Ph.D. Thesis NEW WORLD DIRECT-DEVELOPING FROGS: PHYLOGENETIC RELATIONSHIPS AND BIOGEOGRAPHIC HISTORY By: Lucas S. Barrientos C. Director: Andrew J Crawford, Ph.D. Committee: Carlos Daniel Cadena-Ordoñez, Alejandro Reyes, Jeffrey W. Streicher Referees: Juan Manuel Guayasamín, Ph.D. Juan Armando Sánchez, Ph. D E- mail: [email protected]; [email protected] Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia 1 GENERAL INTRODUCTION The overarching goal of this dissertation to show some patterns and processes involved in the diversification of the New World direct-developing frogs. Extant biodiversity is the result of the interplay between the historical processes of diversification, dispersal (or range shifts), and extinction, understanding mechanisms that drive these processes is essential in evolutionary biology. The lineage-specific phylogenetic baggage of species impinges particularities or trends that may ultimately affect their survival, extinction, and diversification. Moreover, the most important mechanisms generating and maintaining species diversity vary depending on the taxonomic, spatial and temporal scale over which they are quantified (Graham and Fine, 2008). The spatial mechanism could be understood at regional scales, the variation in the timing and rate of lineage diversification, and ecological factors, including the current and past expanse of suitable habitat (Bennett and O’Grady, 2013; Dugo-Cota et al., 2015; Graham et al., 2006; Kozak and Wiens, 2007; Mejía, 2004; Wiens and Donoghue, 2004). Whereas at local scales, biotic interactions and trait evolution in community assembly appear to be the most influential (Hortal et al., 2012; Moen et al., 2009; Pinto-Sánchez et al., 2014). Another way to assess the mechanism underlying the diversification process is by recognizing their characteristics, both intrinsic, e.g., body size, morphological adaptations, or genomic features, and extrinsic, e.g., microhabitat, environmental variation, or range size, both factors play a role in the survival or extinction of the lineage members and are required to understand extant diversity, the diversification process and its current distribution (Bromham et al., 2015; Coyne and Orr, 2004; Gonzalez-Voyer et al., 2011; Morlon, 2014). Our aim is to explore the systematics, biogeography, and phlylogeography at different taxonomic levels of one of the most diverse groups of tetrapods: The New World direct-developing frogs. 2 The New World direct-developing frogs form a single lineage that represents 15% of the global anuran diversity, with more than 1000 described species. These frogs occur in almost every habitat in the Neotropics, including dry and rain forest, savannahs, and high elevation habitats such as páramo and puna (Gonzalez-Voyer et al., 2011; Hedges et al., 2008; Heinicke et al., 2009; Padial et al., 2014). The high diversity and the wide distribution (Figure 1) of this group of frogs could have been driven by the emergence of an evolutionary novelty: direct development, which facilitated their ability to use terrestrial environments that have sufficient moisture for the survival of eggs, hatchlings, and adults in contrast to other frogs that are limited by the availability of water bodies for reproduction and in some instances for the survival of the adults (Duellman and Trueb, 1994). For this reason, direct development releases frogs from the dependency of developing individuals in water bodies such as streams and ponds, and increases the number of habitats and complex landscapes that direct-developing frogs could use (Gonzalez- Voyer et al., 2011; Heinicke et al., 2007; Lynch and Duellman, 1997; Padial et al., 2014) and this could be reflected in the diversification of this group of frogs. 3 Figure 1 – Map of the natural distribution of the 1102 described species of New World direct- developing frogs. the map has been modified from Hedges et al., (2008b). Any evolutionary research questions on New World direct-developing frogs, particularly those that are comparative in scope, face a major problem: the taxonomic and phylogenetic relationships of this group are still incomplete and show conflictive patterns at deep (family- level) and shallow (species-level) scales. Previous taxonomic and phylogenetic research on members of this group were based mostly on morphology, but the almost overwhelming diversity of the group and the absence of clear homologous characters put limits to the power of such analyses. Subsequently, with the development of molecular phylogenetics, studies using 4 mitochondrial and nuclear loci have shed light on the phylogenetic relationships among the major groups of New World direct-developing frogs ( Crawford & Smith, 2005; Frost et al., 2006; Hedges and Heinicke, 2007; Hedges et al., 2008; Heinicke et al., 2009; Padial et al., 2014; Pyron and Wiens, 2011), nonetheless, patterns are still unclear. For instance, the most recent studies (Feng et al., 2017; Heinicke et al., 2018; Pyron, 2014; Streicher et al., 2018) show different positions and compositions of the families and subfamilies of the group based on nuclear and mitochondrial loci. To solve some of these problems we use novel high-throughput DNA sequence data of thousands of loci to estimate phylogenetic relationships. We use this robust phylogenetic framework to re-evaluate the effect of the geology and geography promoted the extant diversity of the New World direct-developing frogs at different levels (at a wide regional level with a biogeography analysis of the family Eleutherodactylidae, and a narrow spatial level with a phylogeographic analysis of the genus Diasporus). Finally, we used the byproduct of the library construction to rescue the mitochondrial genome and compare the changes in genome order of different lineages of some frogs. and is divided into four chapters. the first chapter “Untangling relationships among terraranan frogs: a phylogenomic approach based on 2,665 loci” is focused on solving the phylogenetic relationships of New World direct-developing frogs in deep scale. These phylogenetic relationships will be inferred using new molecular data gathered using massively parallel sequencing technologies and computational analyses that can handle large amounts of data. Specifically, we built genomic libraries enriching ultraconserved elements (UCEs) and a probe set to sequence 1000 loci per sample using sequence-capture and the Illumina platform. This strategy has allowed us to enhance the informative power for phylogenetic inference by gathering data on hundreds of loci at the same time, which would have been prohibited using Sanger sequencing. 5 To solve the deep-level phylogenetic relationships among major groups we applied this procedure to 16 New World direct-developing frogs that represent all the subfamilies and families of the group sensu Heinicke et al., (2018) and five outgroups. The phylogenetic analysis was performed with two strategies concatenate maximum likelihood (ML), and coalescent based analysis under neighbor joining species trees (NJst) and ASTRAL 2. We also evaluate the possible effect of the missing data on the support of the recovered phylogenetic relationships. The second chapter “Integrating ultraconserved elements and mitochondrial DNA sequence data to infer the biogeography of rain frogs (Eleutherodactylidae) across Middle America, South America, and the Caribbean”, focuses on the biogeography of one of the families of direct- developing frogs, the family Eleutherodactylidae. We evaluate the best biogeographic model to explain the present distribution and diversification of the lineages of Eleutherodactylidae and also with and without the founder event parameter +J. For this chapter, we will use the combination of massive sequencing techniques (UCEs) in combination of mitochondrial DNA. The sampling will be composed of 13 frogs of the family Eleutherodactylus, that represents all the genera and almost all of the subgenus (except one Schwartzius) of the family Eleutherodactylidae for the massive sequencing technic. And mitochondrial gene data for 190 samples of all of the genus and subgenus of the family. The included mtDNA genes are the Non-coding mtDNA genes 12S and 16S genes and the protein- coding mtDNA genes including cytochrome b (cytb), and cytochrome c oxidase subunit I (COI). For the biogeographic analysis, we use the package BioGeoBears to select the best model to explain the biogeographic patters of the family The third chapter “Phylogeography of dink frogs (Eleutherodactylidae: Diasporus): Phylogenetics, cryptic diversity, and correlates” is devoted to the phylogeography of dink frogs 6 (Eleutherodactylidae: Diasporus). The species of Diasporus share similar life history strategies, direct development, are semi-arboreal and arboreal and live in rain forests. Hertz et al., (2012) proposed that Diasporus is a genus with a considerable amount of cryptic diversity. We estimate the cryptic diversity and environmental variables as potential drivers of speciation in the dink frogs. For this chapter, we use a combination of massive sequencing techniques (UCEs) in combination of and mitochondrial gene sequences (16S, COI and CytB) to estimate the phylogenetic relationships among Diasporus, and the amount of potential cryptic diversity. To test the environmental effect on the different lineages of the genus, we model their potential distributions with entropy niche models (ENM) and compare the niche divergence between 4 pair of sister species.