

Most bones are present in all examined species. created grooves and pits across the dorsal surface of the Exceptions are the postorbitals, interparietals, and nasals, frontals, and parietal. palpebrals, which are only present in S. alberti. Osteo- Te external nares (Figs. 5 and 6A) are oblong and derms are present in all species. Some are fused to the fattened in T. brevipes, rounded in T. gracilis, oblong skull roofng bones, and digitally removing them has in S. alberti, and oval with a domed dorsal margin in A. Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 9 of 53

Fig. 6 Articulated skulls of S. alberti (left; CAS 263923) and A. occidentalis (right; CAS 196430). A Lateral; B dorsal; C ventral; D anterior; E posterior. c, choanae; en, external nares; fov, fenestra ovalis; inp, interparietal; iptv, interpterygoid vacuity; lf, lacrimal foramen; orb, orbit; ovna, opening for the vomeronasal apparatus; ptfen, posttemporal fenestra; sof, suborbital fenestra; utf, upper temporal fenestra. Scale bars 1 mm = occidentalis. Tey are separated by the premaxilla and prefrontal medially and ventrally, and lacrimal and max- nasals, and extend back to the nasal-maxillary suture. illa laterally. In T. gracilis, the prefrontal does not curve Te orbits are delimited by the prefrontal anteriorly, as far and the lacrimal foramen is delimited by the max- postfrontal posteriorly, frontal medially, jugal laterally illa and jugal ventrally. Te suborbital fenestrae (Figs. 5, (also posteriorly in S. alberti and A. occidentalis), and 6C) are smallest in T. brevipes and largest in S. alberti. maxilla and ectopterygoid ventrally. Te frontal does Tey are bordered by the ectopterygoid laterally, palatine not close the medial wall of the orbit in S. alberti. Te anteromedially, and pterygoid posteromedially (except orbits are circular in S. alberti (21% of skull length), and in A. occidentalis where the pterygoid does not partici- ovoid in A. occidentalis (20%), T. gracilis (18%) and T. pate). In S. alberti, they are also bordered by the maxilla brevipes (13%). Te lacrimal foramen is bordered by the anterolaterally. Te choanae are bordered by the vomer Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 10 of 53

anteromedially and palatine posteriorly, and the openings ventral side of the nasal process. Te vomerine processes for the vomeronasal apparatus are bordered by the vomer extend posterolaterally. Tey are longer in T. brevipes medially and maxilla laterally. Te interpterygoid vacuity where they border the opening to the vomeronasal appa- begins between the medial margins of the palatines and ratus. In T. gracilis, the vomerine process is much shorter. widens posteriorly following the curvature of the ptery- In both species, they are overlapped by the maxillae dor- goids to the sphenoid. It is largest in S. alberti and nar- sally. Lateral to the vomerine processes, the premaxilla rowest in A. occidentalis. Te upper temporal fenestrae has small maxillary processes which articulate with the (Fig. 6B) are narrow and subtriangular in S. alberti. Tey anterolateral processes of the maxilla. Tey are larger and are bordered by the parietal medially, postfrontal and curve dorsally in T. gracilis. Typhlacontias brevipes has 6 postorbital anteriorly, and squamosal laterally. Te pos- tooth loci (6 teeth present) and T. gracilis has 7 tooth loci terior region of the squamosal and parietal contact one (3 teeth present). another, closing the fenestrae and making them smaller Te premaxilla of S. alberti is paired (Fig. 8C). Te compared to non-fossorial skinks [14]. Tese fenestrae nasal process is triangular and broad. It overlaps roughly are absent in Typhlacontias, where they are closed by the one-third of the nasal length. Anteriorly, each premaxilla apposition of the parietal and squamosal, and in A. occi- has two foramina, a smaller dorsal one and a larger ven- dentalis, where the postorbital and squamosal have been tral one. Te vomerine process is small, triangular, and reduced. Te upper temporal arch is present in the scin- overlapped by the maxillae. As in T. gracilis, it reaches cines. Te posttemporal fenestrae (Figs. 5, 6E) are largest the anterior margin of the opening for the vomeronasal in T. brevipes, then in S. alberti, and smallest in T. gracilis apparatus. Laterally, the premaxilla has a small maxil- and A. occidentalis where the parietal nearly contacts the lary process, which has a distinct facet dorsally where the braincase. Te fenestra ovalis (located behind the stapes) maxilla meets it. Sepsina alberti has 9 tooth loci (7 teeth is large and oval in Typhlacontias, small and circular in S. present). Te teeth have slight constrictions near the alberti, and large and elliptical in A. occidentalis. middle and are smaller than in Typhlacontias. Te lower jaw (Fig. 7) consists of four discrete ele- Te premaxilla is paired in A. occidentalis (Fig. 8D). ments: the angular, coronoid, dentary, and splenial, and Te nasal process has a constriction near its anterior end, two fused elements forming the compound bone: the after which it assumes a teardrop shape. Te nasal pro- articular and surangular. Te lower jaw shows the most cess extends roughly halfway up the nasals. Te vomer- curvature in Typhlacontias and the least in S. alberti. Te ine process is broad and triangular. It contacts the vomer Meckelian canal runs open along the lingual side of the dorsally and maxilla laterally. Medially, the premaxillae dentary in the scincines and is then enclosed by the sple- have small ventral knobs which form the incisive process. nial and surangular where it continues to the mandibular Tere is no distinct maxillary process. Instead, the lateral fossa. It is enclosed along its entire length in A. occiden- edge has a slight indent which the maxilla fts into. Tere talis. Te mandibular fossa is larger in T. brevipes and A. are 7 premaxillary tooth loci (7 teeth present). Te teeth occidentalis and smaller in T. gracilis and S. alberti. are similar to Typhlacontias although more robust.

Description of isolated dermatocranial bones Nasal Te nasal (Fig. 9) is thin and paired, with medial Premaxilla Te premaxilla (Fig. 8) is the anterior-most contact between the two. It contacts the premaxilla anter- bone of the skull and can be fused or paired. It consists of omedially, maxilla laterally, frontal posteriorly, prefrontal a curved, ventral alveolar portion and a dorsal nasal pro- posterolaterally, and the septomaxilla ventromedially. cess. It contacts the nasals posterodorsally and the maxil- Te width of a single nasal is about 32% of the length lae posteroventrally. In A. occidentalis, the premaxilla also in Typhlacontias. Te anteromedial premaxillary process contacts the vomer and septomaxilla posteriorly whereas has a shelf facet, which is overlapped by the nasal process in T. gracilis, it contacts the septomaxilla posteriorly. Te of the premaxilla. Tis shelf extends further posteriorly teeth are pleurodont, isodont, and cylindrical. in T. brevipes (Fig. 9A). In T. brevipes, the anteromedial Typhlacontias brevipes has a premaxilla fused at the premaxillary process is more pointed and the anterior base with a slight suture on the nasal process (Fig. 8A) margin bordering the nares is more sharply emarginated whereas T. gracilis possesses a fused premaxilla with no than in T. gracilis (Fig. 9B). Te anteromedial premaxil- trace of a suture (Fig. 8B). Te nasal process is triangular. lary process of T. gracilis is rounded. Te triangular pos- Te base of the nasal process is wider in T. gracilis with terior process overlaps a depressed region of the frontal, rounded corners and narrow in T. brevipes with pointed although this overlap is minimal in T. gracilis. Te pos- corners. Te nasal process overlaps the nasals, extending terior process extends up two-thirds of the frontal in roughly half their length in T. brevipes and one-third in T. brevipes and slightly less than half in T. gracilis. Te T. gracilis. Te septonasal crest is a raised ridge on the maxilla overlaps a small shelf on the dorsolateral surface Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 11 of 53

Fig. 7 Articulated mandibles of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in lateral, medial, dorsal, and ventral views from top to bottom. ang, angular; art, articular of compound bone; cor, coronoid; d, dentary; mc, Meckelian canal; mf, mandibular fossa; san, surangular of compound bone; spl, splenial. Scale bars 1 mm =

of the nasal in T. brevipes, but in T. gracilis, the maxilla margin lacks a distinct posterior process. Anteriorly, the simply abuts the nasal. Both species have a nasal foramen nasal has a very small shelf which is overlapped by the posteriorly. maxilla, but for most of its length, the nasal simply abuts Te width of a single nasal is about 42% of the length the maxilla as in T. gracilis. Tere is a foramen on the left in S. alberti, giving the combined nasals an almost square nasal in the same location as in Typhlacontias although shape (Fig. 9C). Te anteromedial premaxillary process is smaller. triangular and pointed, even more than in T. brevipes. It Te width of a single nasal is about 30% of the length in bears a broad shelf facet, which contacts the nasal pro- A. occidentalis (Fig. 9D). Unlike in the scincines, where cess of the premaxilla, and has a fattened anterior mar- the shelf facet is nearly continuous with the anteromedial gin. Te border with the external nares is emarginated premaxillary process, the shelf facet is not connected to though not as sharply as in T. brevipes. Te posterior the anteromedial premaxillary process in A. occidentalis. Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 12 of 53

Fig. 8 Isolated premaxillae of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in anterior, dorsal, ventral, and lateral views from top to bottom. mp, maxillary process; np, nasal process; pmx-n, nasal facet of the premaxilla; snc, septonasal crest; vp, vomerine process. Scale bars 1 mm =

Te shelf has curved sides to accommodate the premax- the premaxilla. Te anterolateral process of T. gracilis is illa. Te anteromedial premaxillary process is larger too, similar, but the species lacks a distinct anteromedial pro- extending past the shelf. It curves medially, ending in a cess (Fig. 10B). Te medial shelf forms the foor of the sharp point. Te posterior side is slightly curved, and external naris anterodorsally, the anterolateral border of lacks a distinct process. Te nasal also lacks a maxillary the choana, and the lateral border of the opening for the shelf. A small indentation in the lateral margin forms a vomeronasal apparatus. Its curvature follows the shape foramen that is closed ventrally by the maxilla. of the vomer but never contacts it. Te gaps between the vomer and maxilla are larger in T. gracilis (Fig. 5C). Maxilla Te maxilla (Fig. 10) is a paired bone consist- Te medial shelf contacts the palatine posteromedially. ing of a tooth-bearing medial shelf and a large dorsal pro- Te dorsal opening for the superior alveolar canal is at cess that forms the lateral wall of the snout. It contacts the junction of the medial shelf and the dorsal process, the premaxilla anteriorly, nasal dorsomedially, prefrontal and located two-thirds of the length of the maxilla. Te and lacrimal posteriorly, septomaxilla medially, jugal and dorsal process curves dorsomedially to contact the nasal ectopterygoid posteromedially (except in A. occidentalis anteriorly and prefrontal posteriorly. In T. gracilis, there where there is no contact with the jugal), and palatine is greater contact with the prefrontal. Te dorsal pro- ventromedially. Te maxillary teeth are similar to the pre- cess forms the posterolateral border of the external naris maxillary teeth. and part of the lateral border of the lacrimal foramen. It Typhlacontias brevipes has two anterior processes— tapers posteriorly to form the short and blunt posterior the anterolateral process and the anteromedial pro- process. Te posterior process contacts the ectopterygoid cess—which form a U-shaped indentation in ventral posteromedially in T. brevipes and posteriorly in T. graci- view (Fig. 10A). Te anteromedial process curves dor- lis where the posterior process is shorter. It also contacts sally and overlaps the premaxilla’s vomerine process. Te the jugal medially. Tere are three large foramina along anterolateral process contacts the maxillary process of the lateral side in T. brevipes and two in T. gracilis. Te Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 13 of 53

Fig. 9 Isolated nasals of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal (left) and ventral (right) views. apmx, anteromedial premaxillary process; en, external nares; n-mx, maxilla facet of the nasal; n-pmx, premaxilla facet of the nasal; nf, nasal foramen; npp, posterior process of the nasal. Scale bars 1 mm = Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 14 of 53

Fig. 10 Isolated left maxilla of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in lateral, medial, and ventral views from left to right. dsac, dorsal opening for the superior alveolar canal; ipr, incisive process; lf, lacrimal foramen; msh, medial shelf of the maxilla; msh-pal, palatine facet of the medial shelf; mx-ect, ectopterygoid facet of the maxilla; mx-j, jugal facet of the maxilla; mx-pmx, premaxilla facet of the maxilla; mxalp, anterolateral process of the maxilla; mxamp, anteromedial process of the maxilla; mxdp, dorsal process of the maxilla; mxdp-n, nasal facet of the dorsal process; mxdp-prf, prefrontal facet of the dorsal process; mxpp, posterior process of the maxilla; ovna, opening for the vomeronasal apparatus. Scale bars 1 mm =

teeth are limited to the anterior half. Typhlacontias brevi- alveolar canal is about halfway down the maxilla. Te pes has 4 and 4 (left and right) maxillary tooth loci (4 dorsal process curves dorsomedially, as in Typhlacontias, teeth present), and T. gracilis has 5 and 5 maxillary tooth but rather than a straight dorsal edge, the dorsal edge loci (8 teeth present). curves posteriorly. It tapers to form the long posterior As in T. brevipes, the maxilla of S. alberti (Fig. 10C) has process. Te posterior process contacts the jugal antero- two anterior processes although the gap formed between medially and the ectopterygoid posteromedially. Tere them is broader. Te anteromedial process curves dorso- are three large foramina along the lateral side with two medially, resting between the premaxilla and vomer on smaller foramina between them. Te teeth extend up to its ventral side and the septomaxilla on its dorsal side. the posterior process. Tere are more than in Typhlac- It has a smaller area of overlap than in T. brevipes. Te ontias, 16 and 15 tooth loci (27 teeth present), although anterolateral process extends further anteriorly and con- they are similar in shape and size. tacts the maxillary process of the premaxilla. Te medial Like T. brevipes and S. alberti, A. occidentalis has shelf is narrower than in Typhlacontias except for the anterolateral and anteromedial processes, which are expanded posterior lappet which contacts the palatine separated by a shallow W-shaped indent (Fig. 10D). Te dorsally. Tis lappet curves medially, is subtriangular, anteromedial process overlaps the vomerine process of and provides greater contact with the palatine than seen the premaxilla and is overlapped by the septomaxilla. It in Typhlacontias. Te dorsal opening for the superior has a wide base with a triangular point. Te anterolateral Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 15 of 53

process ends in a rounded knob which contacts the pre- A. occidentalis has 8 and 7 maxillary tooth loci (12 teeth maxilla. Te medial shelf is narrower than in the others present). and taken up almost entirely by the tooth loci. Te vomer overlaps the medial shelf, closing the anterior palate Prefrontal Te prefrontal (Fig. 11) is a curved, paired except for the openings for the vomeronasal apparatus bone that forms the anterior wall of the orbit. It contacts and choanae (Fig. 6C). Te depressed shelf for the pala- the maxilla anterolaterally, nasal dorsally, frontal dorsally tine is not as large as in S. alberti, but it is more distinct and medially, and lacrimal laterally. Tere is additional than in Typhlacontias. Te dorsal opening for the supe- ventromedial contact with the frontal in T. brevipes, S. rior alveolar canal is located at two-thirds of the length alberti, and A. occidentalis and posterior contact with the of the maxilla. Te dorsal process has an almost vertical postfrontal in both Typhlacontias. anterior edge. It then curves dorsomedially, contacting Te prefrontal has two dorsal processes in Typhlacon- the nasal anterodorsally, frontal posterodorsally, and pre- tias (Fig. 11A, B). Te anterodorsal process is the shorter frontal posteriorly. It ends in a sharp point. Te posterior of the two, ends in a triangular point, and contacts the process is long, but shorter than in S. alberti. It con- nasal medially. Te posterodorsal process has a rounded tacts the ectopterygoid medially along its entire length. end, which contacts the postfrontal and frontal. Both are Tere are three large foramina on the lateral side, with shorter in T. brevipes. Te anterolateral surface, which is another small foramen near the anterior-most of these. wider in T. gracilis, is overlapped by the dorsal process Te teeth extend up to the posterior process. In total, of the maxilla. Te maxilla extends further dorsally in T.

Fig. 11 Isolated left prefrontal of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in lateral, anteromedial, dorsal, and anterior views from left to right. lf, lacrimal foramen; onf, orbitonasal fange projection; pc, palpebral crest; prf-f, frontal facet of the prefrontal; prf-mx, maxillary facet of the prefrontal; prf-n, nasal facet of the prefrontal; prfadp, anterodorsal process of the prefrontal; prfpdp, posterodorsal process of the prefrontal; prfpvp, posteroventral process of the prefrontal. Scale bars 0.5 mm = Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 16 of 53

brevipes. Ventrally, this region forms the dorsal margin of posteroventral process, which is larger than in the other the lacrimal foramen and ends in a rounded process in T. species. Te palpebral crest is well-defned and forms a brevipes and a triangular point in T. gracilis. Te poster- ridge parallel to the maxilla. Te orbitonasal fange inter- oventral process forms the medial margin of the lacrimal locks with the frontal; it is posterolateral to the frontal foramen. It is small in T. gracilis, only curving ventrally, dorsally but folds and becomes anterior to the frontal whereas it curves back up in T. brevipes. Te palpebral ventrally. Compared to the others, A. occidentalis has the crest is nearly indistinct in T. brevipes whereas in T. graci- smallest orbitonasal fange and the largest gap between lis, it is a notable feature on the lateral side. Te prefron- the two prefrontals. Its orbitonasal fange also terminates tal curves medially to form the orbitonasal fange, which in a blunt end rather than tapering to a point. contacts the frontal dorsally. In anterior view, the paired prefrontals are shaped like bat wings with the orbitona- Palpebral Sepsina alberti is the only one to have palpe- sal fanges sloping down to the center. Tey meet in T. brals (Fig. 12A). Te palpebral is a paired bone that con- brevipes, joined by irregular interlocking projections, but tacts the prefrontal anteromedially and lies in the anterior stop short in T. gracilis. Te medial end in T. gracilis is region of the orbit. It is fattened and roughly triangular simpler, only a single broad knob. Te tube formed by the with a slight medial concavity. orbitonasal fanges is continuous with the cristae cranii of the frontals. Postorbital Te postorbital is absent or fused to the Te prefrontal of S. alberti (Fig. 11C) is more similar to postfrontal in Typhlacontias and A. occidentalis. Without T. gracilis than to T. brevipes. Te anterodorsal process developmental data, the distinction cannot be made. It is is indistinct. Te posterodorsal process is triangular and present as a separate element in S. alberti (Fig. 12B). Te contacts the frontal but not the postfrontal. Te irregu- postorbital contacts the postfrontal anterodorsally along lar edges of the anterolateral surface are likely artifacts of half its length and the squamosal posteriorly. It is a slen- digital segmentation. Ventrally, the prefrontal fares out der bone that curves anteroventrally, extending further and forms the dorsal and medial margins of the lacrimal ventrally than the postfrontal. foramen. Te broad palpebral crest is located on the lat- eral side and is where the palpebral contacts the prefron- Postfrontal Te postfrontal (or possibly postorbitofron- tal. Te orbitonasal fange is triangular and smaller than tal in Typhlacontias and A. occidentalis; Fig. 13A–D) is in Typhlacontias, with a large gap between them. It con- a triradiate paired bone that clasps the frontoparietal tacts the frontal along its entire medial edge. suture. It contacts the frontal anteromedially and the Te anterodorsal process is shorter and wider than the parietal posteromedially. It also contacts the prefrontal posterodorsal process in A. occidentalis (Fig. 11D). Its anteriorly in Typhlacontias and the postorbital ventrally dorsal surface curves medially, more so than in the other in S. alberti. species, and underlies the nasal and frontal. Te pos- Te anterior process is short and broad, and contacts terodorsal process maintains a nearly constant width. It the prefrontal anteriorly in Typhlacontias (Fig. 13A, B). makes up more than half of the dorsal wall of the orbit Tere is a ventral ridge that runs along the ventromedial and contacts the frontal medially. Te entire anterolat- side of the anterior process. It is more clearly defned in eral surface is overlapped by the maxilla. Te prefron- T. brevipes. Te lateral process is shorter than the ante- tal forms the dorsal, medial, and ventral margins of the rior process, curves ventrolaterally, and is triangular. lacrimal foramen. Te ventral margin is closed by the Te posterior process is longer and narrower than the

Fig. 12 Isolated A left palpebral and B left postorbital of S. alberti in lateral view. po-pf, postfrontal facet of the postorbital; po-sq, squamosal facet of the postorbital. Scale bars 0.5 mm = Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 17 of 53

Fig. 13 Isolated postfrontal, jugal, and lacrimal with prefrontal. Left postfrontal of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal (left) and ventral (right) views Left jugal of E T. brevipes; F T. gracilis; G S. alberti; H A. occidentalis; in lateral view. I right lacrimal of T. brevipes; left lacrimal of J T. gracilis; K S. alberti; L A. occidentalis; in posterior view with prefrontal. j-mx, maxillary facet of the jugal; jap, anterior process of the jugal; jf, jugal foramen; jpp, posterior process of the jugal; l-j, jugal facet of the lacrimal; l-mx, maxillary facet of the lacrimal; l-prf, prefrontal facet of the lacrimal; lf, lacrimal foramen; pf-f, frontal facet of the postfrontal; pf-p, parietal facet of the postfrontal; pf-prf, prefrontal facet of the postfrontal; pf-sq, squamosal facet of the postfrontal; pfap, anterior process of the postfrontal; pfp, lateral process of the postfrontal; pfpp, posterior process of the postfrontal; pfr, ventral ridge of the postfrontal; Scale bars 0.5 mm =

other two processes and is pressed close to the parietal. Te posterior process is shorter than in the scincines and It tapers to a point in T. brevipes and to a broad knob in folds medially, ending in a sharp point. T. gracilis. Tere is a triangular facet that articulates with the squamosal on its lateral side. Jugal Te jugal (Fig. 13E–H) is a paired, elongate, and Te anterior process is slender and long in S. alberti curved bone. It does not contact any bone in A. occiden- (Fig. 13C). A well-defned ventral ridge runs alongside talis, but in the others, it contacts the maxilla ventrolater- the ventromedial side. Te lateral process is the longest ally. It also contacts the ectopterygoid ventromedially in of the four species relative to skull size. It is triangular T. gracilis and the lacrimal anteriorly in S. alberti. and narrows to a fne point. Te posterior process tapers Te jugal is highly reduced in Typhlacontias. Te ante- to a rounded end. A small foramen is present halfway on rior process is about the same size as the posterior pro- the posterior process. cess in T. brevipes (Fig. 13E), and smaller in T. gracilis Te postfrontal of A. occidentalis is shaped like a (Fig. 13F). Te jugal has a slight bend where the two pro- checkmark and follows the curvature of the parietal to cesses meet and some tapering near the anterior end in the point of constriction (Fig. 13D). Te anterior process T. brevipes. In T. gracilis, the jugal is constricted where is shorter and broader than the posterior process, but the two processes meet and the anterior process curves narrower than in Typhlacontias. Te ventral ridge runs medially to follow the lateral border of the ectopterygoid. along the entire anterior process and down the poste- Both processes are pointed in T. brevipes and rounded in rior process. Te lateral process is broad and triangular. T. gracilis. Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 18 of 53

Of the species examined, S. alberti is the only one with- Te frontal of Typhlacontias is trapezoidal with out a greatly reduced jugal (Fig. 13G). Te anterior pro- a broad posterior border that narrows anteriorly cess is narrow and extends along the posterior process of (Fig. 14A, B). Te medial process is U-shaped in both the maxilla to the lacrimal. Tere is a small dorsal bump species. Te frontal of T. brevipes has a small, pointed where the anterior process joins the rest of the jugal, after lateral process whereas T. gracilis lacks this. Between which the jugal widens. Te posterior process curves the medial and lateral processes of T. brevipes, there dorsally, narrowing to a point. A jugal foramen is present is a depressed facet, which is overlapped by the nasal. where the jugal begins to curve dorsally. Te posterior border is slightly curved. Te parietal Te jugal is reduced in A. occidentalis (Fig. 13H), but overlaps the frontal at the posteromedial shelf, which rather than reducing the posterior process, A. occiden- is larger and more pronounced in T. brevipes. Te pos- talis has reduced the anterior process. It is a small splint terolateral process is rounded and overlaps the pari- that borders the posterior side of the orbit. It is some- etal. Te cristae cranii curve ventromedially but do not what fattened and tapered at both ends. meet, although they come closer in T. brevipes. In ante- rior view, the tube formed by the cristae cranii is fatter Lacrimal Te lacrimal (Fig. 13I–L) is a small, paired in T. brevipes and more rounded in T. gracilis. Te cris- bone that contacts the prefrontal medially and the maxilla tae cranii are more robust in T. brevipes and have large laterally. It also contacts the maxilla ventrally and the jugal facets anteriorly. Tey are narrower and lack facets in T. posteriorly in S. alberti. Within an individual, contralat- gracilis. eral lacrimals often difer in size. Te frontal of S. alberti is subrectangular, and only Te lacrimal forms the lateral border of the lacrimal slightly narrowed anteriorly (Fig. 14C). Te anterior side foramen in Typhlacontias (Fig. 13I, J). Te size diference forms a large shelf, upon which the nasals sit. Tis gives between the two sides is obvious in T. brevipes where the the frontal an obtuse, triangular shape when viewed in left lacrimal is smaller and closes less than half the lateral the cranium (Fig. 6B). Te medial process is triangular margin of the lacrimal foramen. Te comma-shaped lac- in shape. Te lateral process is also triangular although rimal is larger and the two sides equal in size in T. gracilis. it does not extend as far anteriorly. Te parietal overlaps Te lacrimal of S. alberti (Fig. 13K) is more complex the frontal medially but there is no distinct posteromedial than in the other species. Anteriorly, it forms the ven- shelf, only a slight depression. Te posterolateral process tral and lateral margins of the lacrimal foramen. Te is triangular and overlaps the parietal as in Typhlacon- bone forms an open U-shaped tube with the lateral side tias. Te cristae cranii are triangular and oriented antero- extending further dorsally than the medial side. It con- medially. Tey do not show as much curvature as in the tacts the prefrontal on both sides and the maxilla on the others and do not enter the medial region between the lateral side. Te lacrimal slopes posteroventrally, fatten- orbits. Te anterior side of the crista cranii contacts the ing, and then splits into two posterior processes. Te lat- entire medial length of the prefrontal, closing the ante- eral one is longer. Between these two processes, there is a rior orbit wall. V-shaped indent, into which the jugal fts. Te frontal of A. occidentalis is rectangular (Fig. 14D). Te lacrimal rests along the medial wall of the max- Te medial processes form a triangle. Te lateral process illa for most of its length in A. occidentalis (Fig. 13L), a is large and triangular, extending about as far anteriorly more reclined position compared to the more vertically as the medial process. Between them, there is a large oriented lacrimal of Typhlacontias. At its anterior end, it depressed shelf upon which the nasal sits. Te medial borders the lacrimal foramen ventromedially. Its poste- process is covered by the nasal and the lateral process is rior half runs parallel to the prefrontal without contact- bordered by the nasals medially and the maxillae later- ing it. Te lacrimal tapers posteriorly. ally. Acontias occidentalis lacks posteromedial shelves; the parietal and frontal meet at an angle along the pos- Frontal Te frontal (Fig. 14) is paired with medial con- teromedial side with the parietal slightly overlapping tact with its mate. It contacts the nasals anteriorly, pre- the frontal. Te posterolateral process overlaps the pari- frontal laterally, postfrontal posterolaterally, and parietal etal. Te cristae cranii are much larger in A. occidenta- posteriorly. Tere is additional contact with the prefrontal lis. Tey meet at the midline, creating an enclosed tube ventrally in T. brevipes, S. alberti, and A. occidentalis. It for the olfactory tracts. Anteriorly, they contact the pre- also contacts the maxilla anterolaterally in A. occidentalis. frontal, further reinforcing the skull. Medial processes Te frontal has a fat dorsal surface, which forms part of from the cristae cranii curve ventrally, extending to an the skull roof, and a ventral descending fange called the area between the vomer and palatine without contact- crista cranii. Te two cristae cranii form a canal for the ing either. Two large foramina are present anterior to the olfactory tracts. posterior ends of the cristae cranii. Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 19 of 53

Fig. 14 Isolated frontals of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal, ventral, lateral, and anterior views from left to right. cc, crista cranii; f-n, nasal facet of the frontal; f-p, parietal facet of the frontal; f-pf, postfrontal facet of the frontal; f-prf, prefrontal facet of the frontal; fp, lateral process of the frontal; fmp, medial process of the frontal; fplp, posterolateral process of the frontal. Scale bars 1 mm =

Parietal Te parietal (Fig. 15) is a large, fused bone that squat posteromedial processes. Te posteromedial pro- contacts the frontal anteriorly, postfrontal anterolaterally, cesses of T. brevipes are almost entirely fused, with only a and prootic posteroventrally. It also contacts the squa- small divot between them, to form the dorsal wall of the mosal and supratemporal posterolaterally in the scincines. parietal fossa. Te parietal fossa is a large ovoid opening. Te parietal is composed of a large table, which forms the It narrows anteriorly until it reaches a small foramen on posterior portion of the skull roof, and two descending the dorsal surface. Te cartilaginous processus ascendens processes. On the ventral side, there is a concavity that likely lies inside this tube as documented in Ablepharus accommodates the cerebrum, optic lobe, and cerebellum. [52]. Typhlacontias gracilis has a small parietal fossa vis- Te parietal is a subrectangular bone in Typhlacon- ible posteriorly (Fig. 5E) but lacks the dorsal foramen. tias. Its anterior border is slightly W-shaped in T. brevi- Te rectangular posteromedial processes of T. gracilis are pes (Fig. 15A) and straight in T. gracilis (Fig. 15B). It has clearly separate with a deep parietal notch between them. two small, triangular shelves at the sides, which are over- Te long posterior processes contact the squamosal and lapped by the frontal. Small anterolateral processes form supratemporal laterally, and the prootic ventromedially. the postfrontal facet. A parietal foramen is present. Te Contact with the squamosal extends anteriorly past the parietal has two elongate posterior processes and two posterior process. Te ends of the posterior processes in Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 20 of 53

Fig. 15 Isolated parietal of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal, ventral, and lateral views from left to right. cp, concavity of the parietal; p-pf, postfrontal facet of the parietal; p-st, supratemporal facet of the parietal; p-sq, squamosal facet of the parietal; palp, anterolateral process of the parietal; pdp, descending process of the parietal; pfo, parietal fossa; pfor, parietal foramen; pfsh, parietal shelf for the frontal; pn, parietal notch; ppmp, posteromedial process of the parietal; ppp, posterior process of the parietal; pvr, ventral ridge of the parietal. Scale bars 1 mm =

T. brevipes have a slight bifurcation and a concave sur- species. Te descending process is widest at the base and face whereas the ends in T. gracilis taper to single points narrows ventrally to its tip. It is wider in T. gracilis than and are fat. Despite these diferences in shape, the rela- T. brevipes. In T. gracilis, it extends past the head of the tive length is roughly 36% of the entire parietal in both epipterygoid and contacts its medial surface (Fig. 5A). Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 21 of 53

Due to the uneven size between the two epipterygoids which is larger, also contacts the parietal anterolaterally. It in T. brevipes, only the right descending process extends is triangular and curved on its ventral side. past the epipterygoid although there is no contact. Ven- tral ridges run from the descending process to the pari- Squamosal Except in A. occidentalis, where it is highly etal fossa in T. brevipes. Tey are present, but terminate reduced, the squamosal (Fig. 16) is an elongate, paired more anteriorly in T. gracilis. bone that curves posteriorly and tapers to a point anteri- Although relatively wider, the parietal of S. alberti orly. It contacts the parietal medially, supratemporal pos- (Fig. 15C) is similar in shape to that of Typhlacontias, teromedially, and quadrate posteroventrally. It contacts especially T. gracilis. Te anterior edge is nearly straight the postfrontal anteromedially in Typhlacontias and the except for a small medial curve which overlaps the fron- postorbital anteromedially in S. alberti. tals. Te frontals overlap two small, triangular shelves Te squamosal contacts the parietal for most of its located laterally. Te postfrontal facet extends from the length, closing the upper temporal fenestra in Typhlacon- lateral ridge formed by these shelves to the descending tias (Fig. 5B). It is fattened except where it widens poste- process. Te posteromedial processes are triangular and riorly near where it articulates with the quadrate. Tere form a wide V-shaped parietal notch between them. As is a single condyle in T. brevipes (Fig. 16A), but in T. gra- in T. gracilis, there is a small parietal fossa there (Fig. 6E). cilis, there is both a ventral condyle which fts into the Te long posterior processes contact the squamosal and squamosal notch and a posterior condyle in contact with supratemporal laterally, and the prootic ventromedially. the supratemporal (Fig. 16B). Te squamosal of T. gracilis Contact with the squamosal does not continue anteri- is relatively longer and wider and shows more curvature orly and so the upper temporal fenestrae remain open. than in T. brevipes. Te posterior processes retain the same width for most Overall, the squamosal of S. alberti (Fig. 16C) is more of their length, sharply constricting near the end. Tey slender than in Typhlacontias. It contacts the parietal at account for roughly 40% of the total parietal length. Te its posterior region and forms the lateral border of the descending process is very similar to that in T. brevipes, upper temporal fenestra. Tere is a single condyle at the although not as long. It extends past the head of the epip- posterior end, which articulates with the quadrate. terygoid but does not contact it. Te squamosal of A. occidentalis (Fig. 16D) has Te parietal of A. occidentalis (Fig. 15D) is constricted been greatly reduced to an ovoid stub located near the near its anterior margin, creating an overall hourglass supratemporal. It does not directly contact any bone. shape quite diferent from the other species. Te hori- zontal parietal table is smaller in A. occidentalis as it Septomaxilla Te septomaxilla (Fig. 17A–D) is a paired curves downward. Te anterior margin is slightly curved bone that contacts its counterpart medially, nasals dor- with small lateral shelves which the frontal overlaps. Te sally and maxilla ventrally. It also contacts the premax- parietal foramen is not patent, but there is an indenta- illa anteroventrally in T. gracilis and A. occidentalis, and tion on the ventral surface marking its position. Te vomer posteroventrally in T. gracilis. It is positioned above posteromedial processes are small tabs on either side of the vomer on the foor of the nasal cavity and covers the the U-shaped parietal notch. Similar to T. gracilis and S. vomeronasal apparatus (Jacobson’s organ). alberti, this notch has a small parietal fossa at its center Te ventral concavity occupied by the vomeronasal and there is no dorsal foramen; however, unlike in those apparatus is relatively larger in T. brevipes (Fig. 17A) than two species, ventral ridges run along both sides of the in T. gracilis (Fig. 17B). Te dorsal surface is domed and fossa. On the dorsal surface, there is a ridge and poste- curves vertically to form the nasal septum. In T. gracilis, rior concavity here. Te posterior processes are large the two septomaxillae are joined here; they remain sep- and triangular, tapering to a single point. Teir vertical arate in T. brevipes. Te anterior process projects ante- expansion contributes to the closure of the braincase. rolaterally from the body. Te lateral process projects Tey are only slightly longer (39%) than in Typhlacontias. posterolaterally and forms the widest point. It is triangu- Te descending process has been greatly expanded and lar in T. brevipes and rounded in T. gracilis. In T. brevi- extends from the anterior margin to the posterior pro- pes, the posterior side narrows to the center-line where cess, efectively closing half the skull. It is triangular and the posterior process projects from. Te posterior side extends past the tip of the epipterygoid without contact- is curved in T. gracilis and frst forms the posterolateral ing it. process, which contacts the vomer ventrally. Medial to it, there is a smaller posterior process. Interparietal Te interparietal (inp, Fig. 6B) is only pre- Te ventral concavity makes up most of the septomax- sent in S. alberti. It is a small, paired bone which contacts illa’s length in S. alberti (Fig. 17C). Te dorsal surface is the supraoccipital posteriorly. Te right interparietal, domed and forms a vertical nasal septum stretching from Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 22 of 53

Fig. 16 Isolated left squamosal of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in lateral (left) and dorsal (right) views. sq-pf, postfrontal facet of the squamosal; sq-p, parietal facet of the squamosal; sq-q, quadrate facet of the squamosal; sq-st, supratemporal facet of the squamosal. Scale bars 1 mm = the anterior margin past the center of the bone. Te nasal scincines. In A. occidentalis, it contacts the paroccipital septum tapers to a fne point posteriorly. Te anterior process posteromedially and quadrate ventrally. process projects anterolaterally, although not as much Te contact between the supratemporal and squamosal as in T. brevipes, and the small, triangular lateral pro- is more extensive in T. gracilis (Fig. 17F), continuing for cess projects posterolaterally. Midway between these, the most of the supratemporal’s length, whereas the squa- anterolateral process projects anterolaterally with a slight mosal only contacts the anterior end of the supratem- dorsal curve. It forms a large, rounded knob. From the poral in T. brevipes (Fig. 17E). Te supratemporal of T. lateral process, the septomaxilla turns medially. A small, gracilis is shorter and wider with only a slight fattening triangular posterior process project out at the posterior at the quadrate facet. In T. brevipes, the supratemporal is margin. longer, with a fattened lateral quadrate facet. Tere is a Unlike in Typhlacontias and Sepsina, the ventral con- small ridge anterior to the quadrate facet in T. brevipes. cavity only makes up about half the length of the sep- Te supratemporal of S. alberti (Fig. 17G) is more tomaxilla in A. occidentalis (Fig. 17D). Anteriorly, the robust than in Typhlacontias. It is curved with a pointed septomaxilla has a broad edge, which curls upwards lat- anterior end and a rounded posterior end. It has a large erally. Te triangular lateral process projects postero- indentation near the quadrate articulation. Te contact laterally. Te septomaxilla then curves medially to form with the squamosal is limited to the anterior half of the the triangular posterior process. Tere is a large foramen supratemporal. Medially, it is in continuous contact with at the base of the posterior process. Te dorsal surface the parietal and the paraoccipital. curves to form a small nasal septum. Te supratemporal of A. occidentalis (Fig. 17H) is much broader than in the other species. It is trapezoidal and Supratemporal Te supratemporal (Fig. 17E–H) is a fattened. Te rounded ventral end articulates with the small, paired bone that contacts the parietal anterome- cephalic condyle of the quadrate. Tere is a slight depres- dially, squamosal anterolaterally, paroccipital process sion anteriorly, similar to the squamosal facet seen in the posteromedially, and quadrate posterolaterally in the others, though it does not contact the squamosal. Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 23 of 53

Fig. 17 Isolated septomaxillae and supratemporal. Septomaxillae of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal, ventral, and lateral views from left to right. Right supratemporal of E T. brevipes; F T. gracilis; G S. alberti; H A. occidentalis; in lateral view. ns, nasal septum; smxalp, anterolateral process of the septomaxilla; smxap, anterior process of the septomaxilla; smxlp, lateral process of the septomaxilla; smxplp, posterolateral process of the septomaxilla; smxpp, posterior process of the septomaxilla; st-pop, paroccipital process facet of the supratemporal; st-ppp, posterior process of the parietal facet of the supratemporal; st-q, quadrate facet of the supratemporal; st-sq, squamosal facet of the supratemporal; vnc, ventral concavity for vomeronasal apparatus. Scale bars 1 mm =

Palatine Te palatine (Fig. 18) is a paired bone that T. brevipes having a semicircular notch and T. gracilis a contacts the vomer anteromedially, maxilla anterolater- straight edge. Te pterygoid process extends posteriorly ally, and pterygoid posteriorly. It also contacts the ectop- into the interpterygoid vacuity. It narrows into a slim bar terygoid ventrolaterally in A. occidentalis. Te palatines in T. brevipes and remains broad in T. gracilis. Te ptery- almost contact one another medially, but there remains a goid process has a broad shelf which articulates with the narrow gap in all four species. Each palatine consists of a palatine process of the pterygoid ventrally. From the lat- fat ventral lamina and curved dorsal fange, which meet eral margin, a fange curves dorsomedially, coming near laterally. Te choanal duct runs between them. the center line of the skull and then folding ventrally to Te ventral surface is fat and varies in shape between form the vomerine process. Te vomerine process over- the two Typhlacontias. In T. brevipes, the venter is more laps the vomer dorsally. It is longer in T. brevipes than in gracile and subtriangular (Fig. 18A) whereas in T. gracilis, T. gracilis. Te anterior opening of the choanal duct is it is subrectangular (Fig. 18B). Te medial margin curves elliptical and somewhat fattened. Te posterior opening anterolaterally to form the slender anterolateral process. is more circular. Lateral to this process, there is a small shelf facet which Te ventral surface is narrower and there is a larger gap articulates with the medial shelf of the maxilla. Te ante- between the two palatines in S. alberti (Fig. 18C), leav- rolateral process of T. brevipes is longer than that of T. ing more of the palate open than in Typhlacontias. Te gracilis. Te lateral margin forms the anteromedial bor- venter forms a crescent shape with a broad anterior end der of the suborbital fenestra. Tey difer in shape, with that tapers to a pointed posterior end. Te maxilla covers Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 24 of 53

Fig. 18 Isolated left palatine of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal, ventral, lateral, anterior (top), and posterior (bottom) views from left to right. cd, choanal duct; if, infraorbital foramen; pal-ec, ectopterygoid facet of the palatine; pal-mx, maxillary facet of the palatine; pal-pt, pterygoid facet of the palatine; palp, anterolateral process of the palatine; plp, lateral process of the palatine; ppp, posterior process of the palatine; ppsh, pterygoid process shelf; pptp, pterygoid process of the palatine; sof, suborbital fenestra; vp, vomerine process. Scale bars 1 mm = a semioval region of the anterior end. Laterally, the ante- shape. Te posterior opening, however, is much smaller rior margin curves dorsally to form a small infraorbital and circular. fenestra. Tere are no distinct processes at the ante- Te ventral surface in A. occidentalis is narrower than rior border of the venter. Te pterygoid process, which in Typhlacontias and has an arched rectangular shape extends from the dorsal fange of the palatine and con- (Fig. 18D). Te anterior margin is fat and bears a large, nects with the venter, is broad and triangular. It over- rounded shelf which contacts the maxilla. Directly lat- laps the pterygoid. Te palatine forms the medial border eral to the shelf, the palatine curls in on itself to form of the suborbital fenestra. Te dorsal fange is relatively the large infraorbital foramen. Te palatine forms the longer in S. alberti than in Typhlacontias and has a small entire medial and posterior border of the suborbital foramen near the middle. Anteriorly, it forms the tri- fenestra. Te posterolateral margin forms a half-ellip- angular vomerine process. Te anterior opening of the tical, dorsally oriented lateral process, which articu- choanal duct is larger than in Typhlacontias and oval in lates with the ectopterygoid ventrally and the pterygoid Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 25 of 53

dorsally. Tis process is not seen in the scincines. Te posterior opening is smaller and more compressed. Te pterygoid process is forked, with each part narrow- vomerine process is narrower and sharper. Te poste- ing into a triangular point. Te pterygoid fts between rior end forms a broad posterior process, the ventral them and overlaps the lateral pterygoid process. Tere side of which contacts the pterygoid. It extends fur- is a foramen near the base of the pterygoid process. Te ther posteriorly than the pterygoid process. Tere are anterior opening of the choanal duct is much larger and two foramina on the dorsal surface, one near the lateral more circular than in the other species. However, the process and one posteromedial.

Fig. 19 Isolated vomer of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal, ventral, lateral, anterior (top), and posterior (bottom) views from left to right. mef, medial foramen of the vomer; nr, nasal region of the vomer; nss, space for the nasal septum; v-pal, palatine facet of the vomer; vap, anterior process of the vomer; vae, anterior edge of the vomer; valp, anterolateral process of the vomer; vamp, anteromedial process of the vomer; vle, lateral edge of the vomer; vlp, lateral process of the vomer; vnar, vomeronasal region of the vomer; vp, vomerine process; vplp, posterolateral process of the vomer; vpmp, posteromedial process of the vomer; vs, vomerine septum. Scale bars 1 mm = Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 26 of 53

Vomer Te vomer (Fig. 19) is a fused bone that contacts center line and contact the vomerine process of the pala- the palatine posteriorly. It contacts the premaxilla anteri- tine dorsally. orly in T. gracilis and A. occidentalis, and the septomaxilla Te vomer of A. occidentalis is roughly diamond- dorsally in T. gracilis. Te lateral edge forms the medial shaped (Fig. 19D). Rather than a single anterior process, border of the opening for the vomeronasal apparatus A. occidentalis has two rounded anterolateral processes, anteriorly and the medial border of the choana posteri- which contact the premaxilla ventrally, and a single anter- orly. omedial process, which protrudes ventrally. Two ventral Te anterior process is rounded at the tip in Typhlac- grooves run along either side of this process, ending in ontias. In T. gracilis, it contacts the premaxilla anteri- two medial foramina. Tese foramina also have open- orly and has a ventral knob (Fig. 19B). A ventral groove ings on the dorsal surface. Te anterior margin borders runs from the anterior tip to a small medial foramen. Te but does not contact the premaxilla and maxilla. It termi- medial foramen is located further posteriorly in T. gra- nates with the lateral processes, short but distinct knobs. cilis than in T. brevipes (Fig. 19A). Te anterior margin Te lateral edge fares out, extending past the maxilla at runs parallel to the premaxilla. Te lateral edge fares out its widest point to close the entire roof of the mouth. Te more in T. brevipes, closing more of the palate, whereas in vomeronasal region has been reduced to a slim crescent T. gracilis, the lateral edge is relatively straight. Te dor- and the nasal region is elevated. Tree oval foramina are sal surface is divided into the smaller vomeronasal region located along the space for the nasal septum, two anterior anteriorly and larger nasal region posteriorly by the on either side and one posterior located centrally. Te vomerine septum, a ridge that runs from the edges later- vomer tapers down to form the posteromedial processes. ally to the center and then curves posteriorly. Te vomer- Te posteromedial processes are much smaller than in ine septum is larger and more pointed in T. brevipes, Typhlacontias and only cover the most anterior region of whereas in T. gracilis, the vomerine septum is rounded the palatine. and protrudes less from the dorsal surface. Te nasal region is larger in T. brevipes. Ventrally, this region is con- Pterygoid Te pterygoid (Fig. 20) is a paired bone that cave, and more pronounced in T. gracilis. Te space for contacts the palatine and ectopterygoid anteriorly, epip- the nasal septum runs medially along the dorsal surface terygoid dorsally, sphenoid medially, and quadrate pos- as a distinct ridge. It is interrupted in T. gracilis by the teromedially. It does not contact the quadrate in A. occi- medial foramen. Te posterior region difers between the dentalis. Te general shape is triradiate, though it varies two species. In T. brevipes, the vomer ends in broad lap- substantially between genera. pets whereas in T. gracilis, the lateral edge folds straight Te palatine process is broad and triangular in into the center, completing its squarish appearance. Te Typhlacontias, contacting the palatine on its dorsal side. posteromedial process extends from the centerline to the Te pterygoid fange curves anterolaterally to contact the posterior end of the palatines, further closing the palate. ectopterygoid. It is broader and more robust with a dor- Te palatines contact the vomer on the dorsal surface of sal curve in T. brevipes (Fig. 20A). Te pterygoid fange this process. Te posteromedial process is narrower in T. and palatine process form part of the suborbital fenestra’s brevipes. It is broad and triangular in T. gracilis. posterolateral border. Te fossa columella, which receives Te vomer is wider throughout its entire length in S. the epipterygoid, is located 56% and 44% of the length alberti (Fig. 19C) than in Typhlacontias or A. occiden- from the anterior most point of the pterygoid in T. brevi- talis. Te anterior process is broader. It is pentagonal pes and T. gracilis (Fig. 20B) respectively. Te quadrate with a wide anterior margin which does not contact the process curves laterally in both species to contact the premaxilla. Sepsina alberti has two medial foramina on ventromedial side of the quadrate. It is broader in T. brev- either side of the center line on the anterior process. Te ipes than T. gracilis. At the posterior end in T. brevipes, vomerine septum is more distinct in S. alberti than in the quadrate process fares out vertically. Te quadrate Typhlacontias. Te nasal region is concave, which gives process of T. gracilis retains a similar height throughout the ventral surface a domed appearance. Tis is not seen and ends in a broad rounded tip. in Typhlacontias or A. occidentalis, even though they also As in Typhlacontias, the pterygoid of S. alberti has show concave nasal regions. Te space for the nasal sep- three distinct processes (Fig. 20C). However, it is much tum is distinct in S. alberti and has twin ridges anterior to wider with a diferent shape. Te palatine process is the vomerine septum. Tere is a small foramen posterior broad and rectangular, contacting the palatine dor- to these ridges. Te nasal region ends in dorsally slanted, sally. It has a medial slant, making the posterior ends triangular posterolateral processes. Tey overlap the pal- closer together than the anterior ones. Te pterygoid atines without contacting them. Two short and triangular fange is considerably longer than in Typhlacontias. It posteromedial processes originate from either side of the is covered by the ectopterygoid for most of its ventral Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 27 of 53

Fig. 20 Isolated right pterygoid of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal, ventral, and lateral views from left to right. fco, fossa columellae; pap, palatine process of the pterygoid; pt-bp, basipterygoid facet of the pterygoid; pt-ect, ectopterygoid facet of the pterygoid; pt-pal, palatine shelf of the pterygoid; ptf, pterygoid fange; ptqp, quadrate process of the pterygoid; ptt, pterygoid teeth; sof, suborbital fenestra. Scale bars 1 mm =

surface and part of its medial surface. Its anterior end It has a broad triangular tip, and a fat dorsal surface narrows to a point, which fts into a divot in the ectop- which overhangs the curved ventral side. terygoid. Te pterygoid fange and the palatine process form the posterior margin of the suborbital fenestra. Ectopterygoid Te ectopterygoid (Fig. 21A–D) is a Te fossa columella is located roughly 64% of the length paired, crescent-shaped bone that contacts the maxilla of the pterygoid, which is further posterior than the anteriorly and pterygoid posteriorly. It contacts the jugal other species. Te quadrate process contacts the ven- anterolaterally in T. gracilis and palatine posteriorly in A. tromedial side of the quadrate. It is broader than in T. occidentalis. It forms the lateral border of the suborbital gracilis, but does not fare out as much as in T. brevi- fenestra. pes. Sepsina alberti is the only examined species with Te maxillary process curves medially and ends near pterygoid teeth. It has two small, triangular pterygoid the maxilla-palatine suture. In T. gracilis, the maxil- teeth located near the junction of the palatine process lary process expands anteriorly (Fig. 21B). Te posterior and pterygoid fange. region has a triangular concavity that clasps the ptery- Te pterygoid of A. occidentalis is quite diferent from goid fange dorsally. It is more pronounced in T. brevipes the scincines as it only has two distinct processes, giv- (Fig. 21A). Te posterior region splits into the dorsal and ing it a crescent shape (Fig. 20D). Rather than a palatine ventral processes, which border the pterygoid fange. Te process, A. occidentalis has a palatine shelf, an elliptical, dorsal process is fattened and appears fused to the ven- depressed region upon which the palatine sits. Te ptery- tral process in T. gracilis. Te ventral process has a slight goid fange is longer and curves anterolaterally, contact- medial curve to it. ing the ectopterygoid medially and palatine ventrally. Te Te curvature of the ectopterygoid in S. alberti fossa columella is located about 43% of the length of the (Fig. 21C) is more pronounced than in Typhlacontias. pterygoid. Te quadrate process curves posterolaterally. Te maxillary process curves medially, ending in a Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 28 of 53

Fig. 21 Isolated ectopterygoid and scleral ring. Left ectopterygoid of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in dorsal, ventral, and lateral views from left to right. Left scleral ring of E T. brevipes; F T. gracilis; G S. alberti; H A. occidentalis; in lateral (left) and medial (right) views. ect-pt, pterygoid facet of the ectopterygoid; ectdp, dorsal process of the ectopterygoid; ectmp, maxillary process of the ectopterygoid; ectvp, ventral process of the ectopterygoid; Scale bars 1 mm =

rounded point where the posterior process of the max- Scleral ring Te scleral ossicles (Fig. 21E–H) are small illa splits of. Te lateral margin of the maxillary pro- bones that form a ring within the eye. Segmentation of cess is somewhat fattened. Te dorsal process is fused individual ossicles was challenging, as they were rarely to the ventral process, similar to the condition in T. gra- distinct. Tis may be an artifact of the scan or may indi- cilis. Te diference is that the concavity in which the cate a high level of fusion, as seen in other burrowers pterygoid fange fts is expanded. Te ventral process [53]. curves medially and is broader than in Typhlacontias. Typhlacontias brevipes has at least 8 ossicles, although Te maxillary process is relatively broader and shows some are likely the result of fusion (Fig. 21E). Tey are greater curvature in A. occidentalis than in the others roughly the same size and shape. Individual ossicles in T. (Fig. 21D). Its lateral side is fattened and slightly con- gracilis could not be distinguished (Fig. 21F). Te aper- cave, creating a facet for the maxilla. Te posterior ture diameter is 64% of the external ring diameter in T. region forks into two distinct processes: the longer brevipes and 54% in T. gracilis. dorsal process, which contacts the pterygoid, and the Sepsina alberti has 19 ossicles (Fig. 21G). Tere seems shorter ventral process, which contacts the palatine. to be a lot of fusion, particularly along the interior mar- Both processes have been dorsoventrally compressed, gin. Te ossicles are narrow and rectangular. Te aperture particularly the dorsal process. Te ectopterygoid diameter is roughly 60% of the external ring diameter. is more curved with a larger dorsal process in A. Acontias occidentalis has at least 15 ossicles occidentalis. (Fig. 21H), although there may be more that could not Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 29 of 53

Fig. 22 Isolated left quadrate of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in anterior, dorsal, lateral, medial, and posteroventral views from left to right. icc, intercalary cartilage; mac, mandibular condyle of the quadrate; q-pt, pterygoid facet of the quadrate; qc, quadrate medial column; qcc, cephalic condyle of the quadrate; qco, quadrate conch; qll, lateral lamina of the quadrate; qml, medial lamina of the quadrate; qtc, tympanic crest; sqn, squamosal notch. Scale bars 1 mm = be distinguished. Many are fused together. Te aperture ventrally. It does not contact the squamosal or pterygoid diameter is roughly 59% of the external ring diameter. in A. occidentalis. In lateral view, the quadrate is D-shaped in Typhlac- ontias (Fig. 22A, B). Te conch is fat, with only a slight Description of isolated splanchonocranial bones medial curvature to it. Te cephalic condyle is positioned Quadrate Te quadrate (Fig. 22) plays an important role posteromedially and contacts the paroccipital process in supporting the peripheral auditory system, in cranial of the otoccipital and the supratemporal. It is large and kinesis, and in jaw biomechanics, both as a muscle attach- bulbous, especially in T. brevipes where it forms a ven- ment site and as the sole articulation between the lower tral knob. Te squamosal notch is anterior to the cephalic jaw and cranium [13, 54]. It is a paired bone that contacts condyle and serves as the articulation point for the squa- the squamosal and supratemporal dorsally, the otoccipital mosal. Te mandibular condyle comprises distinct medial dorsomedially, the pterygoid medially, and the articular and lateral condyles, which articulate with the lower jaw. Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 30 of 53

A medial column extends between the cephalic condyle Te epipterygoid has a ventral expansion where it con- and mandibular condyle. Te medial lamina runs along tacts the pterygoid. In T. gracilis, the epipterygoid is thick this column. It is more distinct in T. brevipes. Te lateral and columnar with a slight constriction near the center lamina runs along most of the quadrate in T. gracilis, but and a rounded top (Fig. 23B). Te epipterygoid of T. brev- is only clearly visible on the ventral half in T. brevipes. ipes is smaller, with an irregular, curved shape (Fig. 23A). A pterygoid facet is present slightly dorsal of the medial As this is an endochondral element, it is possible that the mandibular condyle. Te reduced tympanic crest pro- shape is an artifact due to the inability of the scan to cap- jects medially along the conch. Te quadrate is more ture cartilaginous portions of the epipterygoid. recumbent in T. brevipes than in T. gracilis. Te height Te epipterygoid of S. alberti is narrow and columnar, (measured from the cephalic condyle to the mandibular nearly the same width throughout (Fig. 23C). Te base is condyle) is roughly two times the width (measured at the rounded and the top is fattened. widest point of the conch) in both species. Te epipterygoid of A. occidentalis has a slight lateral Te quadrate of S. alberti varies from the quadrate tilt, most visible in anterior view (Fig. 23D). It is thick and of Typhlacontias in many ways, although it has a simi- columnar, with a large paddle-like expansion at the dorsal lar D-shaped silhouette (Fig. 22C). Te conch is more end. Te base is rounded as in the other species. curved, with the tympanic crest curving laterally instead of medially. Te cephalic condyle is oriented medially and Stapes Te stapes (or columella; Fig. 23E–H) forms part contacts the supratemporal and paroccipital process of of the middle ear and is a paired bone. It consists of a foot- the otoccipital. Its medial side is fattened. Directly ante- plate, which sits in the fenestra ovalis, and a shaft which rior, the squamosal notch is a distinct foramen. Te squa- extends perpendicularly from the footplate. mosal articulates with its posterior margin. Te lateral Te footplate is large and roughly circular with a con- side of the squamosal notch is closed by the intercalary cave surface in Typhlacontias. Te short and broad shaft cartilage. Te medial and lateral mandibular condyles are ends with a fat top that nearly contacts the quadrate in narrower than those in Typhlacontias and A. occidenta- T. brevipes (Fig. 23E). In T. gracilis, the shaft has a more lis. Te medial column is more curved in S. alberti than rounded top (Fig. 23F). Typhlacontias. Te lateral lamina is not present, but the Of the examined species, S. alberti has the smallest sta- medial lamina is very distinct. Te pterygoid facet is pre- pes (Fig. 23G). Te footplate is circular and does not fll sent posterior to the medial lamina and slightly dorsal of the entire fenestra ovalis, possibly because part of it is the mandibular condyle. Te quadrate is less recumbent cartilaginous. Te shaft is columnar with a fat end. than in Typhlacontias. Te height is roughly twice the Te stapes of A. occidentalis has an elliptical foot- width. plate (Fig. 23H). Although large, it is still smaller than in Te quadrate of A. occidentalis has a very diferent Typhlacontias. Te shaft is longer and thinner than in the shape than that of the scincines (Fig. 22D). Te reduced other species. It is dorsoventrally compressed but broad- conch is roughly triangular. Its dorsal edge forms the ens out at the fattened end. highly reduced and medially curving tympanic crest, similar to Typhlacontias but smaller. Te cephalic con- Hyoid apparatus Te hyoid apparatus (Fig. 24) consists dyle is oriented posterodorsally and cups the paroccipi- of multiple elongate ossifed and cartilaginous elements tal process and supratemporal. It is slightly concave and located in the posteroventral region of the head and circular. Te squamosal notch is present as an indenta- extending into the neck. Tey do not contact any bones. tion between the cephalic condyle and tympanic crest, Only S. alberti shows the full complement of elements, but it does not contact the squamosal. Te mandibular although in the other species, the missing elements may condyle, made up of medial and lateral condyles, forms be cartilaginous and not visible in the CT scans. a broad base, larger than in the other skinks. Te medial Both Typhlacontias show a single glossohyal and column runs along the posterior side and is more curved basihyal with one pair of ceratobranchials, forming a in the scincines. Te quadrate is less recumbent than in Y-shaped hyoid apparatus. Te glossohyal and basihyal Typhlacontias, but more than in S. alberti. Te height is are stick-like elements that together extend roughly to roughly one and a half times the width. the level of the anterior margin of the sphenoid. In T. brevipes, the glossohyal and basihyal look nearly con- Epipterygoid Te epipterygoid (Fig. 23A–D) is a paired, tinuous with only a slight constriction where they meet rod-like bone that extends from the fossa columella of the (Fig. 24A). In T. gracilis, the basihyal is thicker and there pterygoid to a region near the descending process of the is a bulge where the glossohyal begins (Fig. 24B). Te parietal and the crista alaris of the prootic. In T. gracilis, it glossohyal narrows to a point and the basihyal has a pos- contacts the parietal medially. terior bifurcation. Te tips of the bifurcation arch inwards Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 31 of 53

Fig. 23 Isolated epipterygoid and stapes. Right epipterygoid of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in anterior (left) and lateral (right) views. Left stapes of E T. brevipes; F T. gracilis; G S. alberti; H A. occidentalis; in posterior (left) and lateral (right) views. ept-fco, pterygoid facet of the epipterygoid; fp, oval footplate of the stapes; sh, shaft of the stapes. Scale bars 1 mm =

in T. brevipes. Te ceratobranchials originate here and Of the examined skinks, S. alberti is the only one to arch laterally. Near their center, the ceratobranchials also have all the elements of the hyoid apparatus, including curve dorsally although this is less extreme in T. gracilis. a single basihyal and glossohyal, and paired frst cerato- Te ceratobranchials of T. gracilis are more robust than branchials, second ceratobranchials, ceratohyals, and in T. brevipes. hypohyals (Fig. 24C). Te narrow glossohyal stems from Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 32 of 53

Fig. 24 Isolated hyoid apparatus of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in ventral and lateral view. bhy, basihyal; cbI, frst ceratobranchial; cbII, second ceratobranchial; chy, ceratohyal; ghy, glossohyal; hhy, hypohyal. Scale bars 1 mm =

the basihyal and extends to the posterior margin of the as the lower margin of the orbit. Te ends are broad and palatine. Te basihyal is Y-shaped, with a posterior bifur- fat. Te second ceratobranchial is elongate and extends cation. Tree elements originate from each tip: the hypo- posteriorly with a slight dorsal tilt. It narrows posteriorly. hyal anteriorly, the frst ceratobranchial posterolaterally, Te ceratohyal is anterior to the frst ceratobranchial and and the second ceratobranchial posteromedially. Te lateral to the hypohyal. It has a narrow crescent shape hypohyal has rounded ends and a slight arch. Te pos- and runs parallel to the frst ceratobranchial. A fattened, terior half of the frst ceratobranchial curves dorsolater- trapezoidal chip of bone is present near the anterior end ally, turning vertical and reaching roughly the same level of the ceratohyal. In addition to being more complex than Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 33 of 53

in the other species, the hyoid apparatus of S. alberti has Neurocranium more elongate and gracile elements. Te neurocranium is divided into the orbitotempo- Te hyoid apparatus of A. occidentalis (Fig. 24D) is very ral region (Fig. 25), represented by ossifed and carti- similar to that of Typhlacontias. Te glossohyal extends laginous elements located centrally in the skull, and the to the level of the posterior region of the palatine. Te otoccipital region, represented by a single fused struc- basihyal’s bifurcating ends are larger and narrower than ture called the braincase (Figs. 26, 27). Te orbitotem- in the other species. Te frst ceratobranchial is the most poral region consists of the medial interorbital septum, robust of the elements and retains a similar thickness planum supraseptale, fused or paired orbitosphenoids, throughout. It has a slight dorsal curve, but does not and cultriform process. Many elements are partly or curve as much as in the other species.

Fig. 25 To the left, the orbitotemporal region of the neurocranium within the dermatocranium of T. brevipes, T. gracilis, S. alberti, and A. occidentalis from top to bottom in lateral view. The region is boxed in red. To the right, isolated orbitotemporal elements in lateral (left) and posterodorsal (right) views. A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis. cp, cultriform process; mis, medial interorbital septum; ors, orbitosphenoids. Scale bars 0.5 mm = Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 34 of 53

(See fgure on next page.) Fig. 26 Isolated otoccipital neurocranium of A T. brevipes and B T. gracilis; in dorsal, ventral, anterior, posterior, and lateral views from top to bottom. ascc, anterior semicircular canal; avc, anterior opening of the vidian canal; bo, basioccipital; bp, basipterygoid process; cal, crista alaris; cpro, crista prootica; crif, crista interfenestralis; crs, crista sellae; crt, crista tuberalis; fm, foramen magnum; fov, fenestra ovalis; hscc, horizontal semicircular canal; hvc, lateral head vein; ip, incisura prootica; la, lateral ampulla; lrst, lateral opening of the recessus scalae tympani; occ, occipital condyle; ocr, occipital recess; oto, otooccipital; pop, paroccipital process; pro, prootic; pscc, posterior semicircular canal; pvc, posterior opening of the vidian canal; so, supraoccipital; sopa, facet for the processus ascendens; sph, sphenoid; spht, sphenooccipital tubercle; st, sella turcica; trb, trabecula; VII, foramen of CN VII; X, foramen of CN X; XII, foramen of CN XII. Scale bars 1 mm = entirely cartilaginous in the examined species, with the scincines) the odontoid process of the axis. In A. occiden- planum supraseptale entirely cartilaginous in all four. talis, the odontoid process has been greatly reduced, so Te paired orbitosphenoids of Typhlacontias lie this contact is lost. Te inner ear cavities are formed by directly posterior to the cristae cranii of the frontal. Tey the prootics, supraoccipital, and otooccipitals. Te exter- are well-ossifed. Te orbitosphenoids of T. brevipes are nal opening to the inner ear is the fenestra ovalis, which unfused (Fig. 25A). Tey are y-shaped with the longest is located between the prootic and otooccipital. process extending to the sphenoid. Tey lie in the hori- Te width of the braincase is 63% and 66% of its length zontal plane. Te orbitosphenoids of T. gracilis have fused in T. brevipes and T. gracilis, respectively, making their into a ring structure with two lateral wings (Fig. 25B). braincases relatively longer than in the other species. In Tey lie at a roughly 45° angle from the horizontal. T. brevipes, the walls of the braincase are separated by Te elements of the orbitotemporal region are only a narrow medial gap that continues to the roof of the partially ossifed in S. alberti (Fig. 25C). Te medial inter- supraoccipital (Fig. 26A). In T. gracilis, they meet and orbital septum is a small, rod-like bone located between fuse further down (Fig. 26B). Te foramen magnum is the orbits. Te orbitosphenoids show little ossifcation larger and circular in T. brevipes. In T. gracilis, it is some- with two small, lateral pieces located anterior to a larger what fattened dorsally. Te occipital condyle is larger in medial piece. Tey are oriented at an angle. Te cultri- T. brevipes, but has a smaller anterior notch. form process extends from the sphenoid to a region ven- Te braincase of S. alberti is wider than it is long, with tral to the orbitosphenoids. It is long and narrow, with the width 112% of the length (Fig. 27A). Te brain cav- four ossifed pieces. ity has a medial gap similar to the one in T. brevipes, Te elements of the orbitotemporal region show partial although wider and not as long. Te foramen magnum ossifcation in A. occidentalis (Fig. 25D), although they is more ovoid than in Typhlacontias. Te occipital con- are more ossifed than in S. alberti. Te medial interor- dyle is the smallest of the examined species, but similar bital septum is located directly posterior of the cristae in shape to T. gracilis. cranii. Te orbitosphenoids comprise two ossifed pieces Te width of the braincase is 85% of the length in A. set at an angle. Te cultriform process extends from the occidentalis (Fig. 27B). It is compressed. In anterior view, sphenoid to the orbitosphenoids in three main pieces. the brain cavity has a V-shaped medial gap and appears Anteriorly, there are two elongate, fattened pieces which narrower than in the other species. Te foramen mag- are lateral to one another. Posterior to these pieces, there num is ovoid with a greater height than width. Te occip- is a single, broader piece. ital condyle is very large and sutures can be seen on its Te braincase is formed by the fusion of three medial dorsal surface. unpaired elements (sphenoid, basioccipital, and supraoc- cipital) and two lateral paired elements (prootic and Sphenoid Te sphenoid (Figs. 26, 27) forms the anterior otooccipital). It is covered dorsally by the parietal and region of the braincase. It is formed by the fusion of the contacts the quadrate, pterygoid, supratemporal, sta- basisphenoid and parasphenoid, and contacts the basioc- pes, atlas, and axis (except in A. occidentalis). Te sphe- cipital posteriorly, prootic dorsally, and pterygoid ante- noid and basioccipital form a concave ventral foor. Te rolaterally. Te sphenoid has a wide and deep depression prootics, otooccipitals, and supraoccipital form the roof called the sella turcica, which houses the pituitary gland. and walls of the brain cavity. Te foramen magnum is a Tere is complete fusion between the basioccipital and large, circular opening in the posterior side. Its bounda- sphenoid in Typhlacontias. Anteromedially, the sphe- ries are delimited by the supraoccipital dorsally, otooc- noid is broad with small paired trabeculae in T. brevi- cipitals laterally, and basioccipital ventromedially. Te pes (Fig. 26A). In T. gracilis, the sphenoid narrows to a occipital condyle, located ventral to the foramen mag- fattened tip and there are no trabeculae (Fig. 26B). Te num, is formed by the otooccipitals laterally and basioc- anterior openings for the vidian canal are located ventral cipital medially, and articulates with the atlas and (in the to the trabeculae. Tey are not visible in T. gracilis, likely Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 35 of 53 Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 36 of 53

(See fgure on next page.) Fig. 27 Isolated otoccipital neurocranium of A S. alberti; B A. occidentalis; in dorsal, ventral, anterior, posterior, and lateral views from top to bottom. aa, anterior ampulla; ascc, anterior semicircular canal; avc, anterior opening of the vidian canal; bo, basioccipital; bp, basipterygoid process; bsd, basicranial sesamoid; cal, crista alaris; cpro, crista prootica; crif, crista interfenestralis; crs, crista sellae; crt, crista tuberalis; fm, foramen magnum; fov, fenestra ovalis; hscc, horizontal semicircular canal; hvc, lateral head vein; inp, interparietal; ip, incisura prootica; ipr, inferior process of the prootic; la, lateral ampulla; lrst, lateral opening of the recessus scalae tympani; occ, occipital condyle; ocr, occipital recess; oto, otooccipital; pop, paroccipital process; pro, prootic; pscc, posterior semicircular canal; pvc, posterior opening of the vidian canal; so, supraoccipital; sopa, facet for the processus ascendens; sph, sphenoid; spht, sphenooccipital tubercle; st, sella turcica; trb, trabecula; VII, foramen of CN VII; X, foramen of CN X; XII, foramen of CN XII. Scale bars 1 mm = due to their small size. Te basipterygoid processes point they barely extend further than the trabeculae anteri- anterolaterally in both species, but extend further ante- orly. Te distal ends are broad and rounded. Tey are riorly in T. brevipes. Te distal ends are expanded and a similar width for their entire length. Tey make up contact the pterygoid. Tey are circular in T. brevipes and about 19% of the total braincase length. Te sella tur- more elliptical in T. gracilis. Te basipterygoid processes cica is prominent, as in S. alberti, and is located on the are squat, making up 13% and 18% of the total braincase posterior half of the sphenoid. Te depression is sub- length in T. brevipes and T. gracilis respectively. Te sella rectangular and bordered laterally by distinct ridges. turcica is located at the posterior region. Its posterior Te crista sellae forms a concave wall. Lateral to the border, the crista sellae, forms a slight ridge. Anterior sella turcica, there are half-open canals connecting the to this ridge, the sphenoid of T. brevipes has four lateral openings of the vidian canal. foramina and T. gracilis two foramina. Te posterior set of foramina are the posterior openings of the vidian canal Basioccipital Te basioccipital (Figs. 26, 27) forms the whereas the anterior ones in T. brevipes are the internal posterior portion of the brain cavity’s foor and contacts continuation of the anterior openings of the vidian canal. the sphenoid anteriorly, otooccipital laterally, and atlas Te posterior openings open on the ventrolateral wall of and axis posteriorly. Its dorsal surface is concave and the sphenoid. Overall, the sphenoid of T. brevipes is rela- ventrolaterally it curves out to form the sphenooccipital tively longer than that of T. gracilis. tubercles. Te otooccipitals also participate in forming Te sphenoid of S. alberti (Fig. 27A) is relatively shorter these tubercles. than in Typhlacontias. It is completely fused with the Te basioccipital is wider than it is long in both basioccipital. Te small anterior edge has paired, triangu- Typhlacontias. Ridges form a rounded W-shape on the lar trabeculae. Te anterior openings for the vidian canal ventral side in T. brevipes (Fig. 26A). Tese are not seen are located lateral to the trabeculae. Te basipterygoid in T. gracilis (Fig. 26B). Te sphenooccipital tubercles processes point anterolaterally. Tey are similar in shape protrude out more in T. brevipes. In T. gracilis, they are to those of T. gracilis, but larger, with expanded posterior smaller and have fatter ventral surfaces. sides. Tey contact the basipterygoid facets of the ptery- Te general shape of the basioccipital of S. alberti goid along their fattened medial sides. Te basipterygoid (Fig. 27A) is more similar to the basioccipital of T. gracilis processes make up 23% of the total braincase length. Te than of T. brevipes. Te occipital condyle and sphenooc- sella turcica is very prominent. It is located directly pos- cipital tubercles are much smaller than in Typhlacontias. terior to the anterior margin. Te crista sellae is more Te basioccipital of A. occidentalis (Fig. 27B) is quite distinct than in Typhlacontias and has a slight concave similar to that of the examined scincines, especially T. curvature. On the lateral sides of the sella turcica, there brevipes. Te occipital condyle is large and protrudes are partially open canals, which run between the anterior more than in the other species. Te sphenooccipital and posterior openings of the vidian canal. tubercles are also larger. Tey are capped by basicranial Te sphenoid of A. occidentalis (Fig. 27B) is relatively sesamoids (or element X). Tese sesamoids are elliptical longer than in T. gracilis or S. alberti, but not as long in shape. as T. brevipes. A suture is evident between the sphe- noid and basioccipital on the ventral side. Te sphe- Supraoccipital Te supraoccipital (Figs. 26, 27) forms noid narrows anteromedially and forms two triangular the arched roof of the braincase and the dorsal border of trabeculae. Te anteromedial sides are slightly concave the foramen magnum. It contacts the prootics anteriorly and circular, articulating with the cultriform process. and otooccipitals ventrolaterally. It houses part of the Te anterior openings for the vidian canal are located anterior and posterior semicircular canals as well as the posteroventral to the trabeculae. Te short, broad common crus. Impressions of these can be seen on the basipterygoid processes point anterolaterally, although dorsal surface in Typhlacontias and Sepsina. Te dorsal Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 37 of 53 Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 38 of 53

Fig. 28 Isolated otooccipital region of the neurocranium A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in cut-away medial view. aur, auditory recess of the prootica; cal, crista alaris; endf, endolymphatic foramen; mrst, medial opening of recessus scalae tympani; VII, foramen of CN VII; VIII, foramen of CN VIII; X, foramen of CN X surface is concave and the overall shape is roughly trap- ampulla, lateral ampulla, and the anterior region of the ezoidal to hexagonal. vestibule. It also forms the anterior margin of the fenestra Te supraoccipital has a medial notch in its posterior ovalis. margin. Tis notch is smaller and V-shaped in T. brevipes Te crista alaris (or alar process) extends anteriorly (Fig. 26A) and U-shaped in T. gracilis (Fig. 26B). Typhlac- towards the epipterygoid, but does not contact either it ontias gracilis also has two large foramina, one on either or the descending process of the parietal (Fig. 5A). Te side of the notch. Anteriorly, there is an ovoid medial crista alaris is broader vertically in T. gracilis (Fig. 26B), facet where the cartilaginous processus ascendens con- closing more of the lateral wall of the braincase, than tacts the supraoccipital. Additionally, there are distinct in T. brevipes (Fig. 26A). Ventral to this, there is a deep, ridges running laterally on the dorsal surface. V-shaped notch, the incisura prootica. Te crista proot- Te posterior margin has a rectangular medial notch ica extends anteriorly to contact the sphenoid and pos- in S. alberti (Fig. 27A), larger than in the other species. teriorly to the otooccipital. On its medial side (Fig. 28A, Tere are three small foramina located on the dorsal B), the prootic has openings for the facial (CN VII) and surface. Te facet for the processus ascendens is located auditory (CN VIII) nerves within the auditory recess. Te anteriorly and fanked by the interparietals. endolymphatic foramen is dorsal to the foramen for the Te dorsal surface is not as concave as in the other auditory nerve. Te anterior semicircular canal, hori- skinks, creating a fatter roof in A. occidentalis (Fig. 27B). zontal semicircular canal, and lateral ampulla are visible Te concavity becomes more pronounced at the poste- along the prootic’s surface. rior end. Te medial notch is deep and U-shaped. Te Te prootic of S. alberti (Fig. 27A) is similar in overall facet for the processus ascendens is a circular medial shape to that of Typhlacontias. Te crista alaris extends depression on the anterior side. anteriorly. It curves dorsally, giving it an upturned appearance. Te crista alaris curves posteriorly to form Prootic Te paired prootic (Figs. 26, 27) forms the the dorsal margin of the incisura prootica, a deep, anterodorsal region of the braincase. It contacts the U-shaped notch. Its ventral margin is formed by the infe- sphenoid anteroventrally, supraoccipital posterodorsally, rior process of the prootic. Tis process is either absent and otooccipital posteroventrally. Te prootic houses or greatly reduced in Typhlacontias, but it is large and the anterior parts of the inner ear including the anterior extends to the base of the basipterygoid processes in S. semicircular canal, horizontal semicircular canal, anterior alberti. Te inferior process is uniform in width with Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 39 of 53

a slight bulge near its center. Ventral to this, the crista (Fig. 26). On the other side of the occipital recess, there prootica contacts the sphenoid anteriorly and the otooc- is another crest, the crista tuberalis, which runs ventrally cipital posteriorly. It also forms the anterior margin of the to the sphenooccipital tubercle. Te paroccipital pro- fenestra ovalis and contains the small lateral foramen for cess is posterior to the fenestra ovalis and hosts a broad, the facial nerve. Medially, the prootic has openings for elliptical facet which contacts the quadrate. In posterior the facial and auditory nerves in a similar arrangement view, there are two foramina ventrolateral to the foramen to Typhlacontias (Fig. 28C). Te endolymphatic foramen magnum (only one easily visible in T. gracilis). Te larger is either not present or not visible in the scan. Te same dorsal foramen is the opening for the vagus nerve (CN elements of the inner ear are visible on the prootic’s sur- X) and the ventral foramen is the opening for the hypo- face as in Typhlacontias, with the addition of the anterior glossal nerve (CN XII). On the right side of the posterior ampulla. view of T. brevipes, there is a third small foramen which Te crista alaris of A. occidentalis is narrower than in T. may be a second opening for the hypoglossal nerve. Te gracilis and S. alberti, but more elongate with a blunt end internal opening of the vagus nerve is on the medial wall (Fig. 27B). Te crista alaris forms the dorsal margin of the of the otooccipital, just anterior to the foramen magnum incisura prootica, which is V-shaped and relatively larger (Fig. 28A). Te horizontal semicircular canal is slightly than in the other species. Te inferior process forms the visible, running laterally from the prootic. Te posterior ventral margin of the incisura prootica. Its ventral mar- semicircular canal is more distinct as it runs along the gin is curved, creating a slight uplift at the tip. Te crista dorsal surface of the braincase and then curves ventrally prootica forms the anterior margin of the large fenestra along its posterior side. ovalis. Only a narrow piece of bone separates the facial Te otoccipital is very similar between S. alberti and foramen and fenestra ovalis. Te facial foramen is also Typhlacontias although the back curves out more in S. larger. Medially, the prootic of A. occidentalis has the alberti (Fig. 27A). Te occipital recess is posteroventral to same foramina as seen in Typhlacontias (Fig. 28D). How- the fenestra ovalis. Unlike in Typhlacontias, the occipital ever, the foramen for the facial nerve is located further recess is larger than the fenestra ovalis in S. alberti. Te ventrally from the auditory nerve and the endolymphatic occipital recess is elliptical in shape. Te lateral open- foramen is larger. Of the inner ear elements, the lateral ing of the recessus scalae tympani is located in the ante- ampulla and anterior semicircular canal are the most eas- rior region of the occipital recess. Te medial opening is ily visible on the surface. Te bulge of the lateral ampulla located more dorsally than in Typhlacontias (Fig. 28C). is directly posterior to the crista alaris and the anterior Te crista interfenestralis and crista tuberalis run more semicircular canal is visible on its ventral curve. Tey are diagonally in S. alberti than in Typhlacontias (Fig. 27A). less distinct than in the other species. Te paroccipital process is relatively longer and contacts both the quadrate and supratemporal. Tere are three Otooccipital Te otooccipital (Figs. 26, 27) is a paired small foramina arranged in a triangle ventrolateral to the bone that is the result of fusion between the exoccipital foramen magnum. Te dorsal foramen is the opening for and opisthotic. It forms the lateral border of the foramen the vagus nerve and the two ventral foramina are open- magnum, the posterior wall of the otic capsule, the lateral ings for the hypoglossal nerve. Te internal opening for portion of the occipital condyle, and the posterior border the vagus nerve is located on the posteromedial wall, lat- of the fenestra ovalis. It contacts the prootic anteriorly, eral to the foramen magnum (Fig. 28C). Te horizontal basioccipital ventrally, supraoccipital dorsally, and quad- semicircular canal is visible along the posterolateral wall rate and supratemporal laterally. Te otooccipital houses posterior to the paroccipital process and the posterior the horizontal semicircular canal, the posterior semi- semicircular canal is visible along the dorsal and poste- circular canal, the posterior ampulla, and the posterior rior surface. region of the vestibule. Te otoccipital of A. occidentalis is larger with a poste- Te occipital recess (or recessus scalae tympani) is rior wall that curves out more than in the other species posterior to the fenestra ovalis, which is about twice the (Fig. 27B). Te occipital recess is absent or fused with the height of the occipital recess in Typhlacontias (Fig. 26). fenestra ovalis. Te large fenestra ovalis takes up much of It is elliptical in shape. Te large lateral opening of the ventral region of the lateral wall, is oriented horizon- the recessus scalae tympani (or foramen rotundum) is tally, and is ovoid in shape. It contains the lateral open- located within the occipital recess. Te medial opening of ing of the recessus scalae tympani on its posteromedial the recessus scalae tympani (or perilymphatic foramen) is wall. Te medial opening is set at the junction of the located near the foor of the braincase (Fig. 28A, B). Te posterior and medial walls near the foor of the brain- divide between the fenestra ovalis and occipital recess case (Fig. 28D). Tere is no crista interfenestralis, but the is formed by a crest called the crista interfenestralis crista tuberalis runs along the posterior margin of the Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 40 of 53

fenestra ovalis to the sphenoocipital tubercle (Fig. 27B). and fattens out near the anterior ampulla. Te ampullae Te crista tuberalis is broader than in the other species. are also larger, with the lateral ampulla the largest of the Te paroccipital process is oblong and relatively shorter three. than in S. alberti or T. brevipes. It is overlapped entirely Te osseous labyrinth of A. occidentalis (Fig. 29D) is by the supratemporal and only contacts the quadrate relatively broader than in the other species. Te vesti- ventrally. Acontias occidentalis has fve small foramina bule is larger and nearly circular whereas the endosseous ventrolateral to the foramen magnum. Te medial three cochlear duct is shorter and stumpy in comparison. Te are slightly larger. Te dorsal one is the opening for the fenestra ovalis is large as in Typhlacontias but oriented vagus nerve and the two ventral openings are for the laterally rather than posterolaterally. Te semicircular hypoglossal nerve. Te internal opening for the vagus canals are thick and most similar to S. alberti in appear- nerve is posterodorsal to the medial opening of the reces- ance. One diference is that the anterior semicircular sus scalae tympani (Fig. 28D). Unlike in the other species, canal remains in contact with the vestibule for its entire the horizontal semicircular canal is more distinct and the length. Additionally, the anterior and posterior ampullae posterior semicircular canal is barely visible. are smaller than in S. alberti, with the posterior ampulla hard to distinguish. Te lateral ampulla is of a similar Osseous labyrinth Te osseous labyrinth (Fig. 29, or size. inner ear endocast), which corresponds to the inner ear anatomy, is enclosed within the prootics and otooccipi- Mandibular bones tals. Te body of the inner ear is divided into the vestibule, Dentary Te dentary (Fig. 30) is a long, paired bone which is the dorsal bulbous space where the three semicir- that is the most anterior bone of the lower jaw. It contacts cular canals converge, and the endosseous cochlear duct, the other dentary anteriorly, coronoid and surangular which is ventral to the vestibule. Te anterior semicircular posteriorly, and angular and splenial medially. It houses canal and posterior semicircular canal meet dorsomedi- the Meckelian cartilage in the Meckelian canal and is the ally to form the common crus. Each semicircular canal tooth-bearing element of the lower jaw. expands into a sac called an ampulla. Te fenestra ovalis is Te dentary has three mental foramina in Typhlac- the opening in which the stapes sits. It opens on the lateral ontias, two of which are seen laterally and one which is side of the endosseous cochlear duct. located anteriorly on the ventral side (Fig. 30A, B). Te Te vestibule is rounded in lateral view and only a lit- symphyseal edge slants inward and is the contact site for tle longer than tall in Typhlacontias (Fig. 29A, B). Te the other dentary. Te Meckelian canal runs open along fenestra ovalis is very large and opens on to the ven- the lingual side. It is shadowed by the subdental shelf, a tral region of the vestibule. Te horizontal semicircular well-defned ridge that supports the tooth loci. Where the canal wraps around the lateral side of the vestibule, with subdental shelf ends, there is a coronoid facet where the nearly no space between them. Te anterior semicircu- coronoid connects. Posteriorly, the dentary of T. brevipes lar canal follows the curvature of the vestibule for most bifurcates into the dorsal coronoid process and ventral of its length until it curves out before it joins the ante- angular process. Te dentary of T. gracilis also has these rior ampulla. Tis curve is more pronounced in T. brevi- processes, but there is a third process between them, the pes than in T. gracilis. Te posterior semicircular canal surangular process. Te coronoid process is the shortest is similar, following the vestibule closely for most of its one, and contacts the coronoid. It is highly reduced in length up to the posterior ampulla, where it curves out. T. gracilis and appears like a blunt corner. In T. brevipes, Tere is a larger gap between the posterior semicircular the coronoid process is narrow and ends in a rounded canal and the vestibule in T. brevipes. Each of the semi- point. Te surangular process in T. gracilis is rounded circular canals is tubular with a constant diameter. Te and ends at the anterior foramen of the surangular. Te anterior ampulla and lateral ampulla are in contact with angular process is the longest and stretches along the each other at the anterior side of the vestibule. Te poste- ventral side of the surangular. It also contacts the splenial rior ampulla is located on the posterior side. and angular. Te dentaries have 11 and 11 tooth loci (18 Te vestibule of S. alberti (Fig. 29C) is more oblong teeth present) in T. brevipes and 12 and 12 tooth loci (16 than in Typhlacontias. It is not as enlarged and does not teeth present) in T. gracilis. Te crowns of T. brevipes are contact the semicircular canals as much as in the more slightly more pointed compared to T. gracilis. fossorial skinks. Te fenestra ovalis is a small, circular Te dentary of S. alberti has six mental foramina along opening. It opens to the endosseous cochlear duct, which its lateral side, four smaller ones evenly spaced along the is similar in shape across the scincines. Te arrangement anterior quarter of the bone and two larger ones poste- of the semicircular canals is similar, but they are larger. rior (Fig. 30C). Te symphyseal edge is roughly the same As in T. brevipes, the anterior semicircular canal curves size as in Typhlacontias. Te Meckelian canal runs open Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 41 of 53

Fig. 29 Endocast of inner ear of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in lateral, dorsal, ventral, medial, and posterior views from top to bottom. aa, anterior ampulla; ascc, anterior semicircular canal; cc, common crus; ecd, endosseous cochlear duct; fov, fenestra ovalis; hscc, horizontal semicircular canal; la, lateral ampulla; pa, posterior ampulla; pscc, posterior semicircular canal; vb, vestibule. Scale bars 1 mm =

along the lingual side. Te coronoid process is triangular Overall, the dentary of A. occidentalis is more robust and relatively larger than in Typhlacontias. Te angular than that of the other skinks (Fig. 30D). Te dentary has process is triangular and elongate. It covers a portion of fve mental foramina on the lateral side, four evenly- the angular, and contacts the surangular and splenial. Te sized ones on the frst quarter and one larger, oval- dentaries have 20 and 22 tooth loci (30 teeth present). shaped one posterior to them. Te symphyseal edge Te crowns are rounded. is relatively larger and has a foramen. Te Meckelian canal is enclosed. It has an oval opening through which Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 42 of 53

Fig. 30 Isolated left dentary of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis in lateral, medial, and dorsal views from left to right. d-c, coronoid facet of the dentary; dap, angular process of the dentary; dcp, coronoid process of the dentary; dfor, mental foramina; dsp, surangular process of the dentary; mc, Meckelian canal; sds, subdental shelf; sed, symphyseal edge. Scale bars 1 mm = the anterior process of the splenial runs. Te anterior teardrop-shaped mandibular fossa. Te mandibular process of the coronoid fts into the space created by fossa is larger in T. brevipes, stretching nearly to where the ventrally curved subdental shelf. Compared to the the coronoid sits on the surangular. Tere is a small dor- other species, the coronoid process is much larger. It is sal foramen at the posterior side of the mandibular fossa. triangular. Acontias occidentalis difers from the others Anteriorly, the surangular forms a broad dentary process, in that the most posteriorly-extended point of the den- which contacts the dentary laterally. Te tip is pointed in tary is on the lateral side of the jaw. Te surangular pro- T. brevipes and rounded in T. gracilis. Te coronoid facet cess extends horizontally to this point and then curves is located dorsally on the medial side. Ventral to this, ventrally to form a second smaller point. It contacts the there is an angular facet. Te articular surface curves splenial, angular, and surangular. Te dentaries have posteroventrally to articulate with the quadrate. It is 11 and 12 tooth loci (23 teeth present). Tese teeth are broader in T. brevipes, accommodating the larger quad- similar in shape to Typhlacontias, but larger with more rate. Te retroarticular process extends past the articular rounded crowns. Additionally, the second and third surface and fattens into a concave bowl. Te retroarticu- teeth are much larger than the other teeth and more lar process of T. gracilis is larger and fattens out imme- pointed. diately whereas the retroarticular process of T. brevipes slants downward. Tere is a small foramen beneath the Compound bone (articular and surangular) Te articu- articular surface in T. brevipes. lar and surangular have fused to form a single compound Te surangular of S. alberti has a larger anterior fora- bone (Fig. 31). Te surangular is the larger of the two and men and a smaller posterior foramen on its lateral side houses the posterior portion of the Meckelian cartilage. (Fig. 31C). On its dorsomedial side, there is an ovoid It contacts the dentary anterolaterally, coronoid medi- mandibular fossa. It is relatively smaller than in T. brevi- ally, and splenial and angular ventromedially. It also con- pes, but larger than in T. gracilis. Te posterior foramen tacts the angular laterally in A. occidentalis. Te articular opens into the mandibular fossa, as in Typhlacontias, but contacts the surangular anteroventrally and the quadrate S. alberti lacks a dorsal foramen. Anteriorly, the suran- dorsally. It is the only splanchonocranial element in the gular has a broad triangular dentary process, which mandible. contacts the dentary laterally and splenial medially. Te On its lateral side, the surangular has an anterior coronoid facet is located dorsally and is U-shaped. Te foramen and a posterior foramen in Typhlacontias angular facet begins on the medial side and continues (Fig. 31A, B). Tere is a third foramen located between along the ventral side, ending past the middle of the com- them in T. brevipes. Dorsally, the surangular has the pound bone. Te articular surface is not as prominent as Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 43 of 53

Fig. 31 Isolated left compound bone (articular and surangular) of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in lateral, medial, dorsal, and ventral views from top to bottom. ars, articular surface; art, articular; com-ang, angular facet of the compound bone; com-cor, coronoid facet of the compound bone; com-d, dentary facet of the compound bone; dp, dentary process of the compound bone; mf, mandibular fossa; rap, retroarticular process; rapf, retroarticular process foramen; san, surangular; sanaf, anterior foramen of the surangular; sandf, dorsal foramen of the surangular; sanpf, posterior foramen of the surangular; scp, coronoid process of the surangular; spp, splenial process of the surangular. Scale bars 1 mm = Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 44 of 53

Fig. 32 Isolated coronoid and angular. Left coronoid of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in lateral (left) and medial (right) views. Left angular of E T. brevipes; F T. gracilis; G S. alberti; H A. occidentalis; in medial view. ang-sp, splenial facet of the angular; angap, anterior process of the angular; ce, coronoid eminence; coap, anterior process of the coronoid; copp, posterior process of the coronoid; cor-d, dentary facet of the coronoid; cor-sp, splenial facet of the coronoid; cor-sur, surangular facet of the coronoid; paf, posterior alveolar foramen. Scale bars 0.5 mm =

in Typhlacontias. Te retroarticular process fattens out Te angular facet is located almost entirely on the ventral as in Typhlacontias, but does not form a concave surface. side. Te articular is not entirely fused to the surangular It is broad and oriented more horizontally than in the with a suture visible on the medial side. Te articular sur- other species. face is smaller than the articular surface in the other spe- Te compound bone of A. occidentalis is squatter and cies. Te retroarticular process is broad and fat with a has a diferent anterior shape compared to the scincines slightly concave medial surface. It is oriented vertically. A (Fig. 31D). Tere are two foramina on its lateral side, with small foramen is present directly posterior to the articu- the anterior considerably smaller than the posterior. Dor- lar surface. somedially, the surangular has a large, ovoid mandibular fossa. It extends nearly to the coronoid facet. Te man- dibular fossa is most visible medially in A. occidentalis, Coronoid Te coronoid (Fig. 32A–D) is a paired bone whereas in Typhlacontias, it is closed along its medial set near the midpoint of the lower jaw at the boundary side, especially in T. brevipes. Tere is a small dorsal between the dentary and surangular. It contacts the den- foramen in the mandibular fossa. Anteriorly, the suran- tary anterolaterally, surangular posteroventrally, and sple- gular splits into two processes: the dorsolateral dentary nial anteroventrally. process and the ventral splenial process. Te dentary Te coronoid eminence is reduced in Typhlacontias, process is narrow and triangular. Te splenial process is forming a small, gently sloping peak (Fig. 32A, B). Te broader. Te Meckelian canal runs between them to the anterior process extends along the lingual side of the jaw mandibular fossa. Te surangular has a distinct coro- and terminates where the subdental shelf begins. On its noid process extending from the dorsal side. It is broad lateral side, it has a facet which articulates with the coro- and subrectangular, and contacts the coronoid medially noid process of the dentary. Te posterior process runs and nearly contacts the dentary anteriorly. Te coronoid along the dorsal side of the surangular. It is fattened, par- facet is located on the medial side and covers a broad ticularly in T. brevipes, and ends in a rounded point. Te area from the coronoid process to the mandibular fossa. coronoid of T. brevipes is fat with only mild curvature Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 45 of 53

Fig. 33 Isolated left splenial of A T. brevipes; B T. gracilis; C S. alberti; D A. occidentalis; in medial (left) and lateral (right) views. aiaf, anterior inferior alveolar foramen; dc, dorsal crest; sp-d, dentary facet of the splenial; splvf, ventral foramen of the splenial. Scale bars 0.5 mm = along its ventral side whereas the coronoid of T. gracilis is In ventral view of the mandible (Fig. 7A, B), a small arced and shaped more like a sickle. portion of the angular is visible overlapping the suran- Te coronoid of S. alberti is far more pronounced than gular in Typhlacontias. Te posterior alveolar foramen in Typhlacontias. It is a V-shaped bone with a ridge along is incomplete in T. brevipes (Fig. 32E), with only the dor- its lateral side (Fig. 32C). Te coronoid eminence forms a sal margin present to form a half-circle, and absent in T. triangular peak. Te anterior process expands anteroven- gracilis (Fig. 32F). Although the angular is elongate and trally to the subdental shelf. Its lateral side has two facets: narrow in both species, the anterior process shows more the anterodorsal dentary facet and the ventral surangular expansion in T. brevipes than in T. gracilis. Tis process is facet. Its medial side has a large facet for the splenial. Te slightly overlapped by the splenial in T. brevipes. posterior process is as long as the anterior, but narrower. Te angular of S. alberti (Fig. 32G) is larger and more Te coronoid of A. occidentalis is the largest of the four robust than in Typhlacontias. It extends along the ventral species and has a distinct triradiate shape (Fig. 32D). Te side of the mandible (Fig. 7C), overlapping the surangu- coronoid eminence is large and trapezoidal with a pos- lar. Te posterior alveolar foramen is present at roughly terior curvature. Most of its lateral surface is overlapped two-thirds of the length of the bone. Te anterior pro- by the dentary, leaving only a small fraction visible later- cess is elongate and narrows to a point. It is overlapped ally in the lower jaw (Fig. 7D). Te anterior process is the slightly by the splenial. smallest of the three processes and extends to the sub- Te angular of A. occidentalis (Fig. 32H) is the larg- dental shelf. Te posterior process is longer and broader est and most complex of the examined species. It wraps than the anterior process, extending nearly to the man- around the ventral side of the mandible and extends on dibular fossa. to the lateral side. Its end is broad and rounded. Te pos- terior alveolar foramen is located on the ventral side. It is Angular Te angular (Fig. 32E–H) is a blade-like paired overlapped by the dentary. Te anterior process is large bone mainly located on the medial side of the lower jaw, and rectangular with only a slight narrowing towards its although it extends on to the ventral and lateral side in end. Tere is a distinct splenial facet on the dorsal side of some species. It contacts the surangular dorsally and ven- the anterior process. trally, dentary ventrally, and splenial anteriorly. In A. occi- dentalis, the contact with the splenial is dorsal. Splenial Te splenial (Fig. 33) is a fattened, paired bone located on the medial side of the lower jaw. It contacts the Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 46 of 53

dentary anteriorly, coronoid dorsally (except in A. occi- Te epipterygoid and bones of the braincase showed rela- dentalis), angular posteriorly and ventrally, and surangu- tively less variation. We begin by discussing the results lar posteromedially. It encloses the Meckelian canal. from our morphometric analyses with a focus on the Anteriorly, there are two elongate processes which par- main variables, and then examine major anatomical dif- tially enclose the anterior inferior alveolar foramen in ferences and similarities between four species in light of Typhlacontias. In T. brevipes, the dorsal process is shorter phylogenetic relationships and burrowing specialization. than the ventral one (Fig. 33A) whereas the two processes We also show how broad-scale similarities in traits can are nearly of equal length in T. gracilis (Fig. 33B). Pos- be accomplished through diferent anatomical changes. terior to this, there is a smaller foramen for the anterior mylohyoid nerve. Tis foramen is closed in T. brevipes Allometry and open ventrally in T. gracilis. Tere is a slight dorsal Diferences in allometric scaling between subfamilies crest in T. brevipes. Te splenial is broader and more and genera likely contributed to the variation in shape. robust in T. brevipes than in T. gracilis. For instance, whereas larger acontines have an hour- Te splenial of S. alberti is relatively larger than in glass-shaped parietal, larger scincines tend to have an Typhlacontias, particularly its expanded posterior end almost square parietal. Furthermore, Acontias has the (Fig. 33C). Te anterior side bifurcates into two pro- largest variation in skull size (6.98 mm in A. gariepensis cesses, of which the dorsal process is the larger. Tey par- to 31.00 mm in A. plumbeus) and showed the greatest tially enclose the fnger-shaped anterior inferior alveolar spread in morphospace. Tis distribution roughly aligned foramen. Posteriorly, the ventral foramen for the ante- with size, underlining how variation in size can corre- rior mylohyoid nerve is completely enclosed. It is fat- spond to variation in shape. tened and more ovoid compared to Typhlacontias. It is Allometric scaling often has important functional also very close to the anterior inferior alveolar foramen, implications, including changes to retain equivalent similar to T. gracilis. Te posterior end is much larger and functionality [55, 56]. Te relative increase in the size narrows to a triangular point. Te dorsal edge is relatively of the braincase in smaller species observed here may straight. be related to an increase in the relative size of the semi- Te splenial of A. occidentalis (Fig. 33D) is quite dif- circular canals. Te inner ear must stay above a certain ferent in shape. It narrows to a slim process that extends size to remain functional and a corresponding change between the lateral and medial sides of the dentary to the relative size of the braincase is common in minia- (Fig. 7D). Te ventromedial side of this process is fat- turized species [57]. Tis change in proportion can also tened where it contacts the dentary. Posteriorly, the ven- explain the shift in the quadrate’s relative position; as the tral foramen for the anterior mylohyoid nerve is more braincase expands in relative size, it closes the distance ventrally oriented than in the other species. It is entirely between itself and the quadrate. enclosed and similar in shape, though smaller, to S. Whereas some of the allometric shifts in smaller spe- alberti. Te posterior end has a long, triangular process. cies are likely related to maintaining sensory function, Te dorsal edge forms a small, rounded crest. allometric shifts in larger species are likely related to other functions. Fossorial lizards often show a trade- Discussion of between burrowing speed and bite force, as both are We used geometric morphometrics and comparative related to relative head width [15]. However, in some anatomy to examine skull evolution in African burrow- groups, no diference in bite force is found despite the ing skinks. Overall, we found a strong efect of phyloge- predicted efects of head shape, implying compensatory netic history and a smaller efect of size on skull shape. changes to preserve similar performance [16, 58, 59]. Degree of limb reduction was also correlated with shape, In larger species of Amphisbaenia and Leposternon, the but this disappeared when phylogeny was accounted for. skull is hourglass-shaped, which may allow an increase Substrate played a smaller role, although sand burrowers in adductor muscle mass [31, 60]. Te hourglass-shaped consistently stood out as diferent from other burrowers. parietal in large Acontias may similarly provide greater We also created a detailed anatomical atlas for the skulls space for the adductors and increase bite force while of four skink species, none of which have been examined keeping the head narrow for efcient burrowing. In con- in detail and three of which come from genera that have trast, Feylinia have a square parietal. Feylinia inhabit leaf never been the subject of dedicated osteological study. litter [61], a looser substrate where head width may not Every element showed some degree of variation in shape limit burrowing as much or at all compared to the denser and relative scaling of features, even within the same substrates Acontias is found in. Soft tissue studies and genus. Some of the most variable bones included the performance tests are required to test these hypotheses. skull roofng bones, septomaxilla, vomer, and palatine. Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 47 of 53

Phylogenetic history which are all limbless. However, limbless skinks are con- Phylogenetic history has a strong relationship with cra- sidered the most specialized for burrowing [51], so it fol- nial and mandibular shape in African burrowing skinks lows that they would have diferent skull morphologies with genus being the variable that most explains varia- compared to two and four-limbed skinks. Tis trend can tion in shape. Te pattern is not surprising as phyloge- be observed by examining the anatomy more closely. netic history is important in shaping skull morphology in When looking at qualitative traits, diferences are seen many clades including amphisbaenians [18], geckos [62], between Scelotes possessing diferent numbers of limbs. gymnophthalmids [23], varanids [20], and across lizard Some of the changes observed in some of the two-limbed families generally [17]. Despite the clear diferentiation and all limbless Scelotes compared to the four-limbed between most genera in morphospace, phylogenetic sig- species include an increase in the size of the cristae cra- nal was lower than expected. Tis may be the result of nii, reduction of the squamosal, increase in the fenestra the overlap between Scelotes and Sepsina, Feylinia and ovalis, and reduction of the tympanic crest. Scelotes Typhlacontias having a specialized morphology closer to arenicola, a limbless skink found in sandy soil, has some acontines, and shifts in morphology within genera due to of the most specialized traits among Scelotes including other factors. tube-like cristae cranii, orbitonasal fanges that extend to Scelotes and Sepsina show the most overlap in mor- the midline, larger stapes, thin squamosal, elongation of phospace. Tis is seen even though Sepsina is more the snout, and general fattening of the skull. Tese traits closely related to Typhlacontias and Feylinia than to Sce- are similar to those observed in Typhlacontias and Acon- lotes. Te likely explanation is that Scelotes and Sepsina tias, whose anatomy we described in greater detail, and share an ancestral and conserved scincine morphology. point to a potential progression in skull specialization Although Sepsina’s proximity to Scelotes impacts the low that accompanies limb reduction. phylogenetic signal, the efect is likely diluted as the cur- rent phylogeny only includes one species of Sepsina and Substrate and burrowing specialization is missing four Scelotes. Inclusion of these species would Compared to other studies on other burrowing lizards likely contribute to the low phylogenetic signal. [23, 31], we found a less clear relationship between skull Degree of specialization for burrowing also seems to shape and substrate. Our results do concord with a study play a role in explaining skull morphology, with more on amphisbaenians [18], which suggested that skull mor- specialized burrowers having depressed skulls, rela- phology is largely shaped by phylogenetic history, not soil tively long and pointed snouts, shorter frontals, and type, due to strong stabilizing selection for burrowing. fewer teeth. Other qualitative traits not captured by the Although phylogenetic history is key in explaining skull morphometrics are described later. Many of these traits variation in African burrowing skinks, there is notable have previously been reported in other burrowing squa- convergence between sand-burrowing species of difer- mates [24, 27]. Acontines show the most morphologi- ent genera. cal specialization and are considered the most fossorial. Sand burrowers, including species in the genera Acon- Typhlacontias and Feylinia, both mostly limbless and tias, Typhlosaurus, and Typhlacontias, have a unique highly fossorial, show convergence with these acontine morphology characterized by long and narrow skulls, morphologies and are found in-between them and the wedge-shaped crania with fattened skull roofs, long less specialized scincines in morphospace. snouts, relatively long braincases, small suborbital Finally, morphological shifts within genera that do not fenestrae, and longer dentaries with short tooth rows. refect phylogenetic history and are the result of changes Sand is a complex substrate that possesses both solid and in size or substrate may also play a role in explaining why liquid properties, is composed of smaller grain sizes, and phylogenetic signal is low. For example, large body size is generally unstable [63, 64]. Given its unique physical and its associated traits (like the hourglass-shaped pari- properties, it follows that lizards would require unique etal) evolved multiple times in Acontias, leading to con- adaptations to burrow through it. Some of these adapta- vergence between non-sister species. tions, including a wedge-shaped head and long snout, are also seen in limbed lizards that dive into sand to escape predators [16, 64, 65]. A wedge shape may help to bet- Limb reduction ter part the sand. Many sand burrowers also specialize on We found a strong relationship between the number of termites [66, 67], which may have infuenced the unique limbs and digits with skull morphology, but the relation- mandibular shape, as seen in non-fossorial myrmecopha- ship disappeared when phylogeny was taken into account. gous lizards [68]. Such a relationship could not be directly Most likely, it was driven by the large number of genera tested here because of the paucity of dietary informa- that only exhibit one limb state, especially the acontines, tion in burrowing skinks (although, some are considered Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 48 of 53

termite specialists; [67]). Te variation seen between most diagnostic features [70] are reported here. Previ- sand burrowing species is primarily the result of phylo- ously, Typhlacontias have been regarded as lacking jugals genetic history and secondarily size, but within genera, [70]. We found reduced jugals in both species, highlight- there may also be variation caused by fne-scale difer- ing the value of CT scans in recognizing small structures ences in sandy habitats. For instance, within Typhlacon- that may be lost in a skeletal preparation. tias, species may inhabit dunes, vegetated sandy areas, Te skulls of T. brevipes and T. gracilis, although more and areas with higher rainfall [69]. Each of these micro- similar to one another than to the other species, show habitats is likely to present diferent physical properties variation both in general skull shape and in each indi- and challenges, which may require diferent adaptations. vidual bone. Some of these diferences are related to One caveat to this study is that the full dataset could fossorial adaptations, with T. brevipes showing larger not be analyzed using phylogenetic comparative meth- orbitonasal fanges, more robust cristae cranii, smaller ods. Te reduced dataset showed some variation from suborbital fenestrae, and a more closed palate compared the full dataset in the results, even before phylogenetic to T. gracilis. Tese traits seem to indicate a higher degree correction, so the reduction clearly had an impact. Only of specialization in T. brevipes. However, T. gracilis has scincines were removed and there was a disproportion- a larger crista alaris and smaller posttemporal fenestrae. ate efect on substrate representation with the following Furthermore, T. brevipes retains rudimentary hindlimbs. left: leaf litter (4/8), sand (10/12), sandy soil (10/13), and Tese complex combinations of traits indicate that even soil (4/5). Te sharp reduction of the leaf litter group may within a single genus, the evolution of burrowing adapta- explain the loss of some signifcant diferences in the phy- tions does not follow a single linear path. Some of these logenetic dataset. A fully resolved phylogeny is needed to diferences may also be related to diferences in substrate analyze the full dataset. Tat said, the diference observed or environmental conditions. Both species are described between sand burrowers and other burrowers is likely to as sand-swimmers, but T. brevipes occupies more vege- remain signifcant. tated sandy environments whereas T. gracilis is found in Another limitation, shared by many studies looking Kalahari sand in areas with greater annual rainfall [69], at the relationship between substrate and morphology which may impact the physical properties of the sand. [3, 18, 23, 31], is in substrate characterization. Te most Another source of variation is the development of the accurate way would be to measure mechanical properties epipterygoid. Typhlacontias gracilis retains a prominent of the substrate as specimens are collected. Tis is logis- epipterygoid whereas T. brevipes has a small epiptery- tically challenging as morphological studies tend to use goid. It is unclear if this is because it is partly cartilagi- museum specimens, which lack environmental data. Fur- nous, or if it has been greatly reduced. Furthermore, a ther complicating matters is that the degree of substrate bony epipterygoid is absent in T. punctatissimus. Tis compaction likely varies across ranges and temporally may indicate a reduction of the epipterygoid in Typhlac- based on soil moisture, vegetation, and other variables. ontias. Reduction and loss of the epipterygoid would be We characterized substrate using anecdotal feld observa- novel in skinks, but has been seen in other lizards [14]. tions, condensing them into one of four broad categories. Clearing-and-staining to visualize cartilage (if present) Tese categories captured large variation between sub- and larger number of specimens to look at intraspecifc strates (e.g., leaf litter is loose and relatively easy to move variation is required before defnite conclusions can be through, sand is composed of smaller particles, etc.), but drawn. likely missed out on fne-scale physical diferences. Other Tere are no previous detailed osteological studies studies use location and associated data (bioclimatic fac- of Sepsina to compare our study with. Greer [70] noted tors, soil type) to study the relationship between sub- the following diagnostic skull features: wide separation strate and morphology [18, 31]. Tis works well when of palatines, medially expanded palatine process of the species are all burrowing in soil as with amphisbaeni- pterygoid, pterygoid teeth, postorbital and upper tem- ans. However, this method does not work for our system poral arch, and 12–18 teeth per maxilla. All these are where many species occupy leaf litter rather than the soil observed here in S. alberti. itself. In the future, studies may seek to measure substrate Many of the bones are similar in shape between S. properties and test burrowing performance in the feld to alberti and Typhlacontias and in some cases, more simi- more closely test the relationship between substrate, per- lar between S. alberti and one of the Typhlacontias than formance, and morphology. between the two Typhlacontias. For example, the pari- etal of T. gracilis is more similar to S. alberti due to the Anatomical comparisons across phylogenetic levels development of a complex parietal fossa and bifurcated No complete anatomical descriptions exist for Typhlac- posterior processes in T. brevipes. Such similarities can ontias to compare with the work in this study, although likely be attributed to conserved anatomy. Sepsina alberti Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 49 of 53

also shows many diferences to Typhlacontias. Of the Fossorial adaptations in the skull examined skinks, S. alberti is noteworthy in having pos- All four skinks show skull modifcations seen in other torbitals, interparietals, palpebrals, pterygoid teeth, and a fossorial squamates, although the degree to which these highly ossifed hyoid apparatus. Other diferences include are expressed varies, especially between S. alberti and teeth extending up to the posterior process in the maxilla the more specialized burrowers. Tese modifcations (also seen in A. occidentalis), shorter orbitonasal fanges, include a skull that is twice as long as it is wide, parietal relatively longer frontal, more open palate, wider palatine downgrowths, closure of the lateral wall of the cranium, process of the pterygoid, taller coronoid, larger angu- reduction of the posttemporal fenestrae, and interdigi- lar, and the quadrate, which resembles the more typical tated frontoparietal suture. More adaptations are seen conch shape and retains a prominent tympanic crest. just in Typhlacontias and A. occidentalis including loss of Te skull anatomy of A. occidentalis varies the most the upper temporal fenestrae, closure of the medial wall from the other species despite some convergence with of the orbit, development of the cristae cranii, closure of Typhlacontias. Some key diferences include: the closed the palate, larger occipital condyle and footplate of the Meckelian canal, large anteromedial premaxillary process stapes, reduction of the jugal and tympanic crest, and of the nasal, enlarged anterolateral process of the frontal, enlarged vestibular structures in the inner ear [11]. Acon- greater development of the cristae cranii, hourglass shape tias occidentalis also has large basicranial sesamoids. of the parietal with expanded descending processes, Basicranial sesamoids are found in many squamate fami- reduced squamosal, decreased size of vomeronasal cavity lies, including Scincidae, although previous examination relative to septomaxilla, fattened supratemporal, pres- of an acontine did not fnd any [6, 71]. Tey are typically ence of lateral process of the palatine, absence of occipital small in non-fossorial squamates, but can be large in bur- recess and crista interfenestralis, large coronoid process rowers to provide a larger attachment site for the longus of the dentary, and bifurcated anterior side of the suran- colli muscle [53, 71]. A larger longus colli muscle, which gular. Some of these traits are likely related to increased pulls the head down, may allow for the generation of burrowing specialization or other functional aspects greater forces for burrowing. Typhlacontias lack basicra- (such as the supratemporal, which supports the quadrate) nial sesamoids, but the enlarged sphenooccipital tuber- in A. occidentalis whereas others to phylogenetic history. cles may ofer a similar advantage. Acontias occidentalis shares many of the typical acon- Although many of these traits are broadly convergent, tine features including a scroll-like air passage formed they are often formed through diferent changes to the by the palatines, lateral process of the palatine, robust anatomy, refecting phylogenetic history. One such trait is cristae cranii, broad supratemporal, large crista alaris, the loss of the upper temporal fenestrae. Like many bur- overlap of the maxilla by the vomer, and exclusion of the rowing squamates [24, 72], A. occidentalis loses the upper frontal from the dorsal orbital margin by the prefrontal temporal fenestrae through the reduction of the squa- and postfrontal [10, 43–45]. Te frontals also have anter- mosal. Typhlacontias retains a prominent squamosal, olateral processes, which are seen in most acontines [10]. but loses the upper temporal fenestra through the close Tese processes vary in size between species and A. occi- apposition of the squamosal and parietal. Another trait is dentalis has relatively large anterolateral processes. Like the closure of the lateral wall of the skull, which helps to many Acontias, A. occidentalis has lost the anterior pro- strengthen it [24]. In Typhlacontias, this is accomplished cess of the jugal and the upper temporal arch through the through the expansion of the crista alaris. In A. occiden- reduction of the squamosal. Te jugal is not as reduced talis, the crista alaris plays a role in closing the lateral as in some species where it becomes a stub or absent wall, but the expansion of the parietal is more impor- altogether [10]. Te squamosal is very reduced. Te lat- tant. Similarly, the closure of the anteromedial region, eral wall of the skull is not as closed in A. occidentalis as which strengthens the skull and provides protection for in some species due to a relatively narrow crista alaris the olfactory tracts, is accomplished in diferent ways. and smaller parietal downgrowth. More unique features In Typhlacontias, the orbitonasal fanges of the prefron- include the triradiate checkmark shape of the postfrontal tal are expanded, closing much of the anterior wall. Te (shared with Acontias meleagris and Acontias namaquen- cristae cranii are large in T. brevipes and contribute to the sis), hourglass shape of the parietal (shared with Acon- closure of the region, but in T. gracilis, they do not play a tias gracilicauda, A. namaquensis, and A. plumbeus), large role. In A. occidentalis, the prefrontal is smaller and roughly triangular quadrate, and elliptical stapes with the crista cranii takes the prime responsibility in closing narrow, constricted shaft (most closely resembles A. gra- the region. Te reduction of the jugal is also diferent. In cilicauda). Many of these features appear multiple times Typhlacontias, the posterior process is reduced and the across the acontine phylogeny. anterior process remains. In A. occidentalis, the anterior Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 50 of 53

process is reduced, resulting in a free-foating, splint-like in the feld can all aid in unraveling if and how diferent jugal. skull morphologies yield diferent interactions with the Although skinks have generally been thought to lack environment and may assist in better understanding line- cranial kinesis [73], anatomical studies have pointed age-specifc constraints. to the potential for it in some species, including sev- eral acontines [10, 44], and experimental work has Methods demonstrated it in a small species [52]. Our work pro- Scanning and segmentation vides further evidence for the potential of mesokinesis One adult individual (identifed as such by body size) in some skinks (closure of upper temporal fenestrae, from 39 species was CT scanned at the University of reduction of jugal and postorbital, gap between cris- Florida Nanoscale Research Facility in Gainesville, FL. tae cranii and parietal downgrowths permitting move- All specimens were vouchered museum specimens, ment). While reduction or loss of cranial kinesis may with 30 specimens from the California Academy of Sci- occur when reinforcing the skull, burrowers may also ences (CAS) in San Francisco, CA, and 9 specimens from beneft from a mesokinetic skull that can fatten more the Museum of Comparative Zoology (MCZ) in Cam- (suggested as an adaptation in rupiculous lizards; [74]), bridge, MA. Most specimens were scanned using a GE further streamlining it. Experimental work is needed V|tome|xm240 CT Scanner with the X-ray source set to to confrm whether cranial kinesis is present and if so, 80 kV, 170 µa, and 13.6 W. Four specimens (all Sepsina) whether it difers across substrate types. were scanned earlier using a Phoenix VTome|x M scan- ner with an X-ray source set to 80 kV, 180 µa, and 14.4 W. Two scans were done per specimen: one full body scan Conclusions and one region-of-interest scan of the skull to capture Our work sought to understand the evolution of skull fner detail. Scans were reconstructed on GE’s datosjx morphology in African burrowing skinks and more software. Additional scanning information is available in broadly, how morphology changes for a highly spe- Additional fle 1: Table S17 and all scans are available for cialized and demanding lifestyle like burrowing. Phy- download on Morphosource (https://​www.​morph​osour​ logenetic history is the greatest factor shaping skull ce.​org/​proje​cts/​00000​C482/). morphology in African burrowing skinks, with size We digitally segmented each cranium and right man- and substrate taking on secondary roles. Although dible from the region-of-interest skull scans in Avizo there is broad convergence in both shape and other v.9.4.0 (Visualization Sciences Group, Burlington, Mas- traits related to burrowing, our anatomical compari- sachusetts, USA). Te cranium of Scelotes cafer and the sons reveal these convergences are achieved through mandible of Scelotes arenicola were broken and excluded diferent anatomical modifcations. Our work shows from the cranial and mandibular morphometric analy- that morphological specialization and convergence can ses respectively. For the comparative anatomy, we digi- occur through diferent pathways, even within a sin- tally segmented each bone as a separate element for gle family, adding to the literature on imperfect con- four species: T. brevipes (CAS 224004), T. gracilis (MCZ vergence and lineage-specifc constraints [4, 5]. Te R-18023), S. alberti (CAS 263923), and A. occidentalis atlased species also contribute to a growing body of (CAS 196430). We also segmented the empty space of the detailed skull morphology literature made possible by osseous labyrinth to form an endocast of the inner ear. CT scanning [11, 39, 53, 75] that will facilitate the char- Most images were taken in perspective mode with only acterization of other lizard skulls for phylogenetic and Figs. 5 and 6 taken in orthographic mode. Lengths and functional studies. By using both qualitative and quan- widths of structures were measured using the measure titative methods, we gained a richer understanding of tool in Avizo. When necessary to verify the presence or how phylogenetic history, ecology, and functional pres- absence of sutures that may have been smoothed over in sures infuence morphology and showed that there are the 3D visualization (e.g. suture between two premaxil- many ways to be a burrower. lae), we examined the 2-dimensional tomogram slices. Further work may involve sampling burrowing skinks Identifcation of anatomical structures were based on from elsewhere to test whether these patterns hold up previous studies of skink osteology [11, 39, 75]. across the family. Additionally, functional analyses are necessary to understand the implications of morphologi- Classifers cal variation and how burrowing difers between species Data on microhabitat and number of limbs and dig- from diferent genera, of diferent size, and between dif- its were compiled from the primary literature and feld ferent substrate users. Finite element analysis, perfor- guides [61, 66, 69, 76–80]. We classifed species into four mance tests, and measurements of substrate hardness broad substrate categories: leaf (for species in leaf litter), Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 51 of 53

sand (for sand swimmers and species living in Kalahari, signal in our shape data (Procrustes coordinates) using Namib, coastal, or dune sands), sandy soil (for micro- the physignal function in geomorph. habitats referred to as sandy soil), and soil (for burrow- To test for a relationship between shape and size, we ing in compact soil, humus soil, and alluvial soil). Feylinia ran multivariate and phylogenetic regressions between elegans lacked species-specifc microhabitat information, shape (as characterized by the Procrustes coordinates) but as the other species in the genus are in leaf litter, it and centroid size. A homogeneity of slopes (HOS) test was assumed F. elegans was too. Limb and digit num- and post-hoc pairwise slope comparisons were per- bers were confrmed by examinations of the full body formed on the dataset excluding Mochlus to determine CT scans for each specimen. All classifer information is if the genera and subfamilies had diverged in allomet- available in Additional fle 2. ric relationships. We ran multiple analysis of covari- ance (ANCOVA; [89]) and phylogenetic generalized Geometric morphometrics least squares (PGLS; [90]) to test for a relationship We used 3D geometric morphometrics to characterize between shape and one of the variables (genus, subfam- variation in the overall shape of the cranium and lower ily, number of limbs, number of digits, and substrate) jaw. Fixed landmarks were placed at homologous, repeat- using the Procrustes coordinates, with log-transformed able points using the package geomorph version 3.0.7 [81] centroid size as the covariate. Post-hoc pairwise com- within R version 3.5.1 [82]. Landmark datasets were cre- parisons were run to determine which limb states and ated and analyzed separately for the cranium and man- substrates diverged from one another for all datasets. dible to avoid diferences in orientation and position due Post-hoc pairwise comparisons were also run to deter- to specimen preparation (such as if the jaw was open or mine which genera and subfamilies diverged from one closed). We used 21 landmarks on the cranium and 13 on another using the dataset excluding Mochlus. the mandible (Fig. 2). Written descriptions of the land- mark positions are available for the cranium and mandi- Abbreviations ble (Additional fle 1: Tables S18, S19). BM: Brownian motion; CT: Computed tomography; SVL: Snout–vent length.

Statistics Supplementary Information Cranial and mandibular landmarks were analyzed sepa- The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12862-​021-​01821-w. rately using geomorph. Analyses were run using three dif- ferent datasets for each set of landmarks. Te frst dataset Additional fle 1. Contains 19 additional tables containing results from contained 38 species and was analyzed using only non- statistics, scanning information, and written descriptions of landmarks. phylogenetic methods. Te second contained all species Additional fle 2. Table of classifers used in data analysis. except Mochlus sundevallii, which was removed to allow for pairwise comparisons to be run between genera and Acknowledgements subfamilies. Te third dataset included only the species We would like to thank Lauren Scheinberg (California Academy of Sciences) present on the phylogenetic tree, resulting in a reduced and José Rosado (Museum of Comparative Zoology) for assistance with speci- dataset of 28 species. Tis dataset was analyzed using men loans. Furthermore, we thank the many collectors who collected the specimens making this research possible. We would also like to thank Ed Stan- both phylogenetic comparative methods and non-phylo- ley and Dan Paluh of the University of Florida for CT scanning specimens. We genetic analyses. thank Luis Ceríaco for substrate data for Sepsina. We also thank Todd Jackman We frst performed a Generalized Procrustes analysis and Alyssa Stark for helpful comments on an earlier version of the manuscript as well as Christy Hipsley and an anonymous reviewer for their thoughtful (GPA) on the landmarks to align, rotate, and scale them comments during the review process. to a common coordinate system and unit-centroid size to remove variation in position, orientation, and size [81, Authors’ contributions NS and AMB designed the study. AMB provided funding. NS collected and 83]. To visualize shape variation, we performed a prin- analyzed the data. NS wrote the frst draft of the manuscript and both authors cipal component analysis (PCA) using the Procrustes contributed to revisions. Both authors read and approved the fnal manuscript. coordinates and graphed specimens in two dimensions Funding of tangent space (PC1 and PC2) using the R packages This study was supported by funding from the National Science Foundation ggplot2 version 3.1.0 [84] and ggthemes version 4.0.1 [85]. (DEB 1556255) and Villanova University (Department of Biology and Gerald M. For use in the phylogenetic comparative analyses, we Lemole Endowed Chair Fund). Funding for open access fees was provided by the Falvey Library Scholarship Open Access Reserve (SOAR) Fund at Villanova pruned a multi-locus molecular squamate phylogeny University. [41] to include only the taxa in our dataset using the R package phytools version 0.6.6 [86] and geiger version Availability of data and materials The CT data generated and analyzed are available at Morphosource (https://​ 2.0.6.1 [87]. We calculated the K-statistic’s generalization www.​morph​osour​ce.​org/​proje​cts/​00000​C482). for multivariate data [88] to determine the phylogenetic Stepanova and Bauer BMC Ecol Evo (2021) 21:86 Page 52 of 53

Declarations 17. Stayton CT. Morphological evolution of the lizard skull: a geometric morphometrics survey. J Morphol. 2005;263:47–59. Ethics approval and consent to participate 18. Kazi S, Hipsley CA. Conserved evolution of skull shape in Caribbean head- Not applicable. frst burrowing worm lizards (Squamata: Amphisbaenia). Biol J Linn Soc. 2018;125:14–29. Consent for publication 19. Urošević A, Ljubisavljević K, Jelić D, Ivanović A. Variation in the cranium Not applicable. shape of wall lizards (Podarcis spp.): efects of phylogenetic constraints, allometric constraints and ecology. Zoology. 2012;115:207–16. Competing interests 20. Openshaw GH, Keogh JS. Head shape evolution in monitor lizards The authors declare no competing interests. (Varanus): interactions between extreme size disparity, phylogeny and ecology. J Evol Biol. 2014;27:363–73. Author details 21. Metzger KA, Herrel A. 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