Tracing the Evolutionary History of Two Endemic Ground Frogs of Temperate Forest of Southern Chile, Through Molecular and Cytogenetic Approaches

TRACING THE EVOLUTIONARY HISTORY OF TWO ENDEMIC GROUND FROGS OF TEMPERATE FOREST OF SOUTHERN CHILE, THROUGH MOLECULAR AND CYTOGENETIC APPROACHES TESIS DE MAGISTER CAMILA ANDREA QUERCIA RATY VALDIVIA – CHILE TRACING THE EVOLUTIONARY HISTORY OF TWO ENDEMIC GROUND FROGS OF TEMPERATE FOREST OF SOUTHERN CHILE, TROUGHT MOLECULAR AND CYTOGENETIC APPROACHES Tesis presentada a la Facultad de Ciencias de la Universidad Austral de Chile en cumplimiento parcial de los requisitos para optar al grado de Magíster en Ciencias mención Genética por CAMILA A. QUERCIA RATY Valdivia Chile 2019 Universidad Austral de Chile Facultad de Ciencias INFORME DE APROBACIÓN TESIS DE MAGISTER La Comisión Evaluadora de Tesis comunica a la Directora de la Escuela de Graduados de la Facultad de Ciencias que la Tesis de Magíster presentada por la Candidata CAMILA ANDREA QUERCIA RATY ha sido aprobada en el exámen de defensa de Tesis rendido el día ___de _____ de 20__ como requisito para optar al grado de Magíster en Ciencias menCión Genética y, para que así conste para todos los efectos firman: Profesor Patrocinante Dr. José J. Nuñez Instituto de Ciencias Marinas y Limnológicas __________________________ Profesor Copatrocinante Dr. Elkin Y. Suárez-Villota Instituto de Ciencias Marinas y Limnológicas __________________________ Comisión Evaluadora Dra. Leyla Cárdenas Instituto de Ciencias Ambientales y Evolutivas __________________________ Dr. Guillermo D´Elía Instituto de CienCias Ambientales y Evolutivas __________________________ DEDICATORIA A mis padres y todos quienes se Convirtieron en mi familia De aquellos glaciares y hojarascas de quienes tu no podrás saber. AGRADECIMIENTOS Siento especial gratitud hacia quienes fueron mis mayores guías en este proceso, Dr. José Nuñez y Dr. Elkin Suárez-Villota, Cuyos Consejos y conocimientos fueron el Complemento clave para lograr este trabajo. Más aún, por enseñarme sin reCelo parte de sus experiencias y pensamientos. Por la Confianza y exigencia en mi puesta, y por haber enriqueCido inigualablemente mi camino científico. A todos mis compañeros del Laboratorio de Sistemática UACh, quienes siempre reciben con una sonrisa, dispuestos a contribuir. En especial a la Dr. Leila Díaz por su ayuda en los procedimientos de laboratorio, y al Ingeniero en conservación Nicolás Gonzáles, por su colaboración en la toma de muestras en terreno. AgradezCo también la disposiCión y grata acojida de los investigadores del Laboratorio de Biología e Genética de Peixes (UNESP, Botucatu, Brasil), donde tuve la posibilidad de realizar una pasantía en citogenética. En especial al Dr. Fausto Floresti, Dr. Cristian Araya y Dr. Duilio Zerbinato. A todos aquellas personas que, inCluso sin saberlo, me brindaron inspiraCión y apoyo, mis más sinCeros reConoCimientos. Y por último, pero no menos importante, infinita gratitud a todos los ejemplares de anuros que fueron utilizados para este trabajo. El finanCiamiento para este trabajo fue dado por el proyeCto FONDECYT 3160328 de EYS-V., beca de magíster CONICYT 22180766 de CAQ, y programa de Magister en Ciencias mención GenétiCa, UACh. CONTENT INDEX CONTENT INDEX I-IV TABLES INDEX V-VI FIGURES INDEX VII-VIII ABBREVIATION INDEX IX RESUMEN X-XIII ABSTRACT XIV-XVII GENERAL INTRODUCTION 1-19 1. RESEARCH CONTEXT 1-17 1.1. DETERMINATION OF BIODIVERSITY ON EARTH 1-3 1.2. SPECIES DELIMITATION 3-7 1.3. PLEISTOCENE HISTORY OF SOUTHERN CHILE 7-8 1.4. FROGS OF THE GENUS Eupsophus 9-10 1.5. CYTOGENETIC WITHIN THE GENUS Eupsophus 12-14 1.6. PHYLOGENETIC RELATIONSHIP OF THE 14-16 vertebralis GROUP 2. HYPOTHESIS AND OBJETIVES 18-19 2.1. HYPOTHESIS 18 2.2. GENERAL OBJECTIVE 18 2.3. SPECIFIC OBJECTIVES 19 2.3.1. Specific objectives: CHAPTER 1 19 2.3.2. Specific objectives: CHAPTER 2 19 I CHAPTER 1: MOLECULAR PHYLOGENY, SPECIES 20-81 DELIMITATION, AND TIMING OF DIVERGENCE OF THE vertebralis GROUP (Anura: Alsodidae) IN SOUTHERN CHILE ABSTRACT 20-21 1. INTRODUCTION 21-27 2. MATERIALS AND METHODS 27-35 2.1. SAMPLES AND ETHIC STATEMENTS 27-28 2.2. GENETIC DATA 28-29 2.3. PHYLOGENETIC TREE RECONSTRUCTION 29-31 2.4. SPECIES DELIMITATION 31-34 2.5. TIME OF DIVERSIFICATION 34-35 3. RESULTS 35-39 3.1. PHYLOGENETIC PATTERNS INTO THE 35-37 vertebralis GROUP 3.2. SPECIES DELIMITATION ANALYSES 38-39 3.3. DIVERSIFICATION TIME 39 4. DISCUSSION 40-47 4.1. PHYLOGENETIC RELATIONSHIPS AND 40-43 DIVERGENCE TIME IN THE vertebralis GROUP 4.2. SPECIES DELIMITATION ANALYSES OF THE 44-47 vertebralis GROUP 4.3. FIELDWORKS REMARKS 47 II 5. CONCLUSION 48 ACKNOWLEDGMENTS 49 TABLES 50 FIGURES 51-55 SUPPLEMENTARY TABLES 56-62 SUPPLEMENTARY FIGURES 63-64 6. REFERENCES 65-81 CHAPTER 2: COMPARATIVE CYTOGENETICS OF TWO 82-114 GROUND FROGS OF GENUS Eupsophus: E. Emiliopugini AND E. vertebralis (Alsodidae); WITH COMMENTS ON INTRASPECIFIC POLYMORPHISM IN NORs ABSTRACT 82-83 1. INTRODUCTION 83-86 2. MATERIALS AND METHODS 86-90 2.1. SAMPLES COLLECTION AND CYTOLOGICAL 86-87 PREPARATIONS 2.2. CLASSICAL CYTOGENETIC TECHNIQUES 87-88 2.3. MOLECULAR CYTOGENETIC TECHNIQUES 88-90 3. RESULTS 90-91 3.1. CLASSICAL CYTOGENETIC TECHNIQUES 90-91 3.2. CYTOMOLECULAR TECHNIQUES 91 4. DISCUSSION 92-98 4.1. KARYOTYPES DESCRIPTION OF E. vertebralis 92-93 AND E. emiliopugini SPECIES III 4.2. NUCLEOLAR ORGANIZER REGIONS 93-97 POLYMORPHISM 4.3. HYPOTHESIS ABOUT THE EVOLUTION OF PAIR 97-98 13 5. CONCLUSION 98 ACKNOWLEDGMENTS 99 FIGURES 100-104 SUPPLEMENTARY TABLE 105-106 8. REFERENCES 107-114 GENERAL DISCUSSION AND CONCLUSIONS 115-120 GENERAL REFERENCES 121-139 ANNEX I: Speciation in a biodiversity hotspot: Phylogenetic relationships, speCies delimitation, and divergenCe times of Patagonian ground frogs from the Eupsophus roseus group (Alsodidae) ANNEX II: MitoChondrial genomes of the South AmeriCan Frogs Eupsophus vertebralis and E. emiliopugni (Neobatrachia: Alsodidae) and their phylogenetiC relationships ANNEX III: Resumen taxonómiCo de las espeCies del grupo vertebralis IV TABLES INDEX GENERAL INTRODUCTION Table 1 Classification of Eupsophus groups according to 11 their karyotypes and advertisement Calls. CHAPTER 1: MOLECULAR PHYLOGENY, SPECIES DELIMITATION, AND TIMING OF DIVERGENCE OF THE vertebralis GROUP (Anura: Alsodidae) IN SOUTHERN CHILE Table 1 TaxonomiC index of Congruence (Ctax) among 50 species delimitation analyses. SUPPLEMENTARY TABLES Table 1 Sampling locations of E. vertebralis and E. 56-57 emiliopugini. Table 2 Species delimitation scenarios and results obtained 58 for Tr2 and BPP. Table 3 Bayes faCtor estimates for MoleCular CloCk/Tree 59 priors and SpeCies delimitation scenarios. Table 4 Total nucleotide sites, partition sChemes and 60 nucleotide substitution models for sequences used in this study. Table 5 Nucleotide substitution rates used for sequences of 61 the vertebralis group in this study. Table 6 Likelihood scores and Akaike’s information 62 criterion (AIC) results for STEM analysis. V CHAPTER 2: COMPARATIVE CYTOGENETICS OF TWO GROUND FROGS OF GENUS Eupsophus: E. Emiliopugini AND E. vertebralis (Alsodidae); WITH COMMENTS ON INTRASPECIFIC POLYMORPHISM IN NORs. SUPPLEMENTARY TABLE Table 1 List of the specimens used for cytogenetic analyses 105-106 in this study. VI FIGURES INDEX GENERAL INTRODUCTION Figure 1 MorphologiCal similarities and distribution range of 17 Eupsophus vertebralis and E. emiliopugini. CHAPTER 1: MOLECULAR PHYLOGENY, SPECIES DELIMITATION, AND TIMING OF DIVERGENCE OF THE vertebralis GROUP (Anura: Alsodidae) IN SOUTHERN CHILE Figure 1 Maps of sampling loCations for the vertebralis group. 51 Figure 2 PhylogenetiC relationships among the vertebralis group. 52-53 Figure 3 Phylogenetic dated tree by BEAST and speCies 54-55 delimitation analyses. SUPPLEMENTARY FIGURES Figure 1 PhylogenetiC relationships among speCies of the 63 vertebralis group. CHAPTER 2: COMPARATIVE CYTOGENETICS OF TWO GROUND FROGS OF GENUS Eupsophus: E. emiliopugini AND E. vertebralis (Alsodidae); WITH COMMENTS ON INTRASPECIFIC POLYMORPHISM IN NORs. Figure 1 Map portraying 15 ColleCtion loCalities of the vertebralis 100 group specimens, in Southern Chile. Figure 2 Classical Giemsa and C- banding of the vertebralis group. 101 Figure 3 Results of Ag-NOR staining, and FISH of 28S rDNA probe 102 for the vertebralis group. VII Figure 4. TelomeriC signal obtained by FISH for the vertebralis 103 group. Figure 5 Proposed hypothesis to explain the nucleolar 105 dominance phenomena observed in one specimen from Puyehue locality of E. emiliopugini species. VIII ABBREVIATIONS INDEX 2n Diploid chromosome number Ag-NOR Silver staining for NORs BC Bibrachial chromosomes BFD Bayes factor delimitation BIC Bayesian information criterion BPP Bayesian phylogenetics and phylogeography program Ctax Congruence index DNA Deoxyribonucleic acid e.g. Exampli gratia FISH Fluorescent in situ hybridization FN Fundamental number GMYC General mixed yule coalescent model HPD Highest posterior density interval IB Bayesian inferences i.e. Id est IUCN International union for conservation of nature LGM Last glacial maximum MCMC Markov chain Monte Carlo algorithms ML Maximum likelihood MLE Marginal likelihood estimation MRCA Most recent common ancestor MSC Multispecies coalescent models Mya Million year ago NA Data not available NORs Nucleolar organized region PCR Polymerase chain reaction PP Bayesian posterior probability PTP Multirate Poisson tree processes rDNA Ribosomal genes SC Secondary constriction SexC Presence of sexual chromosomes STEM Estimation using maximum likelihood Tr2 Trinomial distribution model θ = 4Neμ Population size τ Divergence time IX RESUMEN Eupsophus es un género de ranas de hojarasca,

Load more