RESSALVA Atendendo solicitação do(a) autor(a), o texto completo desta dissertação será disponibilizado somente a partir de 30/08/2025. UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU Programa de Pós-Graduação em Ciências Biológicas (Zoologia) MIOLOGIA COMPARADA DE REPRESENTANTES DO GÊNERO AMPHISBAENA LINNAEUS, 1758 (SQUAMATA, AMPHISBAENIDAE) INGRID LIMA E LIMA Botucatu – SP 2024 INGRID LIMA E LIMA MIOLOGIA COMPARADA DE REPRESENTANTES DO GÊNERO AMPHISBAENA LINNAEUS, 1758 (SQUAMATA, AMPHISBAENIDAE) Dissertação apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Mestre no Programa de Pós- graduação em Zoologia. Orientador: Prof. Dr. Ivan Sergio Nunes da Silva Filho Co-Orientadora: Profa. Dra. Angele dos Reis Martins Botucatu – SP 2024 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSANGELA APARECIDA LOBO-CRB 8/7500 Lima e Lima, Ingrid. Miologia comparada de representantes do gênero Amphisbaena Linnaeus, 1758 (Squamata, Amphisbaenidae) / Ingrid Lima e Lima. - Botucatu, 2024 Dissertação (mestrado) - Universidade Estadual Paulista (UNESP), Instituto de Biociências, Botucatu Orientador: Ivan Sergio Nunes Silva Filho Coorientador: Angele dos Reis Martins Capes: 20604009 1. Anatomia comparada. 2. Cobras. 3. Glândulas. 4. Músculos. Palavras-chave: Anatomia; Anfisbênias; Caracteres miológicos; Descrição muscular; Glândulas cefálicas. CERTIFICADO DE APROVAÇÃO Câmpus de Botucatu UNIVERSIDADE ESTADUAL PAULISTA Miologia comparada de representantes do gênero Amphisbaena Linnaeus, 1758 (Squamata, Amphisbaenidae) TÍTULO DA DISSERTAÇÃO: AUTORA: INGRID LIMA E LIMA ORIENTADOR: IVAN SERGIO NUNES SILVA FILHO Aprovada como parte das exigências para obtenção do Título de Mestra em Ciências Biológicas (Zoologia), pela Comissão Examinadora: Prof. Dr. IVAN SERGIO NUNES SILVA FILHO (Participaçao Presencial) Departamento de Ciencias Biologicas e Ambientais / Instituto de Biociencias Campus do Litoral Paulista UNESP Prof. Dr. DANIEL FERNANDES DA SILVA (Participaçao Virtual) Setor de Herpetologia / Universidade Federal do Rio de Janeiro Prof.ª Dr.ª TÂNIA MARCIA COSTA (Participaçao Virtual) Departamento de Ciencias Biologicas e Ambientais / Instituto de Biociencias – Campus do Litoral Paulista – UNESP Botucatu, 30 de agosto de 2024 Instituto de Biociências - Câmpus de Botucatu - Rua Doutor Antonio Celso Wagner Zanin, s/nº, 18618689 https://www.ibb.unesp.br/#!/ensino/pos-graduacao/programas-stricto-sensu/ciencias-biologicas-zoologia/CNPJ: 48031918002259. Maria Victória Ramalho da Cunha Assistente Administrativo II da Seção Técnica de Pós-graduação do Instituto de Biociências 3 AGRADECIMENTOS Gostaria de agradecer primeiramente aos meus pais, que sempre me inspiraram e me apoiaram a buscar e a realizar meus maiores sonhos, por mais loucos que fossem. Em especial minha mãe, que topa qualquer aventura, e sempre me socorreu nos piores momentos. Obrigada por estarem sempre presente quando precisei, sem vocês nada disso seria possível, e eu jamais teria chegado até aqui. E ao meu irmão, que me tira sempre do sério, mas é a pessoa mais companheira que eu poderia ter na vida. Vocês são luz! Agradeço à espiritualidade, pelos momentos de reflexão que me ajudam a buscar minhas melhores versões, e me guiam em direção ao autoconhecimento. Não poderia deixar de agradecer, também, à toda minha família que está no Pará, e mesmo em longas distâncias, estiveram comigo comemorando cada vitória e cada passo dado em minha vida, desde que saí tão pequena da cidade. Esse apoio sempre me deu forças para continuar e com vocês eu aprendi o verdadeiro significado de ter sempre um lar para retornar. Ao meu orientador Ivan Nunes, que me recebeu de volta no LHERP após a graduação e me deu a oportunidade de realizar este trabalho. À minha coorientadora Angele Martins, que abriu as portas do seu laboratório na UnB para que eu pudesse investigar a morfologia dessas quase “minhocas”, e se dispôs a compartilhar informações e tirar dúvidas a respeito do complexo mundo dos músculos desses minis queridos animais. Agradeço aos meus amigos do laboratório, Juan Aquino (Cabron), Bel, Vitany, Camila, Manu e Lívia (Dis), que compartilharam tanto os melhores momentos, quanto momentos não tão bons assim. Muito obrigada pelas horas do café, pelas risadas e descontração, pelas reflexões, ideias compartilhadas, momentos de desabafo, saídas de campo, enfim, por estarem ali dia após dia. Nos apoiamos uns nos outros até aqui e podem continuar contando 4 comigo para qualquer coisa. E aos parceiros Gabriel, Enzo, e Ed, pelas conversas do dia a dia, à técnica de laboratório da UNESP Cláudia, que me ensinou diversas metodologias de extração de DNA, PCR entre outras técnicas moleculares, certamente me será muito útil durante minha jornada acadêmica. À minha namorada e companheira para todos os momentos, que compartilha a vida comigo e tem tanta urgência de viver quanto eu. Muito obrigada por acreditar em mim, pela compreensão, pela paciência quando eu quero falar sobre coisas da biologia, que mesmo sem entender nada, me escuta com todo amor do mundo. Amo você! Agradecimento especial aos meus quatro bebês de quatro patas, que são um momento de conforto em meio ao caos. Sem eles não seria possível aguentar tantos momentos de estresse e ansiedade, acariciar um animal de fato acalma a mente! Agradeço a Caroline e a Carla que lá atrás coletaram essa espécie que hoje estou tendo o privilégio de estudar. A todos os curadores de museus que emprestaram e me permitiram analisar os espécimes de suas coleções. Ao CNPq pela bolsa concedida que me permitiu dedicação integral a este projeto. E a todos que de alguma forma contribuíram para que hoje eu pudesse estar aqui realizando mais uma etapa rumo ao futuro! 5 “All that is gold does not glitter, Not all those who wander are lost; The old that is strong does not wither, Deep roots are not reached by the frost. From the ashes a fire shall be woken, A light from the shadows shall spring; Renewed shall be blade that was broken, The crownless again shall be king.” ― J.R.R. Tolkien 6 RESUMO Dentre os Squamata, a subordem Amphisbaenia possui indivíduos que apresentam diversas adaptações para o modo de vida fossorial, atualmente sendo representada por 202 espécies divididos em seis famílias. Apesar do recente aumento no interesse e progresso acerca do grupo, ainda nos deparamos com a falta de diversos tipos de dados. Sendo assim, devido ao número limitado de descrições miológicas conduzidas neste grupo taxonômico, o presente estudo visa fornecer uma descrição da anatomia muscular cefálica de quatro espécies de Amphisbaena – Amphisbaena dubia, Amphisbaena darwinii, Amphisbaena munoai e Amphisbaena microcephala. Além disso, incorporamos dados de espécies com descrições musculares previamente documentadas na literatura para fornecer uma comparação mais abrangente. Foram descritas variações na musculatura associada ao complexo mandibular e alterações na estrutura dos tendões que sustentam esses músculos. Além disso, foram identificadas variações na forma, origem e inserções do m. adductor mandibulae externus superficialis, medialis e profundus, na disposição das fibras do m. retractor anguli oris e do m. depressor mandibulae. Ademais, novas percepções sobre o sistema glandular das espécies, com a descrição de uma glândula rictal ainda não registrada para este grupo, juntamente com a criação de 24 de caracteres baseados nessas variações, foram fornecidas. Tais resultados traduzidos em caracteres podem ser utilizados para refinar e aprimorar futuras investigações filogenéticas, esclarecendo a evolução do grupo e suas adaptações morfológicas. Palavras-chave: anfisbênias, anatomia, caracteres miológicos, descrição muscular, glândulas cefálicas 7 ABSTRACT Among Squamata, the suborder Amphisbaenia includes individuals that exhibit various adaptations for fossorial lifestyle, currently represented by 202 species divided into six families. Despite the recent surge in interest and progress concerning the morphology of the group, most studies are mainly focused on their skull and hemipenial shape/morphology, with little data on other morphofunctional complexes such as postcranial data, muscles, cephalic glands and viscera. Herein we aim to describe in detail the cephalic myology of four Amphisbaena species – Amphisbaena dubia, Amphisbaena darwinii, Amphisbaena munoai and Amphisbaena microcephala. Furthermore, we incorporate data from species with previously documented muscular descriptions available in the literature to provide a broader comparative framework. Variations in the musculature associated with the mandibular complex and alterations in the structure of tendons supporting these muscles were described. Additionally, variations in shape, origin and insertions of the m. adductor mandibulae externus superficialis, medialis and profundus, iber arrangement of m. retractor anguli oris and m. depressor mandibulae were identified. Moreover, novel insights into the glandular system of the species, with the observation of a rictal gland not yet recorded for this group, along with the creation of a set of characters based on these variations, were provided. The obtained results may supply valuable insights and interesting character information to be used in future phylogenetic investigations of the group. Keywords: amphisbaenians, anatomy, cephalic glands, muscular descriptions, myological characters 8 SUMÁRIO MANUSCRIPT. Comparative myology of representatives of the genus Amphisbaena Linnaeus, 1758………………………………………………………………….………….9 1 INTRODUCTION……………………………………………………………………...10 2 MATERIAL AND METHODS………………………………………………………...14 2.1 Methods of dissection and muscle descriptions.............................................................14 2.2 Construction of the proposed muscular characters........................................................15 3 RESULTS……………………………………………………………………………….16 3.1 Description of the cephalic musculature of round-headed species (A. dubia, A. darwinii e A. munoai)……………………………………………………………………………….16 3.2 Description and variations in cephalic musculature of shovel-headed specie (A. microcephala)……………………………………………………………………………...22 3.3 Cephalic glands………………………………………………………………………..25 3.4. Proposed myological characters………………………………………………………27 4 DICUSSION…………………………………………………………………………….29 5 CONCLUSIONS………………………………………………………………………..33 ACKNOWLEDGMENTS………………………………………………………………..35 REFERENCES…………………………………………………………………………...35 FIGURE LEGENDS……………………………………………………………………..43 FIGURES…………………………………………………………………………………45 APPENDIX S1. List of studied specimens……………………………………………...67 APPENDIX S2. List of abbreviations…………………………………………………...67 This manuscript is formatted according to the requirements of the journal “The Anatomical Records”. For further details see: https://anatomypubs.onlinelibrary.wiley.com/hub/journal/19328494/homepage/forauthors.html. Comparative myology of four species of Amphisbaena (Linnaeus, 1758) (Squamata, Amphisbaenidae) with descriptions on the cephalic glands Short running title: Comparative myology of four Amphisbaena Linnaeus, 1758 Ingrid Lima e Lima¹, ², Ivan Nunes¹, Angele Martins3,4 1Universidade Estadual Paulista, Campus do Litoral Paulista, Instituto de Biociências, Laboratório de Herpetologia 11.330-900, São Vicente, São Paulo, Brazil ²Universidade Estadual Paulista, Campus de Botucatu, Programa de Pós-Graduação em Zoologia, 18618-689, Botucatu, São Paulo Brazil 3 Departamento de Vertebrados, Museu Nacional, Universidade Federal do Rio de Janeiro, Quinta da Boa Vista, 20940-040, Rio de Janeiro, Brazil. 4 Laboratório de Anatomia Comparada de Vertebrados, Departamento de Ciências Fisiológicas, Instituto de Ciência Biológicas, Universidade de Brasília, Asa Norte, Brasília, Distrito Federal, Brazil. *Corresponding author. E-mail: ingridlima.hpt@gmail.com mailto:ingridlima.hpt@gmail.com 1. INTRODUCTION The order Squamata encompasses over 12,000 extant species of lizards, snakes, and amphisbaenians (Uetz et al., 2024), and represent the most diverse clade of terrestrial vertebrates worldwide (Simões & Pyron, 2021), occurring in nearly all habitats (Vitt & Caldwell, 2013). Amphisbaenia is a suborder of Squamata, comprised by fossorial animals commonly known as worm-lizards, currently represented by 202 species divided into six families (Uetz et al., 2024). The geographic range of the group includes North America in eastern USA (Rhineuridae); Central America (Cadeidae and Bipedidae); Europe and North Africa (Blanidae); North Africa and Asia (Trogonophidae; Gans, 2005). The family Amphisbaenidae is the most speciose and has a wide distribution in South America, Central America, and Africa (Costa & Garcia, 2019), even though many species exhibit endemic or restricted distributions and remain poorly understood (Gans, 2005; Costa, 2020). These fossorial Squamata display significant adaptations for subterranean life, including the construction of their own tunnel systems by compressing the soil within the galleries, with horizontal or vertical movements of the head (Gans, 1968). This head-first burrowing behavior is a primary tool for the fossorial lifestyle of Amphisbaenia (Navas et al., 2004), being supported by a suite of morphological features, including cranial adaptations (head shape: rounded, shovel-shaped, spade-shaped, and keel-shaped; Kearney 2003), tegumentary characteristics, and muscular arrangements (Gans, 1986). Round- headed species use a generalized mechanism, while shovel-headed, spade-headed, or keel- headed species employ specialized mechanisms (Gans, 1969; Hohl et al., 2014), this strongly ossified skull enhances resistance to excavation impacts (Gans & Montero, 2008), requiring enduring muscle contractions, especially in dorsal, pectoral and cephalic muscles (Gans, 1968; Navas et al., 2004). Additionally, the tegumentary musculature exhibits a 11 unique configuration, wherein muscles are loosely interconnected with the skin, thereby reducing frictional resistance against the tunnel walls during locomotion (Gans, 1962). Multiple aspects of amphisbaenian biology remain underexplored, with many species having been described only recently, with their natural history receiving limited attention (Colli et al., 2016). This phenomenon is partly attributed to the fossorial behavior of these animals, which can restrict field observations and complicate specimen collection for laboratory research (Navega-Gonçalves & Benites, 2019; Paiva et al., 2024). Consequently, amphisbaenians remain as the group of Squamata less studied and poorly represented in biological collections (Gans, 1967; Kearney, 2003; Colli et al., 2016), which delays the execution of descriptive or comparative studies (Gans, 2005; Pinna et al., 2014; Roberto et al., 2014). This creates a gap in the knowledge of their morphological, anatomical, functional, and phylogenetic characteristics. Which is concerning, as morphological data, especially those related to myology, have proven effective in studies of functional biology and phylogenetic analyses among vertebrates (O’Reilly et al., 2000; Diaz et al., 2022; Lowie et al., 2023). Addressing this gap can provide more detailed answers regarding the musculature involved in burrowing, the influence of mandibular musculature on food acquisition (Cundall, 1983), and the similarity between amphisbaenians and other highly fossorial squamate species (Daza et al., 2011), among other evolutionary questions concerning this group. Some studies in recent decades have focused on the myology analysis of reptiles in the order Squamata (Abdala & Moro, 2006; Bhullar, 2009; Martins et al., 2019; Westphal et al., 2019). However, both older and contemporary studies primarily concentrate on snakes and lizards (e.g., Jayne, 1982; Cundall, 1986; De Souza Leite et al., 2021). One extensively investigated factor, for example, is the limb loss in groups exhibiting this characteristic (Greer, 1991; Abdala et al., 2015; Camaiti et al., 2021). These investigations 12 have demonstrated that osteology and myology provide better insights into the evolutionary patterns of limb loss compared to studies solely focused on external characteristics (Camaiti et al., 2021). Although there are descriptive myology studies on amphisbaenians, they are scarce and mostly outdated (Renous, 1977; Bonin, 1965), leaving a wide-open niche for studies. For example, earlier studies described part of the jaw adductor musculature of Amphisbaena alba Linnaeus, 1758 (Lakjer, 1926), and some observations and descriptions were added for the same species, as well for Amphisbaena microcephala Wagler, 1824 and Trogonophis wiegmanni Kaup, 1830 (Haas et al., 1973). Later, Rieppel (1979) partially described the jaw adductor musculature of A. alba and Amphisbaena fuliginosa Linnaeus 1758. More recently, we have descriptions of muscles located around the neck-trunk region for A. alba (Tsuihiji et al., 2012) and descriptions of pectoral muscles through diceCT visualization for five species of Amphisbaenia (Westphal et al., 2019). This indicates that the descriptions for this group are sporadic and further investigations are needed for better correlations between the homologies of this muscular system. The cranial muscles of amphisbaenians are of special interest due to the complete loss of the upper temporal arch (jugal) and the closure of the cranial box, modifications similar to those seen in snakes (Haas et al., 1973). This cranial structure, associated with powerful temporal musculature, also facilitates food capture and crushing (Gans, 1969). Therefore, descriptive myology studies allow correlating these and other specializations with the different muscular characteristics of these species (Gans & Montero, 2008), providing important data on the functional morphology of amphisbaenians. Furthermore, we must not overlook the knowledge regarding their cephalic glands, as information about their macro and microstructure remain unknow (Foureaux et al., 2010), the conception and 13 integration of these internal characters for a phylogenetic analysis has proven valuable for other Squamata groups (Abdala et al., 2009), yet it has not been applied to amphisbaenians. Among the South American worm-lizards, we highlight four species, Amphisbaena dubia (Müller, 1924), Amphisbaena darwinii (Duméril & Bribon, 1839), Amphisbaena munoai (Klappenbach, 1960) and Amphisbaena microcephala (Wagler, 1824). A. dubia is endemic from Brazil (Evers et al., 2006), but its population density is unknown, and its phylogenetic placement remains unaddressed in extant evolutionary studies (Mott & Vieites, 2009; Gauthier et al., 2012; Pyron et al., 2013). A. munoai and A. darwinii are found under stones in rocky outcrops in the Uruguay and southern Brazil (Perez & Borges- Martins, 2019). All those three share the round-headed pattern, differing only in size (Gans & Montero, 2008). Conversely, A. microcephala is shovel-headed, and has a widespread distribution (Hohl et al., 2017), occurring in different regions of South America (Ribeiro et al., 2011). The diversity, precise identification of species and their boundaries, remain unresolved in many Amphisbaena groups (Perez & Borges-Martins, 2019), being crucial to employ diverse datasets, including myological descriptions, to advance knowledge of the group's evolutionary history. For that reason, herein, we described the cranial myology and its variations in these four species of amphisbaenians. This research involves comparing these anatomical features and discuss with respective cranial shapes, alongside a review of existing literature. Beyond that, we also described the cephalic glands observed and propose a list of myological characters. 33 5. CONCLUSIONS Our study offers a broad description of the cephalic myology and glandular information for four species of Amphisbaena, showing the variation between then and among different head shapes representatives here available. The species with rounded heads (A. darwinii, A. dubia and A. munoai) showed more similarity among themselves than the species with shovel-shaped heads (A. microcephala). Especially the species A. darwinii and A. munoai, which are phylogenetically close (Graboski et al., 2022) and exhibited a nearly identical set of muscles. We identified a novel rictal or supralabial accessory gland in the shovel-headed A. microcephala, that is absent in the other three species. This highlights the importance of investigating the glandular characteristics within other cranial types of Amphisbaenia (e.g., 34 spaded-headed and keel-headed) present in other species, in order to search for patterns associated with different ecological lifestyles and their phylogenetic relationships. While our study lays the groundwork for understanding these morphological traits, it is necessary to test the formulated characters within a broader phylogenetic framework, since new phylogenies investigating various sources of characters, such as osteology and myology, which are rare in in Amphisbaenia to date (Navas et al., 2004; Rieppel, 1979; Westphal et al., 2019), should be conducted. This integrative approach will be crucial for advancing our understanding of the evolution of this group. 35 ACKNOWLEDGMENTS We are grateful to Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPQ) for support and to the curators Carlos Lucena (MCT-PUCRS) and Thiago Silva Soares (INMA), who provided the loan of specimens under their care. REFERENCES Abdala, V., & Moro, S. (2006). Comparative myology of the forelimb of Liolaemus sand lizards (Liolaemidae). Acta Zoologica, 87(1), 1-12. Abdala, V., Grizante, M. B., Diogo, R., Molnar, J., & Kohlsdorf, T. (2015). 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