Molecular analysis and Phylogeography of Neotropical Amphibians Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Marcelo Coelho Miguel Gehara aus Juiz de Fora - MG, Brasilien 1. Referentin oder Referent: Professor Dr. Miguel Vences 2. Referentin oder Referent: Professor Dr. Michael Veith eingereicht am: 01.10.2012 mündliche Prüfung (Disputation) am: 25.01.2013 Drukjahr 2013 2 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Keine Tagungsbeiträge Canedo, C; GEHARA, M ; Vences, M; HADDAD, CFB Molecular and acoustic analyses of Ischnocnema guentheri species complex (Anura: Brachycephalidae). In: IX Congresso Latinoamericano de Herpetologia, 2011 . Resumos do IX Congresso Latinoamericano de Herpetologia, 2011, Curitiba, Brazil (oral presentation) GEHARA, M ; Canedo, C; Haddad, C; Vences, M Molecular analysis of Ischnocnema guentheri highlights a complex of cryptic species. In: XI Congreso Luso-Espanol / XV Congreso Espanol de Herpetología. 2010 . Sevilla, Spain. (oral presentation) 3 When the mind is thinking it is talking to itself Plato 4 Table of contents I. Acknowledgments ................................................................................................................................ 8 II. Abstract ............................................................................................................................................. 10 III. Zusammenfassung ........................................................................................................................... 11 IV. Preface ............................................................................................................................................. 12 V. From widespread to microendemic: molecular and acoustic analyses show that Ischnocnema guentheri (Anura: Brachycephalidae) is endemic to Rio de Janeiro, Brazil ......................................... 22 Abstract ............................................................................................................................................. 22 V.I. Introduction ................................................................................................................................ 24 V.II. Methods .................................................................................................................................... 26 V.II.a. Laboratory Methods ........................................................................................................... 26 V.II.b. Molecular analyses............................................................................................................. 26 V.II.c. Bioacoustic analysis ........................................................................................................... 27 V.III. Results ..................................................................................................................................... 28 V.III.a. Molecular differentiation .................................................................................................. 28 V.III.b. Bioacoustics ...................................................................................................................... 29 V.IV. Discussion ............................................................................................................................... 32 V.IV.a. Species assessment and overall phylogeographycal pattern ............................................. 32 V.IV.b. Current state of the Ischnocnema guentheri complex ...................................................... 33 V.V. Acknowledgments .................................................................................................................... 35 VI. Do continentally widespread species of frogs exist in the Neotropics? Molecular analyses indicate that Dendropsophus minutus is a lineage-rich and biogeographically complex taxon. ........................ 41 VI.I. Introduction ............................................................................................................................... 42 VI.II. Methods ................................................................................................................................... 44 VI.II.a. Data collection .................................................................................................................. 44 VI.II.b. Gene tree inference and genetic distance calculation ....................................................... 44 VI.II.c. Phylogeographic Analysis ................................................................................................ 45 VI.II.d. Substitution rate estimation .............................................................................................. 46 VI.II.e. Demographic analysis ....................................................................................................... 47 VI.III. Results .................................................................................................................................... 48 VI.IV. Discussion .............................................................................................................................. 55 VI.IV.a. Taxonomic implications .................................................................................................. 55 VI.IV.b. Biogeography .................................................................................................................. 56 5 VI.IV.c. Phylogeography of Dendropsophus minutus complex .................................................... 58 VI.V. Conclusions ............................................................................................................................. 60 VII. Finding stable areas with genes: comparative phylogeography of Brazilian Atlantic Forest species. .................................................................................................................................................. 61 VII.I. Introduction ............................................................................................................................. 62 VII.II. Methods .................................................................................................................................. 66 VII.II.a. Data collection ................................................................................................................. 66 VII.II.b. Phylogeographic analysis ................................................................................................ 67 VII.III. Results ................................................................................................................................... 68 VII.IV. Discussion ............................................................................................................................. 73 VII.IV.a. The CNAF diversification center ................................................................................... 73 VII.IV.b. The SEAF diversification center.................................................................................... 73 VII.IV.c. Time of diversification ................................................................................................... 75 VIII. Outlook ......................................................................................................................................... 77 IX. References ....................................................................................................................................... 79 Curriculum Vitae ....................................................................................................................................87 List of Figures Figure 1 Picture of Ischnocnema guentheri ........................................................................................... 13 Figure 2: Distribution range of Ischnocnema guenthei ......................................................................... 15 Figure 3: Picture of Dendropsophus minutus ........................................................................................ 17 Figure 4: Distribution range of Dendrpsophus minutus ........................................................................ 18 Figure 5: Pictures of species analyzed in the third chapter ................................................................... 19 Figure 6 : 50%-majority rule consensus tree derived from Bayesian inference ………………………30 Figure 7: Map showing the distribution of the Ischnocnema guentheri complex in the Atlantic forest of Brazil, and nuclear RAG1 haplotype network. ……………………………………………………….31 Figure 8: 16S Minimum Evolution tree of Dendropsophus.minutus haplotypes with distribution map of each lineage. ...................................................................................................................................... 49 Figure 9: 16S Minimum Evolution tree and distribution maps of mtDNA lineages showing localities where lineages occur in simpatry.. ........................................................................................................ 50 Figure 10 50%-majority rule consensus tree derived from Bayesian inference analysis of 216 16S sequences