Anatomical Network Analyses Reveal Evolutionary Integration and Modularity in the Lizards Skull

Anatomical Network Analyses Reveal Evolutionary Integration and Modularity in the Lizards Skull | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anatomical Network Analyses Reveal Evolutionary Integration and Modularity in the Lizards Skull Yuya Asakura, Soichiro Kawabe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1390987/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract The morphology of lizard skulls is highly diverse, and it is crucial to understand the factors that constrain and promote their evolution to understand how lizards thrive. The results of interactions between cranial bones reflecting these factors can be detected as integration and modularity, and the analysis of integration and modularity allows us to explore the underlying factors. In this study, the integration and modularity of the skulls of lizards and the outgroup tuatara are analyzed using a new method, Anatomical Network Analysis (AnNA), and the factors causing lizards morphological diversity are investigated by comparing them. The comparison of modular structures shows that lizard skulls have high integration and anisomerism, some differences but basically common modular patterns. In contrast, the tuatara shows a different modular pattern from lizards, reflecting underlying developmental factors. In addition, the presence of the postorbital bar by jugal and postorbital (postorbitofrontal) also reflect various functional factors by maintaining low integration. The maintenance of basic structures due to basic functional requirements and changes in integration within the modules play a significant role in increasing the morphological diversity of the lizard skull and in the prosperity of the lizards. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The body parts that constitute the whole morphology of an organism, such as vertebrate skulls, are intricately related to each other. The biological processes that produce interactions between the tissues include development, genetics, function, and evolution (e.g., the sharing of developmental origins, pleiotropic gene effects, the movement in the same direction) 1 . For example, vertebrate skull elements are derived from two sources: neural crest cells and mesodermal cells 2 , so there is a developmental interaction between elements of the same cellular origin. FGFs, SHH, WNT signaling pathway, and BMPs are required for the morphogenesis of the facial cranium derived from neural crest cells (reviewed in Richman et al ., (2006) 3 ). The expression of Fgf8 in the facial epithelium in mammals and birds, for example, is involved in the morphogenesis of multiple bones in the maxilla 4 , 5 , so there is a genetic relationship between these tissue morphologies. Functional interactions also include the coordination between the upper and lower jaw during biting and other activities. The result of the interactions by these complex biological processes can be detected as morphological integration and modularity in a given structure. Therefore, the studies of morphological integration and modularity can provide the relationships between tissues not superficially but potentially and intrinsically, and help to understand the factors that constrained or promoted morphological evolution and the evolvability of the organismal form 1 . Although the fundamental idea of modularity in biology has existed since 1958 6 , morphological modularity is a field that has received much attention in recent years 1 . Among the body tissues of vertebrates, the skull morphology is particularly complex, and its complexity is related to the various functions in protecting the brain and sensory organs and playing roles in foraging and respiration. For this reason, the skull has received a particular attention in studies of morphological modularity 7 . In the study of morphological modularity, the most commonly used method has been geometric morphometrics 7 . On the other hand, network analysis is employed to investigate the modularity of head structures in more recent years 8 , 9 , 10 , 11 , 12 , 13 , 14 . Conventionally used morphometric methods focus on the covariation of size and shape of skeletal parts of interest. In contrast, network analysis is a method that focuses on the interaction between individual bones, which is of interest because it can provide information about the potential function and complement the traditional morphometric approach 7 , 15 , 16 . For example, the geometric morphometrics can only be analyzed from a single side in conventional 2D geometric morphometrics. Additionally, the analysis cannot include non-homologous bones in all specimens as the bones of interest must share a common landmark. Network analysis can overcome these limitations in the geometric morphometrics. Lizards (Lacertilia) belong to Squamata, alongside with snakes, and are the largest group of living reptiles, containing over 7000 species 17 . The lizard skull morphology is extremely diverse. For instance, the group ranges in cranial architecture from amphikinetic skulls in varanids and geckos to the heavily ossified skulls of fossorial taxa 18 , 19 . Therefore, it is essential to understand the factors that constrain and promote lizard skull evolution. However, previous studies on the morphological modularity and integration of lizard skulls are limited 20 , 21 , 22 , 23 , 24 , and network analysis has never been used. Here, we utilize Anatomical Network Analysis 15 , 16 to compare the integration and modularity of skulls across lizards to understand the evolution of their skull morphology and the factors that control it. Using network analysis, which is almost unprecedented to date, this is an important study on the macroevolution of modularity in vertebrate skulls. Results Modularity. The network modules of all species analyzed are shown in Supplementary Figs. 1–58. Despite the morphological diversity of lizard skulls, lizards generally possess separate left and right preorbital (purple and red), postorbital (blue and orange), and mandibular modules (light and dark gray) (Fig. 1 ). Nevertheless, in some taxa, the snout (light purple), including the premaxilla, nasal and frontal (e.g., Basiliscus vitattus , Draco volans , Tupinambis teguixin ), or the braincase elements (yellow) (e.g., Anolis cristatellus , Elgaria panamintina ) form a single module. Furthermore, in the gekkotans, the frontals, parietals, and postorbitals form a skull roof module (pink), while in other taxa, the nasals are included in or the parietals are excluded from the skull roof module. Only in chamaeleonids, the parietals are integrated into the preorbital module, while the iguanians with ornamentation similar to chamaeleonids ( Phrynosom asio , Basiliscus vitattus ) have their parietals integrated into the postorbital module. Rhineura floridana exhibits a unique pattern in which all the cranium bones are integrated into a single module on each side. In two species of geckos, Coleonyx variegatus and Oedura tryoni , the pterygoid forms a separate module with the epipterygoid (see Supplementary Figs. 10 and 32). The boundaries between modules in the dorsal and ventral regions are not constant, and the modules, including the parietal and pterygoid, differ from species to species. Except for Heloderma horridum , Brookesia brygooi , Rhampholeon brevicaudatus , and Oplurus cyclurus , the lateral boundaries are almost constant in the jugal-postorbital (postorbitofrontal). The skull of the tuatara differs significantly from lizards and shows the preorbital module containing the temporal bones, a braincase module, and left and right mandibular modules. Interestingly, the jugal of the tuatara is highly integrated with the postorbital (in the dendrogram, the jugal and postorbital are adjacent to each other (Supplementary Fig. 1)), while in lizards, they are in separate modules (Fig. 1 ; Supplementary Figs. 2–58). Multivariate analyses of network parameters. The PC 1 and PC 2 of the network parameters together account for more than 65% of the total variation (see Supplementary data 4 file). The PC 1 explains most of the parameters except for H. Negative PC 1 values relate to greater N, K, L, Q-modules, S-modules, and Q max , and positive values relate to greater D and C. Amphisbaenia, a fossorial taxon with greater D and less N, K, and L, exhibits larger PC 1 scores. However, within Amphisbaenia, Rhineura floridana (C = 0.5418546) and Bipes biporus (C = 0.4955357) with a greater integration differ from Amphisbaena alba (C = 0.3447368) and Trogonophis wiegmanni (C = 0.3429654), resulting in larger PC 1 score. The basal Lacertoidea, Lacertidae and Teiidae, are intermediate and separated from the derived Lacertoidea, Amphisbaenia, along with PC 1. The PC 1 score for the tuatara was 0.5331 and intermediate. Negative PC 2 values relate to the greater C and H and positive values relate to greater K. Most gekkotans with specialized skulls without postorbital bars and upper temporal bars are plotted on the negative side of PC 2 due to the small value of K. The Mann-Whitney U test strongly supports that Gekkota (n = 14) and other lizards (n = 44) differ from each other in present multivariate analyses (Table 1 ). In other words, only N is lower in the skull of Gekkota than in that of other lizards, and the reduction in connectivity due to the absence of postorbital bars and upper temporal bars in Gekkota does not seem to affect other parameters. Notably, the PC 2 score of the tuatara is the greatest (2.6805), which is due to relatively low C (0.3544974) and the lowest H (0.2763419). This result indicates that lizards evolved skulls that were highly integrated and had greater anisomerism than the tuatara. Table 1. Comparison of network parameters and principal components scores using the Mann-Whitney U test. Values with significant differences are shown in bold. fossorial (n = 6) vs. non-fossorial (n = 52) Gekkota (n = 14) vs. non-Gekkota (n = 44) z-value p-value z-value p-value N 3.30537 0.00095 1.903872 0.05693 K 2.80996 0.00496 3.18151 0.00147 D 3.37071 0.00075 0.890493 0.3732 C 0.178725 0.8582 1.055153 0.2914 L 2.80854 0.00498 1.237532 0.2159 H 0.663846 0.5068 0.981239 0.3265 S-modules 1.707661 0.0877 0.427791 0.6688 Q-modules 2.84809 0.0044 1.912208 0.05585 Q max 3.24258 0.00119 0.620519 0.5349 PC 1 3.24258 0.00119 0.964282 0.3349 PC 2 0.204257 0.8382 2.84763 0.00441 The pPC 1 and pPC 2 of the network parameters together account for about 60% of the total variation (see Supplementary data 4 file). The distributions of pPC1 and pPC2 are essentially unchanged compared to the PCA results, which indicates that there is not much phylogenetic signal in network parameters (Fig. 2 ). However, the plot for most phylogenetically basal and fossorial species, Dibamus novaeguineae , apparently shifts its placement compared to the PCA results, becoming more similar to phylogenetically distant and alike fossorial Amphisbaenia (Fig. 2 a, g). In each ecological category, the network parameters did not differ by diet, but they by habitats and locomotion. Analyses on habitats and locomotion (Fig. 2 c, d) result in greater PC 1 scoring in the fossorial and digger lizards due to their lower N, K, L, Q-Modules, Q max , and higher D than those of other species. Thus, the skulls of fossorial (digger) species are morphologically more complex and have evolved higher functional efficiency and morphological complexity than those of other species. The Mann-Whitney U test supports that fossorial and digger lizards (n = 6) and other species (n = 52) differ from each other (Table 1 ). In the morphological categories, the presence or absence of the upper temporal bars does not appear to be explained in parameters. In the PCA plots, the groups with the upper temporal bars cluster, while those without the upper temporal bars are scattered (Fig. 2 e, k). The FDA on upper temporal bars shows a misclassification error rate of 34.48%, which indicates that the presence of upper temporal bars has no effect on the parameters (Fig. 3 a). On the other hand, the presence or absence of postorbital bars does not have a strong association with the differences in the parameters, where the distribution of each group overlaps in the PCA plots (Fig. 2 f, l). Nonetheless, the group without postorbital bars tends to score greater PC 1 values. On the other hand, the group with postorbital bars scores lower PC 1 and greater PC 2 values, while the group with an incomplete postorbital bar tends to scores lower PC 1 and PC 2 values. The FDA on postorbital bars indicate a misclassification error rate of 20.69%, suggesting that the presence of postorbital bars has a weak effect on the parameters (Fig. 3 b). Discussion Symmetry and asymmetry of modular pattern In AnNA, asymmetric modularity can be detected in the left and right sides of the skulls even in anatomically symmetric structures. Such examples have been detected in the skulls, muscles, and limbs of various taxa 11 , 12 , 13 , 14 . Since the network models and cluster analyses do not distinguish left and right, even if bone connections are coded identically on the left and right, it is expected to result in asymmetric modular structures. A previous study mentions that asymmetrical results of network analysis might be an artifact 14 . In this study, nearly symmetric modules are obtained by virtually dividing an unpaired bone in the median sagittally into the left and right elements and coding it as a pair of bones. Factors causing modularity and integration in lizard skulls The general preorbital and postorbital modular patterns of lizards appear to be largely phylogenetically influenced. However, a comparison of the PCA and pPCA results for the network parameters reveals that the distribution of the plots hardly differs, indicating that factors other than phylogeny play a major role in the preorbital and postorbital modular patterns. The developmental processes shape the morphological structures of an adult. In previous studies on the modularity of the lizard skulls 21 , 23 , one of the developmental factors, cellular origin (neural crest and mesoderm), is adopted from patterns found in mammals. However, skeletal homology between lizards and mammals is not fully appreciated, and it is unknown if the cellular origin patterns of mammals are applicable to lizards 25 . In this regard, we compare the amniote cellular origin patterns of both birds and mammals with the modular patterns of lizards and tuatara, based on Noden and Trainor (2005) 2 . In the case of the mammalian model, the neural-crest-derived elements include the frontals, quadrates, squamosals, and orbitosphenoids, and the mesoderm-derived-elements include the parietals (Fig. 4 ). The mammalian model of cellular origin is discordant with modularity in the lizard skulls demonstrated in this study because the neural-crest-derived quadrates, squamosals, and pterygoids are integrated into the postorbital module with mesoderm-derived bones. In the case of the bird model of cellular origin, the neural-crest-derived elements include the quadrates and squamosals, and the mesoderm-derived elements include the parietals, frontals, postorbitals, and orbitosphenoids (Fig. 4 ). The bird model is also inconsistent with modularity in lizards because, as in the mammalian model, neural-crest-derived quadrates, squamosals, and pterygoids are integrated into the postorbital module with the mesoderm-derived bones. Cellular origins of the temporal elements in lizards is likely indifferent from those of birds and mammals because the cellular origins of birds and mammals coincide in the neural crest despite their very distant phylogenetic relationship. In other words, modularity in the lizard skull is probably caused by factors other than morphogenesis. On the other hand, the modular pattern of the tuatara is almost identical to the amniote cellular origin patterns: mesoderm of neurocranial and skull roof bones and neural crest of facial and mandibular bones (Fig. 4 ). Therefore, it is likely that interactions between skull elements during development strongly influence skull morphology in the tuatara. Another possible factor shaping the modularity of lizard skulls is the difference in ossification sequence patterns. It appears that the ossification sequences of lizard skulls by Khannoon & Evans (2020) 26 and the modular pattern of lizards in this study lacks any correlations (Fig. 5 ). In most species, braincase elements, including the quadrate, tend to ossify later in the ontogeny. Still, parietal, squamosal, and supratemporal, integrated into the same modules as braincase elements, ossify earlier than others in Varanidae and Agamidae. Additionally, when skull roof elements form a separate module, they have the same ossification sequence as the elements of the preorbital module (Phyllodactylidae and Scincidae). Therefore, it is reasonable to assume that the modularity of lizards reflects factors other than ontogenetic development. The general modularity in lizards may reflect functional factors. The modularity would correspond to the functional requirements of the preorbital region of the snout and upper jaw associated with feeding and olfaction, as well as the postorbital region of the braincase and temporal elements associated with brain protection, jaw muscle attachments, and adductor chamber. Independent covariation patterns of anterior and posterior regions in the dorsal skull shape shown in Dactyloids 21 and Lacertids 23 are consistent with present results of a general pre-postorbital modular division. It is concluded that both of their covariation patterns reflect functional demands, which supports present hypothesis described above. Because the fundamental functional requirements do not vary significantly among all taxon, the modularity is likely phylogenetically common to some extent. In contrast to the tuatara and most non-squamate diapsids, lizards lack the lower temporal bar in their skulls. Rieppel & Gronowski 27 proposes that the absence of the quadratojugal and the loss of the lower temporal bar results from the expansion of the external adductor muscle. This observation is consistent with the modular patterns of the lizards reflecting functional factors of feeding, olfaction, muscle attachments, and brain protection more strongly than that of the tuatara. However, it should be noted that the features once considered plesiomorphic, including the lower temporal bar, can be derived or secondarily acquired in Sphenodon 28 , 29 , 30 , 31 , 32 . Whether the modular pattern of the lizards is ancestral or derived remains as a matter for consideration. We found not only general modularity but also variation in modular patterns between lizard taxa. These include the dorsoventral module boundaries, separation of the rostral, skull roof, and braincase modules, and unique modular patterns particularly in chameleons and Rhineura floridana . Again, the covariation patterns of the lizard skulls investigated in previous studies 21 , 22 , 23 are consistent with the general modularity in this study. This also means that the modularity results for lizard skulls in this study may indicate a yet unknown intraspecific covariance pattern in most lizard taxon. Loss of postorbital bar The postorbital bar is composed of the jugal-postorbitofrontal (or postorbital and postfrontal) contact in the tuatara and many lizards. In contrast, Gekkota, Varanidae, Dibamidae, Amphisbaenia, Anguidae, and Anniellidae lack the postorbital bar. The bony jugal-postorbital connection is also lost in Scindoidea and Anguiomorpha and probably articulated by soft tissue. This corresponds to the widely-common jugal-postorbital (postorbitofrontal) lateral module boundaries in other lizards with a postorbital bar, while dorsal and ventral module boundaries are varying. By contrast, the jugal-postorbital contact in the tuatara is highly integrated (these bones are placed on adjacent branches in the network dendrogram (see Supplementary Fig. 1)). It is possible that the low integration of jugal-postfrontal was maintained from the common ancestor of Squamata and that selection pressure caused the loss of the contact in each lineage. The module patterns of the lizard skulls are unaffected by the presence or absence of an upper temporal bar. In contrast, the FDA results indicate that network parameters vary depending on the presence or absence of postorbital bars, which are absent in geckos and fossorial species. Geckos may have reduced connections around the postorbital bone due to structural constraints caused by the enlargement of the eye in the common ancestor 33 , and this is manifested in reduced K. Fully fossorial species tend to degenerate their limbs and use their heads to burrow 34 , 35 , 36 , 37 ; therefore, their heads are subject to large external forces. A solid skull for resistance to such forces is brought about by bone fusion (lower N) and increased connection (greater D and lower L) by the enlargement of the contact surfaces between the bones. In other words, each of these different factors, not the presence or absence of a postorbital bar, is responsible for the differences in the parameters. Cranial kinesis and modularity There seems to be little relevance of cranial kinesis to modularity and network parameters. In taxa with well-developed kinesis (e.g., geckos and varanids), little correspondence exists between the boundaries of modules in which their integration is low in the metakinetic (parietal-supraoccipital), mesokinetic (frontal-parietal), hypokinetic (palatine-pterygoid) axes. Additionally, while Werneburg et al . 13 investigates the cranial kinesis in Tyrannosaurus rex and extant amniotes and argues that species with potential kinesis in their skulls have a larger N and lower D, we do not observe the trend in present dataset. For instance, geckos, known to have well-developed kinesis, did not differ significantly from other taxa except in parameters for K (Table 1 ). The lizard taxa with known degrees of cranial kinesis are indeed limited 19 , 32 , 38 , 39 . However, the degree of cranial kinesis may not be inferred simply by network parameters and modular patterns. Methods Sampling Samples included 57 skulls belonging to 57 species of 38 families in the extant Lacertilia (lizards). Because extant Lacertilia consists of 43 families, the samples in this study nearly cover the entire clade. In addition, we examined tuatara Sphenodon puctatus (Lepidosauria) as an outgroup. All skulls come from adult specimens. Computed tomography (CT) data available at Morphosource ( https://www.morphosource.org/ ) was used for analyses of 45 lacertilian species and Sphenodon (see Supplementary data 1 file). For other 12 specimens, CT images were acquired for the skulls in collections of the National Museum of Nature and Science, Tokyo, or Institute of Dinosaur Research, Fukui Prefectural University (see Supplementary data 1 file). Those CT data were collected by Latheta LCT-200 (Hitachi, Ltd.) or FF35 CT Metrology (Yxlon). Anatomical Network Analysis (AnNA) Following the previous studies 13 , 14 , AnNA was conducted to verify the modularity of the lizard skulls. Unweighted and undirected network matrixes for AnNA of the lizards and tuatara skulls were prepared according to the following method. If two or more skull elements were fused without visible sutures, they were treated as one unit. For a single unpaired bone in the median, we coded it as a pair of bones on the left and right sides by virtually dividing it into two left and right elements sagitally. The presence of contacts between the bones or units was determined by observing the CT images and 3D models on VGStudio MAX 3.3 . Each contact between two bones or units was coded as "1 ", and the absence of contact was coded as " 0 " (see Supplementary data 2 file). As in bone-to-bone/unit-to-unit contacts, articulations were generally coded as "1". However, lizards are equipped with well-developed kinetic joints such as syndesmosis and synovial joints 39 , and it is necessary to recognize the condition of kinetic joints. If they are joined by soft tissues such as ligaments or cartilage, the bones or units may appear to be separated from each other on CT images and 3D models. Although the morphological information of soft tissues should be used to code the joint condition in the skull, studies on the soft tissues of kinetic joints in lizards are very limited 40 . Therefore, in this study, we uniformly coded "1" for joints if the hard bones were in direct contact with each other. Following the script of Plateau & Foth (2020) 14 , the data matrix of each sample was analyzed using the software R-3.6.3 41 and the package igraph 42 . Then, these analyses determined the network parameters for every network 43 , which, in turn, describe the skull anatomy. The number of nodes (N) and connections (K) represent the number of bones and their contacts of each sample, respectively. The density of connections (D) measures the number of existing connections with respect to the maximum possible, where D is interpreted as a proxy of morphological complexity. The mean clustering coefficient (C) measures the average of the sum of connections between all neighbors of each node with respect to the maximum possible, where C is interpreted as a proxy of anatomical integration. The mean shortest path length (L) measures the average of the minimum distance between all nodes, where L is interpreted as a proxy of functional efficiency. The heterogeneity of connectivity (H) is the standard deviation divided by the mean of the number of connections of all nodes in the network, where H is interpreted as a proxy of anisomerism. Modules of the anatomical networks were identified by the hierarchical clustering of the generalized topological overlap similarity matrix among nodes (GTOM). The number of modules, Q-modules, and the identified partition quality, Q max , were determined by the optimization function modularity Q 44 . The S-modules were estimated by performing a two-sample Wilcoxon rank-sum test on internal and external connections of every module. Multivariate analyses of network parameters We performed multivariate analyses of the calculated network parameters to evaluate the factors driving morphological evolution in Lacertilia. Principal component analyses for the network parameters were conducted to compare the distribution of phylogeny, morphological character, and ecology (diet, habitats, and locomotion) in the multivariate data. Ecological and morphological traits of all sampled species are shown in Supplementary data 1 file. The definitions of the ecological traits were adapted from Watanabe et al . (2019) 24 . We also performed phylogenetic principal component analyses (pPCA) 45 to account for phylogenetic effects on network parameters. The phylogenetic hypothesis of Lacertilia for pPCA were based on Pyron et al . (2013) 46 and Mesquite 47 were employed to select species and create NEXUS data for pPCA. In addition, to see if there are statistically significant differences in parameters, taxonomically or ecologically, the Mann-Whitney U test and flexible discriminant analysis (FDA) were conducted. Declarations Acknowledgements We thank Prof. Dr. Hiroshi Nishi (Fukui Prefectural University), Dr. Masateru Shibata (Fukui Prefectural University), Dr. Takuya Imai (Fukui Prefectural University), and Dr. Soki Hattori (Fukui Prefectural University) for their helpful advice. Dr. Takanobu Tsuihiji (National Museum of Nature and Science,Tokyo) and Mis. Chisako Sakata (National Museum of Nature and Science, Tokyo) are thanked for specimens collection assistance. Mizuho Sano (Nagoya University) is thanked for specimen collection assistance and methodological advice. We thank the staff of the Industrial Technology Center of Fukui Prefecture for access to the CT scanner. Author Contributions Y.A. and S.K. designed the project and arranged the materials. Y.A. performed analyzed the data. Y.A. wrote the manuscript with assistance of S.K. Data Availability All data analyzed during this study are included in this published article and its supplementary information files. Additional Information Competing interests The authors declare no competing interests. References Klingenberg, C. P. Morphological integration and developmental modularity. Annu. Rev. Ecol. Evol. Syst . 39 , 115–132 (2008). Noden, D. M., & Trainor, P. A. Relations and interactions between cranial mesoderm and neural crest populations. J. Anat . 207 , 575–601, https://doi.org/10.1111/j.1469-7580.2005.00473.x (2005). Richman, J. M., Buchtová, M., & Boughner, J. C. Comparative ontogeny and phylogeny of the upper jaw skeleton in amniotes. Dev. Dyn . 235 , 1230–1243 (2006). Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M., & Martin, G. R. 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B., & Hanken, J. Evolutionary innovation and conservation in the embryonic derivation of the vertebrate skull. Nat. Commun . 5 , 5661 (2014). Khannoon, E. R., & Evans, S. E. Embryonic skull development in the gecko, Tarentola annularis (Squamata: Gekkota: Phyllodactylidae). J. Anat . 237 , 504–519, https://doi.org/10.1111/joa.13213 (2020). Rieppel, O., & Gronowski, R. W. The loss of the lower temporal arcade in diapsid reptiles. Zool. J. Linn. Soc . 72 , 203–217 (1981). Whiteside, D. I. The head skeleton of the Rhaetian sphenodontid Diphydontosaurus avonis gen. et sp. nov. and the modernizing of a living fossil. Philos. Trans. R. Soc. B: Biol. Sci . 312 , 379–430 (1986). Evans, S. E. At the feet of the dinosaurs: the early history and radiation of lizards. Biol. Rev . 78 , 513–551 (2003). Böhme, W., & Ziegler, T. A review of iguanian and anguimorph lizard genitalia (Squamata: Chamaeleonidae; Varanoidea, Shinisauridae, Xenosauridae, Anguidae) and their phylogenetic significance: comparisons with molecular data sets. J. Zool. Syst. Evol . 47 , 189–202, https://doi.org/10.1111/j.1439-0469.2008.00495.x (2009). Jones, M. E., Curtis, N., O'Higgins, P., Fagan, M., & Evans, S. E. The head and neck muscles associated with feeding in Sphenodon (Reptilia: Lepidosauria: Rhynchocephalia). Palaeontol. Electron . 12 , 7A (2009). Jones, M. E., Curtis, N., Fagan, M. J., O’Higgins, P., & Evans, S. E. Hard tissue anatomy of the cranial joints in Sphenodon (Rhynchocephalia): sutures, kinesis, and skull mechanics. Palaeontol. Electron . 14 , 17A (2011). Herrel, A., Aerts, P., & De Vree, F. Cranial kinesis in geckoes: functional implications. J. Exp. Biol . 203 , 1415–1423 (2000). Greer, A. E. Limb reduction in squamates: identification of the lineages and discussion of the trends. J. Herpetol . 25 , 166–173 (1991). Lee, M. S. Convergent evolution and character correlation in burrowing reptiles: towards a resolution of squamate relationships. Biol. J. Linn. Soc . 65 , 369–453 (1998). Wiens, J. J., Brandley, M. C., & Reeder, T. W. Why does a trait evolve multiple times within a clade? Repeated evolution of snakeline body form in squamate reptiles. Evolution 60 , 123–141 (2006). Benesch, A. R., & Withers, P. C. Burrowing performance and the role of limb reduction in Lerista (Scincidae, Lacertilia). Senckenberg . 82 , 107–114 (2002). Iordansky, N. N. Cranial kinesis in lizards (Lacertilia): origin, biomechanics, and evolution. Biol. Bull . 38 , 868–877 (2011). Mezzasalma, M., Maio, N., & Guarino, F. M. To move or not to move: cranial joints in European gekkotans and lacertids, an osteological and histological perspective. Anat. Rec . 297 , 463–472 (2014). Payne, S. L., Holliday, C. M., & Vickaryous, M. K. An osteological and histological investigation of cranial joints in geckos. Anat. Rec . 294 , 399–405 (2011). R Core Team. R: A language and environment for statistical computing. https://www.r-project.org/ (2020). Csardi, G., & Nepusz, T. The igraph software package for complex network research. Interjournal Complex Syst . 1695 , 1–9 (2006). Esteve-Altava, B., Marugán-Lobón, J., Botella, H., & Rasskin-Gutman, D. Structural constraints in the evolution of the tetrapod skull complexity: Williston’s law revisited using network models. Evol. Biol . 40 , 209–219, https://doi.org/10.1007/s11692-012-9200-9 (2013). Clauset, A., Newman, M. E., & Moore, C. Finding community structure in very large networks. Phys. Rev. E 70 , 066111 (2004). Revell, L. J. phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol . 3 , 217–223 (2012). Pyron, R. A., Burbrink, F. T., & Wiens, J. J. A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evol. Biol . 13 , 93 (2013). Maddison, W. P., & Maddison, D. R. Mesquite: a modular system for evolutionary analysis. Version 3.70. https://www.mesquiteproject.org/ (2021). Dufaure, J. P., & Hubert, J. Table de développement du lézard vivipare-Lacerta (Zootoca) vivipara Jacquin. Arch. anat. microsc. morphol. exp . 50 , 309 (1961). Werneburg, I., Polachowski, K. M., & Hutchinson, M. N. Bony skull development in the Argus monitor (Squamata, Varanidae, Varanus panoptes ) with comments on developmental timing and adult anatomy. Zoology 118 , 255–280 (2015). Ollonen, J., Da Silva, F. O., Mahlow, K., & Di-Poï, N. Skull development, ossification pattern, and adult shape in the emerging lizard model organism Pogona vitticeps : a comparative analysis with other squamates. Front. physiol . 9 , 278 (2018). Jerez, A., Sánchez-Martínez, P. M., & Guerra-Fuentes, R. A. Embryonic skull development in the neotropical viviparous skink Mabuya (Squamata: Scincidae). Acta zool. mex . 31 , 391–402 (2015). Additional Declarations No competing interests reported. Supplementary Files Supplementarydata1.Samplelist.xlsx Supplementarydata2.AnNAdatamatrix.xlsx Supplementarydata3.Skullnetwokdendrograms.pdf Supplementarydata4.NetworkparametersandPCAandpPCAresults.xlsx Supplementarydata5.RscriptforAnNA.txt Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 13 Jun, 2022 Reviews received at journal 04 Jun, 2022 Reviewers agreed at journal 25 May, 2022 Reviews received at journal 17 Apr, 2022 Reviewers agreed at journal 15 Mar, 2022 Reviewers invited by journal 09 Mar, 2022 Editor assigned by journal 09 Mar, 2022 Editor invited by journal 09 Mar, 2022 Submission checks completed at journal 09 Mar, 2022 First submitted to journal 24 Feb, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1390987","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":89411296,"identity":"2d26c93c-4e05-4a19-bcd2-efd58ebc78c8","order_by":0,"name":"Yuya Asakura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYBACgwNgkoFBAkQn/rEBkoyNBwhrqYBq+diQBtLSgFeLZAOIPAPRwjiz4TBYFK8Wfokcww+MbXXykjNyH37m3XHebm37YaAtNTbRuLSwSeQYSzC2HTacLZFuLM175nbytjOJQC3H0nIbcGsxAGo5wDhPIo1BmoftdrLZAaAWxobD+LQY/2D8V2cP1ML8m4ftXLLZ+YcEtZgBbWFOnC2RxiY5s+2AndkNQrbwPCuzSGw7nDyz5xmbxYczyQlmN4C2JODzC3vy5hsf2+psZxxPY76RUGFnb3Y+/eGDDzU2OLUwMHAYMCQgcRPBKhOwqYQD9gcoXHu8ikfBKBgFo2BEAgAuyWJDZIGRAgAAAABJRU5ErkJggg==","orcid":"","institution":"Fukui Prefectural University","correspondingAuthor":true,"prefix":"","firstName":"Yuya","middleName":"","lastName":"Asakura","suffix":""},{"id":89411299,"identity":"d58dc1d5-b262-4334-8176-374cf2df3bd1","order_by":1,"name":"Soichiro Kawabe","email":"","orcid":"","institution":"Fukui Prefectural University","correspondingAuthor":false,"prefix":"","firstName":"Soichiro","middleName":"","lastName":"Kawabe","suffix":""}],"badges":[],"createdAt":"2022-02-24 05:14:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1390987/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1390987/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19131660,"identity":"ee5b31c2-3602-4695-a0ed-759f31978a15","added_by":"auto","created_at":"2022-03-11 15:58:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1239498,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of skull modules in tuatara and some lizards from left and right side with the phylogenetic tree. The phylogenetic tree on the left is based on the molecular phylogenetic information in Pyron \u003cem\u003eet al\u003c/em\u003e. (2013)\u003csup\u003e46\u003c/sup\u003e. Families not covered in this study are grayed out.\u003c/p\u003e","description":"","filename":"Figure1..png","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/9b570c03d665d5f2a9c1bc57.png"},{"id":19131969,"identity":"5a30027a-9ce7-4114-af10-5648b2d4439c","added_by":"auto","created_at":"2022-03-11 16:01:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":888356,"visible":true,"origin":"","legend":"\u003cp\u003eResults of principal component analysis (PCA) and phylogenetic principal component analysis (pPCA). PCA plots colored by (a) taxon, (b) diet, (c) habitat, (d) locomotion, (e) presence of upper temporal bar and (f) presence of postorbital bar. pPCA plots colored by (g) taxon, (h) diet, (i) habitat, (j) locomotion, (k) presence of upper temporal bar and (l) presence of postorbital bar.\u003c/p\u003e","description":"","filename":"Figure2..png","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/e535d3aaddda9b3f0b0e8a15.png"},{"id":19131047,"identity":"5af91ecd-1954-4c65-a986-184b63e62dbc","added_by":"auto","created_at":"2022-03-11 15:55:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":129159,"visible":true,"origin":"","legend":"\u003cp\u003eResults of flexible discriminant analysis (FDA). (a) On the presence of the upper temporal bar. (b) On the presence of the postorbital bar.\u003c/p\u003e","description":"","filename":"Figure3..png","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/673a692772e328b396a3b401.png"},{"id":19131048,"identity":"842bf515-a917-46a7-8a7b-7a4c7d8d4770","added_by":"auto","created_at":"2022-03-11 15:55:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":456493,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the skull modules of lizards and tuatara with the cellular origin patterns. Based on Noden and Trainor (2005)\u003csup\u003e2\u003c/sup\u003e, skulls of lizards \u003cem\u003e(Dipsosaurus dorsalis\u003c/em\u003e) and tuatara are color-coded for mammalian and bird cellular origin patterns (blue, the neural-crest-derived elements; pink, mesoderm-derived elements; gray, non-homologous elements). Abbreviations: f, frontal; os, orbitosphenoid; p, parietal; po, postorbital; q, quadrate; sq, squamosal.\u003c/p\u003e","description":"","filename":"Figure4..png","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/ace6bc59e16447bf39bf886b.png"},{"id":19131659,"identity":"3d1bf598-ee11-4232-8609-d875e80b7335","added_by":"auto","created_at":"2022-03-11 15:58:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":325082,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of ossification sequence and modularity of lizard skulls. These figures are based on comparing the ossification sequence in the developmental stages of Dufaure \u0026amp; Hubert (1961)\u003csup\u003e48\u003c/sup\u003e by Khannoon \u0026amp; Evans (2020)\u003csup\u003e26\u003c/sup\u003e. The ossification sequence patterns with the modules of the same family are compared, respectively\u003csup\u003e26,49,50,51\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Figure5..png","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/a190eee8d9f97e2d8adabc60.png"},{"id":19131970,"identity":"35414382-d289-43af-8325-0ff11def9b05","added_by":"auto","created_at":"2022-03-11 16:01:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1371026,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/ed59b031-ee40-4f16-9ac4-4e37371ce1fe.pdf"},{"id":19131051,"identity":"df62d546-3c04-40e3-9047-b7ccc5c26d32","added_by":"auto","created_at":"2022-03-11 15:55:49","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":31750,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata1.Samplelist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/2f0f84f426e87a52dc77c18d.xlsx"},{"id":19131055,"identity":"1658fbae-d29c-447a-b005-611bdea4d275","added_by":"auto","created_at":"2022-03-11 15:55:49","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":365345,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata2.AnNAdatamatrix.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/cc1ea23f2f4fe8c58a65b94d.xlsx"},{"id":19131056,"identity":"40994231-1f61-47fb-bf7c-c006ed69f753","added_by":"auto","created_at":"2022-03-11 15:55:49","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":5437772,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata3.Skullnetwokdendrograms.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/20d314d3e6d8c17f0510a043.pdf"},{"id":19131662,"identity":"9a4eab46-3df0-428e-bcc5-d4bd0f3be6bf","added_by":"auto","created_at":"2022-03-11 15:58:49","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":39631,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata4.NetworkparametersandPCAandpPCAresults.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/8ff32c780019ce73a791501a.xlsx"},{"id":19131054,"identity":"b7b9826d-c089-4e95-b12e-52ac1fad829d","added_by":"auto","created_at":"2022-03-11 15:55:49","extension":"txt","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":7405,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata5.RscriptforAnNA.txt","url":"https://assets-eu.researchsquare.com/files/rs-1390987/v1/10b2579617d0cc9b84db3f41.txt"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAnatomical Network Analyses Reveal Evolutionary Integration and Modularity in the Lizards Skull\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe body parts that constitute the whole morphology of an organism, such as vertebrate skulls, are intricately related to each other. The biological processes that produce interactions between the tissues include development, genetics, function, and evolution (e.g., the sharing of developmental origins, pleiotropic gene effects, the movement in the same direction)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. For example, vertebrate skull elements are derived from two sources: neural crest cells and mesodermal cells\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, so there is a developmental interaction between elements of the same cellular origin. FGFs, SHH, WNT signaling pathway, and BMPs are required for the morphogenesis of the facial cranium derived from neural crest cells (reviewed in Richman \u003cem\u003eet al\u003c/em\u003e., (2006)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e). The expression of \u003cem\u003eFgf8\u003c/em\u003e in the facial epithelium in mammals and birds, for example, is involved in the morphogenesis of multiple bones in the maxilla\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, so there is a genetic relationship between these tissue morphologies. Functional interactions also include the coordination between the upper and lower jaw during biting and other activities. The result of the interactions by these complex biological processes can be detected as morphological integration and modularity in a given structure. Therefore, the studies of morphological integration and modularity can provide the relationships between tissues not superficially but potentially and intrinsically, and help to understand the factors that constrained or promoted morphological evolution and the evolvability of the organismal form\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although the fundamental idea of modularity in biology has existed since 1958\u003csup\u003e6\u003c/sup\u003e, morphological modularity is a field that has received much attention in recent years\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the body tissues of vertebrates, the skull morphology is particularly complex, and its complexity is related to the various functions in protecting the brain and sensory organs and playing roles in foraging and respiration. For this reason, the skull has received a particular attention in studies of morphological modularity\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In the study of morphological modularity, the most commonly used method has been geometric morphometrics\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. On the other hand, network analysis is employed to investigate the modularity of head structures in more recent years\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Conventionally used morphometric methods focus on the covariation of size and shape of skeletal parts of interest. In contrast, network analysis is a method that focuses on the interaction between individual bones, which is of interest because it can provide information about the potential function and complement the traditional morphometric approach\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For example, the geometric morphometrics can only be analyzed from a single side in conventional 2D geometric morphometrics. Additionally, the analysis cannot include non-homologous bones in all specimens as the bones of interest must share a common landmark. Network analysis can overcome these limitations in the geometric morphometrics.\u003c/p\u003e \u003cp\u003eLizards (Lacertilia) belong to Squamata, alongside with snakes, and are the largest group of living reptiles, containing over 7000 species\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The lizard skull morphology is extremely diverse. For instance, the group ranges in cranial architecture from amphikinetic skulls in varanids and geckos to the heavily ossified skulls of fossorial taxa\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Therefore, it is essential to understand the factors that constrain and promote lizard skull evolution. However, previous studies on the morphological modularity and integration of lizard skulls are limited\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and network analysis has never been used.\u003c/p\u003e \u003cp\u003eHere, we utilize Anatomical Network Analysis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e to compare the integration and modularity of skulls across lizards to understand the evolution of their skull morphology and the factors that control it. Using network analysis, which is almost unprecedented to date, this is an important study on the macroevolution of modularity in vertebrate skulls.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eModularity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe network modules of all species analyzed are shown in Supplementary Figs.\u0026nbsp;1\u0026ndash;58. Despite the morphological diversity of lizard skulls, lizards generally possess separate left and right preorbital (purple and red), postorbital (blue and orange), and mandibular modules (light and dark gray) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Nevertheless, in some taxa, the snout (light purple), including the premaxilla, nasal and frontal (e.g., \u003cem\u003eBasiliscus vitattus\u003c/em\u003e, \u003cem\u003eDraco volans\u003c/em\u003e, \u003cem\u003eTupinambis teguixin\u003c/em\u003e), or the braincase elements (yellow) (e.g., \u003cem\u003eAnolis cristatellus\u003c/em\u003e, \u003cem\u003eElgaria panamintina\u003c/em\u003e) form a single module. Furthermore, in the gekkotans, the frontals, parietals, and postorbitals form a skull roof module (pink), while in other taxa, the nasals are included in or the parietals are excluded from the skull roof module. Only in chamaeleonids, the parietals are integrated into the preorbital module, while the iguanians with ornamentation similar to chamaeleonids (\u003cem\u003ePhrynosom asio\u003c/em\u003e, \u003cem\u003eBasiliscus vitattus\u003c/em\u003e) have their parietals integrated into the postorbital module. \u003cem\u003eRhineura floridana\u003c/em\u003e exhibits a unique pattern in which all the cranium bones are integrated into a single module on each side. In two species of geckos, \u003cem\u003eColeonyx variegatus\u003c/em\u003e and \u003cem\u003eOedura tryoni\u003c/em\u003e, the pterygoid forms a separate module with the epipterygoid (see Supplementary Figs.\u0026nbsp;10 and 32).\u003c/p\u003e\n\u003cp\u003eThe boundaries between modules in the dorsal and ventral regions are not constant, and the modules, including the parietal and pterygoid, differ from species to species. Except for \u003cem\u003eHeloderma horridum\u003c/em\u003e, \u003cem\u003eBrookesia brygooi\u003c/em\u003e, \u003cem\u003eRhampholeon brevicaudatus\u003c/em\u003e, and \u003cem\u003eOplurus cyclurus\u003c/em\u003e, the lateral boundaries are almost constant in the jugal-postorbital (postorbitofrontal).\u003c/p\u003e\n\u003cp\u003eThe skull of the tuatara differs significantly from lizards and shows the preorbital module containing the temporal bones, a braincase module, and left and right mandibular modules. Interestingly, the jugal of the tuatara is highly integrated with the postorbital (in the dendrogram, the jugal and postorbital are adjacent to each other (Supplementary Fig.\u0026nbsp;1)), while in lizards, they are in separate modules (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary Figs.\u0026nbsp;2\u0026ndash;58).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultivariate analyses of network parameters.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PC 1 and PC 2 of the network parameters together account for more than 65% of the total variation (see Supplementary data 4 file). The PC 1 explains most of the parameters except for H. Negative PC 1 values relate to greater N, K, L, Q-modules, S-modules, and Q\u003csub\u003emax\u003c/sub\u003e, and positive values relate to greater D and C. Amphisbaenia, a fossorial taxon with greater D and less N, K, and L, exhibits larger PC 1 scores. However, within Amphisbaenia, \u003cem\u003eRhineura floridana\u003c/em\u003e (C\u0026thinsp;=\u0026thinsp;0.5418546) and \u003cem\u003eBipes biporus\u003c/em\u003e (C\u0026thinsp;=\u0026thinsp;0.4955357) with a greater integration differ from \u003cem\u003eAmphisbaena alba\u003c/em\u003e (C\u0026thinsp;=\u0026thinsp;0.3447368) and \u003cem\u003eTrogonophis wiegmanni\u003c/em\u003e (C\u0026thinsp;=\u0026thinsp;0.3429654), resulting in larger PC 1 score. The basal Lacertoidea, Lacertidae and Teiidae, are intermediate and separated from the derived Lacertoidea, Amphisbaenia, along with PC 1. The PC 1 score for the tuatara was 0.5331 and intermediate.\u003c/p\u003e\n\u003cp\u003eNegative PC 2 values relate to the greater C and H and positive values relate to greater K. Most gekkotans with specialized skulls without postorbital bars and upper temporal bars are plotted on the negative side of PC 2 due to the small value of K. The Mann-Whitney U test strongly supports that Gekkota (n\u0026thinsp;=\u0026thinsp;14) and other lizards (n\u0026thinsp;=\u0026thinsp;44) differ from each other in present multivariate analyses (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In other words, only N is lower in the skull of Gekkota than in that of other lizards, and the reduction in connectivity due to the absence of postorbital bars and upper temporal bars in Gekkota does not seem to affect other parameters. Notably, the PC 2 score of the tuatara is the greatest (2.6805), which is due to relatively low C (0.3544974) and the lowest H (0.2763419). This result indicates that lizards evolved skulls that were highly integrated and had greater anisomerism than the tuatara.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eComparison of network parameters and principal components scores using the Mann-Whitney U test. Values with significant differences are shown in bold.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.607773851590107%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"41.696113074204945%\"\u003e\n \u003cp\u003efossorial (n = 6) vs.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enon-fossorial (n = 52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"41.696113074204945%\"\u003e\n \u003cp\u003eGekkota (n = 14) vs.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enon-Gekkota (n = 44)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.607773851590107%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003ez-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003ez-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.30537\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00095\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e1.903872\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.05693\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.80996\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00496\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.18151\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00147\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.37071\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00075\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.890493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.3732\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.178725\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.8582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e1.055153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.2914\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.80854\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00498\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e1.237532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.2159\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.663846\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.5068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.981239\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.3265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eS-modules\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e1.707661\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.0877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.427791\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.6688\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eQ-modules\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.84809\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0044\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e1.912208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.05585\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003eQ\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.24258\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00119\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.620519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.5349\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003ePC 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.24258\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00119\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.964282\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.3349\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.607773851590107%\"\u003e\n \u003cp\u003ePC 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.204257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e0.8382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.84763\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.848056537102472%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00441\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe pPC 1 and pPC 2 of the network parameters together account for about 60% of the total variation (see Supplementary data 4 file). The distributions of pPC1 and pPC2 are essentially unchanged compared to the PCA results, which indicates that there is not much phylogenetic signal in network parameters (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). However, the plot for most phylogenetically basal and fossorial species, \u003cem\u003eDibamus novaeguineae\u003c/em\u003e, apparently shifts its placement compared to the PCA results, becoming more similar to phylogenetically distant and alike fossorial Amphisbaenia (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, g).\u003c/p\u003e\n\u003cp\u003eIn each ecological category, the network parameters did not differ by diet, but they by habitats and locomotion. Analyses on habitats and locomotion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, d) result in greater PC 1 scoring in the fossorial and digger lizards due to their lower N, K, L, Q-Modules, Q\u003csub\u003emax\u003c/sub\u003e, and higher D than those of other species. Thus, the skulls of fossorial (digger) species are morphologically more complex and have evolved higher functional efficiency and morphological complexity than those of other species. The Mann-Whitney U test supports that fossorial and digger lizards (n\u0026thinsp;=\u0026thinsp;6) and other species (n\u0026thinsp;=\u0026thinsp;52) differ from each other (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn the morphological categories, the presence or absence of the upper temporal bars does not appear to be explained in parameters. In the PCA plots, the groups with the upper temporal bars cluster, while those without the upper temporal bars are scattered (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, k). The FDA on upper temporal bars shows a misclassification error rate of 34.48%, which indicates that the presence of upper temporal bars has no effect on the parameters (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). On the other hand, the presence or absence of postorbital bars does not have a strong association with the differences in the parameters, where the distribution of each group overlaps in the PCA plots (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef, l). Nonetheless, the group without postorbital bars tends to score greater PC 1 values. On the other hand, the group with postorbital bars scores lower PC 1 and greater PC 2 values, while the group with an incomplete postorbital bar tends to scores lower PC 1 and PC 2 values. The FDA on postorbital bars indicate a misclassification error rate of 20.69%, suggesting that the presence of postorbital bars has a weak effect on the parameters (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eSymmetry and asymmetry of modular pattern\u003c/h2\u003e\n\u003cp\u003eIn AnNA, asymmetric modularity can be detected in the left and right sides of the skulls even in anatomically symmetric structures. Such examples have been detected in the skulls, muscles, and limbs of various taxa\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Since the network models and cluster analyses do not distinguish left and right, even if bone connections are coded identically on the left and right, it is expected to result in asymmetric modular structures. A previous study mentions that asymmetrical results of network analysis might be an artifact\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In this study, nearly symmetric modules are obtained by virtually dividing an unpaired bone in the median sagittally into the left and right elements and coding it as a pair of bones.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eFactors causing modularity and integration in lizard skulls\u003c/h2\u003e\n\u003cp\u003eThe general preorbital and postorbital modular patterns of lizards appear to be largely phylogenetically influenced. However, a comparison of the PCA and pPCA results for the network parameters reveals that the distribution of the plots hardly differs, indicating that factors other than phylogeny play a major role in the preorbital and postorbital modular patterns.\u003c/p\u003e\n\u003cp\u003eThe developmental processes shape the morphological structures of an adult. In previous studies on the modularity of the lizard skulls\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, one of the developmental factors, cellular origin (neural crest and mesoderm), is adopted from patterns found in mammals. However, skeletal homology between lizards and mammals is not fully appreciated, and it is unknown if the cellular origin patterns of mammals are applicable to lizards\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In this regard, we compare the amniote cellular origin patterns of both birds and mammals with the modular patterns of lizards and tuatara, based on Noden and Trainor (2005)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In the case of the mammalian model, the neural-crest-derived elements include the frontals, quadrates, squamosals, and orbitosphenoids, and the mesoderm-derived-elements include the parietals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The mammalian model of cellular origin is discordant with modularity in the lizard skulls demonstrated in this study because the neural-crest-derived quadrates, squamosals, and pterygoids are integrated into the postorbital module with mesoderm-derived bones. In the case of the bird model of cellular origin, the neural-crest-derived elements include the quadrates and squamosals, and the mesoderm-derived elements include the parietals, frontals, postorbitals, and orbitosphenoids (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The bird model is also inconsistent with modularity in lizards because, as in the mammalian model, neural-crest-derived quadrates, squamosals, and pterygoids are integrated into the postorbital module with the mesoderm-derived bones. Cellular origins of the temporal elements in lizards is likely indifferent from those of birds and mammals because the cellular origins of birds and mammals coincide in the neural crest despite their very distant phylogenetic relationship. In other words, modularity in the lizard skull is probably caused by factors other than morphogenesis. On the other hand, the modular pattern of the tuatara is almost identical to the amniote cellular origin patterns: mesoderm of neurocranial and skull roof bones and neural crest of facial and mandibular bones (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Therefore, it is likely that interactions between skull elements during development strongly influence skull morphology in the tuatara.\u003c/p\u003e\n\u003cp\u003eAnother possible factor shaping the modularity of lizard skulls is the difference in ossification sequence patterns. It appears that the ossification sequences of lizard skulls by Khannoon \u0026amp; Evans (2020)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and the modular pattern of lizards in this study lacks any correlations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). In most species, braincase elements, including the quadrate, tend to ossify later in the ontogeny. Still, parietal, squamosal, and supratemporal, integrated into the same modules as braincase elements, ossify earlier than others in Varanidae and Agamidae. Additionally, when skull roof elements form a separate module, they have the same ossification sequence as the elements of the preorbital module (Phyllodactylidae and Scincidae). Therefore, it is reasonable to assume that the modularity of lizards reflects factors other than ontogenetic development.\u003c/p\u003e\n\u003cp\u003eThe general modularity in lizards may reflect functional factors. The modularity would correspond to the functional requirements of the preorbital region of the snout and upper jaw associated with feeding and olfaction, as well as the postorbital region of the braincase and temporal elements associated with brain protection, jaw muscle attachments, and adductor chamber. Independent covariation patterns of anterior and posterior regions in the dorsal skull shape shown in Dactyloids\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and Lacertids\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e are consistent with present results of a general pre-postorbital modular division. It is concluded that both of their covariation patterns reflect functional demands, which supports present hypothesis described above. Because the fundamental functional requirements do not vary significantly among all taxon, the modularity is likely phylogenetically common to some extent.\u003c/p\u003e\n\u003cp\u003eIn contrast to the tuatara and most non-squamate diapsids, lizards lack the lower temporal bar in their skulls. Rieppel \u0026amp; Gronowski\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e proposes that the absence of the quadratojugal and the loss of the lower temporal bar results from the expansion of the external adductor muscle. This observation is consistent with the modular patterns of the lizards reflecting functional factors of feeding, olfaction, muscle attachments, and brain protection more strongly than that of the tuatara. However, it should be noted that the features once considered plesiomorphic, including the lower temporal bar, can be derived or secondarily acquired in \u003cem\u003eSphenodon\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Whether the modular pattern of the lizards is ancestral or derived remains as a matter for consideration.\u003c/p\u003e\n\u003cp\u003eWe found not only general modularity but also variation in modular patterns between lizard taxa. These include the dorsoventral module boundaries, separation of the rostral, skull roof, and braincase modules, and unique modular patterns particularly in chameleons and \u003cem\u003eRhineura floridana\u003c/em\u003e. Again, the covariation patterns of the lizard skulls investigated in previous studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e are consistent with the general modularity in this study. This also means that the modularity results for lizard skulls in this study may indicate a yet unknown intraspecific covariance pattern in most lizard taxon.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eLoss of postorbital bar\u003c/h2\u003e\n\u003cp\u003eThe postorbital bar is composed of the jugal-postorbitofrontal (or postorbital and postfrontal) contact in the tuatara and many lizards. In contrast, Gekkota, Varanidae, Dibamidae, Amphisbaenia, Anguidae, and Anniellidae lack the postorbital bar. The bony jugal-postorbital connection is also lost in Scindoidea and Anguiomorpha and probably articulated by soft tissue. This corresponds to the widely-common jugal-postorbital (postorbitofrontal) lateral module boundaries in other lizards with a postorbital bar, while dorsal and ventral module boundaries are varying. By contrast, the jugal-postorbital contact in the tuatara is highly integrated (these bones are placed on adjacent branches in the network dendrogram (see Supplementary Fig.\u0026nbsp;1)). It is possible that the low integration of jugal-postfrontal was maintained from the common ancestor of Squamata and that selection pressure caused the loss of the contact in each lineage.\u003c/p\u003e\n\u003cp\u003eThe module patterns of the lizard skulls are unaffected by the presence or absence of an upper temporal bar. In contrast, the FDA results indicate that network parameters vary depending on the presence or absence of postorbital bars, which are absent in geckos and fossorial species. Geckos may have reduced connections around the postorbital bone due to structural constraints caused by the enlargement of the eye in the common ancestor\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and this is manifested in reduced K. Fully fossorial species tend to degenerate their limbs and use their heads to burrow\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e; therefore, their heads are subject to large external forces. A solid skull for resistance to such forces is brought about by bone fusion (lower N) and increased connection (greater D and lower L) by the enlargement of the contact surfaces between the bones. In other words, each of these different factors, not the presence or absence of a postorbital bar, is responsible for the differences in the parameters.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eCranial kinesis and modularity\u003c/h2\u003e\n\u003cp\u003eThere seems to be little relevance of cranial kinesis to modularity and network parameters. In taxa with well-developed kinesis (e.g., geckos and varanids), little correspondence exists between the boundaries of modules in which their integration is low in the metakinetic (parietal-supraoccipital), mesokinetic (frontal-parietal), hypokinetic (palatine-pterygoid) axes. Additionally, while Werneburg \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e investigates the cranial kinesis in \u003cem\u003eTyrannosaurus rex\u003c/em\u003e and extant amniotes and argues that species with potential kinesis in their skulls have a larger N and lower D, we do not observe the trend in present dataset. For instance, geckos, known to have well-developed kinesis, did not differ significantly from other taxa except in parameters for K (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The lizard taxa with known degrees of cranial kinesis are indeed limited\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, the degree of cranial kinesis may not be inferred simply by network parameters and modular patterns.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSampling\u003c/h2\u003e \u003cp\u003eSamples included 57 skulls belonging to 57 species of 38 families in the extant Lacertilia (lizards). Because extant Lacertilia consists of 43 families, the samples in this study nearly cover the entire clade. In addition, we examined tuatara \u003cem\u003eSphenodon puctatus\u003c/em\u003e (Lepidosauria) as an outgroup. All skulls come from adult specimens. Computed tomography (CT) data available at Morphosource (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.morphosource.org/\u003c/span\u003e\u003c/span\u003e) was used for analyses of 45 lacertilian species and \u003cem\u003eSphenodon\u003c/em\u003e (see Supplementary data 1 file). For other 12 specimens, CT images were acquired for the skulls in collections of the National Museum of Nature and Science, Tokyo, or Institute of Dinosaur Research, Fukui Prefectural University (see Supplementary data 1 file). Those CT data were collected by Latheta LCT-200 (Hitachi, Ltd.) or FF35 CT Metrology (Yxlon).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnatomical Network Analysis (AnNA)\u003c/h2\u003e \u003cp\u003eFollowing the previous studies\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, AnNA was conducted to verify the modularity of the lizard skulls. Unweighted and undirected network matrixes for AnNA of the lizards and tuatara skulls were prepared according to the following method. If two or more skull elements were fused without visible sutures, they were treated as one unit. For a single unpaired bone in the median, we coded it as a pair of bones on the left and right sides by virtually dividing it into two left and right elements sagitally. The presence of contacts between the bones or units was determined by observing the CT images and 3D models on \u003cem\u003eVGStudio MAX 3.3\u003c/em\u003e. Each contact between two bones or units was coded as \"1 \", and the absence of contact was coded as \" 0 \" (see Supplementary data 2 file).\u003c/p\u003e \u003cp\u003eAs in bone-to-bone/unit-to-unit contacts, articulations were generally coded as \"1\". However, lizards are equipped with well-developed kinetic joints such as syndesmosis and synovial joints\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and it is necessary to recognize the condition of kinetic joints. If they are joined by soft tissues such as ligaments or cartilage, the bones or units may appear to be separated from each other on CT images and 3D models. Although the morphological information of soft tissues should be used to code the joint condition in the skull, studies on the soft tissues of kinetic joints in lizards are very limited\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Therefore, in this study, we uniformly coded \"1\" for joints if the hard bones were in direct contact with each other.\u003c/p\u003e \u003cp\u003eFollowing the script of Plateau \u0026amp; Foth (2020)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, the data matrix of each sample was analyzed using the software \u003cem\u003eR-3.6.3\u003c/em\u003e\u003csup\u003e41\u003c/sup\u003e and the package \u003cem\u003eigraph\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Then, these analyses determined the network parameters for every network\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, which, in turn, describe the skull anatomy. The number of nodes (N) and connections (K) represent the number of bones and their contacts of each sample, respectively. The density of connections (D) measures the number of existing connections with respect to the maximum possible, where D is interpreted as a proxy of morphological complexity. The mean clustering coefficient (C) measures the average of the sum of connections between all neighbors of each node with respect to the maximum possible, where C is interpreted as a proxy of anatomical integration. The mean shortest path length (L) measures the average of the minimum distance between all nodes, where L is interpreted as a proxy of functional efficiency. The heterogeneity of connectivity (H) is the standard deviation divided by the mean of the number of connections of all nodes in the network, where H is interpreted as a proxy of anisomerism. Modules of the anatomical networks were identified by the hierarchical clustering of the generalized topological overlap similarity matrix among nodes (GTOM). The number of modules, Q-modules, and the identified partition quality, Q\u003csub\u003emax\u003c/sub\u003e, were determined by the optimization function modularity Q\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The S-modules were estimated by performing a two-sample Wilcoxon rank-sum test on internal and external connections of every module.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMultivariate analyses of network parameters\u003c/h2\u003e \u003cp\u003eWe performed multivariate analyses of the calculated network parameters to evaluate the factors driving morphological evolution in Lacertilia. Principal component analyses for the network parameters were conducted to compare the distribution of phylogeny, morphological character, and ecology (diet, habitats, and locomotion) in the multivariate data. Ecological and morphological traits of all sampled species are shown in Supplementary data 1 file. The definitions of the ecological traits were adapted from Watanabe \u003cem\u003eet al\u003c/em\u003e. (2019)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We also performed phylogenetic principal component analyses (pPCA)\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e to account for phylogenetic effects on network parameters. The phylogenetic hypothesis of Lacertilia for pPCA were based on Pyron \u003cem\u003eet al\u003c/em\u003e. (2013)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eMesquite\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e were employed to select species and create NEXUS data for pPCA. In addition, to see if there are statistically significant differences in parameters, taxonomically or ecologically, the Mann-Whitney U test and flexible discriminant analysis (FDA) were conducted.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Dr. Hiroshi Nishi (Fukui Prefectural University), Dr. Masateru Shibata (Fukui Prefectural University), Dr. Takuya Imai (Fukui Prefectural University), and Dr. Soki Hattori (Fukui Prefectural University) for their helpful advice. Dr. Takanobu Tsuihiji (National Museum of Nature and Science,Tokyo) and Mis. Chisako Sakata (National Museum of Nature and Science, Tokyo) are thanked for specimens collection assistance. Mizuho Sano (Nagoya University) is thanked for specimen collection assistance and methodological advice. We thank the staff of the Industrial Technology Center of Fukui Prefecture for access to the CT scanner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.A. and S.K. designed the project and arranged the materials. Y.A. performed analyzed the data. Y.A. wrote the manuscript with assistance of S.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKlingenberg, C. P. Morphological integration and developmental modularity. \u003cem\u003eAnnu. Rev. Ecol. Evol. Syst\u003c/em\u003e. \u003cstrong\u003e39\u003c/strong\u003e, 115\u0026ndash;132 (2008).\u003c/li\u003e\n \u003cli\u003eNoden, D. M., \u0026amp; Trainor, P. A. Relations and interactions between cranial mesoderm and neural crest populations. \u003cem\u003eJ. Anat\u003c/em\u003e. \u003cstrong\u003e207\u003c/strong\u003e, 575\u0026ndash;601, \u003ca href=\"https://doi.org/10.1111/j.1469-7580.2005.00473.x\"\u003ehttps://doi.org/10.1111/j.1469-7580.2005.00473.x\u003c/a\u003e (2005).\u003c/li\u003e\n \u003cli\u003eRichman, J. M., Buchtov\u0026aacute;, M., \u0026amp; Boughner, J. C. Comparative ontogeny and phylogeny of the upper jaw skeleton in amniotes. \u003cem\u003eDev. Dyn\u003c/em\u003e. \u003cstrong\u003e235\u003c/strong\u003e, 1230\u0026ndash;1243 (2006).\u003c/li\u003e\n \u003cli\u003eTrumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M., \u0026amp; Martin, G. R. 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Bony skull development in the Argus monitor (Squamata, Varanidae, \u003cem\u003eVaranus panoptes\u003c/em\u003e) with comments on developmental timing and adult anatomy. \u003cem\u003eZoology\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, 255\u0026ndash;280 (2015).\u003c/li\u003e\n \u003cli\u003eOllonen, J., Da Silva, F. O., Mahlow, K., \u0026amp; Di-Po\u0026iuml;, N. Skull development, ossification pattern, and adult shape in the emerging lizard model organism \u003cem\u003ePogona vitticeps\u003c/em\u003e: a comparative analysis with other squamates. \u003cem\u003eFront. physiol\u003c/em\u003e. \u003cstrong\u003e9\u003c/strong\u003e, 278 (2018).\u003c/li\u003e\n \u003cli\u003eJerez, A., S\u0026aacute;nchez-Mart\u0026iacute;nez, P. M., \u0026amp; Guerra-Fuentes, R. A. Embryonic skull development in the neotropical viviparous skink \u003cem\u003eMabuya\u003c/em\u003e (Squamata: Scincidae). \u003cem\u003eActa zool. mex\u003c/em\u003e. \u003cstrong\u003e31\u003c/strong\u003e, 391\u0026ndash;402 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1390987/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1390987/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe morphology of lizard skulls is highly diverse, and it is crucial to understand the factors that constrain and promote their evolution to understand how lizards thrive. The results of interactions between cranial bones reflecting these factors can be detected as integration and modularity, and the analysis of integration and modularity allows us to explore the underlying factors. In this study, the integration and modularity of the skulls of lizards and the outgroup tuatara are analyzed using a new method, Anatomical Network Analysis (AnNA), and the factors causing lizards morphological diversity are investigated by comparing them. The comparison of modular structures shows that lizard skulls have high integration and anisomerism, some differences but basically common modular patterns. In contrast, the tuatara shows a different modular pattern from lizards, reflecting underlying developmental factors. In addition, the presence of the postorbital bar by jugal and postorbital (postorbitofrontal) also reflect various functional factors by maintaining low integration. The maintenance of basic structures due to basic functional requirements and changes in integration within the modules play a significant role in increasing the morphological diversity of the lizard skull and in the prosperity of the lizards.\u003c/p\u003e","manuscriptTitle":"Anatomical Network Analyses Reveal Evolutionary Integration and Modularity in the Lizards Skull","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-11 15:55:47","doi":"10.21203/rs.3.rs-1390987/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-06-13T07:15:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-04T13:42:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a5ce2b00-dc50-473b-92aa-118f6f672f30","date":"2022-05-25T09:46:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-04-17T10:09:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"593bf6af-82db-4f27-82fc-90d7e2dc1a4b","date":"2022-03-15T13:47:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-03-09T20:05:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-03-09T19:59:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-03-09T10:18:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-03-09T10:13:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-02-24T05:05:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b4af9423-2bf2-4ace-ae18-53c63616d7de","owner":[],"postedDate":"March 11th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-08-08T06:59:33+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-11 15:55:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1390987","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1390987","identity":"rs-1390987","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}