Ph.D. Thesis

NEW WORLD DIRECT-DEVELOPING : 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 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 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 : 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 , and a narrow spatial level with a phylogeographic analysis of the 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.

Finally, the chapter 4 – “Mitochondrial genomes of frogs captured using massive sequencing techniques”. Report the capture of 16 almost complete mitogenomes obtained from byproduct of the library construction for the target of ultraconserved elements (UCEs). The almost complete genomes ranged from approximately 12 to 16 Kb in length. We found that the mitochondrial genomes are similar to previously assembled genomes for neobatrachian frogs in terms of gene content and gene reorders. This short report wants to show the utility of using off- target sequences from sequence-capture methods, to obtain mitochondrial information.

7 Chapter 1 - Untangling relationships among terraranan frogs: a phylogenomic approach based on 2,665 loci

Lucas S. Barrientos1 *, Jeffrey W. Streicher2,3, Elizabeth C. Miller3, Marcio R. Pie4, John J.

3 1 Wiens , Andrew J. Crawford

1 Department of Biological Sciences, Universidad de los Andes, Bogotá, código postal 1117711,

Colombia.

2 Department of Life Sciences, The Natural History Museum, South Kensington, London SW7

5BD, England, UK.

3 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721-

088, USA.

4 Departamento de Zoologia, Universidade Federal do Paraná, Curitiba, Paraná, 81531-980,

Brazil.

Email addresses: [email protected] (LSB), [email protected] (JWS), [email protected] (ECM), [email protected] (MRP), [email protected] (JJW), [email protected] (AJC), * Corresponding author: Department of Biological Sciences Universidad de los Andes Carrera 1a # 18A-10 Bogotá, D.C., A.A. 4976, Colombia, South America E-mail: [email protected] Cell phone: +57 3158918734

1 ABSTRACT

Terraranae is a large clade of New World direct-developing frogs that includes 3–5 families and

>1,000 described species, encompassing ~15% of all named species. The relationships among major groups of terraranan frogs have been highly contentious, including conflicts among three recent phylogenomic studies utilizing 95 loci, 389 loci, and 2,214 nuclear loci, respectively.

In this paper, we re-evaluate relationships within Terraranae using a novel genomic dataset for 16 ingroup species representing most terraranan families and subfamilies. The preferred data matrix consisted of 2,665 nuclear loci from ultra-conserved elements (UCEs), with a total of 743,419 aligned base pairs and 57% missing data. Concatenated likelihood analyses and coalescent-based species-tree analyses both recovered strong statistical support for the following relationships among terraranan families: (Brachycephalidae, (Eleutherodactylidae, (“” +

Strabomantidae)). Our results place Strabomantis with (or within) , a result which suggests that these genera belong in a single family (Craugastoridae), since recognizing

Strabomantidae would make Craugastoridae non-monophyletic. These results conflict with two previous phylogenomic studies which placed Strabomantis closer to Pristimantis. Furthermore, our results suggest that Pristimantinae is paraphyletic with respect to Holoadeninae, and should be subsumed into Holoadeninae. Our placement of Brachycephalidae agrees with two previous phylogenomic studies but conflicts with another. We also found that using matrices of UCE loci with less missing data (and concomitantly fewer loci) generally decreased support for most nodes on the tree. Overall, our results strongly resolve controversial relationships within one of the largest clades of frogs, with a dataset ~7 times larger than previous studies focused on this clade.

Keywords: , anurans, missing data, phylogenomics, Terraranae

2 1. Introduction

Terraranae (sensu Heinicke et al. 2018) is one of the most species-rich clades of frogs. It contains ~1,000 described species, equivalent to ~15% of all known species of anurans (Frost,

2017). Terraranae is characterized by several soft anatomical characters (Taboada et al., 2013) and by direct development, which involves the evolutionary loss of the larval stage, such that 4- legged hatchlings emerge from fully terrestrial eggs (Duellman and Trueb, 1994). The clade occurs from the southern United States to southern , including the West Indies (Gonzalez-

Voyer et al., 2011; Hedges et al., 2008). Terraranan frogs are found in a variety of habitats, from deserts to rainforests and from tropical islands to high-elevation páramo and puna habitats

(Gonzalez-Voyer et al., 2011; Hedges et al., 2008). In the Neotropics, terraranans have been estimated to make up (on average) >40% of all frog species in local communities, with especially high richness in mesic habitats of Middle America, the Andes of South America, and on

Caribbean islands (Pinto-Sánchez et al., 2014).

Terraranans have had a controversial and dynamic , largely because different studies support contrasting phylogenetic hypotheses (Fig. 1). Prior to 2005, traditional taxonomy placed all terraranans in the tribe Eleutherodactylini within the family (Lynch,

1971). Based on a parsimony analysis of nuclear and mitochondrial DNA data, Frost et al. (2006) subdivided Leptodactylidae and placed all terraranans in the family Brachycephalidae. Hedges et al. (2008) proposed a new phylogeny for this group of frogs, to which they gave the unranked clade name of Terrarana (latter amended to superfamily Terraranae, see below). They divided this clade into four families: Brachycephalidae, Craugastoridae, Eleutherodactylidae, and

Strabomantidae (Fig. 1A). Subsequently, Heinicke et al. (2009) added a fifth family,

Ceuthomantidae (Fig. 1B). Pyron and Wiens (2011) performed a large-scale maximum likelihood

3 analysis of 12 concatenated nuclear and mitochondrial genes for many species, which placed

Strabomantidae within Craugastoridae (Fig. 1C). Padial et al. (2014) added two more nuclear and three more mitochondrial genes to the previous data sets, and analyzed relationships using dynamic homology (Wheeler, 2001, 1996) under a parsimony criterion. This analysis placed

Eleutherodactylidae as the sister taxon to Brachycephalidae plus Craugastoridae [including

Strabomantidae], and placed Ceuthomantidae within Craugastoridae [including Strabomantidae]

(Fig. 1D). Pinto-Sánchez et al. (2014) combined data from two previous studies (Pinto-Sánchez et al., 2012; Pyron and Wiens, 2011) to analyze relationships in terraranan frogs. They found weak support for placing Brachycephalidae as the sister taxon to Eleutherodactylidae (Fig. 1E), unlike Pyron and Wiens (2011). Pyron (2014) added published data to the matrix of Pyron and

Wiens (2011), and also found weak support for placing Eleutherodactylidae with

Brachycephalidae instead of Craugastoridae [including Strabomantidae] (Fig. 1F). Feng et al.

(2017) analyzed 95 nuclear loci to infer relationships among anurans and found support for the sister relationship between Eleutherodactylidae and Craugastoridae plus Strabomantidae (Fig.

1G), with Ceuthomantidae as sister to all other terraranans. Heinicke et al. (2018) analyzed 389 nuclear loci and placed Eleutherodactylidae as the sister taxon to a clade including

Brachycephalidae and Craugastoridae plus Strabomantidae (Fig. 1H), again with Ceuthomantidae as sister to all other terraranans. Hutter et al. (2017) performed a Bayesian analysis of 158 hyloid genera including 13 nuclear and 7 mitochondrial genes that showed strong support for the relationships (Ceuthomantidae, (Brachycephalidae, (Craugastoridae [including Strabomantidae],

Eleutherodactylidae))). Finally, Streicher et al. (2018) analyzed 2,214 nuclear loci for hyloid frogs (including five terraranan species) and also found support for Brachycephalidae as the sister taxon of Craugastoridae (including Strabomantidae) plus Eleutherodactylidae (Fig. 1J).

4 The results from these studies can be summarized as follows. There are three main competing hypotheses regarding relationships among terraranan families: (1) Eleutherodactylidae as the sister taxon to Craugastoridae + Strabomantidae plus Brachycephalidae (Hedges et al.,

2008; Heinicke et al., 2018, 2009, 2007; Padial et al., 2014); (2) Brachycephalidae as the sister taxon to Craugastoridae + Strabomantidae + Eleutherodactylidae (Feng et al., 2017; Hutter et al.,

2017; Pyron and Wiens, 2011; Streicher et al., 2018); and (3) Craugastoridae + Strabomantidae as sister to Brachycephalidae + Eleutherodactylidae (Pinto-Sánchez et al., 2014; Streicher et al.,

2018). Another difference among studies is that the genus Ceuthomantis was assigned to its own family by Heinicke et al. (2009; see also Feng et al., 2017; Pyron, 2014; Pyron and Wiens, 2011), but Padial et al. (2014) placed Ceuthomantis inside Craugastoridae. With the exception of Feng et al.(2017), Heinicke et al. (2018), and Streicher et al. (2018), all previous molecular phylogenetic studies were based on fewer than 10 mitochondrial and 14 nuclear loci. Furthermore, most data were shared among studies. These studies differed somewhat in their sampling of genes and taxa

(Table 1), both of which might have contributed to the conflicting results. Another potential cause of conflict among studies is the contrasting inference methods used (Table 1), including parsimony (with a dynamic optimization criterion), maximum likelihood, Bayesian analysis of concatenated data, and coalescent-based species-tree analyses.

In recent years, a new class of molecular markers has been developed based on ultraconserved genomic elements (UCEs; Bejerano et al., 2004). Because of their conserved nature, researchers are able to enrich and capture DNA sequences from thousands of nuclear loci, even from distantly related taxa (McCormack et al., 2012). UCEs have been used to address relationships within many vertebrate clades, including major groups of non-avian reptiles

(Crawford et al., 2015) and among families of fishes (Faircloth et al., 2013), frogs (Streicher et

5 al., 2018), lizards (Streicher et al., 2016; Streicher and Wiens, 2017), and snakes (Streicher and

Wiens, 2016). UCEs have also been applied to phylogenetic questions at lower taxonomic levels, such as among bird genera (Meiklejohn et al., 2016) and among species of birds and frogs

(Alexander et al., 2016; McCormack et al., 2016; Smith et al., 2014; Trujillo-Perez, 2015).

A potential disadvantage of UCEs is that the data matrices generated may include considerable missing data. Specifically, including more UCE loci in a given dataset typically requires increasing levels of missing data. Using UCE data from iguanian lizards, Streicher et al.

(2016) examined the impacts of including different levels of missing data and different numbers of loci on the performance of concatenated and species-tree analyses. These authors found that the recovery of well-established clades (and overall branch support levels) were maximized by including loci with an intermediate level of missing data (up to 50% missing taxa per locus).

However, these authors only examined data matrices containing 20−60% missing taxa per locus.

Here, we use data from thousands of UCE loci to resolve higher-level relationships within

Terraranae using both concatenated and species-tree analyses. We also evaluate the effect of missing data on phylogenetic inference in UCEs across a wider range of sampling strategies

(allowing 10−90% missing taxa per locus).

2. Material and Methods

2.1. Taxon sampling

Taxon sampling included 16 ingroup species of Terraranae featuring representatives of four of the five terraranan families, following the taxonomy of AmphibiaWeb (2018), plus five outgroup species. Data for four of the ingroup species and the five outgroup species were taken from Streicher et al. (2018; Table 2). From the family Eleutherodactylidae, we included samples

6 from both subfamilies (Eleutherodactylinae and Phyzelaphryninae) and all four genera

(, Diasporus, Phyzelaphryne, and ). Brachycephalidae contains only two genera, Brachycephalus and , and we included the former. From

Craugastoridae, Craugastorinae was represented by one of the two genera (Craugastor). Within

Craugastor our samples represented the subgenera Campbellius, Craugastor, and Hylactophryne.

From Strabomantidae we sampled four of the eighteen genera: Barycholos from the subfamily

Holoadeninae, and representatives of Lynchius, Pristimantis, and from the subfamily

Pristimantinae (see Table 2). We lacked a sample of Ceuthomantis, which has been considered a distinct family (e.g., Feng et al., 2017; Heinicke et al., 2018, 2009; Pyron, 2014; Pyron and

Wiens, 2011). We also lacked Hypodactylus, which has been considered a monogeneric subfamily of Strabomantidae (Heinicke et al., 2018). However, our data are adequate to address a major debate among studies of terraranan phylogeny: the relationships among Brachycephalidae,

Craugastoridae, Eleutherodactylidae, and Strabomantidae.

Many previous phylogenetic studies have demonstrated that Terraranae is nested within

Hyloidea (Feng et al., 2017; Frost et al., 2006; Pyron, 2014; Pyron and Wiens, 2011; Streicher et al., 2018). However relationships among hyloid families have generally been only weakly supported in previous studies. For outgroups, we included representatives of five hyloid families

(Centrolenidae, Dendrobatidae, , , Leptodactylidae). Based on the well- supported tree of Streicher et al. (2018), the sister group to Terraranae includes Centrolenidae,

Dendrobatidae, and Leptodactylidae. These plus additional families form the clade

Commutabirana, and the sister group to Commutabirana includes Hemiphractidae and Hylidae

(members of the clade Amazorana).

7 Samples were provided by the Círculo Herpetológico de Panamá (CH), Museo de Historia

Natural ANDES at the Universidad de los Andes in Bogotá (ANDES), the Museum of Vertebrate

Zoology at the University of California, Berkeley (MVZ), and Reptile Diversity

Research Center at the University of Texas at Arlington (UTA), Museum of Comparative

Zoology at Harvard University (MCZ), and the Biodiversity Institute and Natural History

Museum at the University of Kansas (KU).

2.2. DNA extraction, library preparation, and sequencing

Genomic DNA (gDNA) was extracted using DNeasy® Blood and Tissue kits (Qiagen) or using magnetic beads (Rohland and Reich 2012; Sera-Mag Speedbeads, Fisher Scientific).

Samples were digested overnight in 20 µL proteinase K in 180 µL of lysis buffer. Genomic DNA was captured with ca. 360 µL magnetic beads, cleaned with two 700 µL washes of 70% EtOH, and eluted in 70 µL of 10 mM Tris (pH 8). After extraction, we quantified the amount of gDNA via fluorometry using double-stranded DNA high-sensitivity assay kits (Qubit, Life

Technologies).

For capture and library preparation we followed the protocol of Faircloth et al. (2012; available at http://ultraconserved.org), with the modifications used by Streicher et al. (2016).

Template gDNA (~150 ng) was fragmented by either physical shearing with a Bioruptor

(Diagenode) using 6 cycles of high-speed agitation for 30 s on and 90 s off, or by enzymatic digestion using NEBNext dsDNA Fragmentase (New England Biolabs) at 37°C for 25 m. The post-hybridization PCR was conducted with NEB Phusion DNA polymerase and TruSeq primers

(Streicher et al., 2016). Enriched libraries were visualized for fragment-size distribution and abundance using a Bioanalyzer 7500 (Agilent®). We sequenced the three capture libraries on

8 three runs, each with 48 individuals (not all individuals were included in the present study). We performed 600-cycle paired-end sequencing runs on an Illumina MiSeq at the genomics core facility of the University of Texas at Arlington (Arlington, TX, USA; http://gcf.uta.edu/).

2.3. Sequence quality control, assembly, and alignment

UCE data were processed with the pipeline provided by Faircloth et al. (2012) available at http://phyluce.readthedocs.org/en/latest/tutorial-one.html#preparing-data-for-raxml-and-examl.

We trimmed sequences to remove adapters and low-quality bases using the Trimmomatic package implemented in Illumiprocessor (Bolger et al., 2014; Faircloth, 2013)

(https://github.com/faircloth-lab/illumiprocessor). We assembled contigs de novo for each sample using Velvet 1.2.10 with a kmer length of 75 and a coverage cutoff of 10. Following contig assembly, we processed the data using programs available from PHYLUCE 1.5.0

(http://phyluce.readthedocs.org/en/latest/tutorial-one.html#preparing-data-for-raxml-and-examl).

We identified the UCE contigs from de novo assemblies on a sample-by-sample basis. Resulting

UCEs were aligned using MAFFT 7.130 (Katoh et al., 2002) with default settings.

2.4. Concatenated phylogenetic analyses

We inferred phylogenetic relationships from each concatenated data matrix using maximum likelihood (ML) analysis as implemented in RAxML version 8.0.19 (Stamatakis,

2014). We used the standard GTRGAMMA substitution model. We did not search the data for partitions, given the very large number of loci. Furthermore, there are few obvious a priori partitions for UCE data (e.g. many loci are not protein coding, so most sites cannot be assigned to codon positions). We ran two RAxML analyses. First, we made 20 replicate searches for the

9 optimal ML tree. Second, we performed bootstrapping using the autoMRE option, which automatically determines a sufficient number of bootstrap replicates. The bootstrap support values are shown on the inferred best ML tree, and all trees were rooted using the outgroup species (see above).

2.5. Species-tree analyses

We used two coalescent-based species-tree approaches designed to work on large phylogenomic datasets. First, we used a species-tree approach (NJst) based on a matrix of internode distances across gene trees (Liu and Yu, 2011), which approximates the species tree under the multi-species coalescent. To build our species tree, we generated 100 bootstrap samples per locus using RAxML version 8.0.19 and the GTRGAMMA model. To obtain bootstrap support values we used a two-stage bootstrap procedure in which genes were randomly resampled followed by random resampling of base pairs within the resampled genes (Seo, 2008).

We ran all NJst analyses using the Species Tree Analysis Web (STRAW) Server (Shaw et al.,

2013). We also used the Accurate Species TRee ALgorithm (ASTRAL-II 5.5.9; Mirarab et al.,

2014; Mirarab and Warnow, 2015). This method estimates an unrooted species tree given a set of unrooted gene trees, under a multi-species coalescent model. Branch support for both NJst and

ASTRAL-II analyses was estimated using the same bootstrap method proposed by Seo (2008).

Species trees were rooted using the outgroups.

2.6. Missing data

We also evaluated the effect of varying the number of loci included and the amount of missing data included (by changing the maximum amount of missing taxa allowed for a locus to

10 be included). We used PHYLUCE to filter the alignments to create nine data matrices that differed based on the number of loci included, where the decision to include a locus was based on the maximum percentage of taxa (including outgroups) that were missing data for that locus. We created nine matrices, each allowing different maximum amounts of missing taxa per locus (10%,

20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%). For example, the 10% matrix included only those loci that had data for 90% or more of all taxa. The overall amount of missing data in a matrix could be quite different from the maximum amodunt of missing taxa allowed per locus.

For example, allowing 60% to 90% missing taxa per locus generates matrices with only 49–58% missing data overall (Table 3). We estimated the overall number of parsimony-informative sites and the amount of missing data in each dataset (Table 3) using Geneious pro 8.1 using Perl scripts available from http://phyluce.readthedocs.io/en/latest/tutorial-one.html. We generated alignment statistics using the script get_align_summary_data.py implemented in alignment_assessment_v1 (Portik et al., 2016).

Following Streicher et al. (2016, 2018), we used the mean bootstrap value of all nodes across the tree to evaluate how missing data and number of loci impacted the phylogenetic results. We did not consider any clades within terraranans to be well-established based on non- molecular data. Therefore, we did not focus on the bootstrap score for any particular clade.

However, previous studies (Streicher et al. 2016, 2018) found that bootstrap support for well- established clades was strongly correlated with mean bootstrap support for other nodes across the tree. Therefore, we used mean support across nodes as a provisional index of performance of each approach. Nevertheless, we readily acknowledge that there are conditions under which mean bootstrap support could be misleading as a measure of method performance (Hillis and Bull,

1993).

11

3. Results

3.1. Phylogenetic results

Using concatenated maximum likelihood, the tree inferred from the matrix allowing up to

80% missing data per locus (Fig. 2) provided the overall highest bootstrap support (bs) values

(Table 4). We describe this tree and then compare it with those obtained based on other matrices and other inference methods. Terraranans formed a monophyletic group with 100% bootstrap support, confirming previous phylogenetic results (Crawford and Smith, 2005; Feng et al., 2017;

Frost et al., 2006; Hedges et al., 2008; Heinicke et al., 2009; Padial et al., 2014; Pyron and

Wiens, 2011; Streicher et al., 2018). In our results, Brachycephalidae, represented by

Brachycephalus quiririensis, was recovered as the sister taxon to a strongly supported clade

(bs=100%) uniting all other sampled terraranan taxa. Eleutherodactylidae was also strongly supported as monophyletic (bs=100%). Within Eleutherodactylidae, the subfamilies

Eleutherodactylinae (Diasporus, Eleutherodactylus) and Phyzelaphryninae (Adelophryne,

Phyzelaphryne) were each strongly supported as monophyletic (bs=100%). Our results show strong support for a clade uniting Craugastoridae (in our phylogeny represented by Craugastor augusti, C. daryi, and C. longirostris) and Strabomantidae (sensu Heinicke et al., 2018) represented by Barycholos pulcheus, Lynchius nebulanastes, Oreobates quixensis, Pristimantis miyatai, P simonsii, Strabomantis anomalus (bs=100%). However, we find that Craugastoridae is non-monophyletic if Strabomantidae is recognized as a separate family, because Strabomantidae is nested within Craugastoridae. There is moderate support (bs=73%) for a clade uniting

Craugastor (Craugastorinae) and Strabomantis (Strabomantidae). The monophyly of Craugastor was strongly supported (bs=98%). The sister relationship between the subgenera Hylactophryne

12 (represented by C. augusti) and Craugastor (represented by C. longirostris) relative to

Campbellius (represented by C. daryi) was also well supported (bs=93%). There was a strongly supported clade (bs=100%) uniting Lynchius, Oreobates, Pristimantis, and Barycholos. These genera were previously assigned to the subfamilies Pristimantinae (Lynchius, Oreobates, and

Pristimantis) and Holoadeninae (Barycholos). The genera Lynchius and Oreobates together formed the sister clade (bs=100%) to Barycholos plus Pristimantis (bs =100%; Fig. 2). Thus,

Pristimantinae was paraphyletic with respect to Holoadeninae.

Coalescent-based species-tree analyses using NJst and ASTRAL-II applied to the 80% missing-data matrix yielded phylogenetic trees that generally agreed with each other and with the concatenated ML results (see above) in terms relationships among families and among subfamilies (Figs. 2 and S1). We compare the statistical support provided by these two methods, and then highlight one topological difference with respect to the concatenated ML results. NJst and ASTRAL-II provided strong support for the monophyly of Terraranae (bs=100 in both cases), and for the clade uniting Eleutherodactylidae, Craugastoridae, and Strabomantidae (bs=92 in NJst, bs=85 in ASTRAL-II). Support was strong for monophyly of Eleutherodactylidae (bs=96 in NJst, bs=100 in ASTRAL-II), Eleutherodactylinae (bs=99 in NJst, bs=100 in ASTRAL-II), and Phyzelaphryninae (bs=98 in NJst, bs=100 in ASTRAL-II). Monophyly of the clade uniting

Craugastoridae and Strabomantidae was somewhat weaker (bs=85 in NJst, bs=96 in ASTRAL-

II). Relationships among Barycholos, Lynchius, Oreobates, and Pristimantis were the same with

NJst and ASTRAL-II as with concatenated ML, and remained strongly supported (bs=96–100).

Importantly, Barycholos (Holoadeninae) was again nested inside of Pristimantinae (Lynchius,

Oreobates, and Pristimantis) with strong support (bs=97 in NJst, bs=100 in ASTRAL-II).

13 The only major topological difference we observed among methods was that, whereas concatenated ML trees placed Strabomantis as sister to Craugastor, under NJst and ASTRAL-II,

S. anomalus and C. daryi were placed as sister taxa relative to the other two species of

Craugastor (bs=92 in NJst, bs=73 in ASTRAL-II). Thus, Craugastorinae was paraphyletic in the

NJst and ASTRAL analyses. The contrasting results for the placement of Strabomantis relative to

C. daryi may reflect the low number of UCE loci recovered for these taxa (136 and 71 loci for S. anomalus and C. daryi, respectively; Table 2).

3.2. Impact of missing data

As more missing data were allowed per locus (from 10% up to 90%), both the number of

UCE loci and base pairs included increased (Table 3). However, the range of variation in the overall amount of missing data was relatively limited (no more than 58% missing data overall).

The number of parsimony-informative sites also increased with increased number of missing bases (Table 4; Fig 3; Spearman’s rank correlation, rs = 0.976; P < 0.0001). This may explain why bootstrap support values increased when higher levels of missing data were allowed. This relationship is also revealed by the strong association between the number of loci and mean support values for all three methods (Spearman’s rank correlation, ML rs = 0.95, P < 0.0001; NJst rs = 0.92, P = 0.0004; ASTRAL-II rs = 0.88, P = 0.0017).

Phylogenetic results reported above based on the 80% missing data matrix. With matrices with 50% to 90% missing data per locus, all analyses (ML, NJst, and ASTRAL-II; Figs. S1, S2,

S3, S4) supported the monophyly of Terraranae, Eleutherodactylidae, and Strabomantidae +

Craugastoridae sensu Feng et al. (2017). All of these analyses also placed Brachycephalidae as the sister taxon to Eleutherodactylidae + Craugastoridae + Strabomantidae. The number of loci

14 included varied from 1,262 when allowing 50% missing taxa per locus to 2,745 UCEs when allowing 90% missing taxa per locus (Table 3).

The phylogenetic results were more variable when allowing ≤ 40% missing taxa per locus. When loci were included with a maximum of 40% missing taxa, the data matrix included only 632 loci, and the ASTRAL-II analysis placed Brachycephalus quiririensis inside the clade of Craugastoridae plus Strabomantidae (bs=7; Fig. S4-E), whereas ML and NJst topologies were unaffected. The ML concatenated and NJst analyses based on matrices with 40% or more missing data placed Brachycephalidae as sister to Eleutherodactylidae plus Craugastoridae. Thus, our

ASTRAL-II results may be more sensitive than NJst and ML to the limited number of sites. The maximum 30% missing taxa per locus matrix included only 202 loci. Mean bootstrap values dropped precipitously across the three inference methods (Table 4). The ML, NJst, and

ASTRAL-II analyses did not recover the monophyly of Eleutherodactylidae or Craugastoridae

(Fig. S2) in the data matrices that allowed only 10 or 20% missing taxa per locus, the total number of loci was very low (4 and 22 UCEs, respectively). The topologies inferred under concatenated ML, NJst, and ASTRAL-II were incongruent with some previously well-established relationships. For example, with 10% or 20% missing data, Terraranae was non-monophyletic

(Figs. S1G, S1H, S2G, S2H, S3G, S3H).

4. Discussion

4.1. Phylogenetic relationships and comparisons with previous studies

Higher-level phylogenetic relationships among terraranan frogs are highly uncertain (Fig.

1). Based on concatenated ML and species-tree analyses, our phylogenomic dataset of 2,665 loci provides a well-supported hypothesis that may resolve much of this uncertainty (Fig. 2). The 80%

15 matrix (including loci with up to 80% missing taxa per locus) provided the overall best-supported phylogeny for two of the three methods used (ML, ASTRAL-II), and the inferred relationships were generally consistent across datasets regardless of the inference method employed (Figs. S1,

S2 A to E, S3 A-F, S4 A to E, Table 4).

The family-level phylogenetic relationships recovered here were most similar to those of

Pyron and Wiens (2011), Feng et al. (2017), Hutter et al. (2017), and Streicher et al. (2018).

Specifically, we placed the family Brachycephalidae as the sister to Eleutherodactylidae +

Craugastoridae + Strabomantidae, with relatively strong support (bs=100 from concatenated ML, bs=92 for NJst, and bs=85 for ASTRAL-II). This is consistent with Pyron and Wiens (2011), based on concatenated ML analysis using a dataset of 3 nuclear and 9 mitochondrial genes from

340 terraranan species. Using a dataset of 95 nuclear genes from 16 terraranan species, Feng et al. (2017) also placed Brachycephalidae as the sister to Eleutherodactylidae + Craugastoridae +

Strabomantidae (ML bs=90%). This same topology was also recovered with strong support

(bs=100%) in a study that used 2,214 UCEs but included only five terraranan taxa (Streicher et al., 2018). Our results contradict Pyron (2014), who used 3 nuclear and 9 mitochondrial loci with

418 species, and placed Craugastoridae + Strabomantidae as sister to Brachycephalidae +

Eleutherodactylidae. Our findings also contradict Heinicke et al. (2018) who placed

Eleutherodactylidae as sister to Brachycephalidae + Craugastoridae + Strabomantidae. They used a dataset of 389 genes for 30 terraranan species. Among all these studies, our results are based on the largest number of loci. Our results are also strongly supported by both concatenated analyses and coalescent-based species-tree analyses.

Another conflict among recent molecular phylogenetic studies of Terraranae is the placement of Strabomantis and the recognition of the family Strabomantidae. Our results support

16 synonymizing Strabomantidae with Craugastoridae. Within Craugastoridae, Pyron and Wiens

(2011) recognized the monogeneric subfamily Strabomantinae, which is consistent with our ML result, but conflicts with our species-tree results which places Strabomantis within Craugastor

(Figs. S1, S3, S4). While its exact placement is uncertain, Strabomantis should be placed within

Craugastorinae inside the family Craugastoridae (Fig 2). In contrast, Heinicke et al. (2018) recognized Strabomantidae as the sister taxon of Craugastoridae. Within Strabomantidae, they placed Strabomantis as the sister taxon of a clade including Barycholos, Oreobates, and

Pristimantis, among other genera. Feng et al. (2017) also recognized Strabomantidae, placing

Strabomantis, Pristimantis, Hypodactylus and Barycholos in a clade that was the sister taxon of

Craugastor. In our concatenated ML tree, Strabomantis is placed as the sister taxon of

Craugastor (bs=73%), rather than with the other sampled genera of Strabomantidae (Barycholos,

Lynchius, Oreobates, Pristimantis). In both species tree methods Strabomantis is nested inside

Craugastor, as sister to C. daryi (NJst bs=89%; ASTRAL-II bs=73%). Either topology would render Strabomantidae non-monophyletic with respect to Craugastoridae. Therefore, we support

Pyron and Wiens (2011), Gomez-Mestre et al. (2012), and others in placing Strabomantidae sensu Heinicke et al. (2018) within Craugastoridae.

The relationships among genera of Holoadeninae and Pristimantinae also differ between our study and previous studies. Here, we recovered the following relationships among the four sampled genera: ((Lynchius, Oreobates), (Barycholos, Pristimantis)), with strong support from all three methods (Fig. 2). Importantly, this topology renders Pristimantinae (represented here by

Lynchius, Oreobates, and Pristimantis) paraphyletic with respect to Holoadeninae (represented here by Barycholos). In contrast, Heinicke et al. (2018) and Pyron and Wiens (2011) recovered the following relationships among these genera (Barycholos, (Pristimantis, (Lynchius,

17 Oreobates))). They found moderately strong support for the clade excluding Barycholos [ML bs=89% and ML bs=81%, in Heinicke et al. (2018) and Pyron and Wiens (2011), respectively].

Our support here for the relationships among these genera is much higher and is based on many more loci. However, we note that taxon sampling was more extensive in Pyron and Wiens

(2011). Our results that recover Holoadeninae (Barycholos) nested inside Pristimantinae

(Lynchius, Pristimantis, and Oreobates) suggest that one of these two subfamilies is not valid.

We note that the name Holoadeninae proposed by Hedges et al. (2008) is available and also has the priority over Pristimantinae Pyron and Wiens (2011), following Article 23.1 of the

International Code of Zoological Nomenclature (ICZN, 1999).

Other phylogenetic relationships inferred here were less controversial. Within

Eleutherodactylidae, the two subfamilies Eleutherodactylinae and Phyzelaphryninae were monophyletic with strong support (bs=100%) as found in all previous studies with relevant sampling (Fouquet et al., 2012; Hedges et al., 2008; Heinicke et al., 2018; Padial et al., 2014;

Pyron, 2014; Pyron and Wiens, 2011).

In summary, our analyses using ML concatenated analysis and coalescent-based species- tree methods (NJst and ASTRAL-II) resulted in high support for Brachycephalidae as the sister taxon to the clade Craugastoridae + Eleutherodactylidae, as also found by Pyron and Wiens

(2011), Feng et al. (2017), Hutter et al. (2017), and Streicher et al. (2018), but contra Hedges et al. (2008a), Heinicke et al. (2007, 2009, 2018), Pyron (2014), and Padial et al. (2014). Our results also show that Strabomantidae is nested inside of Craugastoridae and should not be recognized as a distinct family. We also find that Holoadeninae is nested inside Pristimantinae, and that

Pristimantinae should be synonymized with Holoadeninae.

18 We resolved some controversial issues in terraranan phylogenetics, but much additional work is needed. We suggest that the highest priority for future studies will be to include more taxa. For example, because we had no samples of Ceuthomantis in this study, we could not evaluate the hypothesis that this genus is sister to all other Terrananae. Nevetheless, this hypothesis has been consistently supported by all other recent studies (Feng et al., 2017; Heinicke et al., 2009, 2018; Pyron, 2014; Pyron and Wiens, 2011), except Padial et al. (2014). It will also be crucial to include several genera that have not yet been included in molecular phylogenetic analyses (i.e., Atopophrynus, Dischiodactylus, Geobatrachus, and ).

4.2. Missing data

A major concern in phylogenomic analyses is the possible impact of missing data on phylogenetic inference (Jiang et al., 2014; Philippe et al., 2004; Roure et al., 2013; Streicher et al., 2016; Xi et al., 2016; Wiens and Morrill, 2011). Here, we found that support values from concatenated ML, ASTRAL-II, and NJst analyses increased with larger datasets containing more, not fewer, missing data (Table 3 and 4; Spearman’s rank correlation, ML rs = 0.95, P < 0.0001;

NJst rs = 0.92, P < 0.0004; ASTRAL-II rs = 0.88, P < 0.0017). This may occur because allowing more loci, although with more missing data per locus, allows the inclusion of proportionally more parsimony-informative sites (Fig. 3). In the present study, allowing a maximum of 90% missing taxa per locus generated matrices with only 58% missing data overall, and a maximum of 60% missing taxa per locus resulted in 49% missing data overall (Table 3). ML bootstrap support was maximized at 80% missing taxa per locus, not 90%. For the NJst analyses, mean bootstrap support values were tied for highest at 70% and 90%. For ASTRAL-II, mean bootstrap support was high between 50% and 90% missing data, peaking at 80% of missing data per locus (Table

19 4). Lowering the threshold to a maximum of 40% missing data per locus caused lower bootstrap support in a few branches (Fig. S3E). Our phylogenetic results were generally similar across levels of missing data in the range of 40% to 90% missing taxa per locus (Table 4). The gains in bootstrap support achieved using data matrices that allowed more missing data may be explained by the increasing number of informative sites that are sampled when allowing more missing data

(Jiang et al., 2014; Wiens and Morrill, 2011; Xi et al., 2016). Many simulation and empirical studies have now shown that increasing the number of characters, despite the increase in missing data, may increase the accuracy of phylogenetic analyses (Jiang et al., 2014; Streicher et al.,

2016; Wiens and Morrill, 2011; Xi et al., 2016).

5. Conclusions

Many previous studies on the phylogenetic relationships within terraranan frogs were in disagreement and often had weak support for key branches (Fig. 1, Table 1). Our results provide a generally well-supported estimate of relationships among most terraranan families and subfamilies based on concatenated and species-tree methods, including the largest number of genetic loci considered so far. At the family level, our results show that Brachycephalidae is the sister taxon of Eleutherodactylidae + “Craugastoridae” + Strabomantidae. This result conflicts with some previous studies (e.g. Hedges et al., 2008; Heinicke et al., 2014; Padial et al., 2014;

Pinto-Sanchez et al., 2014; Pyron, 2014) but is congruent with others (Feng et al., 2017; Pyron and Wiens, 2011; Streicher et al., 2018). Within these families, we suggest that Strabomantidae should be placed in the synonymy of Craugastoridae (otherwise Craugastoridae is paraphyletic).

Within Craugastoridae, we find that Pristimantinae should be placed in the synonymy of

Holoadeninae (otherwise Pristimantinae is polyphyletic). Finally, we explored the effect of

20 including loci with progressively more missing data on three phylogenetic methods (concatenated likelihood, NJst, ASTRAL-II). We found that including loci with more missing data generally increased mean bootstrap values for all three methods, given that including more missing data allowed more loci to be included. Our results add additional support to the idea that the benefits of including more loci can potentially overcome any negative consequences of including more missing data in phylogenomic analyses.

Acknowledgements

We are grateful for tissue loans from Círculo Herpetológico de Panamá (CH), Museo de

Historia Natural ANDES at the Universidad de los Andes in Bogotá (ANDES), the Museum of

Vertebrate Zoology at the University of California, Berkeley (MVZ), Amphibian and Reptile

Diversity Research Center at the University of Texas at Arlington (UTA), Museum of

Comparative Zoology at Harvard University (MCZ), and the Biodiversity Institute & Natural

History Museum at Kansas University (KU), Museo de Herpetología de la Universidad de

Antioquia (MHUA). Funding for this project came from Colciencias grant number 567 for PhD studies and Crédito Condonable of the Universidad de los Andes to LSB, the FAPA fund of the

Universidad de los Andes to AJC, and The University of Arizona and U.S. National Science

Foundation Grant DEB 1655690 to JJW. We thank Andres M. Cuervo, Luisa Dueñas, Carlos E.

Guarnizo, Santiago Herrera, Andrea Paz, and Camila Plata for providing valuable comments on earlier versions of this manuscript. Special thanks to David O. Botero, Catalina Ramirez-Portilla, and Fanny Gonzáles for help with script writing and bioinformatic advice. Thanks to Carolina

Amorocho and Luisa Castellanos for help in the lab and with paperwork, to Jorge Avendaño for his correction to the figures and to Mauricio Rivera-Correa and Carlos Henrique Luz Nunes de

21 Almeida for given access to use their photos in the graphical abstract.

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30 Table 1. Summary of previous studies of the phylogenetic relationships within Terraranae using

molecular data. The abbreviation for the inference methods are: maximum parsimony (MP),

maximum likelihood (ML), Bayesian inference (Bayes), parsimony under direct /dynamic

optimization criterion (POY), and coalescent-based species-tree estimation using Accurate

Species TRee Algorithm (ASTRAL) and NJst.

Study Terraranan Outgroup Mitochondrial Nuclear Inference taxa taxa genes genes method Heinicke et al. (2007) 276 4 3 2 MP, ML, Bayes

Hedges et al. (2008) 344 18 2 2 ML, Bayes Heinicke et al. (2009) 42 4 6 11 MP, ML, Bayes

Pyron and Wiens (2011) 340 2533 3 9 ML

Padial et al. (2014) 405 25 9 11 POY Pyron (2014) 418 2892 3 9 ML Pinto-Sánchez et al. (2014) 363 7 3 9 Bayes Feng et al. (2017) 16 278 -- 97 ML, ASTRAL

Heinicke et al. (2018) 30 5 -- 389 ML, ASTRAL

Streicher et al. (2018) 5 45 -- 2214 ML, NJst, ASTRAL Hutter et al. (2017) 610* 1708* 7 13 Bayes

* Includes well-supported but undescribed species.

31

Table 2. Voucher information and amount of DNA data produced for each sample, including number of contigs assembled using Velvet (v1.2.10), and the resulting number of aligned ultraconserved elements (UCEs) obtained. Sequence Read Archive (SRA) accession numbers provide access to all reads obtained for each individual. Family-level taxonomy follows Heinicke et al. (2018). Accession numbers starting with SAMN were published previously

Streicher et al. (2018). Museum numbers are from the following natural history collections: Museo Herpetológico de la Universidad de Antioquia

(MHUA), Royal Ontario Museum (ROM), Museo de Historia Natural, Universidad Nacional Mayor de San Marcos (MHNSM), Círculo Herpetológico de Panamá (CH), Museo de Historia Natural ANDES at the Universidad de los Andes in Bogotá (ANDES), the Museum of Vertebrate Zoology at the

University of California, Berkeley (MVZ), Amphibian and Reptile Diversity Research Center at the University of Texas at Arlington (UTA), Museum of

Comparative Zoology at Harvard University (MCZ), and the Biodiversity Institute & Natural History Museum at Kansas University (KU), Coleção

Herpetológica do Departamento de Zoologia, Universidade Federal do Paraná, Curitiba, state of Paraná (DZUP). Three samples currently have only field numbers: Jonathan A. Campbell (JAC), Erik R. Wild (ERW), William E. Duellman (WED).

Species Family Museum Contigs UCEs SRA accession number Brachycephalus quiririensis Brachycephalidae DZUP 522 5415 886 SAMN05559884 Craugastor augusti Craugastoridae UTA A-60654 3790 738 - Craugastor daryi Craugastoridae UTA A-62648 375 71 - Craugastor longirostris Craugastoridae MHUA 4809 2569 1301 SAMN05559889 Eleutherodactylus johnstonei Eleutherodactylidae ANDES-A 1912 4588 1792 - Eleutherodactylus longipes Eleutherodactylidae JAC 29834 2710 1243 - Diasporus gularis Eleutherodactylidae ANDES-A 3833 19214 427 - Diasporus vocator Eleutherodactylidae CH 4786 8247 429 - Adelophryne adiastola Eleutherodactylidae ANDES-A 2560 3680 1515 SAMN05559873 Phyzelaphryne miriame Eleutherodactylidae ANDES-A 3834 3875 512 -

32

Strabomantis anomalus Strabomantidae ANDES-A 1416 1034 136 - Barycholos pulchrus Strabomantidae KU 217782 1261 610 - Lynchius nebulanastes Strabomantidae ERW 86 11368 2233 SAMN05559921 Oreobates quixensis Strabomantidae ANDES-A 1954 4975 1198 - Pristimantis simonsii Strabomantidae WED 56667 5268 2118 - Pristimantis miyatai Strabomantidae ANDES-A 1776 4897 1429 - Espadarana prosoblepon Centrolenidae MVZ 149741 6094 1851 SAMN05559886 coxi Hemiphractidae ROM 39478 3350 1826 SAMN05559931 Dendropsophus leali Hylidae KU 215259 6094 1767 SAMN05559892 Hyloxalus nexipus Dendrobatidae KU 211806 368 1456 SAMN05559914 Leptodactylus didymus Leptodactylidae MHNSM 14643 5814 1909 SAMN05559919

33 Table 3. Summary of data matrices based on ultraconserved elements (UCEs), organized by the maximum percentage of missing data allowed per locus in each matrix. The summary includes maximum the total number of UCE loci per matrix, total number of characters in aligned base pairs (bp), number of parsimony-informative sites across all UCEs, total number of missing data cells in each matrix, percent of sites across all loci that contain parsimony-informative variation

(excluding sites with gap characters) in relation to the total length of the alignment, and the overall percentage of missing data cells in each matrix.

Maximum Number of Total DNA Parsimony- Missing % % missing missing UCEs sequence informative bases informative data in data per length in bp sites sites matrix locus 90% 2,745 754,266 24,079 9,264,413 3.10% 58% 80% 2,665 743,419 24,079 8,844,779 3.20% 57% 70% 2,368 639,195 22,083 7,126,259 3.50% 53% 60% 1,906 503,502 17,390 5,166,952 3.50% 49% 50% 1,262 337,741 11,217 3,097,704 3.30% 44% 40% 632 172,052 5,513 1,359,560 3.20% 38% 30% 202 53,240 1,243 342,194 2.30% 31% 20% 22 5,565 83 23,879 1.50% 20% 10% 4 1,265 14 944 1.10% 4%

34 Table 4. Mean bootstrap support across all nodes in trees inferred from nine data matrices, each allowing different amounts of missing taxa per locus (10% to 90%). Results are given for and three phylogenetic inference methods (concatenated likelihood using RAxML, coalescent-based species tree using NJst and ASTRAL-II).

Missing Average bootstrap support values across all nodes taxa/locus RAxML NJst ASTRAL-II 90% 94.9 91.6 93.0 80% 95.7 90.9 94.1 70% 95.4 91.6 89.6 60% 93.8 91.3 93.0 50% 91.8 88.9 93.0 40% 91.6 80.3 83.4 30% 51.7 56.3 56.0 20% 41.3 15.0 13.0 10% 18.0 16.4 12.5

35 FIGURE LEGENDS

Fig. 1. Summary of hypotheses of higher-level phylogenetic relationships among terraranan frogs. (A) Hedges et al. (2008) concatenated maximum likelihood (ML) analysis of two mitochondrial genes and two nuclear genes, totaling 2578 base pairs (bp); numbers adjacent to nodes represent bootstrap support. (B) Heinicke et al. (2009) concatenated ML analysis of 6 mitochondrial and 11 nuclear genes totaling 10739 bp; numbers adjacent to nodes represent bootstrap support. (C) Pyron and Wiens (2011) concatenated ML analysis of 3 mitochondrial and

9 nuclear genes totaling 12712 bp; numbers adjacent to nodes represent bootstrap support (D)

Padial et al. (2014) parsimony analysis of 9 mitochondrial and 12 nuclear genes totaling 17233 bp; numbers adjacent to nodes represent jackknife support. (E) Pinto-Sánchez et al. (2014) concatenated Bayesian analysis of 3 mitochondrial and 9 nuclear genes totaling 12712 bp; asterisks represent Bayesian posterior probabilities of 0.95 or greater. (F) Pyron (2014) concatenated ML analysis of 3 mitochondrial and 9 nuclear genes totaling 12712 bp; numbers adjacent to nodes represent bootstrap support. (G) Feng et al. (2017) concatenated ML analysis and coalescent-based species tree (ASTRAL) of 95 nuclear protein-coding genes; numbers adjacent to nodes represent bootstrap support. (H) Hutter et al. (2017) concatenated Bayesian analysis of 7 mitochondrial and 13 nuclear genes; numbers adjacent to nodes represent Bayesian posterior probabilities. (I) Heinicke et al. (2018) concatenated ML analysis and coalescent-based species tree (ASTRAL) based on 389 nuclear protein-coding genes; numbers adjacent to nodes represent ML bootstrap support before the slash, followed by local posterior probabilities for

ASTRAL. (J) Streicher et al. (2018) concatenated ML analysis based on 2,214 UCEs (802,167 base pairs), including loci with up to 60% missing taxa per UCE. Numbers next to each branch

36 show support from concatenated analysis (left) and from NJst. The branch lengths in each topology are arbitrary and not representative of inferred amounts of evolution.

37 Fig. 2. Relationships among terraranan frogs based on a concatenated ML analysis, the family- level taxonomy follows AmphibiaWeb (2018). The data matrix included 2,665 UCE loci for a total of 743,419 aligned base pairs, and included loci with up to 80% missing taxa. Numbers next to each node indicate the bootstrap support values from the ML analysis (top) and coalescent- based species-tree analyses from NJst (middle) and ASTRAL-II (bottom number). The black squares indicate the clades that were recovered in all concatenated ML and in NJst and

ASTRAL-II analyses of this data matrix. The white square indicates a node (monophyly of

Craugastorinae) that was not recovered in the NJst or ASTRAL-II analyses, and thus has only the

ML bootstrap score. In the NJst and ASTRAL-II analyses, Strabomantis is placed inside

Craugastor (see Fig. S1). Note that Strabomantidae and Pristimantinae are paraphyletic in all of these trees. The full trees from each method (including branch lengths and outgroups) are provided in Fig. S1.

Fig. 3. A) Percentage of parsimony-informative sites (ignoring sites with gaps) in the data matrix relative to the maximum number of missing taxa allowed per locus. B) Percentage of missing data per matrix relative to the maximum number of missing taxa per locus included in the. In both graphs, the proportion is calculated at the total number of characters of a given type

(parsimony-informative or containing of missing data) over the total length of the alignment in base pairs.

Fig. S1. Phylogeny of terraranan frogs based on three methods, using the data matrix including loci with up to 80% missing data per locus and including 2,665 ultraconserved element (UCE) loci for a total of 743,419 concatenated base pairs. (A) Maximum likelihood tree inferred from

38 concatenated data using RAxML version 8.0.19. (B) Coalescent-based species tree inferred using

NJst. (C) Coalescent-based species tree inferred using ASTRAL-II. The numbers adjacent to the nodes represent bootstrap support (see Methods).

Fig. S2. Concatenated maximum likelihood trees inferred using RAxML, with bootstrap support values indicated next to each branch. The eight trees are based on increasingly complete data matrices that have fewer UCE loci. (A) Tree inferred from a data matrix allowing up to 90% missing taxa per UCE locus, including 2,745 loci; (B) allowing up to 70% missing taxa per UCE locus, and including 2,368 loci; (C) allowing up to 60% missing taxa per UCE locus, including

1,906 loci; (D) allowing up to 50% missing taxa per UCE locus, including 1,262 loci; E) allowing a maximum of 40% missing taxa per UCE locus, including 632 loci; (F) allowing up to

30 % missing taxa per UCE locus, including 202 loci; (G) allowing up to 20% missing taxa per

UCE locus, thus including 22 loci; (F) allowing up to 10% missing taxa per UCE locus, including

4 loci. The numbers next to each node represent bootstrap support (see Methods). Note that the tree based on the 80% missing data matrix is shown in Fig S1.

Fig. S3. Coalescent-based species trees inferred using NJst, with bootstrap support values indicated next to each branch. The eight trees are based on increasingly complete data matrices that have fewer UCE loci. (A) Tree inferred from a data matrix allowing up to 90% missing taxa per UCE locus, including 2,745 loci; (B) allowing up to 70% missing taxa per UCE locus, and including 2,368 loci; (C) allowing up to 60% missing taxa per UCE locus, including 1,906 loci;

(D) allowing up to 50% missing taxa per UCE locus, including 1,262 loci; (E) allowing up to

40% missing taxa per UCE locus, including 632 loci; (F) Allowing up to 30 % missing taxa per

39 UCE locus, including 202 loci; (G) Allowing up to 20% missing taxa per UCE locus, thus including 22 loci; (F) allowing a maximum of 10% missing taxa per UCE locus, thus including only 4 loci. The numbers next to each node represent the bootstrap support (see Methods). Note that the tree based on the 80% missing data matrix is shown in Fig S1.

Fig. S4. Coalescent-based species trees inferred using ASTRAL-II, with bootstrap support values indicated next to each branch. The eight trees are based on increasingly complete data matrices that have fewer UCE loci (A) Tree inferred from a data matrix allowing up to 90% missing taxa per UCE locus, including 2,745 loci; (B) allowing up to 70% missing taxa per UCE locus, including 2,368 loci; (C) allowing up to 60% missing taxa per UCE locus, including 1,906 loci;

(D) allowing up to 50% missing taxa per UCE locus, including 1,262 loci; (E) allowing up to

40% missing taxa per UCE locus, including 632 loci; (F) allowing up to 30 % missing taxa per

UCE locus, including 202 loci; (G) allowing up to 20% missing taxa per UCE locus, thus including 22 loci; (F) allowing a maximum of 10% missing taxa per UCE locus, including 4 loci.

The numbers next to each node represent the bootstrap support (see Methods). Note that the tree based on the 80% missing data matrix is shown in Fig S1.

40 A. Hedges et al. (2008) B. Heinicke et al. (2009)

100 Eleutherodactylinae Ceuthomantidae Eleutherodactylidae 97 Phyzelaphryninae Eleutherodactylidae 29 100 Brachycephalidae Brachycephalidae Craugastoridae 68 35 84 Craugastoridae 54 Holoadeninae Strabomantidae 76 Strabomantidae 55 Strabomantinae

C. Pyron & Wiens (2011) D. Padial et al. (2014)

Ceuthomantidae 99 Eleutherodactylinae Eleutherodactylidae Brachycephalidae Phyzelaphryninae 98 89 Eleutherodactylinae Eleutherodactylidae 99 Brachycephalidae Phyzelaphryninae 97 Ceuthomantinae Strabomantinae 95 Holoadeninae Craugastoridae <50 Craugastorinae 92 Craugastoridae 91 86 Holoadeninae Craugastorinae <50 Pristimantinae E. Pinto-Sánchez et al. (2014) F. Pyron (2014) Brachycephalidae Ceuthomantidae 40 Eleutherodactylinae Brachycephalidae Eleutherodactylidae 99 Phyzelaphryninae Eleutherodactylinae Eleutherodactylidae Craugastorinae 100 Phyzelaphryninae * Holoadeninae Craugastoridae Craugastorinae Holoadenina e * Pristimantinae 99 Craugastoridae 93 Pristimantinae

G. Feng et al. (2017) H. Hutter et al. (2017)

Ceuthomantidae Ceuthomantidae 0.95 94 Brachycephalidae Brachycephalidae 1 90 Eleutherodactylidae Eleutherodactylinae Eleutherodactylidae Craugastoridae 1 1 Phyzelaphryninae 68 Craugastoridae 82 Strabomantidae

I. Heinicke et al. (2018) J. Streicher et al. (2018) Ceuthomantidae Eleutherodactylinae Brachycephalidae 100/0.74 Eleutherodactylidae Phyzelaphryninae Craugastoridae 100/0.83 100/100 Brachycephalidae Craugastoridae 100/97 Eleutherodactylidae 100/0.51 Hypodactylinae 100/0.64 Strabomantinae 100/0.53 Strabomantidae

--/0.38 Holoadeninae 95/0.37 Pristimantina e

41 Brachycephalus quiririensis Brachycephalidae 100 96 Phyzelaphryne miriame 100 Phyzelaphryninae 100 Adelophryne adiastola 98 100 Eleutherodactylus longipes 100 100 Eleutherodactylus johnstonei Eleutherodactylidae 100 100 Eleutherodactylinae 100 73 100 100 Diasporus gularis 89 100 99 70 99 100 100 Diasporus vocator

100 Strabomantis anomalus 92 9 8 85 Craugastor daryi Craugastorinae Craugastor augusti 93 Craugastor longirostris 99 100 93 Lynchius nebulanastes Craugastoridae 85 96 100 Oreobates quixensis 99 Holoadeninae 100 100 Barycholos pulcheus 96 Pristimantis miyatai 94 100 97 100 Pristimantis simonsii 100 100 100

substitutions / site 0.005

A. B.

3.5 60

50 3.0

40 2.5

30

2.0

Proportion missing data 20

Proportion parsimony-informative sites 1.5 10

1.0 10% 20% 30% 40% 50% 60% 70% 80% 90% 10% 20% 30% 40% 50% 60% 70% 80% 90% Missing taxa / locus Missing taxa / locus

42 Chapter 2 – Integrating ultraconserved elements and mitochondrial DNA sequence data to infer the biogeography of rain frogs (Eleutherodactylidae) across Middle

America, South America, and the Caribbean

Lucas S. Barrientos1 *, Jeffrey W. Streicher2, 3, Elizabeth C. Miller3, John J. Wiens3,

1 Andrew J. Crawford

1 Department of Biological Sciences, Universidad de los Andes, Bogotá, código postal 111711, Colombia. 2 Department of Life Sciences, The Natural History Museum, South Kensington, London SW7 5BD, England, UK. 3 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721-088, USA.

Email addresses: [email protected] (LSB), [email protected] (JWS), [email protected] (ECM), [email protected] (JJW), [email protected] (AJC).

* Corresponding author: Department of Biological Sciences Universidad de los Andes Calle 19 No. 1–60 Bogotá, código postal 111711, Colombia, South America E-mail: [email protected] Cell phone: +57 315-891-8734

1 ABSTRACT

Aim: Infer the phylogenetic relationships among frogs of the family Eleutherodactylidae

(Anura: Terraranae) and assess the biogeographic history of the genera and subgenera of the family.

Location: Southern North America, Middle America, the Caribbean islands, and northern

South America.

Methods: Relationships among eleutherodactylid frogs were inferred based on a concatenated data matrix containing 1,909 ultraconserved elements (UCE loci) and four mitochondrial genes (12S, 16S, COI, Cyt b) for a total of 722,106 aligned base pairs. We used RAxML 8.0.19 to infer a maximum likelihood tree that was time-calibrated of the phylogenetic tree was performed using TreePL. And the historical biogeographic reconstruction was implemented in BioGeoBears under a stratified and no stratified analysis.

2 Results: The phylogenetic results recover the same phylogenetic relationships among the genus of the family Eleutherodactylidae ((Adelophryne + Phyzelaphryne) +

(Eleutherodactylus + Diasporus). The most remarkable differences in our results to previous studies are inside the internal relationships among the subgenera of Eleutherodactylus. The first finding is the no monophyly of the subgenera Syrrhophus and Euhyas. The second is are relationships in between the subgenera of the included Eleutherodactylus: (Pelorius

((Syrrhophus (Syrrhophus + Ehuyas)) + (Eleutherodactylus + Schwartzius)). The biogeographic results show that the best explanatory model was DIVALIKE, under that model the most probable area of the most recent common ancestor (MRCA) of

Eleutherodactylidae was South America (100%), followed by colonization of the Caribbean around 58 Ma (95% confidence interval (CI) of 60 – 29 Ma), then two independent dispersal events from the Caribbean to Middle America around 48 (95%) and 31 Ma (95%

CI interval of 32 – 27 Ma). The genus Diasporus is likely of South American origin (up to

70%), with a dispersal event to Middle America around 20 Ma (95% CI interval of 22 – 16

Ma). Finally, the lately biogeographic event in the family, the MRCA genus Phyzelaphryne colonize the Amazon basin around 12.5 Ma.

3 Main Conclusions: A) While the origin of the family is in South America, the vast family diversity is in Caribbean, B) Some of the diversification events in the genera support some of the hypothesized geological events. The colonization of the Eleutherodactylus overlap whit the end of the end of the proto-Antilles, but the radiation of the genera happened between 50 to 30 Ma. The origin of the MRCA of Diasporus is South America, and colonize Middle America near 20 Ma, that coincides with the closure time for the Panama

Isthmus proposed by Montes et al. (2015). The less diverse genus of the family is

Phyzelaphryne, that diversify near to 10 Ma and is distributed in the Amazon basin, is possible that the draining of the Pebas lake trigger that last event in the biogeography of the genera of the family, C) The diversification of the genera of the family Eleutherodactylidae using a combination of genomic and mtDNA data, give support to some of the geological hypothesis and the possible influence for some diversification events in northern South

America, the Caribbean, and Middle America.

KEYWORDS: Ancestral area reconstruction, Dispersal, GAARlandia, Great American

Biotic Interchange, Vicariance.

4 INTRODUCTION

The two primary processes to explain the distribution of the organisms are dispersal and vicariance events. In the 20th Century, vicariance explanation dominated historical biogeography for decades, in part due to the claim that hypotheses of dispersal were not falsifiable, that is, dispersal can explain any distribution. Vicariance, therefore, was the classical explanation for the disjunctions in the distribution of tropical organisms. However, phylogenetic inference and divergence-time estimation have indicated that long-distance dispersal has often played a significant role in shaping current distributions, resulting in a substantial shift in our view of historical biogeography (De Queiroz, 2005; Michalak et al.,

2010; Renner et al., 2010). Even iconic examples of vicariance such as the plant genera

Araucaria and Nothofagus have been demonstrated to disperse over long distances

(Setoguchi et al., 1998; Swenson et al., 2001; Cook & Crisp, 2005).

The Neotropics comprise one of the most species-rich region in the world. One explanation for this richness is the dynamic geological history and complex geography of this region (Smith et al., 2014). To understand the origin of the continental-scale patterns of

Neotropical biodiversity, we also need to consider the history of the connections between

South America, Middle America, and North America over the past 100 million years, as well as the dynamic history of the Caribbean islands. About 100 million years ago (Ma)

South America began to separate from Africa moving westward, leaving South America in

‘splendid isolation’ during most of the Cenozoic (Simpson, 1980).

The Neotropics can be divided up into three broad areas: tropical South America

(SA), tropical Middle America (MA), and the Caribbean islands (C). The extant biota in each of these three regions is the sum of endemic lineages plus colonists from the other two areas (Hedges and Heinicke, 2007; Heinicke et al., 2007). To explain these distributions

5 biologists using the advances in dating divergence times using molecular data and fossil calibrations show interchange events, have proposed hypotheses of temporal land bridges and island chains between North and South America that existed periodically from the Late

Cretaceous to the Oligocene. Each hypothesis is based on where the leading or trailing edge of the Caribbean Plate would have been located relative to the NA and SA continents. First, the Proto-Antilles (Rosen, 1985, 1975) is an hypothesized connection between SA and MA that may have existed around 80 to 65 Ma when the leading (eastern) edge of the Caribbean

Plate would have been located where Middle America is now. As the Caribbean Plate moved eastward, the Proto-Antilles would have been severed. Much later, around 35 to 33

Ma, the leading edge may have a second land-bridge connection between NA and SA called the Greater Antilles-Aves Ridge land bridge, or GAARlandia (Macphee & Iturralde- vinent, 1994; Woods & Sergile, 2001). Specifically, GAARlandia would have linked South

America (around modern-day ) to the Greater Antillean Islands in the Eocene–

Oligocene transition (Bacon et al., 2013; Iturralde-Vinent, 2006). This second connection, too, would have been quickly severed. The final connection involves the trailing or western edge of the Caribbean Plate forming the current Panamanian Isthmus. Of course, the exist of this connection is a fact, but controversy surrounds the date of final closure of the

Isthmus and separation of the Pacific Ocean from the Caribbean Sea. A recent hypothesis suggests this closure happened around 15 Ma (Montes et al., 2015), while the more traditional estimate is placed at 3 Ma (Coates and Obando, 1996). Here we use a group of frogs distributed in all three realms, NA, SA, and the Caribbean, to evaluated these three historical, geological hypotheses.

Amphibians have strong physiological and ecological restrictions that limit their dispersals, such as poikilothermy, permeable skin, and lack of cleidoic eggs (Duellman and

6 Trueb, 1994). Most species of frogs also have water-dependent , but here we focus on members of a clade of Neotropical frogs with direct-development.) These characteristics increase amphibians’ risk of dehydration and limit their ability to cross xeric terrestrial environments or salt water barriers (Blaustein and Bancroft, 2007; Cruz-Piedrahita et al.,

2018; Navas and Otani, 2007; Paz et al., 2015). For that reasons, this close relationship with their environments and low dispersal make amphibians a great model to study the drivers of diversity distributions.

We use the frog family Eleutherodactylidae (Anura: Terraranae) which is thought to have originated around 80 Ma, and is currently distributed in South America (13 species),

Middle America (13 species), and has the bulk of species diversity in the Caribbean (187 species, Figure S1). The frogs of the family Eleutherodactylidae have direct developing 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. For that reason the direct development release the dependency of developing individuals from water bodies such as streams and ponds, and increase 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). That’s make it an excellent model to determine whether the current distribution could be explained by the land connection and disconnection between South and Middle America during the last 80 Ma or if the better explanation is the overseas dispersal. And try to infer which ones must be explained by the migration along the land bridges, oversea dispersal, or a combination of these could describe the actual distribution of the members of the family.

7 MATERIALS AND METHODS

SAMPLING

DNA sequence data to resolve phylogenies are becoming increasingly abundant but often heterogeneous across taxa. For some molecular phylogenetic studies, the sampling includes large numbers of genes for a relatively small sample of species (Chiari et al., 2012;

Jarvis et al., 2014; Streicher et al., 2018; Streicher and Wiens, 2016). Other studies include hundreds or thousands of species but for a much smaller number of genes (Frost et al.,

2006; Pyron and Wiens, 2011).

Simulation and empirical studies have demonstrated that one can combine these two strategies, such that the data matrix has many genes for few taxa, and few genes for most taxa. Such a matrix would have widespread missing data, of course, but the benefits in terms of phylogenetic resolution appear to outweigh possible costs (Burleigh et al., 2009;

Wiens and Morrill, 2011; Zheng and Wiens, 2016).

Here we divide the sampling in two stages to improve the number of genes and taxa included. First, to obtain genomic-scale data, we obtained UCE data from 14

Eleutherodactylidae ingroup operational taxonomic units (OTUs), and four out-groups

(Leptodactylus mystaceus, Craugastor daryi, Espadarana prosoblepon, and Pristimantis miyatai; Table1). Our eleutherodactylid ingroup OTUs represented all major lineages

(subfamilies, genus, and subgenus), with the exception of the monotypic subgenus,

Schwartzius. To increase taxonomic sampling, we then added short mitochondrial DNA sequence data from 170 eleutherodactylids, including the subgenera Schwartzius (Table 2).

TARGETED SEQUENCE CAPTURE

8 We obtained genomic DNA (gDNA) of 14 eleutherodactylid frogs and 4 outgroups

(Table 1), from tissues that were storage in ethanol 96% or 20% DMSO in 0.25 M EDTA and NaCl saturated (Seutin et al., 1991). DNA extractions were made with NDeasy® Blood and tissue kit from Qiagen or with SeraPure beads after being digested overnight in 20 µL proteinase K and 180µL of lysis buffer. We used 360µL of SeraPure beads to capture the gDNA, and cleaned it with two baths of 700µL of 70 % ETOH. Finally, the gDNA was eluted in 70 µL of 10 mM Tris at PH 8.

To construct a phylogenomic data set for Eleutherodactylidae, we obtained UCEs data using the 5000 probes and published protocols Faircloth et al. (2012) using the modifications performed from Streicher et al. (2016). A post-hybridization PCR was conducted using Phusion enzyme and Truseq primers for 18. Enriched libraries were visualized for fragment size distribution and concentration using Bioanalyzer 7500 DNA chip sets (Agilent). We sequenced the capture library (48 individuals at a time, the library was mixed with different frogs of other studies) using 600 cycles sequencing paired-end run (i.e., PE300) on an Illumina MiSeq at the University of Texas at Arlington genomics core facility (Arlington, TX, USA; www.gcf.uta.edu).

DATA ANALYSIS

After using Trimommatic (http://dx.doi.org/10.1093/bioinformatics/btu170.) implemented in Illumiprocesor (https://github.com/faircloth-lab/illumiprocessor) to remove adapter contamination and to quality-trim sequence reads (Faircloth, 2013), we assembled reads into contigs using Velvet (version 2.1.7; Zerbino & Birney, 2008). We used phyluce version 1.5.0; Faircloth, 2015) to identify those contigs that were UCE loci, remove

9 putatively duplicate UCE loci, create a database of UCE loci recovered, and prepare

FASTA files for sequence alignment. We filtered the resulting alignments to create ‘de novo assembled’ data matrix: 80% of allowance of missing taxa per locus based on the amount of parsimony informative sites (Table 1).

MITOCHONDRIAL DATA

Mitochondrial DNA sequences from Eleutherodactylidae species plus four out- groups (Leptodactylus mystaceus, Craugastor daryi, Espadarana prosoblepon, and

Pristimantis miyatai), were obtained from GenBank (Table 2), representing 82% of the estimated species richness of the clade and including representatives from all recognized genera and subgenera. The selected genes for this study are the mitochondrial 12S rRNA

(12S), 16S rRNA (16S), cytochrome oxidase I (COI), and cytochrome oxidase b (cyt b).

PHYLOGENETIC METHODS

We used a concatenation of the genomic data (UCEs) and the concatenation of the genomic data (UCEs) plus the mitochondrial data (that were manually combined using

Geneious pro 8.0.5 Biomatters Ltd.). They were analyzed using maximum likelihood (ML) as implemented in RAxML v. 8.0.19 (Stamatakis, 2014), assuming the GTRGAMMA substitution model. We ran RAxML in two steps. First, we ran the ‘best’ tree search using

20 replicates. Second, we ran a bootstrap (bs) analysis using the autoMRE option, allowing for automatic determination of the sufficient number of bootstrap replicates.

ESTIMATING DIVERGENCE DATES

10 The combination of 100’s f taxa and 1000’s of genes makes inviable the implementation of calibration strategies based on Bayesian MCMC inference. For this reason, the optimal tree inferred from the combined dataset was assumed to estimate divergence times using penalized likelihood method (Sanderson, 2002) as implemented in treePL (Smith and O’Meara, 2012). Penalized likelihood uses a tree with branch lengths and age constraints without prior parametric distributions. The age constraints were taken from previous studies and implemented here as secondary calibrations using minimum and maximum ages based on robust divergence-time estimates for major amphibian lineages

(Heinicke et al. 2009; Gomez-Mestre et al. 2012; Pinto-Sánchez et al. 2012; Fouquet et al.

2012, 2013; Zhang et al. 2013; Pyron 2014; Frazão et al. 2015; Table 3). This ensures that age estimates for the large-scale tree presented here fit broadly within existing fossil- calibrated, temporal frameworks for amphibian evolution (Table 3). We were careful to avoid implementing any temporal estimates based on the same biogeographic events under investigation here, for that propose we identified the nodes that covered the temporal and taxonomic breadth of the stem-group age of the most recent common ancestor (MRCA) of the subfamilies and genus. We fixed the following internal nodes using estimated ages from

Heinicke et al. (2009); Gomez-Mestre et al. (2012); Pinto-Sánchez et al. (2012); Fouquet et al. (2012), (2013); Zhang et al. (2013); Pyron (2014); Frazão et al. (2015; Table 3)

BIOGEOGRAPHICAL RECONSTRUCTION

Eleutherodactylidae frogs are distributed throughout South America, Middle

America, and the Caribbean. All the species preserve their “natural” distribution with the exception of E. coqui and E. jonstonei that have several invasions to South and North

America, and different islands in the last 200 years (e.g. Hawaii, Guam, New Zeland;

11 CABI, 2018). We defined three distribution areas for our analyses based on the distribution of each genera defined by the UICN red list maps. The selected areas are the north of South

America (SA), the south of North America and Middle America (MA), and finally the

Caribbean islands (C). Numerous analytical methods for historical biogeography exist, all of these models contain similar elements, some of them have been unified in the R package

“BioGeoBEARS” (Matzke, 2013). This provides a flexible framework for comparing alternative models in a statistical context to explore alternative biogeographic scenarios, and also proposes a model of founder-event speciation (‘+J’) and allows the fit of models to be compared using a model choice procedure. We use two different approaches for the biogeographical analysis, the first one is a no stratified and the second a stratified analysis using geological events how could affect the availability of areas in a certain time (e.g. the

Proto-Antilles, the Pebas lake, or the formation of the amazon river; Table 4 to compare the impact of the inclusion of parameters in the analysis and to select the best fit model to explain the distribution of the frogs of the family.

RESULTS

SYSTEMATICS

Tree topologies from the phylogenetic analyses of the individual UCEs (Fig. 1) and the combined and concatenated (mtDNA + UCEs; Fig 1, S2) showed broad similarities among the subfamilies, genera, and subgenera. The only exception between the two topologies is Eleutherodactylus (Schwartzius) because that sample is in the mtDNA +

UCEs but not in the UCE data set. Our time-calibrated phylogeny obtained by the combination of mtDNA and UCEs includes 170 (Fig. S2) species of eleutherodactylid

12 frogs, or about 82% of current estimated diversity of the family. Overall, 67.2% of nodes are recovered with at least 70% bootstrap support.

Using the concatenated maximum likelihood, the tree inferred from the matrix allowing up to 80% missing data per locus, the phylogenetic relationships among

Eleutherodactylidae recovered the family as a monophyletic group (bs = 100%). Each of the subfamilies of Eleutherodactylidae (Eleutherodactylinae and Phyzelaphryninae) also has strong support (bs = 100%). Each of the genera of Eleutherodactylinae (Diasporus and

Eleutherodactylus) has strong support (bs = 100%), and also the genera of

Phyzelaphryninae represented by Phyzelaphryne miriame and Adelophryne adiastola

(Figure 1). In the recovered relationships among the subgenus of Eleutherodactylus,

Euhyas is the sister to Syrrhophus (bs = 100%) and this group is sister to Eleutherodactylus

(bs = 98%), and finally, Pelorius is the sister to all of the other Eleutherodactylus subgenera (bs = 100%).

The combined super-matrix of mitochondrial and UCE data recover the same relationships among the genera and subgenera, the main difference is the inclusion of the subgenus Schwartzius. The position of the subgenus Schwartzius is the sister to the genus

Eleutherodactylus (Eleutherodactylus), but the support of this relationship is low (bs =

55%). The subgenus Syrrhophus has recovered as a paraphyletic group; this subgenus is inserted in two different positions inside the subgenus Euhyas, the support of the group of

Syrrhophus and Euhyas is high (bs = 99%).

TIME DIVERGENCE ESTIMATIONS

The divergence time estimations based on the analysis of TreePL placing the ancestor of all the frogs of the family Eleutherodactylidae dates to the early Paleocene

13 approximately 80 Mya. The members of the subfamily Eleutherodactylinae 60 to 58 Mya, and Phyzelaphryninae near to 55 Mya. The ancestor of the genera Eleutherodactylus appear near to 57 to 55 Mya late Paleocene to the early Eocene. Diasporus ancestor near to 20

Mya late Oligocene. Phyzelaphryne 13 to 10 Mya middle of the Miocene, and Adelophryne

55 to 50 Mya late Paleocene to the early Eocene.

BIOGEOGRAPHIC AREA RECONSTRUCTION

Our biogeographic analyses using BioGeoBEARS validated the implementation of the DIVALIKE+J model under a stratified scenario (Landis, Matzke, Moore, &

Huelsenbeck, 2013; Figure 2, and S3, Table 5). The DIVALIKE model was recovered with

-lnL = 36.9. Marginally the DIVALIKE+J model performs better than the DIVALIKE only model (Table 5), suggesting that accounting for founder-event speciation in this clade had some important effects, although this was not the only process in operation (d = 0.004; e =

0; j = 0.0177; AICws = 0.492; Table 5). The examination of the graphical outputs suggests that there tends to be higher probabilities (=less ambiguity) for ancestral state area reconstructions under the DIVALIKE+J model than under the DIVALIKE only model

(Figure S3). Molecular calibration through TrePL sets the origin of the Eleutherodactylidae clade (Adelophryne + Eleutherodactylus + Diasporus + Phyzelaphryne) in the early

Paleocene near to 70 Mya, and the diversification within these lineages beginning in the late Paleocene to the early Eocene 60 to 55 Mya.

Ancestral area reconstructions using BioGeoBEARS suggest a South American

MRCA for the Eleutherodactylidae clade (Figure 2 and S3). Subsequent late Paleocene to the early Eocene divergences resulted in two major clades, Eleutherodactylinae and

Phyzelaphryninae (Figure 2). Results from both the DIVALIKE and DIVALIKE+J models

14 suggest that these clades have South American ancestral areas, and show evidence of southern to northern expansion in Eleutherodactylus (Figures 2-S3) and the MCRA of the genus Adelophryne continues in South America with a subsequent radiation to the MRCA of Phyzelaphryne close to 10 Mya to the Amazon basin. In the Eleutherodactylinae, the

Eleutherodactylus clade has an expansion from its MRCA is primarily related to the eleutherodactylid subgenus Pelorius (endemic to the Hispaniola Island) from mainland

(SA) to the Caribbean islands. Following expansion of the MRCA of the rain-frogs belong to the subgenus Eleutherodactylus and Schwartzius to the Caribbean, today Schwartzius is monotypic and endemic Hispaniola and Eleutherodactylus is distributed across the

Antillean Islands in its natural range. There is a first expansion of a MRCA to Middle

America from the Caribbean in the middle of the Eocene (near to 50 Mya) of E.

(Syrrhophus) longipeps, and a second incursion from the Caribbean Islands to Middle

America of the MCRA of frogs of the subgenus E. (Syrrhophus) happened next to 40 Mya. the remaining subgenera of the Eleutherodactylus expand to all the Caribbean islands. Over

23 to 20 Mya the MRCA of the remaining genera member of the subfamily

Eleutherodactylinae Diasporus expand its distribution for firs time from South America to

Middle America.

DISCUSSION

Phylogenetic and biogeographic history

The phylogenetic analysis of the rain-frogs that we present in this paper contains samples covering 82% of the described species of the family Eleutherodactylidae, and all the ranges of the genera and subgenera that composite the family. In general, the topology we recover is consistent with the classical taxonomy based on few or several loci (Feng et

15 al., 2017; Heinicke et al., 2018; Padial et al., 2014; Pyron and Wiens, 2011), the most remarkable difference is that our analysis reveals that the subgenus Pelorius is in fact paraphyletic with the subgenus Euhyas of Eleutherodactylus (Figure S2).

The biogeographical reconstruction that we infer shows South America as the most likely regions of origin of the rain-frogs next to 66.9 Mya. The MRCA of the subfamily

Phyzelaphryninae stay in South America and it origin is near to 57.4 Mya. and the MRCA of the subfamily Eleutherodactylinae being originated 59.7 Mya and have an early expansion to the Caribbean near to 58.6 Mya, possibly promoted by a fonder even, because for that time the Proto-Antilles formation has been ended, and the AVES ridge have not started. A fist expansion of Eleutherodactylus from the Caribbean to Middle America happened near to 48.5 Mya. a second group of Eleutherodactylus expanded to Middle

America from the Caribbean near to 31.2 Mya. The MRCA of the genus Diasporus has a first movement from South America to Middle America near to 23.5 Mya, and posterior expansion to Middle America near to 18 Mya. the MRCA of the genus Phyzelaphryne expands its distribution to the Amazon Basin near to 12.6 Mya.

The distribution of different frog’s lineages was attributed to an ancient overseas dispersal from South America towards Middle America and the proto Caribbean already has been discussed by Crawford and Smith, (2005), Heinicke et al. (2007), Vences et al.,

(2003), over the vicariant hypothesis. The vicariant model is unsupported by the fossil record and the model does not agree with the prevailing opinions of tectonic and faunal history (Pregill, 1981; Pregill et al., 1988). We found mixed support for vicariance and oceanic dispersion as the processes explaining the current distribution of the

Eleutherodactylidae family. However, the most important process was dispersal as suggested by the DIVALike model. This study sheds new light into the importance of

16 considering both vicariance and dispersal as potential drivers of current distributions that likely acted together in shaping the current patterns of biodiversity.

ACKNOWLEDGEMENTS

We are grateful for tissue loans from Museo Herpetológico de la Universidad de

Antioquia (MHUA), Museo de Historia Natural, Universidad Nacional Mayor de San

Marcos (MHNSM), Círculo Herpetológico de Panamá (CH), Museo de Historia Natural

ANDES at the Universidad de los Andes in Bogotá (ANDES), the Museum of Vertebrate

Zoology at the University of California, Berkeley (MVZ), Amphibian and Reptile Diversity

Research Center at the University of Texas at Arlington (UTA) and Museo de Zoología

Universidad de Costarica (UCR).The research and collecting permission was provided by the Autoridad Nacional de Licencias Ambientales (ANLA) de Colombia (permiso marco resolución No 1177 to the Universidad de los Andes)." This work was supported by the

Colciencias doctoral fellowship 567, Proyecto semilla Universidad de los Andes, FAPA grant AJC, and The University of Arizona and U.S. National Science Foundation Grant

DEB 1655690 to JJW. We thank to Camila Plata, Andres M. Cuervo, Luisa Castellanos,

Luisa Dueñas, Santiago Herrera, Laura Céspedes, Catalina Palacios, and Camila Gomez for providing valuable comments on earlier versions of this manuscript and to all of the

Biomi|cs lab members.

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26

TABLE 1 Voucher information and amount of DNA data produced for each sample, including number of contigs assembled using Velvet (v1.2.10), and the resulting number of aligned ultraconserved elements (UCEs) obtained. Sequence Read Archive (SRA) accession numbers provide access to all reads obtained for each individual. Museum numbers are from the following natural history collections: Museo Herpetológico de la

Universidad de Antioquia (MHUA), Círculo Herpetológico de Panamá (CH), Museo de

Historia Natural ANDES at the Universidad de los Andes in Bogotá (ANDES), the

Museum of Vertebrate Zoology at the University of California, Berkeley (MVZ),

Amphibian and Reptile Diversity Research Center at the University of Texas at Arlington

(UTA), Museum of Comparative Zoology at Harvard University (MCZ), and the

Biodiversity Institute & Natural History Museum at Kansas University (KU).

Field Museum SRA Taxon Number Contigs UCEs Number accession

Adelophryne AJC SAMN055598 adiastola 2463 ANDES-A 2560 3680 1515 73

Diasporus anthrax 4446 1038

Diasporus quidditus 2499 852

Diasporus gularis 3082 1000

Diasporus vocator 185 84

Diasporus tinker 2441 933

27 Eleutherodactylus atkinsi 1981 1081

Eleutherodactylus inoptatus 1597 935

Eleutherodactylus AJC johnstonei 3902 ANDES-A 1912 2970 1792

Eleutherodactylus JAC longipes 29834 UTA A- 2171 1243

Eleutherodactylus monensis 4362 1842

Eleutherodactylus zugi 1763 1027

Phyzelaphryne AJC miriame 3606 ANDES-A 3834 2170 512

Espadarana SAMN055598 prosoblepon MVZ 149741 6094 1851 86

Craugastor daryi MEA 622 UTA A-62648 191 71

Leptodactylus mystaceus 3458 1909

AJC

Pristimantis miyatai 3499 ANDES-A 1776 2562 1429

28 TABLE 2 Voucher information mtDNA data included for each sample, Gene Bank

accession numbers provide access for the mitochondrial loci included in this study.

Species Museum ID 12S 16S cytb coI Phyzelaphryne miriamae MTR12700 JX298268 JX298304 JX298395 JX298344 Adelophryne adiastola 1 AJC2463 JX298263 JX298299 JX298391 JX298340 Phyzelaphryne sp. 1a MTR19206 JX298270 JX298309 JX298399 JX298349 Phyzelaphryne sp. 1b JMP2058 JX298271 JX298311 JX298400 JX298355 Adelophryne sp. 7 MTR13808 JX298295 JX298389 JX298336 Adelophryne patamona PK1969 JX298260 JX298296 JX298390 JX298337 Adelophryne adiastola 2 AJC ------Adelophryne gutturosa PK1168 JX298266 JX298302 JX298393 JX298342 Adelophryne patamona 1 ROM43034 JX298261 JX298297 JX298338 Adelophryne pachydactyla MTR16244 JX298259 JX298294 JX298388 JX298335 Adelophryne sp. 5 CFBHE234 JX298254 JX298288 JX298383 JX298328 Adelophryne sp. 4 MTR13570 JX298256 JX298290 JX298384 JX298331 Adelophryne sp. 6 CFBH23672 JX298257 JX298292 JX298386 JX298333 Adelophryne maranguapensis CFBHT14119 JX298253 JX298286 JX298381 JX298326 Adelophryne sp. 1 CFBHT11716 JX298251 JX298284 JX298380 JX298324 Adelophryne baturitensis MTR14012 JX298248 JX298280 JX298375 JX298320 Adelophryne sp. 2 PEU80 JX298379 JX298323 Adelophryne gutturosa EU186679 EU186679 ------Craugastor daryi EF493531 EF493531 ------Diasporus diastema EU186682 EU186682 ------Diasporus vocator** JN991484 JN991419 ------Eleutherodactylus abbotti EF493540 EF493540 ------Eleutherodactylus acmonis EF493787 EF493637 ------Eleutherodactylus albipes EF493386 EF493386 ------Eleutherodactylus alcoae EF493382 EF493382 ------Eleutherodactylus alticola EF493768 EF493620 ------Eleutherodactylus amadeus EF493805 EF493644 ------Eleutherodactylus amplinympha EF493732 EF493560 ------Eleutherodactylus andrewsi EF493544 EF493623 ------Eleutherodactylus antillensis EF493728 EF493556 ------Eleutherodactylus apostates EF493811 EF493650 ------Eleutherodactylus armstrongi EF493778 EF493611 ------Eleutherodactylus atkinsi EF493797 EF493598 ------Eleutherodactylus audanti EU186662 EU186662 ------Eleutherodactylus auriculatoides EF493754 EF493572 ------

29 Eleutherodactylus auriculatus EF493344 EF493344 ------Eleutherodactylus bakeri EF493808 EF493647 ------Eleutherodactylus barlagnei EF493735 EF493563 ------Eleutherodactylus bartonsmithi EF493736 EF493576 GQ426514 --- Eleutherodactylus blairhedgesi EF493371 EF493606 ------Eleutherodactylus bothroboans EU186655 EU186655 ------Eleutherodactylus bresslerae EF493785 EF493635 ------Eleutherodactylus brevirostris EF493819 EF493657 ------Eleutherodactylus brittoni EF493727 EF493554 ------Eleutherodactylus caribe EF493385 EF493385 ------Eleutherodactylus casparii EF493788 EF493599 ------Eleutherodactylus cavernicola EF493763 EF493614 ------Eleutherodactylus chlorophenax EF493543 EF493589 ------Eleutherodactylus cochranae EF493725 EF493555 ------Eleutherodactylus cooki EF493539 EF493539 ------Eleutherodactylus coqui GQ345176 GQ345176 EF637038 --- Eleutherodactylus corona EF493807 EF493645 ------Eleutherodactylus counouspeus EF493719 EF493719 ------Eleutherodactylus cubanus EF493796 EF493594 ------Eleutherodactylus cundalli EF493761 EF493612 ------Eleutherodactylus cuneatus EF493775 EF493608 ------Eleutherodactylus darlingtoni EF493777 EF493610 ------Eleutherodactylus dimidiatus EF493802 EF493640 ------Eleutherodactylus dolomedes EF493809 EF493648 ------Eleutherodactylus eileenae EF493740 EF493577 GQ426518 --- Eleutherodactylus emiliae EF493368 EF493638 ------Eleutherodactylus eneidae EF493729 EF493557 ------Eleutherodactylus etheridgei EF493794 EF493593 ------Eleutherodactylus eunaster EF493804 EF493646 ------Eleutherodactylus flavescens EF493731 EF493559 ------Eleutherodactylus fowleri EF493752 EF493568 ------Eleutherodactylus furcyensis EF493814 EF493654 ------Eleutherodactylus fuscus EF493769 EF493618 ------Eleutherodactylus glamyrus EF493737 EF493575 GQ426515 --- Eleutherodactylus glandulifer EF493816 EF493655 ------Eleutherodactylus glanduliferoides EF493546 EF493364 ------Eleutherodactylus glaphycompus EF493383 EF493383 ------Eleutherodactylus glaucoreius EF493762 EF493613 ------Eleutherodactylus goini EF493791 EF493604 ------Eleutherodactylus gossei EF493716 EF493716 ------

30 Eleutherodactylus grabhami EF493772 EF493624 ------Eleutherodactylus grahami EF493781 EF493632 ------Eleutherodactylus greyi EF493801 EF493628 ------Eleutherodactylus griphus EF493381 EF493381 ------Eleutherodactylus gryllus EF493724 EF493552 ------Eleutherodactylus guanahacabibes EF493789 EF493600 ------Eleutherodactylus guantanamera EF493749 EF493565 ------Eleutherodactylus gundlachi EF493798 EF493597 ------Eleutherodactylus haitianus EF493743 EF493583 ------Eleutherodactylus hedricki EF493726 EF493553 ------Eleutherodactylus heminota EF493806 EF493649 ------Eleutherodactylus hypostenor EF493757 EF493585 ------Eleutherodactylus iberia EF493374 EF493591 ------Eleutherodactylus inoptatus EF493380 EF493380 ------Eleutherodactylus intermedius EF493799 EF493595 ------Eleutherodactylus ionthus EF493748 EF493564 ------Eleutherodactylus jamaicensis EF493770 EF493621 ------Eleutherodactylus jaumei EU186672 EU186672 ------Eleutherodactylus johnstonei EF493733 EF493561 ------Eleutherodactylus jugans EF493810 EF493652 ------Eleutherodactylus junori EF493764 EF493617 ------Eleutherodactylus klinikowskii EF493547 EF493363 ------Eleutherodactylus lamprotes EF493379 EF493379 ------Eleutherodactylus leberi EF493342 EF493342 ------Eleutherodactylus lentus EF493717 EF493717 ------Eleutherodactylus leoncei EF493375 EF493715 ------Eleutherodactylus limbatus EF493795 EF493590 ------Eleutherodactylus locustus EF493730 EF493558 ------Eleutherodactylus luteolus EF493545 EF493545 ------Eleutherodactylus maestrensis EF493369 EF493639 ------Eleutherodactylus mariposa EF493738 EF493573 GQ426516 --- Eleutherodactylus marnockii DQ283102 DQ283101 ------Eleutherodactylus martinicensis EF493343 EF493343 ------Eleutherodactylus melacara EF493751 EF493567 ------Eleutherodactylus minutus EF493741 EF493578 ------Eleutherodactylus monensis EF493783 EF493633 ------Eleutherodactylus nitidus EU186730 EU186712 ------Eleutherodactylus nortoni EF493760 EF493588 ------Eleutherodactylus nubicola EF493771 EF493622 ------Eleutherodactylus orcutti EF493767 EF493619 ------

31 Eleutherodactylus orientalis EF493373 EF493592 ------Eleutherodactylus oxyrhyncus EF493812 EF493651 ------Eleutherodactylus pantoni EF493766 EF493616 ------Eleutherodactylus parabates EF493746 EF493581 ------Eleutherodactylus parapelates EF493758 EF493587 ------Eleutherodactylus patriciae EF493755 EF493570 ------Eleutherodactylus paulsoni EF493815 EF493659 ------Eleutherodactylus pentasyringos EF493765 EF493615 ------Eleutherodactylus pezopetrus EF493793 EF493601 ------Eleutherodactylus pictissimus EF493782 EF493631 ------Eleutherodactylus pinarensis EF493792 EF493607 ------Eleutherodactylus pinchoni EF493734 EF493562 ------Eleutherodactylus pipilans EU186729 EU186711 ------Eleutherodactylus pituinus EF493747 EF493582 ------Eleutherodactylus planirostris EF493346 EF493346 ------Eleutherodactylus poolei EF493742 EF493579 ------Eleutherodactylus portoricensis EF493720 EF493548 EF636947 --- Eleutherodactylus principalis FJ527411 GQ426528 GQ426513 --- Eleutherodactylus probolaeus EF493784 EF493634 ------Eleutherodactylus rhodesi EF493779 EF493629 ------Eleutherodactylus richmondi EF493541 EF493541 ------Eleutherodactylus ricordii EF493786 EF493636 ------Eleutherodactylus riparius Y10944 X86310 ------Eleutherodactylus rivularis EF493376 EF493626 ------Eleutherodactylus rogersi EF493372 EF493603 ------Eleutherodactylus ronaldi EF493739 EF493574 GQ426517 --- Eleutherodactylus rufifemoralis EF493813 EF493653 ------Eleutherodactylus ruthae EF493759 EF493586 ------Eleutherodactylus schmidti EU186653 EU186653 ------Eleutherodactylus schwartzi EF493723 EF493551 ------Eleutherodactylus sciagraphus EF493817 EF493656 ------Eleutherodactylus sisyphodemus EF493773 EF493625 ------Eleutherodactylus sommeri EU186654 EU186654 ------Eleutherodactylus symingtoni EF493821 EF493643 ------Eleutherodactylus thomasi EF493370 EF493605 ------Eleutherodactylus thorectes EF493384 EF493384 ------Eleutherodactylus toa EF493774 EF493627 ------Eleutherodactylus tonyi EF493790 EF493602 ------Eleutherodactylus turquinensis EF493776 EF493609 ------Eleutherodactylus unicolor EF493542 EF493542 ------

32 Eleutherodactylus varians EF493750 EF493566 ------Eleutherodactylus varleyi EF493800 EF493596 ------Eleutherodactylus ventrilineatus EF493818 EF493658 ------Eleutherodactylus weinlandi EF493780 EF493630 ------Eleutherodactylus wetmorei EU186652 EF493569 ------Eleutherodactylus wightmanae EU186651 EU186651 ------Eleutherodactylus zeus EF493718 EF493718 ------Eleutherodactylus zugi EF493347 EF493347 ------

33 TABLE 3 Nodes used for the calibration times estimated in TreePL, maximum and minimum age for the steam age of each genus, and the source reference.

Min age Max age Node node Source node (Ma) (Ma)

Gomez-Mestre et al., 2012; Pyron, Eleutherodactylus 29 58 2014

Pinto-Sánchez et al., 2012; Pyron, Diasporus 16.4 22.2 2014

Phyzelaphryne 13.2 13.2 Fouquet et al., 2012

Adelophryne 26 42 Fouquet et al., 2012; Pyron, 2014

Fouquet et al., 2013; Frazão et al.,

2015; Gomez-Mestre et al., 2012;

Heinicke et al., 2009; Heinicke, Craugastoridae 52 79 Duellman, & Hedges, 2007; Pinto-

Sánchez et al., 2012; Pyron, 2014;

Zhang et al., 2013b

Frazão et al., 2015; Gomez-Mestre

Centrolenidae 26 33 et al., 2012; Pyron, 2014; Zhang et

al., 2013

34 Fouquet et al., 2013; Frazão et al.,

Leptodactylidae 59 68 2015; Gehara et al., 2014; Gomez-

Mestre et al., 2012; Pyron, 2014

35 TABLE 4 Geological events used to stratify the analysis in BioGeoBEARS with the

estimated time of each.

Geological event Time Source

Panama land bridge

sensu Coates and 5 Coates & Obando, 1996

Obando.

Draining the Pebas 11-13.6 Hoorn et al. 2010 lake

Panama land bridge 25-15 Montes et al., 2015

Macphee & Iturralde-

GAARlandia 35-33 vinent, 1994; Woods &

Sergile, 2001

Proto-Antilles 80-65 Rosen, 1975, 1985

TABLE 5 BioGeoBears model results to select the best model scenario. All the models

were analyzed with and without stratification. In color grey is indicated the best model

selected under the –LnL, AIC, and AICwt.

Founder Dispersion Biogeographic Number of Extinction event -LnL probability AIC AIC wt model parameters probability probability (d) (e) (j)

No stratification analysis

DEC -75.5500 3 0.02000 0.042000 0.10000 157.1000 5.00E-17

36 DEC+J -39.7100 3 0.00220 1.00E-12 0.04000 85.4200 0.180000

DIVALIKE -43.5400 2 0.00350 1.00E-12 0 91.0800 0.011000

DIVALIKE+J -40.6600 3 0.00260 1.00E-12 0.03700 87.3200 0.071000

BAYAREALIKE -47.9800 2 0.00250 0.018000 0 99.9600 0.000100

BAYAREALIKE+J -38.3300 3 0.00110 1.00E-12 0.04600 82.6600 0.730000

Stratification analysis

DEC -36.1942 2 0.00436 1.00E-12 0 76.3884 0.204040

DEC+J -35.1981 3 0.00347 1.00E-12 0.01579 76.3963 0.203230

DIVALIKE -36.8991 2 0.00583 1.00E-12 0 77.7983 0.100820

DIVALIKE+J -34.3142 3 0.00425 1.00E-12 0.01768 74.6284 0.491910

BAYAREALIKE -62.4424 2 0.00338 0.002102 0 128.8848 8.13E-13

BAYAREALIKE+J -46.5856 3 0.00297 0.000268 0.02484 99.1712 2.30E-06

37 TABLE 6. Comparison of the fit of the biogeographical reconstruction models under stratification BayArea (BAYEAREALIKE),

dispersal– extinction–cladogenesis (DEC) and dispersal–vicariance analysis (DIVALIKE), all with the possibility of founder-event

speciation (‘+J’). We used these models to infer the ancestral geographical ranges of rain-frogs on our phylogeny. Results are ordered

by model fit. The log-likelihood (lnL) of each model is given as well as the Akaike information criterion (AIC) values.

AIC AIC

LnL DF DF AIC weight weight alt null LnL alt DF D statistic p-val AIC 1 AIC 2 AIC wt2 null alt null wt1 ratio ratio

model1 model2

DEC+J DEC -35.2 -36.19 3 2 1 1.99 0.16 76.4 76.39 0.5 0.5 1 1

74.6

DIVALIKE+J DIVALIKE -34.31 -36.9 3 2 1 5.17 0.023 3 77.8 0.83 0.17 4.88 0.2

1.80E- 99.1 283281

BAYAREALIKE+J BAYAREALIKE -46.59 -62.44 3 2 1 31.71 08 7 128.9 1 3.50E-07 9 3.50E-07

38

FIGURE 1 Relationships among Eleutherodactylidae frogs based on a concatenated ML inferred in RAxML 8.0.19. Numbers below nodes indicate bootstrap analysis. The data matrix included 1909 UCE loci for a total of 718,354 aligned base pairs, and included loci with up to 80% missing data. Numbers next to each node indicate the bootstrap support value.

FIGURE 2 Simplified BioGeoBears tree DIVALIKE+J (d=0.004; e=0 j=0.0177; LnL= -

34.31) concatenated ML tree of the Eleutherodactylidae inferred in RAxML 8.0.19. and dated using TreePL, this program doesn’t give error bars because this program does no estimate it. Letters from A to C represent the areas (see inlayed map) used for the biogeographical reconstructions. the north of South America (SA), the south of North

America and Middle America (MA), and finally the Caribbean islands (C). Colored pie charts show the probability of each area on specific nodes. Outgroup species were only used to root the tree and are not shown.

39 Fig 1

Phyzelaphryne miriame Phyzelaphryninae 100 Adelophryne adiastola

44 Diasporus gularis

Diasporus tinker 100 100 Diasporus anthrax

4 5 Diasporus quidditus

6 1 100 Diasporus vocator Eleutherodactylus (Pelorius) inoptatus Eleutherodactylinae Eleutherodactylus (Eleutherodactylus)i johnstonei 100

9 8 Eleutherodactylus (Syrrhophus) longipes

100 Eleutherodactylus (Euhyas) monensis

9 9 Eleutherodactylus (Euhyas) zugi

9 4 Substitutions/site Eleutherodactylus (Euhyas) atkinsi 0.005

40 DIVALIKE+J A South America d=0.004 e=0 B Middle America j=0.0177 C Antilles LnL= -34.31 AB South America + Middle America AC South America + Antilles BC Middle America + Antilles

Paleocene Eocene Oligocene Miocene 70 60 50 40 30 20 10 0

60 Ma 40 Ma 20 Ma 5 Ma

B Diasporus diastema

A Diasporus anthrax

AB Diasporus vocator

B Diasporus aff. hylaeformis

B Diasporus citrinobapheus

B Diasporus tigrillo

AB Diasporus quidditus

AB Diasporus sp CH4717

A Diasporus cf gularis

C Eleutherodactylus (Eleutherodactylus)

C Eleutherodactylus (Schwartzius)

C Eleutherodactylus (Ehuyas) 1

C Eleutherodactylus (Ehuyas) albipes

C Eleutherodactylus (Ehuyas) maestrensis

C Eleutherodactylus (Ehuyas) emiliae

C Eleutherodactylus (Ehuyas) dimidiatus

C Eleutherodactylus (Ehuyas) schmidti

B Eleutherodactylus (Syrrhophus) marnockii

B Eleutherodactylus (Syrrhophus) pipilans

B Eleutherodactylus (Syrrhophus) nitidus

C Eleutherodactylus (Ehuyas) symingtoni

C Eleutherodactylus (Ehuyas) zeus C Eleutherodactylus (Ehuyas) 2 C Eleutherodactylus (Ehuyas) zugi

C Eleutherodactylus (Ehuyas) klinikowskii

B Eleutherodactylus (Syrrhophus) longipes C Eleutherodactylus (Pelorius)

A Adelophryne sp.2

A Adelophryne baturitensis

A Adelophryne sp.1

A Adelophryne maranguapensis

A Adelophryne sp.6

A Adelophryne sp.4

A Adelophryne sp.5

A Adelophryne pachydactyla

A Adelophryne patamona

A Adelophryne gutturosa

A Adelophryne adiastola 2

A Adelophryne adiastola 1

A Adelophryne patamona

A Adelophryne sp. 7

A Phyzelaphryne sp.1b

A Phyzelaphryne miriame

Paleocene Eocene Oligocene Miocene 70 60 50 40 30 20 10 0 Millions of years ago A -> C A -> B C -> B C -> B A -> B 41 APENDIX

FIGURE S1 Actual distribution of the frog genera of the family Eleutherodactylidae based on the UICN red list distribution maps. Number of species for each genus next to each distribution. Red represent the distribution of Eleutherodactylus, green is the actual distribution of Diasporus, dark blue Adelophryne, light blue Phyzelaphryne.

FIGURE S2 Relationships among Eleutherodactylidae frogs based on a concatenated

UCEs (The data matrix included 1909 UCE loci Included loci with up to 80% missing data) and three mitochondrial loci (12S, 16S, COI, CytB) for a total of 722,106 aligned base pairs. the ML analysis inferred in RAxML 8.0.19. Numbers next to each node indicate the bootstrap support value.

FIGURE S3 Biogeographic analysis performed in BioGeoBears for four different models plus the founder event (J) DEC; DEC+I; DIVALIKE; DIVALIKE+J; BAYESAREALIKE; and BAYESAREALIKE+J, based on the ultrametric tree of the relationships among

Eleutherodactylidae frogs based on a concatenated UCEs (The data matrix included 1909

UCE loci Included loci with up to 80% missing data) and three mitochondrial loci (12S,

16S, COI, CytB) for a total of 722,106 aligned base pairs. the ML analysis inferred in

RAxML 8.0.19. The time calibration was established using TreePL and secondary calibration nodes obtained from the literature (see Table 3).

42 Fig. S1

N

191

15

6

1

0 800

Kilometers

43 Fig S2

100 Phyzelaphryne miriamae Phyzelaphryne sp.1b Adelophryne sp.7 9 8 100 Adelophryne patamona 6 4

100 Adelophryne gutturosa 100 9 7 Adelophryne adiastola Adelophryne pachydactyla 100

7 2 Adelophryne sp.6 8 3 Adelophryne sp.5 8 8 Adelophryne sp.4

4 0 Adelophryne maranguapensis

100 Adelophryne sp.1

4 5 Adelophryne baturitensis 100 Adelophryne sp.2 Diasporus diastema 9 9 4 4 Diasporus gularis Diasporus sp CH4717 “tinker” 100 Diasporus anthrax 4 7 Diasporus vocator 5 7 Diasporus aff. hylaeformis 5 6

8 0 Diasporus quidditus 100 Diasporus tigrillo Diasporus citrinobapheus Eleutherodactylus inoptatus 100 100 Eleutherodactylus nortoni Eleutherodactylus chlorophenax Pelorius 8 6 100 Eleutherodactylus ruthae

100 Eleutherodactylus bothroboans

9 2 Eleutherodactylus hypostenor 100 Eleutherodactylus parapelates Eleutherodactylus counouspeus Schwartzius 9 8 Eleutherodactylus unicolor Eleutherodactylus richmondi

7 5 100 Eleutherodactylus barlagnei

100 Eleutherodactylus pinchoni Eleutherodactylus johnstonei

9 4 100 Eleutherodactylus martinicensis 5 1 Eleutherodactylus amplinympha

100 Eleutherodactylus auriculatoides Eleutherodactylus patriciae

100 Eleutherodactylus fowleri 9 8 4 7 Eleutherodactylus lamprotes

9 9 100 Eleutherodactylus wetmorei 5 5 8 6 Eleutherodactylus sommeri

100 Eleutherodactylus leberi

100 Eleutherodactylus melacara

100 Eleutherodactylus varians 9 9 Eleutherodactylus guantanamera Eleutherodactylus ionthus

9 2 Eleutherodactylus flavescens

100 Eleutherodactylus cooki 100 100 Eleutherodactylus eneidae Eleutherodactylus locustus 9 7 Eleutherodactylus antillensis 8 8

9 5 Eleutherodactylus brittoni 7 5 Eleutherodactylus hedricki Eleutherodactylus 8 1 Eleutherodactylus cochranae Eleutherodactylus gryllus 7 4 6 7 8 2 9 4 Eleutherodactylus schwartzi

7 7 Eleutherodactylus coqui Eleutherodactylus portoricensis 7 0 Eleutherodactylus wightmanae

8 1 Eleutherodactylus poolei Eleutherodactylus minutus Eleutherodactylus pituinus 100 6 9 1 8 Eleutherodactylus haitianus

6 9 Eleutherodactylus abbotti

3 4 Eleutherodactylus parabates 3 8 Eleutherodactylus audanti Eleutherodactylus eileenae

7 1 100 Eleutherodactylus ronaldi

100 Eleutherodactylus mariposa Eleutherodactylus glamyrus

5 2 9 5 Eleutherodactylus auriculatus Eleutherodactylus bartonsmithi Eleutherodactylus longipes

100 Eleutherodactylus klinikowskii Eleutherodactylus zugi

100 Eleutherodactylus zeus

9 9 Eleutherodactylus symingtoni

100 Eleutherodactylus marnockii 100 Eleutherodactylus nitidus 6 9 9 9 Eleutherodactylus pipilans Eleutherodactylus schmidti 100 Eleutherodactylus albipes 100 Eleutherodactylus maestrensis 44 6 6 6 8 100 Eleutherodactylus dimidiatus Eleutherodactylus emiliae

100 Eleutherodactylus glandulifer 9 2 9 4 Eleutherodactylus sciagraphus Eleutherodactylus brevirostris 6 3 100 Eleutherodactylus ventrilineatus Eleutherodactylus paulsoni

100 Eleutherodactylus furcyensis 7 4 Eleutherodactylus rufifemoralis

100 Eleutherodactylus apostates 7 7 Eleutherodactylus oxyrhyncus

8 9 Eleutherodactylus jugans Eleutherodactylus glanduliferoides 4 6 8 6 Eleutherodactylus glaphycompus 7 4 3 1 Eleutherodactylus dolomedes 5 9 Eleutherodactylus thorectes

9 Eleutherodactylus bakeri

1 1 Eleutherodactylus heminota

1 9 Eleutherodactylus amadeus

3 8 Eleutherodactylus corona 2 5 Eleutherodactylus caribe 9 5 Eleutherodactylus eunaster Eleutherodactylus greyi

3 1 Eleutherodactylus probolaeus Eleutherodactylus monensis 100 Eleutherodactylus pictissimus 100 Eleutherodactylus grahami

8 0 Eleutherodactylus lentus

7 3 9 2 Eleutherodactylus weinlandi 8 1 Eleutherodactylus rhodesi Eleutherodactylus atkinsi

9 6 Eleutherodactylus gundlachi 100 Eleutherodactylus intermedius 6 7 Eleutherodactylus varleyi 9 0 8 2 Eleutherodactylus cubanus 6 8 Eleutherodactylus orientalis

4 4 Eleutherodactylus etheridgei 7 8

100 Eleutherodactylus iberia 8 3 100 Eleutherodactylus limbatus Eleutherodactylus jaumei

100 Eleutherodactylus guanahacabibes 100 Eleutherodactylus casparii Eleutherodactylus planirostris 8 8 4 3 Eleutherodactylus goini

9 3 Eleutherodactylus rogersi 6 8 4 4 Eleutherodactylus tonyi Eleutherodactylus pezopetrus

2 8 8 8 Eleutherodactylus pinarensis 100 Eleutherodactylus blairhedgesi Eleutherodactylus thomasi

100 Eleutherodactylus acmonis Eleutherodactylus ricordii 7 5 Eleutherodactylus bresslerae

9 7 Eleutherodactylus alcoae 8 5 Euhyas 7 9 Eleutherodactylus armstrongi + 100 Eleutherodactylus leoncei Eleutherodactylus darlingtoni Syrrhopus

3 4 100 Eleutherodactylus turquinensis Eleutherodactylus cuneatus Eleutherodactylus toa 6 8 4 4 Eleutherodactylus rivularis 5 6 Eleutherodactylus riparius

4 6 7 4 Eleutherodactylus sisyphodemus 7 8 Eleutherodactylus cavernicola

6 4 Eleutherodactylus grabhami 4 4 Eleutherodactylus jamaicensis

4 5 Eleutherodactylus nubicola Eleutherodactylus andrewsi

2 7 Eleutherodactylus orcutti Eleutherodactylus fuscus 3 3 4 2 Eleutherodactylus luteolus

1 5 Eleutherodactylus alticola

2 8 Eleutherodactylus junori

6 5 Eleutherodactylus gossei Eleutherodactylus griphus 3 9 Eleutherodactylus pantoni 3 2 7 8 Eleutherodactylus pentasyringos 2 3 0.04 9 6 Eleutherodactylus cundalli Eleutherodactylus glaucoreius 100 Phyzelaphryne miriamae Phyzelaphryne sp.1b Adelophryne sp.7 9 8 100 Adelophryne patamona 6 4

100 Adelophryne gutturosa 100 9 7 Adelophryne adiastola Adelophryne pachydactyla 100

7 2 Adelophryne sp.6 8 3 Adelophryne sp.5 8 8 Adelophryne sp.4

4 0 Adelophryne maranguapensis

100 Adelophryne sp.1

4 5 Adelophryne baturitensis 100 Adelophryne sp.2 Diasporus diastema 9 9 4 4 Diasporus gularis Diasporus sp CH4717 “tinker” 100 Diasporus anthrax 4 7 Diasporus vocator 5 7 Diasporus aff. hylaeformis 5 6

8 0 Diasporus quidditus 100 Diasporus tigrillo Diasporus citrinobapheus Eleutherodactylus inoptatus 100 100 Eleutherodactylus nortoni Eleutherodactylus chlorophenax Pelorius 8 6 100 Eleutherodactylus ruthae

100 Eleutherodactylus bothroboans

9 2 Eleutherodactylus hypostenor 100 Eleutherodactylus parapelates Eleutherodactylus counouspeus Schwartzius 9 8 Eleutherodactylus unicolor Eleutherodactylus richmondi

7 5 100 Eleutherodactylus barlagnei

100 Eleutherodactylus pinchoni Eleutherodactylus johnstonei

9 4 100 Eleutherodactylus martinicensis 5 1 Eleutherodactylus amplinympha

100 Eleutherodactylus auriculatoides Eleutherodactylus patriciae

100 Eleutherodactylus fowleri 9 8 4 7 Eleutherodactylus lamprotes

9 9 100 Eleutherodactylus wetmorei 5 5 8 6 Eleutherodactylus sommeri

100 Eleutherodactylus leberi

100 Eleutherodactylus melacara

100 Eleutherodactylus varians 9 9 Eleutherodactylus guantanamera Eleutherodactylus ionthus

9 2 Eleutherodactylus flavescens

100 Eleutherodactylus cooki 100 100 Eleutherodactylus eneidae Eleutherodactylus locustus 9 7 Eleutherodactylus antillensis 8 8

9 5 Eleutherodactylus brittoni 7 5 Eleutherodactylus hedricki Eleutherodactylus 8 1 Eleutherodactylus cochranae Eleutherodactylus gryllus 7 4 6 7 8 2 9 4 Eleutherodactylus schwartzi

7 7 Eleutherodactylus coqui Eleutherodactylus portoricensis 7 0 Eleutherodactylus wightmanae

8 1 Eleutherodactylus poolei Eleutherodactylus minutus Eleutherodactylus pituinus 100 6 9 1 8 Eleutherodactylus haitianus

6 9 Eleutherodactylus abbotti

3 4 Eleutherodactylus parabates 3 8 Eleutherodactylus audanti Eleutherodactylus eileenae

7 1 100 Eleutherodactylus ronaldi

100 Eleutherodactylus mariposa Eleutherodactylus glamyrus

5 2 9 5 Eleutherodactylus auriculatus Eleutherodactylus bartonsmithi Eleutherodactylus longipes

100 Eleutherodactylus klinikowskii Eleutherodactylus zugi

100 Eleutherodactylus zeus

9 9 Eleutherodactylus symingtoni

100 Eleutherodactylus marnockii 100 Eleutherodactylus nitidus 6 9 9 9 Eleutherodactylus pipilans Eleutherodactylus schmidti 100 Eleutherodactylus albipes 100 Eleutherodactylus maestrensis

6 6 6 8 100 Eleutherodactylus dimidiatus Eleutherodactylus emiliae

100 Eleutherodactylus glandulifer 9 2 9 4 Eleutherodactylus sciagraphus Eleutherodactylus brevirostris 6 3 100 Eleutherodactylus ventrilineatus Eleutherodactylus paulsoni

100 Eleutherodactylus furcyensis 7 4 Eleutherodactylus rufifemoralis

100 Eleutherodactylus apostates 7 7 Eleutherodactylus oxyrhyncus

8 9 Eleutherodactylus jugans Eleutherodactylus glanduliferoides 4 6 8 6 Eleutherodactylus glaphycompus 7 4 3 1 Eleutherodactylus dolomedes 5 9 Eleutherodactylus thorectes

9 Eleutherodactylus bakeri

1 1 Eleutherodactylus heminota

1 9 Eleutherodactylus amadeus

3 8 Eleutherodactylus corona 2 5 Eleutherodactylus caribe 9 5 Eleutherodactylus eunaster Eleutherodactylus greyi

3 1 Eleutherodactylus probolaeus Eleutherodactylus monensis 100 Eleutherodactylus pictissimus 100 Eleutherodactylus grahami

8 0 Eleutherodactylus lentus

7 3 9 2 Eleutherodactylus weinlandi 8 1 Eleutherodactylus rhodesi Eleutherodactylus atkinsi

9 6 Eleutherodactylus gundlachi 100 Eleutherodactylus intermedius 6 7 Eleutherodactylus varleyi 9 0 8 2 Eleutherodactylus cubanus 6 8 Eleutherodactylus orientalis

4 4 Eleutherodactylus etheridgei 7 8

100 Eleutherodactylus iberia 8 3 100 Eleutherodactylus limbatus Eleutherodactylus jaumei

100 Eleutherodactylus guanahacabibes 100 Eleutherodactylus casparii Eleutherodactylus planirostris 8 8 4 3 Eleutherodactylus goini

9 3 Eleutherodactylus rogersi 6 8 4 4 Eleutherodactylus tonyi Eleutherodactylus pezopetrus

2 8 8 8 Eleutherodactylus pinarensis 100 Eleutherodactylus blairhedgesi Eleutherodactylus thomasi

100 Eleutherodactylus acmonis Eleutherodactylus ricordii 7 5 Eleutherodactylus bresslerae

9 7 Eleutherodactylus alcoae 8 5 Euhyas 7 9 Eleutherodactylus armstrongi + 100 Eleutherodactylus leoncei Eleutherodactylus darlingtoni Syrrhopus

3 4 100 Eleutherodactylus turquinensis Eleutherodactylus cuneatus Eleutherodactylus toa 6 8 4 4 Eleutherodactylus rivularis 5 6 Eleutherodactylus riparius

4 6 7 4 Eleutherodactylus sisyphodemus 7 8 Eleutherodactylus cavernicola

6 4 Eleutherodactylus grabhami 4 4 Eleutherodactylus jamaicensis

4 5 Eleutherodactylus nubicola Eleutherodactylus andrewsi

2 7 Eleutherodactylus orcutti Eleutherodactylus fuscus 3 3 4 2 Eleutherodactylus luteolus

1 5 Eleutherodactylus alticola

2 8 Eleutherodactylus junori

6 5 Eleutherodactylus gossei Eleutherodactylus griphus 3 9 Eleutherodactylus pantoni 3 2 7 8 Eleutherodactylus pentasyringos 2 3 0.04 9 6 Eleutherodactylus cundalli Eleutherodactylus glaucoreius

45 Chapter 3 – Phylogeography of dink frogs (Eleutherodactylidae: Diasporus):

Phylogenetics, cryptic diversity, and correlates.

Lucas S. Barrientos1, Andrea Paz2,3, Cesar Jaramillo3, 4, Juan M. Daza5, Adrian García6, Abel Batista7, 8, Andreas Hertz9, 10, Mason Ryan11, 12, Roberto Ibañez 3, 4, Andrew J. Crawford1, 3, 4

1 Department of Biological Sciences, Universidad de los Andes, Bogotá, código postal 111711, Colombia.

2 Biology program, The Graduate Center, City University of New York, New York, USA

3 Biology Department, City College of New York, New York, USA

3 Smithsonian Tropical Research Institute; Apartado 0843–03092, Panamá, Republic of Panama.

4 Círculo Herpetológico de Panamá, Apartado 0824-00122, Panama City, Republic of Panama.

5 Instituto de Biología; Universidad de Antioquia; Medellín, Colombia

6 Laboratório de Biogeografia, Macroecologia e Biologia Evolutiva; Departamento de Ecologia, Universidade Federal do Rio Grande do Norte; Natal - RN, Brasil, 59078-900.

7 Universidad XYZ, David, Apartado 0426-01459, Chiriquí, Republic of Panama.

8 Instituto Senckenberg. Senckenberganlage 25, D-60325, Frankfurt am Main, Germany.

9 Department of Herpetology, Senckenberg Research Institute and Nature Museum, Senckenberganlage 25, 60325 Frankfurt am Main, Germany

10 Johann Wolfgang Goethe-University. Institute for Ecology, Evolution & Diversity. BioCampus – Westend. Siesmayerstr. 70. 60323 Frankfurt am Main, Germany

11 Department of Biology and Museum of Southwestern Biology, University of New Mexico, Albuquerque, New Mexico 87131-0001 USA

12 Tropical Forestry Initiative, Tres Piedras, Costa Rica

Email addresses: [email protected] (LSB), [email protected] (AP), [email protected] (CJ), [email protected] (JMD), [email protected] (AG),

1 [email protected] (AB), [email protected] (AH), [email protected] (MR), [email protected] (RI), ), [email protected] (AJC).

Corresponding author:

Address for correspondence:

2 ABSTRACT

Diasporus also called dink frogs is a genus of Neotropical direct-developing frogs currently composed of 15 nominal species ranging throughout the wet, biographic Chocó region from Honduras to Ecuador. The taxonomy and systematics of this group represents a challenge because the high variability in traditional taxonomical characters

(morphology, advertisement calls, and coloration) used to describe and distinguish frog species. Previous studies have suggested that additional, cryptic species may exist under the current nomenclature, emphasizing the necessity to provide a clear delimitation of species within Diasporus. Here, we provide a novel combination of mitochondrial DNA

(mtDNA) data for 230 samples distributed from Honduras to Colombia integrated with a massively parallel sequencing of 2,477 ultraconserved genomic elements (UCE loci) from 15 individuals. Using the resulting mtDNA + UCEs maximum likelihood phylogenetic tree, we delimit dink frog species using a Bayesian analysis of a mixed

Yule-coalescent model (bGMYC) of genetic divergence. For sister pairs of species with >

5 number of different locality records we evaluated the role of environmental heterogeneity in speciation by testing for significant differences in environmental niche models (ENM). bGMYC analyses of mtDNA recovered 26 operational taxonomic units

(OTUs), suggesting the existence of 11 undescribed species and we clearly recover 9 of the previously described species that uses morphological and genetic data for its decriptions, the remaining 6 described species has been described only under morphological characters and we do not assign to a specific genetic group because the uncertainty to identify some of the genetic group to the specie. The ENMs in 5 sister- species pairs are significantly differentiated in 5 cases but indistinguishable in 1 pair.

3 Support for the role of niche divergence via local ecological factors promoting speciation in dink frogs is therefore weak. We show that cryptic species in the genus Diasporus are prevalent and widespread through the Chocoan forests, including multiple cases of sympatric cryptic species.

4 INTRODUCTION

Understanding the patterns and processes underlying the uneven distribution of biodiversity across space and time constitutes a major scientific challenge in evolutionary biology. Spatial distribution of genetic variation within and among populations or closely related species can help us understand the drivers of the diversification processes responsible for extant diversity (Avise et al. 1987; Avise 2000). A common driver of genetic divergence is vicariance either by ways of physical barriers as mountains or rivers and/or ecological barriers as environmental differences (Mallet et al., 2009; Rundle and

Nosil, 2005). The environmental heterogeneity can generate areas unsuitable for species survival, and divide populations into spatially segregated genetic populations (Gerick et al., 2014; Haffer, 2008, 1969; Huang et al., 2006). Characteristics of organismal life history as territoriality, habitat preference and environmental tolerances (Antonelli et al.,

2009; Kozak and Wiens, 2007; Pabijan et al., 2015; Rodríguez et al., 2015) facilitate or prevent the gene flow, and finally shaping biodiversity patterns. But also, population connectivity is influenced by landscape features (Valderrama et al., 2014; Wang, 2013;

Wang et al., 2013). When comparing phylogeographic results for multiple species often no single barrier seems to explain the conspecific differentiation observed among co- distributed species (Soltis et al., 2006). This lack of spatially congruous phylogeographic patterns is likely due to the fact that environmental barriers do not have the same impact on all species (Paz et al., 2015). Dispersal limitation is caused not by the environment alone, but by the interaction between environment and the organisms’ physiology and habitat requirements (Pabijan et al., 2015; Paz et al., 2015; Rodríguez et al., 2015). A goal in comparative phylogeography is identifying common sets of historical vicariant

5 events that have geographically structured a group of ancestrally co-distributed organisms in a similar way. The distribution of genetic variation is likely shaped by historical events as well as could be shaped by ecological constraints (Condamine et al., 2012; Hatchwell and Komdeur, 2000).

Ecological divergence among populations may increase the rate of climatic-niche differentiation and promote speciation (Kozak and Wiens, 2007). Under a model of neutral divergence, more closely related species will have more similar ecological or abiotic niches than more diverged species (Pyron et al., 2015), especially for geographically restricted populations and species (Rissler and Apodaca, 2007). Species could also be subject to niche conservatism, perhaps due to stabilizing selection, that prevents divergence in abiotic niche characteristics (Peterson, 1999; Wiens, 2004).

Environmental niche models have been used to address important questions such as whether recently diverged taxa that show limited genetic differences are also ecologically distinct, supporting the idea that they are separately evolving lineages (Rissler &

Apodaca, 2007; Raxworthy et al., 2007), and to examine the role that ecological differences have played as a mechanism influencing speciation in particular groups

(Peterson et al., 1999; Graham et al., 2004; Knouft et al., 2006; Kozak & Wiens, 2006;

McCormack et al., 2010). Combining ENM with phylogeographic inference has become a powerful tool to test hypotheses about historical and ecological drivers of isolation and divergence, and to help define cryptic species (Richards et al., 2007). For phylogeographic studies amphibians are regarded as useful model systems because they are physiologically constrained to particular environmental conditions (Feder and

Burggen, 1992), show low dispersal ability, and often show high philopatry to natal sites

6 (Vences and Wake, 2007; Zeisset and Beebee, 2008). These factors make explain why amphibians typically displaying deep phylogeographic structure over short distances, especially in Neotropical frogs with direct development (Crawford et al., 2013; Elmer et al., 2007).

Neotropical direct-developing frogs comprise a single clade of about 1,000 species knowns as Terraranae (Hedges et al., 2008). Within this large clade, the morphologically and behaviorally distinctive genus Diasporus also called “dink frogs” is composed of 15 described species (Frost, 2017) of minuscule arboreal and semi-arboreal frogs of 12.2 mm to 26.0 mm in length (Hertz et al., 2012). This genus inhabits the rainforest of the biogeographical Choco and ranges from the northern Pacific coast of

Ecuador, through Colombia (including Colombia’s Magdalena Valley) and Lower

Middle America to southeastern Honduras. Fouquet et al. (2012) has suggested, based on molecular data, that Diasporus and its sister genus Eleutherodactylus shared a most recent common ancestor (MRCA; between 26 – 30 million years ago (Ma). Batista et al.,

(2016), Hertz et al. (2012), and Lynch and Duellman (1997) based on the wide variation in morphology, body size, advertisement call, and coloration within and among species of

Diasporus suggest that there remain several undescribed species, but Batista et al. (2016,

2012) and Hertz et al. (2012) also claim that the morphology and bioacoustics are not the best source of information to describe and identify species of Diasporus. Unfortunately, more than the 50% of species of Diasporus have been described using morphology. In this particular case the morphological data must be integrated with genomic data and

SDM to solve the taxonomic problems within this genus (Ojanguren-Affilastro et al.,

2016; Pirie et al., 2017).

7 Identify the units of biodiversity is important step in how the diversity evolves.

The members of a this unit also called species not only share evolutionary ancestry, also shared evolutionary processes and patterns (Coyne and Orr, 2004). But many species concepts has been developed to define the species as a real, biological entities (Wheeler and Meier, 2000). In our particular case, we have the previous inferences that suggest high diversity inside Diasporus with relatively wide, discontinuous distributions.

Although their effective population sizes are likely to be large, and the preliminary data suggest that populations are strongly structured, but not all the lineages have available species names and its respective descriptions. Therefore, some of those could be species no yet described, for practical propose we will going to use the Operational or taxonomic unit (OUT) to define our groups and to could test our hypotheses.

Here we present a new combination of mitochondrial DNA (mtDNA) and genomic data (UCEs) densely sampled across the geographic distribution of the genus to estimate the cryptic diversity among dink frogs and to evaluate patterns of divergence in an ecological niche modeling framework. We expect that the lineage diversification is accompanied by niche divergence and should be reflected in geographically congruent patterns of ecological, morphological and molecular variation. Alternatively, a null hypothesis of niche conservatism would suggest that allopatric and divergent sister lineages share similar (i.e. more similar than random), if not identical, niches (Warren et al., 2008). We consider that Diasporus is a good study case to test this hypothesis.

8 METHODS

TAXONOMIC SAMPLING

For our molecular phylogenetic analyses, we sampled most named species, including multiple localities for many species, for a total of 228 individuals of Diasporus from Honduras to Colombia, with particularly dense sampling in Panama, Colombia, and

Costa Rica (Figure 1). Following Streicher et al. (2018) we include five species as outgroups, including some of hyloid lineages close to the direct developing frogs of the

New World Leptodactylus didymus, Espadarana prosoblepon and also sister lineages to

Diasporus represented by Adelophryne adiastoa, Phyzelaphryne miriame, and

Eleutherodactylus johnstonei . Tissue samples and voucher specimens are mostly housed at the Smithsonian Tropical Research Institute (STRI) and at the Círculo Herpetológico de Panamá, in Panama, the Museo de Zoologia of the Universidad de Costa Rica (UCR), and in Colombia at the Museo Herpetológico de la Universidad de Antioquia (MHUA),

Instituto de Ciencias Naturales (ICN) of the Universidad Nacional de Colombia, and at the Museo de Historia Natural ANDES at the Universidad de los Andes, Bogotá (Table 1, table S1).

Most molecular phylogenetic studies either sample 100’s of tissues at few loci, or

100’s of loci from few specimens. Here we decided to combine these two approaches by creating a data matrix based on three traditional mitochondrial DNA mt(DNA) Sanger- sequenced mitochondrial loci representing 235 samples (97 samples for cytB, 120 samples for COI, and 199 samples for 16S), and genomic-scale DNA sequence data based on 5472 proves to capture ultraconserved elements (or UCEs; Faircloth et al.,

2012) from 15 samples. Simulation and empirical studies have demonstrated that one can

9 combine these two strategies, such that the data matrix has many genes for few taxa, and few genes for most taxa. Such a matrix would have widespread missing data, of course, but the benefits in terms of phylogenetic resolution appear to outweigh possible costs

(Burleigh et al., 2009; Wiens and Morrill, 2011; Zheng and Wiens, 2016).

SANGER SEQUENCING

Total genomic DNA was extracted from live tissue samples using a DNeasy Blood and Tissue Extraction Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s protocol. We amplified and sequenced fragments of the following three mitochondrial genes: 16S ribosomal RNA subunit (16S), cytochrome oxidase b (Cyt b), and the 3’ end of the cytochrome oxidase subunit I (COI) (Table 2). We chose these mitochondrial genes because of their fast rate of evolution, their standardized usage in molecular phylogenetics, and their proven utility in amphibian phylogeography; (Crawford et al.,

2010; Mendoza et al., 2015; Pabijan et al., 2015). The PCR amplification of gene fragments was performed in 30 µl reactions using 12.5 µl GoTaq Green Master Mix 2x

(Promega), 0.25 µl each of forward and reverse primers at 10 mM (Table 2), 1 µl of genomic DNA extract, and 16 µl of dd H2O. Standard reaction conditions were an initial denaturation 95 °C (5 min) followed by 32 cycles of 95 °C (30 s), annealing at 50 °C (for

16S) for 30 s, and extension at 72 °C for 60 s plus one additional second each cycle.

Annealing temperature was lowered to, 48 °C for COI and 42 °C for Cyt b. A final extension of 72 °C for 10 min was performed. PCR products were cleaned by

Exonuclease I (Exo I)/Shrimp Alkaline Phosphatase (Werle et al., 1994). Sanger sequencing of DNA was performed on an ABI 3730 multi-capillary genetic analyzer.

10 Chromatograms were cleaned and assembled using Sequencher v. 5.0.1. The protein-coding genes COI and cytb were translated to amino acids to check for premature stop codons in Geneious Pro version 5.5.7. To align the 16S rRNA gene fragments we used the secondary structure of Xenopus laevis deposited in The Comparative RNA Web

Site and Project the Gutell lab

(http://www.rna.icmb.utexas.edu/DAT/3C/Structure/index.php), and the sequences were folded using the online mfold web server (Zuker, 2003)

(http://unafold.rna.albany.edu/?q=mfold). COI and Cyt b were aligned using Clustal W implemented in MEGA 6.06 (Tamura et al., 2013), while 16S was aligned based on the secondary structure using 4Sale v. 1.7.2 (Seibel et al., 2006).

ULTRACONSERVED ELEMENTS LIBRARY PREPARATION

For capture and library preparation we followed the protocol of Faircloth et al.

(2012; available at http://ultraconserved.org), with the modifications used by Streicher et al. (2016) and also implemented in Barrientos et al (in prep). The template gDNA was selected in a initial concentration of ~150 ng, later was fragmented by either physical shearing with a Bioruptor (Diagenode) using 6 cycles of high-speed agitation for 30 s on and 90 s off, or by enzymatic digestion using NEBNext dsDNA Fragmentase (New

England Biolabs) at 37°C for 25 m. The post-hybridization PCR was conducted with

NEB Phusion DNA polymerase and TruSeq primers (Streicher et al., 2016). We sequenced the three capture libraries on three runs, each with 48 individuals (not all individuals were included in the present study). We performed 600-cycle paired-end

11 sequencing runs on an Illumina MiSeq at the genomics core facility of the University of

Texas at Arlington (Arlington, TX, USA; http://gcf.uta.edu/).

UCE ASSEMBLY AND ALIGNMENT

The raw reads of the UCE library data were processed following the pipeline provided by Faircloth et al. (2012) available at http://phyluce.readthedocs.org/en/latest/tutorial-one.html#preparing-data-for-raxml-and- examl. We trimmed sequences to remove adapters and low-quality bases using the

Trimmomatic package implemented in Illumiprocessor (Bolger et al., 2014; Faircloth,

2013) (https://github.com/faircloth-lab/illumiprocessor). The contigs where assembled de novo using Velvet 1.2.10 the parameters for the ensemble where kmer length of 75 and coverage cutoff of 10. Following contig assembly, we processed the data using programs available from PHYLUCE 1.5.0 (http://phyluce.readthedocs.org/en/latest/tutorial- one.html#preparing-data-for-raxml-and-examl). We identified the UCE contigs from de novo assemblies on a sample-by-sample basis. with default settings. The resulting UCEs were aligned using MAFFT 7.130 (Katoh et al., 2002). We filtered the resulting alignments to create ‘de novo assembled’ data matrices to select the best data matrix for our analysis we select a data matrix that allow 80% of missing taxa per loci (e.g. a minimum 4 of 18 individuals must have data for each UCE locus). Our decision was based in the previous experience exposed in Barrientos et al. (in prep) that shows the inclusion of more loci increases the overall number of parsimony informative sites, despite the inclusion of more missing data.

12

PHYLOGENETIC ANALYSES

For the mitochondrial data, we aligned the 3 loci (cytB, COI, 16S) using Mafft

7.130 with default settings. To select the best substitution model and partitioning schemes prior to ML analysis we used the software PartitionFinder (Lanfear et al., 2012) via the corrected Akaike Information Criterion (AICc). We performed ML phylogenetic inference and non-parametric bootstrapping using the program RAxML version 8.0.19

(Stamatakis 2006) and assuming the partition scheme recommended by PartitionFinder.

We conducted 100 maximum-likelihood (ML) searches on the concatenated data set.

Following the best-tree search, we generated nonparametric bootstrap replicates using the autoMRE option of RAxML, which runs bootstrapping until bootstrap replicates converge. Following the best tree and bootstrap analyses, we used RAxML to reconcile the best-fitting ML tree with the bootstrap replicates.

For phylogenetic analyses of UCEs + mtDNA loci, we combine the UCE 20% missing taxa per locus matrix and the 3-gene mitochondrial DNA (mtDNA) data. Using

RAxML version 8.0.19 (Stamatakis 2006) and assuming a GTRGAMMA substitution model, we conducted 100 maximum-likelihood (ML) searches on the concatenated data set. Following the best-tree search, we generated nonparametric bootstrap replicates using the autoMRE option of RAxML, which runs bootstrapping until bootstrap replicates converge. Following the best tree and bootstrap analyses, we used RAxML to reconcile the best-fitting ML tree with the bootstrap replicates.

DIVERGENCE TIME

13 To estimate the number of genetic clusters (OTUs) and to test the effect of environment on the diversification process of Diasporus, a time-calibrated tree is needed.

Given our large data matrix with substantional missing data, we were unable to estimate a timetree using the popular BEAST software package. Instead, a timetree for Diasporus was estimated using a penalized likelihood algorithm implemented in a C++ implementation of r8s (Sanderson 2002) called “treePL” (Smith and O’Meara 2012). This algorithm estimates evolutionary rates and divergence dates on a tree given a set of date constraints and a smoothing factor determining the amount of among- branch rate heterogeneity. We used a secondary data to calibrate our time tree, the minimum age of

40 Ma to a maximum age 70 Ma for the origin of the family Eleutherodactylidae

(Fouquet et al., 2013, 2012; Frazão et al., 2015; Gomez-Mestre et al., 2012; Pyron, 2014).

OPERATIONAL TAXONOMIC UNIT DEFINITION

To estimate the number of genetic clusters or potential species within the genus

Diasporus we used a Bayesian implementation of the general mixed Yule-coalescence model (Pons et al., 2006) available in the package bGMYC (Reid and Carstens, 2012).

Starting from our time-calibrated tree, bGMYC models the transition in rates of branching (or coalescence, in reverse-time) from a within-population coalescent process to a between-species distribution basd on a Yule process (Pons et al., 2006). The number of distinct coalesenct clusters isolated by long branches provides an estimate of the number of potential species. We used the function bgmyc.singlephy to sample from the

ML tree. The parameters that we use to run bGMYC eas 500,000 iterations with a burn-in set of 9,000 and sampling every 1,000th step. We als used ABGD software (Puillandre et

14 al., 2012) to sort the sequences into genetic clusters or candidate species on the basis of their genetic distance using the Web interface at http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html. The genetic clusters were identified using a range of intraclade divergence between 0.001 and 0.1, the Kimura 2- parameter (K2P) model, and remaining parameters by default (Steps = 10, X = 1.5, Nb bins = 20).

ENVIRONMENTAL NICHE MODELING

We used environmental niche modeling (ENM; Warren et al., 2010) to estimate the geographic distribution of each potential species identified by the bGMYC analysis.

This list of species included named species but excluded the potential species based on unique samples due to uncertainty in assigning locality records to these ‘singleton’ taxa.

Locality data for modeling consisted of only samples with genetic data assigned to one of our “potential species” with mor than 5 distinguishable localities (Table S1), no external data was added as we could not determine the cluster they belonged to. We modeled the area for the remaining actual or potential species using a presence-only approach implemented in Maxent 3.3.3 (Phillips et al., 2004

[http://biodiversityinformatics.amnh.org/open_source/maxent/]; Merow et al., 2013). As environmental predictor variables in the modelling procedure we used the 19 bioclimatic layers available in the Worldclim database (http://www.worldclim.org/methods.htm) derived from temperature and precipitation data.

To reduce spatial biases in ENM estimation we thinned our dataset by removing

‘conspecific’ locality records that were closer than 1 km. Using the filtered locality

15 points, we generated a buffered minimum convex polygon using a 10 degree buffer for each taxon to use as background area for the model. Models were tuned using the

ENMeval package for R (Muscarella et al., 2014) to select the best combination of parametrs to be used. For that, we built models for each OTU using different feature classes (“L”, “LQ”, “H”, “LQH”, “LQHP” and “LQHPT”) and regularization multipliers

(0.5-4 in 0.5 steps) thus avoiding the use of Maxent defaults that often lead to overfitting the data points. The lowest AICc was used to select the model with the best combination of parameters (feature classes and regularization multipliers). The final model for each

OUT was converted to binary presence/absence maps using the minimum presence threshold minus 10% of the observations (T10, Pearson et al. 2007).

ENVIRONMENTAL ANALYSIS.

To evaluate whether pairs of named or potential species of Diasporus have diverged in their environmental niches we compared the ENM’s generated for each sister-species pair. We ran a niche identity test as implemented in the ENMtools package for R (Warren et al., 2008). This test operates under the null hypothesis that the two considered species presence points are a random draw from the environmental space where they both occur and thus takes random samples of combined points from both species to build models and uses a pooled background for the creation of species distribution models (Warren et al. 2014). Identity tests were run among 6 pairs of sister taxa using in each case the Maxent algorithm and 99 replicates.

16 RESULTS

PHYLOGENETIC ANALYSES

We obtained a wide geographical sampling of dink frogs covering almost their complete distribution, from Honduras to Colombia, including 230 samples representing 9 of 15 described species (Fig 1). We sequenced 97 individuals for cytochrome b (cyt b),

120 for cytochrome oxidase I (coi), 199 individuals for 16S, and 18 individuals for UCEs

(Table S1). For the three concatenated mtDNA loci we obtain a total of 1,874 bp. We obtain 2,477 UCE loci using a data matrix that allows the 80% of missing taxa per loci, the concatenated data set obtained was 696,951 bp in length. The resulting data matrix of the combined mtDNA + UCEs have 698,825 bp in length.

We found no conflicting phylogenetic signal between the mtDNA data and the

UCEs, justifying the use of the combined data in maximum likelihood (ML) analyses.

Both, concatenated ML analysis of the mtDNA + UCEs data produced trees identical in all major topological details (Fig 2 and Fig 3). The reconstruction of the phylogenetic relationships using the concatenated mtDNA data + UCEs in ML tree of dink frogs, shows strong support (bs = 83%) for the monophyly of the genus in relationship to the sister genus (Eleutherodactylidae) and to the other members of the family

Eleutherodactylidae included as outgroups (Phyzelaphryne and Adelophryne). Most of the support to the Diasporus groups that we found is relatively high (£ 70 % bs), but the relationships between groups are not well supported. In the combined mtDNA + UCEs

ML tree, Diasporus is divided in two major groups with good support (bs = 83%), the first group is composed exclusively by lineages distributed in Middle America (Clade A).

The second group is more complex because has lineages from Middle and South America

17 (Clade B). The time estimation using TreePL analysis indicated that the crown age of

Diasporus dates from the early Miocene, between 23 and 16 Ma. Based on the topology, both Clades (A and B) lineages diverged between 21 and 15 Ma, which is roughly coincident with the hypothesis of Montes et al. (2015) to the closure of panama seaway

(Fig. 4).

OPERATIONAL TAXONOMIC UNIT DEFINITION

The species delimitation analysis under the bGMYC algorithm indicated the presence of at least 26 genetic groups (OTUs), 19 conformed by clusters at least 2 individuals and 7 singletons. ABGD indicated the presence 32 OTUs, 22 conformed by clusters of at least 2 individuals plus 10 singletons (Fig. 4). In the present study, we decide to follow the bGMYC result because we found more consistence to some of the previous described species. All the bGMYC assigned OTUs have a bootstrap support above 83% on the concatenated ML tree, with the exception of the OTUs “1” (bs = 55

%), “2” (bs = 28 %), and “5” (bs = 35 %). In our bGMYC analysis we recover the following species previously described with morphological and molecular data: OTU 3 = that corresponds to D. vocator, OTU 19 = that corresponds to D. anthrax, the OTU 21 was selected as a genetic unity but we divide it in two OTU 21a = that corresponds to D. sapo, OTU 21b = that corresponds to D. darinensis based in the clear morphological differences and to test if this clades have environmental differences that support our separation, OTU 16 = D. pequeno, OTU 5 = that corresponds to D. tinker, OTU 7 = that corresponds to D. tigrillo, OTU 8 = that corresponds to D. citrinobapheus, and the OTU

20 and 22 = that corresponds to D. majeensis,. The remaining OTUs couldn’t be assigned

18 to a clear specie name because under the same OTU there are several individuals under different species names for that reason and the no comparison to museum material in this work to compare to the typological description, we couldn’t assign to one specific specie name.

SPECIES DISTRIBUTION MODELLING

Using the categorization under different OTUs obtained by the bGMYC analysis and the occurrence distribution points for dink fogs (Fig. 1). The predicted distribution of each OTU is based on analysis of the environmental data associated with these points

using Maxent (Fig. 5). These projections represent a spatially explicit estimation of habitat suitability using information from all 19 bioclimatic variables that were considered. To evaluate the nature of ecological niche differentiation among species pairs

(including undescribed OTUs identified by bGMYC) as identified by our ML tree (Fig.

4). The sister taxa we analyzed were: OTU 4 D. aff. quidditus vs. OTU 5 D. tinker, OTU

8 D. citrinobapheus vs. 9 D. aff. diastema, OTU 11 vs. 12, OTU 21a D. sapo vs. 21b D. darinensis. the Comparisons of Schoener’s D (1968) and the Warren et al.’s I statistic

(2008) values based on the overall ENMs. There were no differences in the niche model for the OTU 4 and OTU 5 (D = 0.72, p = 0.84; I: = 0.89, p = 0.81), and significant niche differentiation for OTU 8 vs. OTU 9 (D = 0.375, p = 0.01; I = 0.71, p = 0.01), OTU 11 vs. OTU 12 (D = 0.39, p = 0.04; I = 0.74, p = 0.03), OTU 21a vs. OTU 21b (D = 0.37, p

= 0.02; I = 0.65, p = 0.02; Table 1; Fig. 6)

DISCUSSION

19 In this study, we realize for first time a phylogeny that try to include a wide amount of species of dink frogs that covers almost all the distribution of the genus, with the exception of the northern Ecuador. Also, we use an extended amount of genetic information, a total of 2,480 loci with 699,100 bp (three mtDNA loci and 2,477 UCEs).

The genera rising was estimated close to 40 M.a. and the diversification of most of the lineages c.a. 23 M.a. to 15 M.a. Although we do not know where is the exact point of the encounter of the land masses, and for that reason we can’t explore the colonization direction in this particular case. We found that the estimated time for the radiation of most of the lineages of dink frogs matches with the estimated time of the closure of the

Panamá Isthmus sensu Montes et al. (2015).

The genetic barcode analysis positively distinguishes some of the previous described species with high support D. vocator (OTU 3, bs = 94), D. hylaeformis (OTU

1, bs = 73), D. tinker (OTU 5, bs = 95), D. sapo (OTU 21a, bs = 100), D. darinensis

(OTU 21b, bs = 100), D. pequeno (OTU 16, bs = 95), D. tigrillo (OTU 7, bs = 100), D. citrinobapheus (OTU 8, bs = 99), unfortunately some of the previously described species of dink frogs are not clearly assignable to a particular OTU in our analysis, that species are D. gularis (Boulenger, 1898), D. diastema (Cope, 1875), D. igneus Batista, Ponce, and Hertz, 2012, D. ventrimaculatus Chaves, García-Rodríguez, Mora, and Leal, 2009, and D. quidditus (Lynch, 2001), and finally D. majeensis that is non-recovered as monophyletic (OTUs 20 and 22). For this genus of frogs is required to refine the diagnostic characters and complement it with genetic data, to clearly could be assigned the remaining species in one of the genetic groups founded and also is required assign

20 names to the orphan groups (species candidate to will be described) to solving the taxonomy and systematics of this genus.

Some of the relationships between groups that we found are similar as some of the results of previous authors using few loci. We found that the clade conformed by D. citrinobapheus, D. aff. citrinobapheus, and D. tigrillo are a monophyletic clade as

García-Rodríguez et al. (2016) found, but on the other hand we have a different phylogenetic relationship for another species i.e. D. vocator in comparison to García-

Rodríguez et al. (2016) and Hertz et al. (2012), but coincide as the sister taxa to all other

Diasporus in congruence to Batista et al. (2016) unfortunately in our case the branch support is low (bs = 19 %). In our study is D. sapo and D. darinensis, the gap analysis

(bGMYC) grouped under the same OTU 21, or several genetic lineages under ABGD analysis, but we decide to analyze it as independent entities based on the clear morphological characters that difference both entities and also use as a different source of support to that hypothesis. The ENMs analysis that shows that species uses different environmental space (Fig 6D,We also recover with high support (bs = 95) to the “Darien highlands” sensu Batista et al. (2016) clade shaped by D. majeensis, D. sapo, and D. darinensis, in our analysis this Panamanian clade appear as sister to the South American specie D. anthrax (OTU 19, bs = 69). Regardless of the measure of similarity used

Shoener’s D or Warren et al.’s I, the background tests supported ecological differentiation between some of the sister pairs. These pairs are clearly separated in the bioclimatic space and the estimates of niche overlap based on independent niche axes also support strong niche displacement between sister taxa.

21 Using a species distribution model approach, our data variably supports niche divergence and niche conservatism (Warren et al., 2008; Wiens, 2004). From a climatic perspective, our background similarity test found evidence supporting ecological differentiation between OTUs 8 and 9, 11 and 12, and 21a and 21b, but on the other hand,

OTU 4 and 5 are indistinguishable in their environmental model area variables support niche conservatism. We understand that niche divergence is an important process leading to the diversification of dink frogs. But we cannot conclude that is the main mechanism that promoted diversification within dink frogs, mainly because (a) we only tested the climatic niche similarity for four pairs from the total of 26 lineages, (b) we admittedly used rough bioclimatic variables to describe the climatic niche, which might explain the fact that the background similarity test was unable to detect significant differences in the climatic niches between lineages.

ACKNOWLEDGMENTS We are grateful for tissue loans from Museo Herpetológico de la Universidad de

Antioquia (MHUA), Museo de Historia Natural, Universidad Nacional Mayor de San

Marcos (MHNSM), Círculo Herpetológico de Panamá (CH), Museo de Historia Natural

ANDES at the Universidad de los Andes in Bogotá (ANDES), the Museum of Vertebrate

Zoology at the University of California, Berkeley (MVZ), and Museo de Zoología

Universidad de Costarica (UCR).The research and collecting permission was provided by the Autoridad Nacional de Licencias Ambientales (ANLA) de Colombia (permiso marco resolución No 1177 to the Universidad de los Andes)." This work was supported by the

Colciencias doctoral fellowship 567 and the Colciencias grant Purdue grant 655, Proyecto semilla Universidad de los Andes, FAPA grant AJC, and The University of Arizona and

22 U.S. National Science Foundation Grant DEB 1655690 to JJW. We thank to Camila

Plata, Andres M. Cuervo, Luisa Castellanos, Luisa Dueñas, Santiago Herrera, Laura

Céspedes, Catalina Palacios, and Camila Gomez for providing valuable comments on earlier versions of this manuscript and to all of the Biomi|cs lab members.

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Tables:

Table 1. Voucher information and amount of DNA data produced for each sample, including number of contigs assembled using

Velvet (v1.2.10), and the resulting number of aligned ultraconserved elements (UCEs) obtained. Sequence Read Archive (SRA) accession numbers provide access to all reads obtained for each individual. Accession numbers starting with SAMN were published previously Streicher et al. (2018). Museum numbers are from the following natural history collections: Museo Herpetológico de la

Universidad de Antioquia (MHUA), Museo de Historia Natural, Universidad Nacional Mayor de San Marcos (MHNSM), Círculo

Herpetológico de Panamá (CH), Museo de Historia Natural ANDES at the Universidad de los Andes in Bogotá (ANDES), the

Museum of Vertebrate Zoology at the University of California, Berkeley (MVZ). The samples currently have only field numbers:

Adolfo Amezquita (AA), Jhon Jairo Sarria (JSS), Marco Rada (MAR).

Velvet Species ID number UCE loci SRA accession contigs Diasporus diastema CH 4794 2210 533 Diasporus anthrax MHUA 7237 4446 1038 Diasporus quidditus CH 5373 4446 852 ANDES-A 6675 1634 Diasporus gularis 3833 Diasporus sp. AA 2178 4762 1224 Diasporus quidditus CH 4717 3082 1000

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Diasporus sp. CH 4751 1110 481 Diasporus quidditus CH 4760 379 165 Diasporus diastema CH 4794 309 163 Diasporus aff. gularis ICN (JJS 065) 185 84 Diasporus aff. gularis ICN (JJS 074) 2538 953 Diasporus aff. gularis MAR 689 2097 811 Diasporus aff. gularis MHUA 7414 3062 1087 Diasporus vocator CH 4786 2441 933 Outgroups MHNSM 6019 1851 Leptodactylus didymus 14643 SAMN05559919 Espadarana prosoblepon MVZ 149741 6094 1851 SAMN05559886 ANDES-A 3680 1515 Adelophryne adiastola 2560 SAMN05559873 ANDES-A 2170 512 Phyzelaphryne miriame 3834 Eleutherodactylus ANDES-A 2970 1792 johnstonei 1912

Table 2. Schoener’s D (1968) and the Warren et al.’s I statistic (2008) values for Diasporus selected sister OTUs calculated using

ENMTools (Warren et al., 2008).

D I OTU 4 vs. 5 0.84 0.81 OTU 8 vs. 9 0.01 0.01 OTU 11 vs. 12 0.04 0.03

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OTU 21a vs. 21b 0.02 0.02

Figures

Figure 1. Map of the included Diasporus samples of this study. Blue dots represent the samples with mtDNA data, red dots represent the samples whit UCEs and mtDNA data.

Figure 2. Phylogenetic relationships among Diasporus concatenate of mtDNA + UCEs loci using maximum likelihood (ML). The numbers next to each node represents the bootstrap support. The gray bars represent the Cade A and B.

Figure 3. Relationships among eleutherodactylid frogs based on a concatenated ML analysis. The data matrix included 1,851 UCE loci for a total of 696,951 aligned base pairs, and included loci with up to 80% missing taxa. Numbers next to each node indicate the bootstrap support values from the ML analysis.

Figure 4. Time-tree estimated with TreePL, based on the phylogenetic relationships among Diasporus concatenate of mtDNA + mtDNA loci using maximum likelihood (ML). The numbers next to each node represents the bootstrap support. The bars in front

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represents the clustering groups by ABGD (blue) and bGMYC (red, orange and yellow). The grey bar that overlaps the topology represents the time period of the closure of the Panamá Isthmus sensu Montes et al. (2015).

Figure 5. Potential distribution areas of the OTUs. The figures A), B), and C) the colored area corresponds to potential distribution models and the dots symbolize the specimens used for the model construction. In figures D), E), F) are dots represents the localities that correspond to each OTU. There are no models to those lineages because have few data to could build the distribution model.

Figure 6. Frequency histograms from tests of identity test (Shoener’s D) of niche conservatism and divergence from an analysis of observed and ‘null’ niche models using ENMTools. The dash line represents expected niche overlap value compared with the background divergence. Each histogram represents a pairwise comparison between two sister OTUs, where the environmental niche models (ENM) for taxon A is compared with the background points from taxon B and vice versa.

Supplements

Table S1. Diasporus samples included in the phylogenetic analysis. And the localities of the dink frogs included for the potential distribution and for the comparison between environmental variables between sister OTUs.

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Collecto province/st longitud Genus species Inst.# r # locality/Municipio ate country latitude e - FMNH AJC 83,9552 Diasporus vocator 257769 0127 Las Cruces Puntarenas Costa Rica 8,7911111 778 - AJC 83,5673 Diasporus diastema UCR 16404 0369 EARTH, Pocora, Guácimo Limón Costa Rica 10,2357 167 - AJC 83,5673 Diasporus diastema UCR 16405 0370 EARTH, Pocora, Guácimo Limón Costa Rica 10,2357 167 - AJC 83,5673 Diasporus diastema UCR 16406 0371 EARTH, Pocora, Guácimo Limón Costa Rica 10,2357 167 - AJC Alfombra, along Dominical-- 83,7720 Diasporus vocator UCR 16411 0377 SanIsidro Hwy. San José Costa Rica 9,3122833 333 - AJC 10,043283 83,5486 Diasporus diastema UCR 16407 0414 Guayacan, Siquirres Limón Costa Rica 3 333 "Vuelta de Queque" --> Rio - AJC Siquirres trail, Guayacan, 10,043283 83,5486 Diasporus diastema UCR 16408 0418 Siquirres Limón Costa Rica 3 333 Estación Biológica Alberto Ml. - AJC Brenes, Reserva Biológica San 10,218766 84,5969 Diasporus diastema UCR 16409 0466 Ramón Alajuela Costa Rica 7 833 Estación Biológica Alberto Ml. - hylaefor AJC Brenes, Reserva Biológica San 10,218766 84,5969 Diasporus mis UCR 16263 0467 Ramón Alajuela Costa Rica 7 833 Estación Biológica Alberto Ml. - hylaefor AJC Brenes, Reserva Biológica San 10,218766 84,5969 Diasporus mis UCR 16264 0468 Ramón Alajuela Costa Rica 7 833

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Estación Biológica Alberto Ml. - hylaefor AJC Brenes, Reserva Biológica San 10,218766 84,5969 Diasporus mis UCR 16265 0469 Ramón Alajuela Costa Rica 7 833 hylaefor AJC Diasporus mis UCR 16266 0474 Tapantí, Cantón Paraíso Cartago Costa Rica 9,7494 -83,7816 - AJC 10,202233 84,1626 Diasporus diastema UCR 16410 0482 La Paz Waterfall Gardens Heredia Costa Rica 3 667 200m east of Rio Gacho, Los hylaefor AJC Juncos, Cascajal, Cantón 10,033333 Diasporus mis UCR 16403 0515 Vazquez de Coronado San José Costa Rica 3 -83,,95 Finca Sergio Jimenez, 6.5 km - diastema AJC by air SSE of Aguitas, Drake, 83,6247 Diasporus (vocator) UCR 16412 0540 Osa Puntarenas Costa Rica 8,6485 5 - hylaefor AJC Top of V. Tenorio, P. N. 10,673333 85,0116 Diasporus mis UCR 16413 0560 Tenorio, Bijagua Alajuela Costa Rica 3 667 - hylaefor AJC Top of V. Tenorio, P. N. 10,673333 85,0116 Diasporus mis UCR 16414 0561 Tenorio, Bijagua Alajuela Costa Rica 3 667 - AJC Cana, Pirre high camp, trail to 77,7302 Diasporus sp.nov. MVUP 0595 summit Darien Panama 7,7711111 778 - AJC Bocas del 82,1100 Diasporus diastema 0867 Isla Popa (NNE shore) Toro Panama 9,2234444 833 MVUP AJC Bocas del Diasporus diastema 1871 0871 Isla Colón (near reservoir) Toro Panama 9,3643889 -82,245 - diastema AJC Unión Emberá, Río Majé, 78,7580 Diasporus /sp.nov. 0993 south of Lago Bayano Panamá Panama 9,034267 5 AJC field camp on lower Río Urtí - Diasporus vocator 0998 (feeds into lower Río Majé), Panamá Panama 9,00343 78,7487

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south of Lago Bayano 33 field camp on lower Río Urtí - AJC (feeds into lower Río Majé), 78,7487 Diasporus diastema MVUP 1056 south of Lago Bayano Panamá Panama 9 33 Burbayar Lodge, El Llano- AJC Cartí Rd., Distrito de Chepo, Diasporus diastema CH 6020 1176 Corrigimiento El Llano Panamá Panama 9,3128 -79 Burbayar Lodge, El Llano- cf. AJC Cartí Rd., Distrito de Chepo, Diasporus vocator CH 6018 1177 Corrigimiento El Llano Panamá Panama 9,3128 -79 Estación Biológica El Amargal (Fundación Inguedé), AJC Corregimiento Arusí, Diasporus gularis? 1183 Municipio de Nuquí Chocó Colombia 5,5705 -77,5026 Estación Biológica El Amargal AJC (Fundación Inguedé), 1183/toe Corregimiento Arusí, Diasporus gularis? s 3 Municipio de Nuquí Chocó Colombia 5,5705 -77,5026 Estación Biológica El Amargal (Fundación Inguedé), AJC Corregimiento Arusí, Diasporus gularis? 1187 Municipio de Nuquí Chocó Colombia 5,5705 -77,5026 Estación Biológica El Amargal (Fundación Inguedé), AJC Corregimiento Arusí, Diasporus gularis? 1192 Municipio de Nuquí Chocó Colombia 5,5705 -77,5026 Estación Biológica El Amargal (Fundación Inguedé), cf. AJC Corregimiento Arusí, Diasporus vocator 1194 Municipio de Nuquí Chocó Colombia 5,5705 -77,5026 Hacienda Santa Barbara, Área - AJC de Reserva del "Distrito de 74,6402 Diasporus diastema MHUA 1335 Manejo Integral de los Antioquia Colombia 6,54413 8

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Recursos Naturales Cañón del Río Alicante," Vereda Las Brisas, Municipio de Maceo. - AJC Comunidad de Guayacan, Bocas del 82,4729 Diasporus sp. 1732 Corregimiento Valle de Risco Toro Panama 9,07372 5 Estación Biológica El Amargal (Fundación Inguedé), cf. AJC Corregimiento Arusí, Diasporus gularis 2100 Municipio de Nuquí Chocó Colombia 5,5705 -77,5026 - aff. AJC Estacion Biologica El 77,5025 Diasporus gularis ANDES-A 2287 Amargal, Arusi, Nuqui Chocó Colombia 5,57046 4 - aff. AJC Estacion Biologica El 77,5025 Diasporus gularis ANDES-A 2291 Amargal, Arusi, Nuqui Chocó Colombia 5,57046 4 Parque Nacional Carara, Bajo Carara, Jct of Quebrada - Surtubal and Quebrada 84.5312 Diasporus diastema MF 4651 DL 373 Chancho San José Costa Rica 9.7777333 833 Parque Nacional Carara, Bajo Carara, Jct of Quebrada - Surtubal and Quebrada 84.5312 Diasporus diastema MF 4639 DL 374 Chancho San José Costa Rica 9.7777333 833 Area de Conservacion Guanacaste, Estacion Cacao, 50 trail meters S of Jct of - Sendero La Cima and Sendero 10.930805 85.4609 Diasporus diastema MF 6033 DL 684 Casa Fran. Alajuela Costa Rica 6 444 Area de Conservacion - Guanacaste, Estacion Cacao, 10.931222 85.4562 Diasporus diastema MF 6014 DL 685 Sendero La Cima. Alajuela Costa Rica 2 222 Diasporus diastema UCR FB?&03- Charcos cerca del Serpentario Alajuela Costa Rica 9,9730556 -

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PARA- Rio Azul. (Same spot as in 84,0936 0367 AJC field notes 22 May 111 2003?!) - 83,1663 Diasporus diastema FB2630 Fila Asunción Limón Costa Rica 9,8994444 889 FB4315 &03- Guanacaste- PARA- Camino a la Cima de Vulcán Alajuela 10,929166 Diasporus diastema UCR 0372 Cacao border Costa Rica 7 -85,45 FB 4329&03 Guanacaste- -PARA- Camino a la Cima de Vulcán Alajuela 10,929166 Diasporus diastema UCR 0377 Cacao border Costa Rica 7 -85,45 "Main Trail," b/n lodge & - MVUP KRL creek, Parque Nacional Omar 80,5916 Diasporus vocator 1782 0688 Torrijos H., El Copé Coclé Panama 8,6666667 667 diastema "cheese- MVUP KRL Parque Nacional Omar Torrijos Diasporus ball" 1783 0694 H., El Copé Coclé Panama 8,667 -80,592 Gracias a - LDW Dios 14,933333 84,5333 Diasporus diastema 12453 Bodega de Rio Tapalwas Province Honduras 3 333 Gracias a USNM LDW quebrada between Rio Dios Diasporus diastema 549352 12461 Tapalwas and Rio Rus Rus Province Honduras 14,7 -84,45 - cf. 77,7222 Diasporus diastema CH 6431 CH 6431 Serrania de Pirre, Cloud Forest Darien Panama 7,763667 67 Centro Cristo Misionero (Padre Wally's), Carretera - AJC Panamericana, Wacuco, 78,4572 Diasporus diastema 1504 Corregimiento Torti Panamá Panama 8,96367 8

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Centro Cristo Misionero (Padre Wally's), Carretera - AJC Panamericana, Wacuco, 78,4572 Diasporus diastema 1524 Corregimiento Torti Panamá Panama 8,96367 8 Centro Cristo Misionero (Padre Wally's), Carretera AJC Panamericana, Wacuco, Diasporus diastema 1531 Corregimiento Torti Panamá Panama 8,9637 -78,4573 Centro Cristo Misionero (Padre Wally's), Carretera AJC Panamericana, Wacuco, Diasporus diastema 1532 Corregimiento Torti Panamá Panama 8,9637 -78,4573 Centro Cristo Misionero (Padre Wally's), Carretera - AJC Panamericana, Wacuco, 78,4572 Diasporus diastema 1534 Corregimiento Torti Panamá Panama 8,96367 8 Centro Cristo Misionero (Padre Wally's), Carretera AJC Panamericana, Wacuco, Diasporus diastema 1536 Corregimiento Torti Panamá Panama 8,9637 -78,4573 Centro Cristo Misionero (Padre Wally's), Carretera - Panamericana, Wacuco, 78,4572 Diasporus diastema AJC1538 Corregimiento Torti Panamá Panama 8,96367 8 Centro Cristo Misionero (Padre Wally's), Carretera - AJC Panamericana, Wacuco, 78,4572 Diasporus diastema 1545 Corregimiento Torti Panamá Panama 8,96367 8 Diasporus diastema CH 6353 Camino del camp laguna Darien Panama 7,722167 -77,6555 - 77,7222 Diasporus diastema CH 6439 Cana, tent camp Darien Panama 7,763667 67 Diasporus diastema AJC Refugio ANAM, Cerro Panamá Panama 9,319848 -

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1940 Brewster "hacia Cerro 79,2889 Guajaral". Límite P.N. 21 Chagres, Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus diastema CH 6676 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus diastema CH 6730 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus diastema CH 6744 Chagres, Panamá Panama 9,319848 21 Tent camp, Reserva Natural Privada Chucanti, diastema Corregimiento Rio Congo Diasporus group CH 6297 Arriba, Distrito de Chepigana Darien Panama 8,7972 -78,4625 Urbanizacion de los Altos de - grupo AJC Cerro Azul, Distrito de 79,4028 Diasporus diastema 1762 Chilibre Panama Panama 9,23051 3 Urbanizacion de los Altos de - grupo AJC Cerro Azul, Distrito de 79,4028 Diasporus diastema 1765 Chilibre Panama Panama 9,23051 3 Urbanizacion de los Altos de - grupo AJC Cerro Azul, Distrito de 79,4191 Diasporus diastema 1789 Chilibre Panama Panama 9,22787 7 - Grupo AJC 77,6841 Diasporus diastema 1819 Cana, Main Camp Darien Panama 7,7561 2 Grupo AJC - Diasporus diastema 1822 Cana, Darien Darien Panama 7,7561 77,6841

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2 - Grupo AJC 77,6841 Diasporus diastema 1832 Cana, Main Camp Darien Panama 7,7561 2 - Grupo AJC 77,7240 Diasporus diastema 1854 Stream, Rio cana, Tent Camp Darien Panama 7,762233 5 - Grupo AJC 77,7326 Diasporus diastema 1862 Serrania de Pirre, Cloud Forest Darien Panama 7,773567 17 - Grupo AJC 77,7326 Diasporus diastema 1864 Serrania de Pirre, Cloud Forest Darien Panama 7,773567 17 - Grupo AJC 77,7326 Diasporus diastema 1866 Serrania de Pirre, Cloud Forest Darien Panama 7,773567 17 - Grupo AJC 77,7222 Diasporus diastema 1874 Serrania de Pirre Darien Panama 7,763667 67 - Grupo AJC 77,7240 Diasporus diastema 1895 Serrania de Pirre (Quebrada) Darien Panama 7,762233 5 Refugio ANAM, Cerro Brewster "hacia Cerro - grupo AJC Guajaral". Límite P.N. 79,2889 Diasporus diastema 1963 Chagres, Panamá Panama 9,319848 21 - Grupo 77,6841 Diasporus diastema CH 6326 Cana, Main Camp Darien Panama 7,7561 2 - Grupo 77,7326 Diasporus diastema CH 6385 Serrania de Pirre Darien Panama 7,773567 17 Diasporus Grupo CH 6387 Serrania de Pirre Darien Panama 7,773567 -

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diastema 77,7326 17 - Grupo 77,7326 Diasporus diastema CH 6391 Serrania de Pirre Darien Panama 7,773567 17 - Grupo 77,7222 Diasporus diastema CH 6395 Serrania de Pirre Darien Panama 7,763667 67 - Grupo 77,7326 Diasporus diastema CH 6403 Serrania de Pirre Darien Panama 7,773567 17 - Grupo 77,7240 Diasporus diastema CH 6420 Serrania de Pirre (Quebrada) Darien Panama 7,762233 5 - Grupo 77,7240 Diasporus diastema CH 6425 Serrania de Pirre, Cloud Forest Darien Panama 7,762233 5 - Grupo 77,7222 Diasporus diastema CH 6442 Serrania de Pirre, Cloud Forest Darien Panama 7,763667 67 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4191 Diasporus diastema CH 6647 Chilibre Panama Panama 9,22787 7 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4191 Diasporus diastema CH 6648 Chilibre Panama Panama 9,22787 7 Refugio ANAM, Cerro Brewster "hacia Cerro - grupo Guajaral". Límite P.N. 79,2889 Diasporus diastema CH 6719 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro - grupo Brewster "hacia Cerro 79,2889 Diasporus diastema CH 6720 Guajaral". Límite P.N. Panamá Panama 9,319848 21

47

Chagres, Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4028 Diasporus diastema CH 6759 Chilibre Panama Panama 9,23051 3 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4028 Diasporus diastema CH 6762 Chilibre Panama Panama 9,23051 3 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4028 Diasporus diastema CH 6775 Chilibre Panama Panama 9,23051 3 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4191 Diasporus diastema CH 6786 Chilibre Panama Panama 9,23051 7 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4243 Diasporus diastema CH 6792 Chilibre Panama Panama 9,23051 2 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4191 Diasporus diastema CH 6800 Chilibre Panama Panama 9,23051 7 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4191 Diasporus diastema CH 6802 Chilibre Panama Panama 9,23051 7 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4191 Diasporus diastema CH 6803 Chilibre Panama Panama 9,23051 7 Urbanizacion de los Altos de - grupo Cerro Azul, Distrito de 79,4191 Diasporus diastema CH 6804 Chilibre Panama Panama 9,23051 7 Refugio ANAM, Cerro Brewster "hacia Cerro - AJC Guajaral". Límite P.N. 79,2889 Diasporus quidditus 1915 Chagres, Panamá Panama 9,319848 21 Diasporus quidditus AJC Refugio ANAM, Cerro Panamá Panama 9,319848 -

48

1935 Brewster "hacia Cerro 79,2889 Guajaral". Límite P.N. 21 Chagres, Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus quidditus CH 6674 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus quidditus CH 6682 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus quidditus CH 6725 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus quidditus CH 6726 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus quidditus CH 6733 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus quidditus CH 6738 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro Brewster "hacia Cerro - Guajaral". Límite P.N. 79,2889 Diasporus quidditus CH 6739 Chagres, Panamá Panama 9,319848 21 Refugio ANAM, Cerro - Diasporus quidditus CH 6741 Brewster "hacia Cerro Panamá Panama 9,319848 79,2889

49

Guajaral". Límite P.N. 21 Chagres, Estación Biológica El Amargal (Fundación Inguedé), Corregimiento Arusí, Diasporus gularis ANDES-A AJC3438 Municipio de Nuquí Chocó Colombia 5,5042 -77,5208 - cf. AJC 77,3302 Diasporus gularis 3417 Coquí, Quebrada la Brava Chocó Colombia 5,62169 1 - cf. AJC Arusí, Estación biológica "El 77,5025 Diasporus gularis 3419 Amargal" Chocó Colombia 5,57046 4 Centro Cristo Misionero (Padre Wally's), Carretera AJC Panamericana, Wacuco, Diasporus diastema MVUP 1536 Corregimiento Torti Panamá Panama 8,9637 -78,4573 - AJC 77,7326 Diasporus quidditus MVUP 1864 Serrania de Pirre, Cloud Forest Darien Panama 7,773567 17 - Phyzaleph cf. AJC 69,9510 ryne miriame 3606 Km 11/Leticia Amazonas Colombia -4,11949 4 - cf. MAR 76,9938 Diasporus gularis ANDES-A 689 Baudó Chocó Colombia 5,1416667 889 - 77,3321 Diasporus sp. ANDES-A LEP 264 Aguacate Antioquia Colombia 8,600476 84 - Valle del 76,9011 Diasporus sp. JSS 074 San Cipriano Cauca Colombia 3,8452778 111 MAA - Diasporus sp. 299 Coqui Chocó Colombia 5,6063889 77,3297

50

222 - MAA 77,3166 Diasporus sp. 300 Coqui Chocó Colombia 5,6 667 - MAR 77,3302 Diasporus sp. 777 Baudo Chocó Colombia 5,62169 1 - Valle del 76,9011 Diasporus sp. JSS 065 San Cipriano Cauca Colombia 3,8452778 111 MHUA-A Valle del Diasporus sp. 7264 Buenaventura Cauca Colombia 3,9084 -77,3582 - MHUA-A 75,0233 Diasporus sp. 5129 Qda. Cañal, Nuquí Chocó Colombia 5,38871 54 - MHUA-A Vereda El Respaldo, Bosque la 75,0233 Diasporus sp. 7412 Arenosa II, Alejandría Antioquia Colombia 5,38871 54 - MHUA-A Vereda El Jague, Vivero, San 75,0023 Diasporus sp. 7237 Rafael Antioquia Colombia 6,34818 73 - MHUA-A 74,6403 Diasporus anthrax 7305 Maceo Antioquia Colombia 6,55195 73 - MHUA-A 76,4195 Diasporus sp. 6789 Vereda Chibuga, Dabeiba Antioquia Colombia 7,068822 71 - MHUA-A 75,0444 Diasporus sp. 5379 Colombia 6,978611 41 - MHUA-A Vereda El Respaldo, Bosque la 75,0233 Diasporus sp. 7405 Arenosa II, Alejandría Antioquia Colombia 6,38871 54

51

- MHUA-A 74,2211 Diasporus sp. 7374 Vereda Mina seca, Norosí Bolívar Colombia 8,4151 53 - MHUA-A 75,0233 Diasporus sp. 7414 Alejandría Antioquia Colombia 6,38871 54 Comarca citrinoba Ngöbe- Diasporus pheus SMF 89814 AH 449 Buglé Panama 8.485 -81.173 citrinoba Diasporus pheus SMF 89820 AH 211 Veragua Panama 8.569 -81.099 Comarca citrinoba MHCH Ngöbe- Diasporus pheus 2370 AH 450 Buglé Panama 8.485 -81.173 Comarca citrinoba MHCH Ngöbe- Diasporus pheus 2371 AH 452 Buglé Panama 8.485 -81.173 - Golfito, Refugio Nacional de 82,9588 Diasporus CH 4792 Fauna Silvestre Puntarenas Panama 8,785278 9 - Golfito, Refugio Nacional de 82,9588 Diasporus CH 4794 Fauna Silvestre Puntarenas Panama 8,785278 9 - Jardin Botanico, Wilson, Agua 82,2417 Diasporus vocator CH 4786 Buena, Coto Brus Puntarenas Panama 9,340992 71 Diasporus sp. CH 4760 Nusagandi. Sendero Ina Igar Panama 9,3167 -78,9833 Diasporus sp. CH 4751 Serrania del Jungurudo Darien Panama 7,55 -77,95 Diasporus sp. CH 4760 Nusagandi. Sendero Ina Igar Panama 9,3167 -78,9833 - 79,2889 Diasporus sp. CH 4717 Cerro Brewster, P.N. Chagres. Panama Panama 9,319848 21 Diasporus sp. AA 2178 Valle del Colombia 3,845 -76,901

52

Cauca Diasporus quidditus CH 5373 Quebrada la Lajosa, Santa Maria, Cano Sucio Panama 7,5833 -80,1833

53

Table S3. Mitochondrial and Nuclear primers used to test the purification of mtDNA.

Gene Primer sequence (5’– region Primer name 3’) Source

GGTCAACAAATCAT

COI dgLCO-1490 AAAGAYATYGG (Meyer et al., 2005)

TAAACTTCAGGGTG

dgHCO-2198 ACCAAARAAYCA (Meyer et al., 2005)

CCATCCAACATCTC

CB1-L AGCATGATGAAA

CytB (Palumbi, 1996)

GGCGAATAGGAAG

CB3-H TATCATTC

(Palumbi, 1996)

CGCCTGTTTATCAA

16S Sar-L AAACAT (Palumbi et al., 1991)

CCGGTCTGAACTCA

Sbr-H GATCACGT (Palumbi et al., 1991)

Fig. 1.

54

55

Fig. 2.

Adelophryne_adiastola 7 1 Phyzelaphryne sp.1b AF-2012 9 3 Phyzalephryne sp. AJC 3606 Eleutherodactylus johnstonei

2 4 Diasporus vocator AB 564 Diasporus vocator CH 4786 Diasporus sp. UCR 21844 9 9 Diasporus vocator UCR 21966 Diasporus vocator UCR 21857 4 1 Diaiasporus vocator Pichi 248 Diasporus vocator UCR 20479 3 6 Diasporus vocator AJC 0127 Diasporus vocator CH 4791

3 6 Diasporus diastema CH 4794 Diasporus sp. AB 476 100 Diasporus sp.6 MHCH 1678 100 3 0 Diasporus diastema Pichi 216

6 6 Diasporus diastema UCR 22968 Diasporus sp. UCR 21676

7 2 Diasporus hylaeformis Pichi 229 Diasporus aff. diastema USNM 572456 Diasporus sp. KRL 0782 100 Diasporus sp. KRL 0831 Diasporus aff. diastema MVUP 1826 Diasporus sp. CH 5900 8 6 9 8 Diasporus diastema CH 5370 5 8 Diasporus diastema CH 5031 Diasporus diastema CH 5004 50 Diasporus hylaeformis KRL 0831

8 3 Diasporus aff. diastema AAB 3745 Diasporus hylaeformis KRL 0782 3 3 Diasporus diastema CH 5712

6 6 Diasporus vocator UCR 21946 100 Diasporus sp. UCR 21843

Diasporus sp. UCR 21953 Clade A Diasporus sp. MHCH 1298 8 6 Diasporus aff. hylaeformis SMF 89875 Diasporus aff. hylaeformis SMF 89872 Diasporus hylaeformis Pichi 240 5 0 Diasporus hylaeformis Pichi 241 9 1 Diasporus aff. hylaeformis SMF 89868 Diasporus aff. hylaeformis SMF 89869 Diasporus diastema CH 5695 Diasporus sp. CH 5694 6 4 Diasporus hylaeformis AJC 0468 7 9 Diasporus hylaeformis Pichi 234 Diasporus sp. DL 685 Diasporus hylaeformis AJC 468 6 2 Diasporus diastema Pichi 218 Diasporus hylaeformis UCR 21935

6 4 Diasporus diastema Pichi 224 Diasporus diastema MVZ 203844 3 1 Diasporus sp. AJC 414 Diasporus sp. AJC 369 5 4 Diasporus diastema Pichi 223 Diasporus sp. AB 032 8 3 Diasporus sp. CH 6525 6 1 Diasporus sp. CH 6121 Diasporus sp. CH 6578 Diasporus hylaeformis CH 6520 Diasporus hylaeformis CH 6134 Diasporus cf. hylaeformis AJC 1732 Diasporus sp. CH 6577 Diasporus sp. CH 6583 Diasporus sp. CH 6617 Diasporus vocator CH 4773 Diasporus vocator CH 4772 Diasporus sp. CH 5918 Diasporus sp. MHCH 1288 Diasporus sp.5 AB032 Diasporus hylaeformis CH 4774 Diasporus sp. CH 6505 Diasporus hylaeformis CH 6124 Diasporus sp. CH 6251 100 Diasporus antrax MHUA 7237 Diasporus sp. MHUA 7305 9 7 Diasporus antrax MHUA 7374 4 7 Diasporus aff. quidditus AB 1030

8 Diasporus sp. AB 1065 100 Diasporus sp. CH 6297 Diasporus sapo AB 429 56

9 4 Diasporus sapo AB 435 9 9 Diasporus sapo AB 430 Diasporus sp.4 AB 439 Diasporus sp.4 AB 431 3 2 Diasporus sp. CH 9142 100 Diasporus sp. AB 1268 Diasporus darienensis AB 329 5 8 Diasporus sp. AJC 595 9 7 Diasporus sp. CH 6425 Diasporus sp. CH 6431 5 6 Diasporus darienensis AB 1185

4 0 Diasporus sp.3 AB 159

9 6 Diasporus darienensis AB 1134 Diasporus darienensis AB 1144 Diasporus darienensis AB 151 Diasporus sp. JJS 065 5 2 Diasporus sp. AJC 2119/MAR1486 9 3 Diasporus quidditus AJC 1194 Diasporus gularis MAA 300 6 4 Diasporus gularis MAA 299 Diasporus pequeno AB 860 Diasporus pequeno AB 822 9 7 Diasporus pequeno AB 857 Diasporus pequeno AB 856 Diasporus pequeno AB 861 Diasporus sp.2 AB 823 100 Diasporus sp. CH 4718 Diasporus sp. CH 4721 Diasporus quidditus CH 5807 Diasporus quidditus CH 4760 Diasporus sp. CH 4741 Diasporus vocator CH 4780 Diasporus quidditus CH 5340 Diasporus quidditus CH 5338 Diasporus quidditus CH 5792 Diasporus quidditus CH 5337 Diasporus quidditus CH 5341 Diasporus quidditus CH 5339 Diasporus quidditus CH 5802 Diasporus quidditus CH 5543

1 4 Diasporus quidditus CH 5540 8 3 6 7 Diasporus quidditus AB 689 4 4 Diasporus quidditus KRL 0856 9 6 Diasporus quidditus KRL 0647 9 0 3 9 Diasporus aff. quidditus AB 931 Diasporus quidditus AB 1130 Diasporus quidditus AB 138 Diasporus quidditus AB 131 Diasporus quidditus AB 158 Diasporus quidditus CH 4748 Diasporus sp. CH 4966 100 Diasporus quidditus CH 4717 Diasporus vocator CH 5392 Diasporus vocator CH 5391 Diasporus vocator CH 5390 Diasporus quidditus CH 6804 3 0 Diasporus diastema CH 6648 Diasporus quidditus AJC 1789 Diasporus diastema CH 4830 Diasporus quidditus CH 6803 Diasporus sp. AJC 600 Diasporus quidditus CH 5624 100 Diasporus quidditus CH 5544 Diasporus quidditus CH 5557 100 4 3 Diasporus quidditus CH 5556 Diasporus sp. AJC 1866

9 9 Diasporus sp. CH 6439 Diasporus sp. CH 9147 Diasporus sp. AJC 1866 7 8 Diasporus tinker AB 308 Diasporus sp. CH 4751

100 Diasporus tinker AB 1270 Clade B Diasporus tinker AB 1269 Diasporus tinker AB 1271 Diasporus tinker AB 1272 Diasporus quidditus CH 5373 Diasporus quidditus CH 5371 100 Diasporus quidditus CH 5372 Diasporus sp. CH 5214 Diasporus vocator AB 028

2 6 Diasporus quidditus CH 5314 Diasporus quidditus CH 5213 Diasporus vocator CH 5400 Diasporus vocator CH 5401 Diasporus sp. AA2178 9 9 Diasporus gularis MHUA 7264 7 9 Diasporus sp. JJS 074 100 Diasporus gularis LEP 264 Diasporus gularis VCK13 100 Diasporus gularis VCK14 9 2 Diasporus quidditus MHUA 6789 Diasporus gularis MHUA 5379 Diasporus sp. MHUA 7405 Diasporus gularis MHUA 7414 2 1 Diasporus sp. MHUA 7412 6 1 Diasporus aff. gularis AJC 3417 3 7 Diasporus gularis MAR 777 Diasporus sp. MHUA 5129

6 3 Diasporus gularis AJC 3419 Diasporus gularis AJC 1183

5 5 Diasporus sp. AJC 2287 Diasporus sp. AJC 1192 Diasporus gularis Toe 1 8 5 Diasporus gularis Toe 2 Diasporus gularis Toe 3 Diasporus sp. AJC 2291 Diasporus diastema AB 073

100 Diasporus diastema AB 084 Diasporus diastema AB 637 Diasporus gularis MAR 689 Diasporus diastema AB 818 2 0 9 5 Diasporus diastema AJC 1536 9 6 Diasporus aff. diastema AB 035 9 7 Diasporus diastema AB 675

8 100 Diasporus aff. diastema AB 218

7 7 Diasporus sp. CH 6503 100 Diasporus sp. CH 6556 Diasporus tigrillo UCR 22364 Diasporus tigrillo UCR 22365

1 8 100 Diasporus tigrillo UCR 22366 Diasporus tigrillo UCR 22367 Diasporus tigrillo UCR 22368 Diasporus diastema CH 5799 Diasporus diastema AJC 1940 Diasporus sp. CH 4764 100 Diasporus diastema CH 5806 Diasporus diastema CH 6676 3 6 Diasporus diastema AB 979

100 Diasporus diastema CH 6786 Diasporus quidditus CH 4726 Diasporus diastema AB 602 Diasporus diastema CH 4834 9 9 Diasporus quidditus CH 5745 Diasporus diastema CH 4746 Diasporus quidditus CH 6792 Diasporus diastema CH 4719 4 8 6 7 Diasporus diastema CH 6800 Diasporus diastema CH 6802

6 6 Diasporus citrinobapheus SMF 89820 Diasporus citrinobapheus SMF 89814

8 5 Diasporus citrinobapheus MHCH 2370 Diasporus citrinobapheus MHCH 2371 Diasporus diastema CH 5302 5 0 9 5 Diasporus diastema CH 5270 Diasporus diastema CH 5225 Diasporus diastema CH 4992 Diasporus diastema CH 5226 9 7 Diasporus diastema CH 5221 Diasporus diastema CH 5211 Diasporus citrinobapheus KRL 0694 Diasporus citrinobapheus KRL 1181

0.03 Diasporus citrinobapheus KRL 0902 Diasporus citrinobapheus KRL 0900 Diasporus citrinobapheus KRL 0901 Diasporus citrinobapheus KRL 0840 Adelophryne_adiastola 7 1 Phyzelaphryne sp.1b AF-2012 9 3 Phyzalephryne sp. AJC 3606 Eleutherodactylus johnstonei

2 4 Diasporus vocator AB 564 Diasporus vocator CH 4786 Diasporus sp. UCR 21844 9 9 Diasporus vocator UCR 21966 Diasporus vocator UCR 21857 4 1 Diaiasporus vocator Pichi 248 Diasporus vocator UCR 20479 3 6 Diasporus vocator AJC 0127 Diasporus vocator CH 4791

3 6 Diasporus diastema CH 4794 Diasporus sp. AB 476 100 Diasporus sp.6 MHCH 1678 100 3 0 Diasporus diastema Pichi 216

6 6 Diasporus diastema UCR 22968 Diasporus sp. UCR 21676

7 2 Diasporus hylaeformis Pichi 229 Diasporus aff. diastema USNM 572456 Diasporus sp. KRL 0782 100 Diasporus sp. KRL 0831 Diasporus aff. diastema MVUP 1826 Diasporus sp. CH 5900 8 6 9 8 Diasporus diastema CH 5370 5 8 Diasporus diastema CH 5031 Diasporus diastema CH 5004 50 Diasporus hylaeformis KRL 0831

8 3 Diasporus aff. diastema AAB 3745 Diasporus hylaeformis KRL 0782 3 3 Diasporus diastema CH 5712

6 6 Diasporus vocator UCR 21946 100 Diasporus sp. UCR 21843

Diasporus sp. UCR 21953 Clade A Diasporus sp. MHCH 1298 8 6 Diasporus aff. hylaeformis SMF 89875 Diasporus aff. hylaeformis SMF 89872 Diasporus hylaeformis Pichi 240 5 0 Diasporus hylaeformis Pichi 241 9 1 Diasporus aff. hylaeformis SMF 89868 Diasporus aff. hylaeformis SMF 89869 Diasporus diastema CH 5695 Diasporus sp. CH 5694 6 4 Diasporus hylaeformis AJC 0468 7 9 Diasporus hylaeformis Pichi 234 Diasporus sp. DL 685 Diasporus hylaeformis AJC 468 6 2 Diasporus diastema Pichi 218 Diasporus hylaeformis UCR 21935

6 4 Diasporus diastema Pichi 224 Diasporus diastema MVZ 203844 3 1 Diasporus sp. AJC 414 Diasporus sp. AJC 369 5 4 Diasporus diastema Pichi 223 Diasporus sp. AB 032 8 3 Diasporus sp. CH 6525 6 1 Diasporus sp. CH 6121 Diasporus sp. CH 6578 Diasporus hylaeformis CH 6520 Diasporus hylaeformis CH 6134 Diasporus cf. hylaeformis AJC 1732 Diasporus sp. CH 6577 Diasporus sp. CH 6583 Diasporus sp. CH 6617 Diasporus vocator CH 4773 Diasporus vocator CH 4772 Diasporus sp. CH 5918 Diasporus sp. MHCH 1288 Diasporus sp.5 AB032 Diasporus hylaeformis CH 4774 Diasporus sp. CH 6505 Diasporus hylaeformis CH 6124 Diasporus sp. CH 6251 100 Diasporus antrax MHUA 7237 Diasporus sp. MHUA 7305 9 7 Diasporus antrax MHUA 7374 4 7 Diasporus aff. quidditus AB 1030

8 Diasporus sp. AB 1065 100 Diasporus sp. CH 6297 Diasporus sapo AB 429

9 4 Diasporus sapo AB 435 9 9 Diasporus sapo AB 430 Diasporus sp.4 AB 439 Diasporus sp.4 AB 431 3 2 Diasporus sp. CH 9142 100 Diasporus sp. AB 1268 Diasporus darienensis AB 329 5 8 Diasporus sp. AJC 595 9 7 Diasporus sp. CH 6425 Diasporus sp. CH 6431 5 6 Diasporus darienensis AB 1185

4 0 Diasporus sp.3 AB 159

9 6 Diasporus darienensis AB 1134 Diasporus darienensis AB 1144 Diasporus darienensis AB 151 Diasporus sp. JJS 065 5 2 Diasporus sp. AJC 2119/MAR1486 9 3 Diasporus quidditus AJC 1194 Diasporus gularis MAA 300 6 4 Diasporus gularis MAA 299 Diasporus pequeno AB 860 Diasporus pequeno AB 822 9 7 Diasporus pequeno AB 857 Diasporus pequeno AB 856 Diasporus pequeno AB 861 Diasporus sp.2 AB 823 100 Diasporus sp. CH 4718 Diasporus sp. CH 4721 Diasporus quidditus CH 5807 Diasporus quidditus CH 4760 Diasporus sp. CH 4741 Diasporus vocator CH 4780 Diasporus quidditus CH 5340 Diasporus quidditus CH 5338 Diasporus quidditus CH 5792 Diasporus quidditus CH 5337 Diasporus quidditus CH 5341 Diasporus quidditus CH 5339 Diasporus quidditus CH 5802 Diasporus quidditus CH 5543

1 4 Diasporus quidditus CH 5540 8 3 6 7 Diasporus quidditus AB 689 4 4 Diasporus quidditus KRL 0856 9 6 Diasporus quidditus KRL 0647 9 0 3 9 Diasporus aff. quidditus AB 931 Diasporus quidditus AB 1130 Diasporus quidditus AB 138 Diasporus quidditus AB 131 Diasporus quidditus AB 158 Diasporus quidditus CH 4748 Diasporus sp. CH 4966 100 Diasporus quidditus CH 4717 57 Diasporus vocator CH 5392 Diasporus vocator CH 5391 Diasporus vocator CH 5390 Diasporus quidditus CH 6804 3 0 Diasporus diastema CH 6648 Diasporus quidditus AJC 1789 Diasporus diastema CH 4830 Diasporus quidditus CH 6803 Diasporus sp. AJC 600 Diasporus quidditus CH 5624 100 Diasporus quidditus CH 5544 Diasporus quidditus CH 5557 100 4 3 Diasporus quidditus CH 5556 Diasporus sp. AJC 1866

9 9 Diasporus sp. CH 6439 Diasporus sp. CH 9147 Diasporus sp. AJC 1866 7 8 Diasporus tinker AB 308 Diasporus sp. CH 4751

100 Diasporus tinker AB 1270 Clade B Diasporus tinker AB 1269 Diasporus tinker AB 1271 Diasporus tinker AB 1272 Diasporus quidditus CH 5373 Diasporus quidditus CH 5371 100 Diasporus quidditus CH 5372 Diasporus sp. CH 5214 Diasporus vocator AB 028

2 6 Diasporus quidditus CH 5314 Diasporus quidditus CH 5213 Diasporus vocator CH 5400 Diasporus vocator CH 5401 Diasporus sp. AA2178 9 9 Diasporus gularis MHUA 7264 7 9 Diasporus sp. JJS 074 100 Diasporus gularis LEP 264 Diasporus gularis VCK13 100 Diasporus gularis VCK14 9 2 Diasporus quidditus MHUA 6789 Diasporus gularis MHUA 5379 Diasporus sp. MHUA 7405 Diasporus gularis MHUA 7414 2 1 Diasporus sp. MHUA 7412 6 1 Diasporus aff. gularis AJC 3417 3 7 Diasporus gularis MAR 777 Diasporus sp. MHUA 5129

6 3 Diasporus gularis AJC 3419 Diasporus gularis AJC 1183

5 5 Diasporus sp. AJC 2287 Diasporus sp. AJC 1192 Diasporus gularis Toe 1 8 5 Diasporus gularis Toe 2 Diasporus gularis Toe 3 Diasporus sp. AJC 2291 Diasporus diastema AB 073

100 Diasporus diastema AB 084 Diasporus diastema AB 637 Diasporus gularis MAR 689 Diasporus diastema AB 818 2 0 9 5 Diasporus diastema AJC 1536 9 6 Diasporus aff. diastema AB 035 9 7 Diasporus diastema AB 675

8 100 Diasporus aff. diastema AB 218

7 7 Diasporus sp. CH 6503 100 Diasporus sp. CH 6556 Diasporus tigrillo UCR 22364 Diasporus tigrillo UCR 22365

1 8 100 Diasporus tigrillo UCR 22366 Diasporus tigrillo UCR 22367 Diasporus tigrillo UCR 22368 Diasporus diastema CH 5799 Diasporus diastema AJC 1940 Diasporus sp. CH 4764 100 Diasporus diastema CH 5806 Diasporus diastema CH 6676 3 6 Diasporus diastema AB 979

100 Diasporus diastema CH 6786 Diasporus quidditus CH 4726 Diasporus diastema AB 602 Diasporus diastema CH 4834 9 9 Diasporus quidditus CH 5745 Diasporus diastema CH 4746 Diasporus quidditus CH 6792 Diasporus diastema CH 4719 4 8 6 7 Diasporus diastema CH 6800 Diasporus diastema CH 6802

6 6 Diasporus citrinobapheus SMF 89820 Diasporus citrinobapheus SMF 89814

8 5 Diasporus citrinobapheus MHCH 2370 Diasporus citrinobapheus MHCH 2371 Diasporus diastema CH 5302 5 0 9 5 Diasporus diastema CH 5270 Diasporus diastema CH 5225 Diasporus diastema CH 4992 Diasporus diastema CH 5226 9 7 Diasporus diastema CH 5221 Diasporus diastema CH 5211 Diasporus citrinobapheus KRL 0694 Diasporus citrinobapheus KRL 1181

0.03 Diasporus citrinobapheus KRL 0902 Diasporus citrinobapheus KRL 0900 Diasporus citrinobapheus KRL 0901 Diasporus citrinobapheus KRL 0840 Adelophryne_adiastola 7 1 Phyzelaphryne sp.1b AF-2012 9 3 Phyzalephryne sp. AJC 3606 Eleutherodactylus johnstonei

2 4 Diasporus vocator AB 564 Diasporus vocator CH 4786 Diasporus sp. UCR 21844 9 9 Diasporus vocator UCR 21966 Diasporus vocator UCR 21857 4 1 Diaiasporus vocator Pichi 248 Diasporus vocator UCR 20479 3 6 Diasporus vocator AJC 0127 Diasporus vocator CH 4791

3 6 Diasporus diastema CH 4794 Diasporus sp. AB 476 100 Diasporus sp.6 MHCH 1678 100 3 0 Diasporus diastema Pichi 216

6 6 Diasporus diastema UCR 22968 Diasporus sp. UCR 21676

7 2 Diasporus hylaeformis Pichi 229 Diasporus aff. diastema USNM 572456 Diasporus sp. KRL 0782 100 Diasporus sp. KRL 0831 Diasporus aff. diastema MVUP 1826 Diasporus sp. CH 5900 8 6 9 8 Diasporus diastema CH 5370 5 8 Diasporus diastema CH 5031 Diasporus diastema CH 5004 50 Diasporus hylaeformis KRL 0831

8 3 Diasporus aff. diastema AAB 3745 Diasporus hylaeformis KRL 0782 3 3 Diasporus diastema CH 5712

6 6 Diasporus vocator UCR 21946 100 Diasporus sp. UCR 21843

Diasporus sp. UCR 21953 Clade A Diasporus sp. MHCH 1298 8 6 Diasporus aff. hylaeformis SMF 89875 Diasporus aff. hylaeformis SMF 89872 Diasporus hylaeformis Pichi 240 5 0 Diasporus hylaeformis Pichi 241 9 1 Diasporus aff. hylaeformis SMF 89868 Diasporus aff. hylaeformis SMF 89869 Diasporus diastema CH 5695 Diasporus sp. CH 5694 6 4 Diasporus hylaeformis AJC 0468 7 9 Diasporus hylaeformis Pichi 234 Diasporus sp. DL 685 Diasporus hylaeformis AJC 468 6 2 Diasporus diastema Pichi 218 Diasporus hylaeformis UCR 21935

6 4 Diasporus diastema Pichi 224 Diasporus diastema MVZ 203844 3 1 Diasporus sp. AJC 414 Diasporus sp. AJC 369 5 4 Diasporus diastema Pichi 223 Diasporus sp. AB 032 8 3 Diasporus sp. CH 6525 6 1 Diasporus sp. CH 6121 Diasporus sp. CH 6578 Diasporus hylaeformis CH 6520 Diasporus hylaeformis CH 6134 Diasporus cf. hylaeformis AJC 1732 Diasporus sp. CH 6577 Diasporus sp. CH 6583 Diasporus sp. CH 6617 Diasporus vocator CH 4773 Diasporus vocator CH 4772 Diasporus sp. CH 5918 Diasporus sp. MHCH 1288 Diasporus sp.5 AB032 Diasporus hylaeformis CH 4774 Diasporus sp. CH 6505 Diasporus hylaeformis CH 6124 Diasporus sp. CH 6251 100 Diasporus antrax MHUA 7237 Diasporus sp. MHUA 7305 9 7 Diasporus antrax MHUA 7374 4 7 Diasporus aff. quidditus AB 1030

8 Diasporus sp. AB 1065 100 Diasporus sp. CH 6297 Diasporus sapo AB 429

9 4 Diasporus sapo AB 435 9 9 Diasporus sapo AB 430 Diasporus sp.4 AB 439 Diasporus sp.4 AB 431 3 2 Diasporus sp. CH 9142 100 Diasporus sp. AB 1268 Diasporus darienensis AB 329 5 8 Diasporus sp. AJC 595 9 7 Diasporus sp. CH 6425 Diasporus sp. CH 6431 5 6 Diasporus darienensis AB 1185

4 0 Diasporus sp.3 AB 159

9 6 Diasporus darienensis AB 1134 Diasporus darienensis AB 1144 Diasporus darienensis AB 151 Diasporus sp. JJS 065 5 2 Diasporus sp. AJC 2119/MAR1486 9 3 Diasporus quidditus AJC 1194 Diasporus gularis MAA 300 6 4 Diasporus gularis MAA 299 Diasporus pequeno AB 860 Diasporus pequeno AB 822 9 7 Diasporus pequeno AB 857 Diasporus pequeno AB 856 Diasporus pequeno AB 861 Diasporus sp.2 AB 823 100 Diasporus sp. CH 4718 Diasporus sp. CH 4721 Diasporus quidditus CH 5807 Diasporus quidditus CH 4760 Diasporus sp. CH 4741 Diasporus vocator CH 4780 Diasporus quidditus CH 5340 Diasporus quidditus CH 5338 Diasporus quidditus CH 5792 Diasporus quidditus CH 5337 Diasporus quidditus CH 5341 Diasporus quidditus CH 5339 Diasporus quidditus CH 5802 Diasporus quidditus CH 5543

1 4 Diasporus quidditus CH 5540 8 3 6 7 Diasporus quidditus AB 689 4 4 Diasporus quidditus KRL 0856 9 6 Diasporus quidditus KRL 0647 9 0 3 9 Diasporus aff. quidditus AB 931 Diasporus quidditus AB 1130 Diasporus quidditus AB 138 Diasporus quidditus AB 131 Diasporus quidditus AB 158 Diasporus quidditus CH 4748 Diasporus sp. CH 4966 100 Diasporus quidditus CH 4717 Diasporus vocator CH 5392 Diasporus vocator CH 5391 Diasporus vocator CH 5390 Diasporus quidditus CH 6804 3 0 Diasporus diastema CH 6648 Diasporus quidditus AJC 1789 Diasporus diastema CH 4830 Diasporus quidditus CH 6803 Diasporus sp. AJC 600 Diasporus quidditus CH 5624 100 Diasporus quidditus CH 5544 Diasporus quidditus CH 5557 100 4 3 Diasporus quidditus CH 5556 Diasporus sp. AJC 1866

9 9 Diasporus sp. CH 6439 Diasporus sp. CH 9147 Diasporus sp. AJC 1866 7 8 Diasporus tinker AB 308 Diasporus sp. CH 4751

100 Diasporus tinker AB 1270 Clade B Diasporus tinker AB 1269 Diasporus tinker AB 1271 Diasporus tinker AB 1272 Diasporus quidditus CH 5373 Diasporus quidditus CH 5371 100 Diasporus quidditus CH 5372 Diasporus sp. CH 5214 Diasporus vocator AB 028

2 6 Diasporus quidditus CH 5314 Diasporus quidditus CH 5213 Diasporus vocator CH 5400 Diasporus vocator CH 5401 Diasporus sp. AA2178 9 9 Diasporus gularis MHUA 7264 7 9 Diasporus sp. JJS 074 100 Diasporus gularis LEP 264 Diasporus gularis VCK13 100 Diasporus gularis VCK14 9 2 Diasporus quidditus MHUA 6789 Diasporus gularis MHUA 5379 Diasporus sp. MHUA 7405 Diasporus gularis MHUA 7414 2 1 Diasporus sp. MHUA 7412 6 1 Diasporus aff. gularis AJC 3417 3 7 Diasporus gularis MAR 777 Diasporus sp. MHUA 5129

6 3 Diasporus gularis AJC 3419 Diasporus gularis AJC 1183

5 5 Diasporus sp. AJC 2287 Diasporus sp. AJC 1192 Diasporus gularis Toe 1 8 5 Diasporus gularis Toe 2 Diasporus gularis Toe 3 Diasporus sp. AJC 2291 Diasporus diastema AB 073

100 Diasporus diastema AB 084 Diasporus diastema AB 637 58 Diasporus gularis MAR 689 Diasporus diastema AB 818 2 0 9 5 Diasporus diastema AJC 1536 9 6 Diasporus aff. diastema AB 035 9 7 Diasporus diastema AB 675

8 100 Diasporus aff. diastema AB 218

7 7 Diasporus sp. CH 6503 100 Diasporus sp. CH 6556 Diasporus tigrillo UCR 22364 Diasporus tigrillo UCR 22365

1 8 100 Diasporus tigrillo UCR 22366 Diasporus tigrillo UCR 22367 Diasporus tigrillo UCR 22368 Diasporus diastema CH 5799 Diasporus diastema AJC 1940 Diasporus sp. CH 4764 100 Diasporus diastema CH 5806 Diasporus diastema CH 6676 3 6 Diasporus diastema AB 979

100 Diasporus diastema CH 6786 Diasporus quidditus CH 4726 Diasporus diastema AB 602 Diasporus diastema CH 4834 9 9 Diasporus quidditus CH 5745 Diasporus diastema CH 4746 Diasporus quidditus CH 6792 Diasporus diastema CH 4719 4 8 6 7 Diasporus diastema CH 6800 Diasporus diastema CH 6802

6 6 Diasporus citrinobapheus SMF 89820 Diasporus citrinobapheus SMF 89814

8 5 Diasporus citrinobapheus MHCH 2370 Diasporus citrinobapheus MHCH 2371 Diasporus diastema CH 5302 5 0 9 5 Diasporus diastema CH 5270 Diasporus diastema CH 5225 Diasporus diastema CH 4992 Diasporus diastema CH 5226 9 7 Diasporus diastema CH 5221 Diasporus diastema CH 5211 Diasporus citrinobapheus KRL 0694 Diasporus citrinobapheus KRL 1181

0.03 Diasporus citrinobapheus KRL 0902 Diasporus citrinobapheus KRL 0900 Diasporus citrinobapheus KRL 0901 Diasporus citrinobapheus KRL 0840 Adelophryne_adiastola 7 1 Phyzelaphryne sp.1b AF-2012 9 3 Phyzalephryne sp. AJC 3606 Eleutherodactylus johnstonei

2 4 Diasporus vocator AB 564 Diasporus vocator CH 4786 Diasporus sp. UCR 21844 9 9 Diasporus vocator UCR 21966 Diasporus vocator UCR 21857 4 1 Diaiasporus vocator Pichi 248 Diasporus vocator UCR 20479 3 6 Diasporus vocator AJC 0127 Diasporus vocator CH 4791

3 6 Diasporus diastema CH 4794 Diasporus sp. AB 476 100 Diasporus sp.6 MHCH 1678 100 3 0 Diasporus diastema Pichi 216

6 6 Diasporus diastema UCR 22968 Diasporus sp. UCR 21676

7 2 Diasporus hylaeformis Pichi 229 Diasporus aff. diastema USNM 572456 Diasporus sp. KRL 0782 100 Diasporus sp. KRL 0831 Diasporus aff. diastema MVUP 1826 Diasporus sp. CH 5900 8 6 9 8 Diasporus diastema CH 5370 5 8 Diasporus diastema CH 5031 Diasporus diastema CH 5004 50 Diasporus hylaeformis KRL 0831

8 3 Diasporus aff. diastema AAB 3745 Diasporus hylaeformis KRL 0782 3 3 Diasporus diastema CH 5712

6 6 Diasporus vocator UCR 21946 100 Diasporus sp. UCR 21843

Diasporus sp. UCR 21953 Clade A Diasporus sp. MHCH 1298 8 6 Diasporus aff. hylaeformis SMF 89875 Diasporus aff. hylaeformis SMF 89872 Diasporus hylaeformis Pichi 240 5 0 Diasporus hylaeformis Pichi 241 9 1 Diasporus aff. hylaeformis SMF 89868 Diasporus aff. hylaeformis SMF 89869 Diasporus diastema CH 5695 Diasporus sp. CH 5694 6 4 Diasporus hylaeformis AJC 0468 7 9 Diasporus hylaeformis Pichi 234 Diasporus sp. DL 685 Diasporus hylaeformis AJC 468 6 2 Diasporus diastema Pichi 218 Diasporus hylaeformis UCR 21935

6 4 Diasporus diastema Pichi 224 Diasporus diastema MVZ 203844 3 1 Diasporus sp. AJC 414 Diasporus sp. AJC 369 5 4 Diasporus diastema Pichi 223 Diasporus sp. AB 032 8 3 Diasporus sp. CH 6525 6 1 Diasporus sp. CH 6121 Diasporus sp. CH 6578 Diasporus hylaeformis CH 6520 Diasporus hylaeformis CH 6134 Diasporus cf. hylaeformis AJC 1732 Diasporus sp. CH 6577 Diasporus sp. CH 6583 Diasporus sp. CH 6617 Diasporus vocator CH 4773 Diasporus vocator CH 4772 Diasporus sp. CH 5918 Diasporus sp. MHCH 1288 Diasporus sp.5 AB032 Diasporus hylaeformis CH 4774 Diasporus sp. CH 6505 Diasporus hylaeformis CH 6124 Diasporus sp. CH 6251 100 Diasporus antrax MHUA 7237 Diasporus sp. MHUA 7305 9 7 Diasporus antrax MHUA 7374 4 7 Diasporus aff. quidditus AB 1030

8 Diasporus sp. AB 1065 100 Diasporus sp. CH 6297 Diasporus sapo AB 429

9 4 Diasporus sapo AB 435 9 9 Diasporus sapo AB 430 Diasporus sp.4 AB 439 Diasporus sp.4 AB 431 3 2 Diasporus sp. CH 9142 100 Diasporus sp. AB 1268 Diasporus darienensis AB 329 5 8 Diasporus sp. AJC 595 9 7 Diasporus sp. CH 6425 Diasporus sp. CH 6431 5 6 Diasporus darienensis AB 1185

4 0 Diasporus sp.3 AB 159

9 6 Diasporus darienensis AB 1134 Diasporus darienensis AB 1144 Diasporus darienensis AB 151 Diasporus sp. JJS 065 5 2 Diasporus sp. AJC 2119/MAR1486 9 3 Diasporus quidditus AJC 1194 Diasporus gularis MAA 300 6 4 Diasporus gularis MAA 299 Diasporus pequeno AB 860 Diasporus pequeno AB 822 9 7 Diasporus pequeno AB 857 Diasporus pequeno AB 856 Diasporus pequeno AB 861 Diasporus sp.2 AB 823 100 Diasporus sp. CH 4718 Diasporus sp. CH 4721 Diasporus quidditus CH 5807 Diasporus quidditus CH 4760 Diasporus sp. CH 4741 Diasporus vocator CH 4780 Diasporus quidditus CH 5340 Diasporus quidditus CH 5338 Diasporus quidditus CH 5792 Diasporus quidditus CH 5337 Diasporus quidditus CH 5341 Diasporus quidditus CH 5339 Diasporus quidditus CH 5802 Diasporus quidditus CH 5543

1 4 Diasporus quidditus CH 5540 8 3 6 7 Diasporus quidditus AB 689 4 4 Diasporus quidditus KRL 0856 9 6 Diasporus quidditus KRL 0647 9 0 3 9 Diasporus aff. quidditus AB 931 Diasporus quidditus AB 1130 Diasporus quidditus AB 138 Diasporus quidditus AB 131 Diasporus quidditus AB 158 Diasporus quidditus CH 4748 Diasporus sp. CH 4966 100 Diasporus quidditus CH 4717 Diasporus vocator CH 5392 Diasporus vocator CH 5391 Diasporus vocator CH 5390 Diasporus quidditus CH 6804 3 0 Diasporus diastema CH 6648 Diasporus quidditus AJC 1789 Diasporus diastema CH 4830 Diasporus quidditus CH 6803 Diasporus sp. AJC 600 Diasporus quidditus CH 5624 100 Diasporus quidditus CH 5544 Diasporus quidditus CH 5557 100 4 3 Diasporus quidditus CH 5556 Diasporus sp. AJC 1866

9 9 Diasporus sp. CH 6439 Diasporus sp. CH 9147 Diasporus sp. AJC 1866 7 8 Diasporus tinker AB 308 Diasporus sp. CH 4751

100 Diasporus tinker AB 1270 Clade B Diasporus tinker AB 1269 Diasporus tinker AB 1271 Diasporus tinker AB 1272 Diasporus quidditus CH 5373 Diasporus quidditus CH 5371 100 Diasporus quidditus CH 5372 Diasporus sp. CH 5214 Diasporus vocator AB 028

2 6 Diasporus quidditus CH 5314 Diasporus quidditus CH 5213 Diasporus vocator CH 5400 Diasporus vocator CH 5401 Diasporus sp. AA2178 9 9 Diasporus gularis MHUA 7264 7 9 Diasporus sp. JJS 074 100 Diasporus gularis LEP 264 Diasporus gularis VCK13 100 Diasporus gularis VCK14 9 2 Diasporus quidditus MHUA 6789 Diasporus gularis MHUA 5379 Diasporus sp. MHUA 7405 Diasporus gularis MHUA 7414 2 1 Diasporus sp. MHUA 7412 6 1 Diasporus aff. gularis AJC 3417 3 7 Diasporus gularis MAR 777 Diasporus sp. MHUA 5129

6 3 Diasporus gularis AJC 3419 Diasporus gularis AJC 1183

5 5 Diasporus sp. AJC 2287 Diasporus sp. AJC 1192 Diasporus gularis Toe 1 8 5 Diasporus gularis Toe 2

Diasporus gularis Toe 3 Diasporus sp. AJC 2291 Diasporus diastema AB 073

100 Diasporus diastema AB 084 Diasporus diastema AB 637 Diasporus gularis MAR 689 Diasporus diastema AB 818 2 0 9 5 Diasporus diastema AJC 1536 9 6 Diasporus aff. diastema AB 035 9 7 Diasporus diastema AB 675

8 100 Diasporus aff. diastema AB 218

7 7 Diasporus sp. CH 6503 100 Diasporus sp. CH 6556 Diasporus tigrillo UCR 22364 Diasporus tigrillo UCR 22365

1 8 100 Diasporus tigrillo UCR 22366 Diasporus tigrillo UCR 22367 Diasporus tigrillo UCR 22368 Diasporus diastema CH 5799 Diasporus diastema AJC 1940 Diasporus sp. CH 4764 100 Diasporus diastema CH 5806 Diasporus diastema CH 6676 3 6 Diasporus diastema AB 979

100 Diasporus diastema CH 6786 Diasporus quidditus CH 4726 Diasporus diastema AB 602 Diasporus diastema CH 4834 9 9 Diasporus quidditus CH 5745 Diasporus diastema CH 4746 Diasporus quidditus CH 6792 Diasporus diastema CH 4719 4 8 6 7 Diasporus diastema CH 6800 Diasporus diastema CH 6802

6 6 Diasporus citrinobapheus SMF 89820 Diasporus citrinobapheus SMF 89814

8 5 Diasporus citrinobapheus MHCH 2370 Diasporus citrinobapheus MHCH 2371 Diasporus diastema CH 5302 5 0 9 5 Diasporus diastema CH 5270 Diasporus diastema CH 5225 Diasporus diastema CH 4992 Diasporus diastema CH 5226 9 7 Diasporus diastema CH 5221 Diasporus diastema CH 5211 Diasporus citrinobapheus KRL 0694 Diasporus citrinobapheus KRL 1181

0.03 Diasporus citrinobapheus KRL 0902 Diasporus citrinobapheus KRL 0900 Diasporus citrinobapheus KRL 0901 Diasporus citrinobapheus KRL 0840

59

Fig. 3

Eleutherodactylus johnstonei

Diasporus vocator CH 4786 Panamá 100 Diasporus sp. CH 4794 Panamá 100 3 7 Diasporus anthrax MHUA 7237 Colombia

4 5 Diasporus sp. CH 4760 Panamá

6 3 Diasporus sp. CH 4751 Panamá

3 9 Diasporus quidditus CH 5373 Panamá

Diasporus tinker CH 4717 Panamá 1 2 3 3 Diasporus sp. JSS 065 Colombia

3 2 Diasporus sp. MAA 299 Colombia

6 4 Diasporus gularis MHUA 7414 Colombia

3 7 Diasporus gularis MAR 689 Colombia

4 7 Diasporus gularis JJS 074 Colombia

9 6 Diasporus gularis AA 2178 Colombia

Substitutions/site 0.006

60

Fig. 4

Montes et al., 2015 Panamá closre

ABGDbGMC Eleutherodactylus johnstonei Diasporus vocator CH 4786 9 4 Diasporus vocator UCR 21966 9 3 Diasporus vocator UCR 21857 Diaiasporus vocator Pichi 248 Diasporus vocator UCR 20479 OTU 3 Diasporus vocator CH 4791 D. vocator Diasporus sp. UCR 21844

1 9 Diasporus vocator AJC 0127 Diasporus diastema CH 4794 OTU 24 2 7 100 Diasporus sp. AB 476 Diasporus sp.6 MHCH 1678 OTU 2 2 8 7 1 Diasporus diastema Pichi 216 8 7 Diasporus diastema UCR 22968 Diasporus sp. UCR 21676 6 9 Diasporus hylaeformis Pichi 229 OTU 27 Diasporus sp. CH 5900 9 9 Diasporus diastema CH 5370 Diasporus diastema CH 5031 9 4 Diasporus diastema CH 5004 5 5 Diasporus hylaeformis KRL 0831 8 1 9 9 Diasporus aff. diastema AAB 3745 Diasporus hylaeformis KRL 0782 100 Diasporus aff. diastema MVUP 1826 Diasporus aff. diastema USNM5 72456 7 3 Diasporus diastema CH 5712 3 4 100 Diasporus vocator UCR 21946 Diasporus sp. UCR 21843 Diasporus sp. UCR 21953

8 1 Diasporus hylaeformis Pichi 234 Diasporus hylaeformis AJC 468

7 5 5 2 Diasporus sp. DL 685 Diasporus diastema Pichi 218 8 9 Diasporus sp. AJC 414 Diasporus diastema Pichi 223 Diasporus hylaeformis UCR 21935 Diasporus diastema Pichi 224

7 7 Diasporus diastema MVZ 203844 Diasporus sp. MHCH 1298 Diasporus aff. hylaeformis SMF 89869 9 3 Diasporus aff. hylaeformis SMF 89868 OTU 1 Diasporus diastema CH 5695 D. hylaeformis 9 2 Diasporus sp. CH 5694 9 9 Diasporus aff. hylaeformis SMF 89872 Diasporus aff. hylaeformis SMF 89875 1 5 Diasporus hylaeformis Pichi 240 Diasporus hylaeformis Pichi 241 Diasporus sp. CH 6525 7 3 Diasporus vocator CH 4773 Diasporus sp. CH 6121 Diasporus sp. CH 6578 1 Diasporus hylaeformis CH 6134 bGMYC cluster probabilities Diasporus hylaeformis CH 6520 Diasporus sp. CH 6583 p=0 - 0.05 Diasporus sp. CH 6617 Diasporus hylaeformis AJC 1732 p= 0.05-0.5 Diasporus sp. CH 6577 Diasporus sp. CH 6251 p= 0.5 - 0.9 Diasporus hylaeformis CH 6124 Diasporus hylaeformis CH 4774 p= 0.9 - 0.95 Diasporus sp. CH 6505 Diasporus vocator CH 4772 Diasporus sp. CH 5918 p= 0.95 - 1 Diasporus sp. MHCH 1288 Diasporus sp.5 AB 032

Million years ago 4 0 3 0 2 0 1 0 0

61

Diasporus sp.5 AB 032 Diasporus vocator AB 564 OTU 23 Diasporus antrax MHUA 7237 100 OTU 19 100 Diasporus sp. MHUA 7305 D. anthrax Diasporus antrax MHUA 7374 6 9 OTU 20 Diasporus majeensis AB 1030 D. majeensis 100 Diasporus sp. CH 6297 OTU 22 9 5 Diasporus majeensis AB 1065 D. majeensis Diasporus sapo AB 435 4 1 100 Diasporus sapo AB 429 OTU 21a Diasporus sapo AB 430 D. sapo Diasporus sp.4 AB 439 5 9 Diasporus sp.4 AB 431 Diasporus sp. CH 9142 9 9 6 0 8 1 Diasporus darienensis AB 329 Diasporus sp. AB 1268 100 9 5 Diasporus sp. CH 6425 Diasporus sp. AJC 595 OTU 21b Diasporus sp. CH 6431 D. darinensis 6 1 Diasporus darienensis AB 1185 Diasporus sp.3 AB 159 5 4 9 8 Diasporus darienensis AB 1134 Diasporus darienensis AB 151 Diasporus darienensis AB 1144 Diasporus sp. JJS 065 OTU 18 5 1 Diasporus sp. AJC 2119/MAR 1486 OTU 15

9 9 9 9 Diasporus quidditus AJC 1194 9 8 7 4 Diasporus gularis MAA 299 OTU 17 Diasporus gularis MAA 300 Diasporus pequeno AB 860 9 5 Diasporus pequeno AB 822 Diasporus pequeno AB 861 OTU 16 Diasporus pequeno AB 856 D. pequeno Diasporus pequeno AB 857 Diasporus sp. AB 823 Diasporus sp. CH 4718 100 Diasporus sp. CH 4721 Diasporus quidditus CH 4760 Diasporus quidditus CH 5807 Diasporus vocator CH 4780 Diasporus sp. CH 4741 Diasporus quidditus CH 5338 Diasporus quidditus CH 5340 Diasporus quidditus CH 5337 Diasporus quidditus CH 5792 Diasporus quidditus CH 5341 Diasporus quidditus CH 5339 OTU 4 D. af. quidditus Diasporus quidditus CH 5802 Diasporus quidditus CH 5540 9 9 Diasporus quidditus CH 5543 Diasporus quidditus AB 138 bGMYC cluster probabilities 9 9 Diasporus quidditus AB 689 Diasporus quidditus KRL 0856 p=0 - 0.05 Diasporus quidditus KRL 0647 Diasporus aff. quidditus AB 931 p= 0.05-0.5 Diasporus quidditus AB 1130 Diasporus quidditus AB 158 p= 0.5 - 0.9 Diasporus quidditus AB 131

p= 0.9 - 0.95

p= 0.95 - 1

Million years ago 4 0 3 0 2 0 1 0 0

62

Diasporus quidditus AB 131 Diasporus quidditus CH 4748 100 Diasporus sp. CH 4966 Diasporus quidditus CH 4717 Diasporus vocator CH 5392 Diasporus vocator CH 5390 Diasporus vocator CH 5391 Diasporus quidditus CH 6804 Diasporus quidditus CH 6803 3 5 Diasporus diastema CH 4830 Diasporus quidditus AJC 1789 Diasporus diastema CH 6648 Diasporus sp. AJC 600 OTU 5 100 Diasporus quidditus CH 5544 D.tinker Diasporus quidditus CH 5624 Diasporus quidditus CH 5556 5 3 100 Diasporus quidditus CH 5557

9 9 Diasporus sp. AJC 1866 Diasporus sp. CH 6439 Diasporus sp. CH 9147

9 2 Diasporus tinker AB 308 Diasporus sp. CH 4751 100 Diasporus tinker AB 1270 Diasporus tinker AB 1271 Diasporus tinker AB 1269 Diasporus tinker AB 1272 Diasporus quidditus CH 5373 100 Diasporus quidditus CH 5371 7 8 Diasporus quidditus CH 5372 Diasporus sp. CH 5214 6 4 Diasporus vocator AB 028 OTU 14 Diasporus quidditus CH 5213 Diasporus quidditus CH 5314 Diasporus vocator CH 5401 Diasporus vocator CH 5400 Diasporus sp. AA 2178 100 100 9 5 Diasporus gularis VCK 14 100 Diasporus gularis VCK 13 OTU 13 4 4 Diasporus gularis LEP 264 9 9 Diasporus gularis MHUA 7264 Diasporus sp. JJS 074 Diasporus quidditus MHUA 6789 9 7 Diasporus gularis MHUA 5379

6 6 Diasporus sp. MHUA 7405 OTU 12 9 9 7 3 Diasporus sp. MHUA 7412 5 3 6 2 Diasporus gularis MHUA 7414 7 8 Diasporus aff. gularis AJC 3417 bGMYC cluster probabilities Diasporus sp. MHUA 5129 9 1 Diasporus gularis AJC 3419 p=0 - 0.05 9 4 Diasporus sp. AJC 2287 Diasporus gularis AJC 1183 OTU 11 p= 0.05-0.5 Diasporus sp. AJC 1192 Diasporus sp. AJC 2291 p= 0.5 - 0.9 100 Diasporus gularis Toe 3 Diasporus gularis Toe 1 p= 0.9 - 0.95 Diasporus gularis Toe 2

p= 0.95 - 1

Million years ago 4 0 3 0 2 0 1 0 0

63

Diasporus gularis Toe 2

100 Diasporus diastema AB 073 Diasporus diastema AB 637 OTU 6 Diasporus diastema AB 084 D. af. diastema Diasporus gularis MAR 689 OTU 25 Diasporus diastema AB 818 5 2 9 4 100 Diasporus diastema AB 675 100 Diasporus aff. diastema AB 218 OTU 10 9 4 Diasporus aff. diastema AB 035 D. af. diastema 1 1 Diasporus diastema AJC 1536 100 Diasporus sp. CH 6556 Diasporus sp. CH 6503 8 3 Diasporus tigrillo UCR 22364 OTU 7 100 Diasporus tigrillo UCR 22367 D. tigrillo 2 8 Diasporus tigrillo UCR 22366 Diasporus tigrillo UCR 22368 Diasporus tigrillo UCR 22365

6 5 Diasporus citrinobapheus SMF 89820 9 0 Diasporus citrinobapheus MHCH 2370 Diasporus citrinobapheus MHCH 2371 Diasporus diastema CH 5302 9 9 Diasporus diastema CH 5270 5 7 5 1 Diasporus diastema CH 4992 Diasporus diastema CH 5225 OTU 8 9 4 Diasporus diastema CH 5226 Diasporus diastema CH 5221 D. citrinobapheus Diasporus diastema CH 5211

6 5 Diasporus citrinobapheus KRL 0694 Diasporus citrinobapheus KRL 1181 Diasporus citrinobapheus KRL 0902 Diasporus citrinobapheus KRL 0900 7 8 Diasporus citrinobapheus KRL 0901 Diasporus citrinobapheus KRL 0840 Diasporus sp. CH 4764 bGMYC cluster probabilities Diasporus diastema CH 5806 100 Diasporus diastema CH 5799 p=0 - 0.05 Diasporus diastema AJC 1940 Diasporus diastema AB 979 p= 0.05-0.5 Diasporus diastema CH 6786 100 Diasporus diastema CH 6676 p= 0.5 - 0.9 Diasporus quidditus CH 6792 Diasporus diastema CH 4719 OTU 9 p= 0.9 - 0.95 Diasporus diastema CH 6802 D. af. diastema 100 Diasporus diastema CH 6800 Diasporus diastema AB602 p= 0.95 - 1 Diasporus quidditus CH 4726 Diasporus diastema CH 4746 Diasporus quidditus CH 5745 Diasporus diastema CH 4834 Million years ago 4 0 3 0 2 0 1 0 0

64

Fig. 5

B. A. OTU 1 D. hylaeformis OTU 5 D. tinker OTU 4 D. af. quidditus OTU 7 D. tigrillo OTU 11 OTU 14

D. OTU 2 OTU 3 D. vocator C. OTU 6 D. af. diastema OTU 10 D. af. diastema OTU 8 D. citrinobapheus OTU 12 OTU 9 OTU 13

E. OTU 16 D. pequeno OTU 17 F. OTU 18 OTU 22 D. majeensis OTU 19 D. anthrax OTU 23 OTU 20 D. majeensis OTU 24 OTU 21a D. sapo OTU 25 OTU 21b D. darinensis OTU 26

65

Fig. 6

Identity test: Identity test: A. OTU 4 vs. OTU 5 D. tinker B. OTU8 vs. OTU9

15

15

10 10 count count

5 5

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 D D C. Identity test: Identity test: OTU 11 vs. OTU 12 D. OTU 21a D. sapo vs. OTU 21b D. darinensis

20 25

20 15

15

count 10 10

5 5

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 D D

66 Chapter 4 - Mitochondrial genomes of frogs captured using massive sequencing techniques.

Lucas S. Barrientos1, Jeffrey. W. Streicher2, John J. Wiens3, Andrew. J. Crawford1,4,5

1 Department of Biological Sciences, Universidad de los Andes, Bogotá, código postal

111711, Colombia

2 Department of Life Sciences. The Natural History Museum. Present Address. Darwin

Centre, DC1 209B. Cromwell Road, South Kensington. London SW7 5BD UK

3 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ

85721-088, USA.

1 ABSTRACT

Over the past few decades, the mitochondrial genome has been a marker widely used in population genetics, phylogeography, and phylogenetic studies of due its high mutation rate, rare or non-existent recombination, maternal inheritance, and easy amplification using PCR. Recent improvements in DNA sequencing technologies have made massively parallel sequencing (MPS) commonplace, and obtaining nearly complete mitochondrial genome sequences relatively easy, even as a byproduct of MPS efforts to obtain other genomic information. Here, we report the capture of 16 almost complete mitogenomes obtained from byproduct of the library construction for the target of ultraconserved elements (UCEs). The almost complete mitochondrial genome sequence for

11 frogs (lacking only the control region), plus long fragments of over 5 kilobases (Kb) for

5 additional frogs, for a total of 16 partial mitochondrial genomes. The almost complete genomes ranged from approximately 12 to 16 Kb in length. We found that the mitochondrial genomes are similar to previously assembled genomes for neobatrachian frogs in terms of gene content and gene reorders. tRNAs near the nad4–nad5 junction and close to the control region are thought to be more mobile than in other mitogenome regions.

They contain a conserved structure including 13 protein-coding genes, 2 ribosomal RNA

(rRNA) genes, and 22 tRNA genes. This study highlights the utility of using off-target sequences from sequence-capture methods.

2 INTRODUCTION

The typical animal mitochondrial genome (mitogenome) structure is a circular DNA molecule ranging from 14 kilobases (Kb) to 25 Kb (Wolstenholme, 1992; Sano et al.,

2005), composed of two ribosomal RNA genes (rRNA-S and rRNA-L, also known as 12S and 16S genes, respectively), 22 transfer RNAs (tRNAs), and 13 protein-coding genes that belong to four enzyme complexes of the respiration chain: Cytochrome oxidase b (CytB), two subunits of ATP synthase (ATP 6,8), and seven subunits of NADH dehydrogenase

(NAD1–6, 4L; Fig 1; Bernt et al., 2013a; Boore, 1999). Substantial heterogeneity in substitution rates among sites within genes and among genes make mitochondrial DNA

(mtDNA) a practical data source for studying molecular evolution, phylogeography, and phylogenetics (Wolstenholme, 1992). Mitochondrial DNA is also used widely as a proxy for species diversity in DNA barcoding applications (Hebert et al., 2003). Contrary to early reports that mitogenomes were largely invariant in structure up to the level of phylum, animal mitogenomes exhibit variation over multiple levels of organization, from gene content and gene order to variation in nucleotide sequences, facilitating phylogenetic inference of both recent and ancient divergence events (Boore, 1999)

Recently, complete animal mitochondrial genomes have been recovered as a byproduct of massively parallel sequences (MPS) of genomic libraries (Hung et al. 2013, from ancient DNA) or gene capture libraries, e.g, ultraconserved elements (UCEs; Pie et al., 2017). Thus, the de novo assembly of short reads generated by shotgun sequencing could be an efficient approach to sequence mitogenomes. Here, we report the partial mitogenomes from 16 specimens of frog species, recovered as a byproduct of the genome capture libraries produced in previous studies of hyloid frogs (Streicher et al. 2018;

3 Barrientos et al. in prep.) and briefly describe the mitochondrial genomes of 16 species of frogs obtained from off-target MST sequencing technology.

The mtDNA sequences were obtained as off-target regions from previous ongoing studies on the phylogenetic relationships among hyloids (Streicher et al., 2017) and terraranan frogs (Barrientos et al., in prep) both focusing on target capture of ultraconserved elements (UCEs; Faircloth et al. 2012). The taxon sampling included representatives of ten taxonomic families of frogs: Calyptocephalellidae, Centrolenidae,

Ceratophryidae, Craugastoridae, Cycloramphidae, Hemiphractidae, Hylidae,

Leptodactylidae, Odontophrynidae, Telmatobiidae, and (Table 1, Fig. 1). Samples were provided by the Museo de Historia Natural ANDES at the Universidad de los Andes in

Bogotá (ANDES), the Museum of Vertebrate Zoology at the University of California,

Berkeley (MVZ), Amphibian and Reptile Diversity Research Center at the University of

Texas at Arlington (UTA), Museum of Comparative Zoology at Harvard University

(MCZ), and the Biodiversity Institute and Natural History Museum at the University of

Kansas (KU).

Genomic DNA (gDNA) was extracted using DNeasy® Blood and Tissue kits

(Qiagen) or using magnetic beads (Rohland & Reich 2012 Rohland and Reich 2012; Sera-

Mag Speedbeads, Fisher Scientific). Samples were digested overnight at 55ºC in 20 µL proteinase K in 180 µL of lysis buffer. Genomic DNA was captured with ca. 360 µL magnetic beads, cleaned with two 700 µL washes of 70% EtOH, and eluted in 70 µL of 10 mM Tris (pH 8). After extraction, we quantified the amount of gDNA via fluorometry using double-stranded DNA high-sensitivity assay kits (Qubit, Life Technologies).

4 For capture and library preparation we followed Faircloth et al. (2012; available at http://ultraconserved.org), with the modifications used by Streicher et al. (Streicher et al.,

2016) as part of diferent studies to capture ultraconserved elements (UCEs). 150 ng of starting gDNA was fragmented by either physical shearing with a Bioruptor (Diagenode) using 6 cycles of high-speed agitation for 30 s on, 90 s off, or by enzymatic digestion using

NEBNext dsDNA Fragmentase (New England Biolabs) at 37°C for 25 m. The post- hybridization PCR was conducted with NEB Phusion DNA polymerase and TruSeq primers (Streicher et al., 2016). Enriched libraries were visualized for fragment-size distribution and abundance using a Bioanalyzer 7500 (Agilent). We sequenced the three capture libraries on three runs, each with 48 individuals (not all individuals were included in the present study). We performed 600-cycle paired-end sequencing runs on an Illumina

MiSeq at the genomics core facility of the University of Texas at Arlington (Arlington, TX,

USA; http://gcf.uta.edu/).

The obtained raw reads were de novo assembled using Velvet 1.2.10 (Zerbino and

Birney, 2008), which is part of the common UCE-processing pipeline. To filter out mitochondrial sequences we compared the merged reads with a dataset of 3 mitochondrial genomes (downloaded from GenBank on 20 October 2017, Espadarana prosopblepon

JX564857, Eleutherodcatylus atkinsi JX564864, Xenopus laevis M10217) using BLAST

(blastn, e-value: 10e-4; Altschul et al. 1990). Some of the longest contigs included nearly complete mitogenomes (qv. Pie et al. 2017; Hung et al., 2013). Genome annotation was carried out using MITOS 2 (Bernt et al., 2013b) at the online webserver http://mitos2.bioinf.uni-leipzig.de/index.py under the reference data set for the annotation

RefSeq 81 (ftp://ftp.ncbi.nlm.nih.gov/refseq/release/release-notes/), and assuming the standard vertebrate genetic code.

5 RESULTS

From the Velvet assembled contigs we found one or two contigs per species containing the mitochondrial DNA. Each of the contigs recovered varied from 48% to 97% of the total complement of loci relative to the standard vertebrate mitogenome (Boore,

1999; Wolstenholme, 1992). The read coverage on the contigs containing mitochondrial genes varied from 10.49X to 292.55X, and the length of mtDNA-containing contigs was between 2,169 base pairs (bp) and 15,718 bp. The total length of the partial mitochondrial genomes reported here varied from 7,198 to 15,718 bp (Table 1, Table S1). Within contigs, the order of protein-coding genes was had the same to mitochondrial gene order that the previous reported neobatrachian mitogenomes (Fig. 1), also shared estructural similarities whit the rearrangements for the tRNAs previously described for neobatrachians (Zhang et al., 2013). In general, tRNAs in the near the nad4–nad5 junction and close to the control region are thought to be more mobile than in other mitogenome regions (Boore 1999). Our data support this finding as most tRNA rearrangements occurred between the nad4–nad5 and the control region, moves the tRNAs close to the rrnS (12S).

The mitogenomes are highly informative markers, not only in terms of sequence data, also gene order (Zhang et al., 2013). With the more frequent use of next-generation sequencing, the acquisition of the mitochondrial genomes could will become an inexpensive complementary marker. We highlight the utility of using byproduct sequences from massive sequence-capture methods, such as UCEs to explore and describe the possible presence of mitochondrial genomes.

ACKNOWLEDGEMENTS

6 We are grateful for tissue loans from Museo de Historia Natural ANDES at the

Universidad de los Andes in Bogotá (ANDES), the Museum of Vertebrate Zoology at the

University of California, Berkeley (MVZ), Amphibian and Reptile Diversity Research

Center at the University of Texas at Arlington (UTA), Museum of Comparative Zoology at

Harvard University (MCZ), and the Biodiversity Institute & Natural History Museum at

Kansas University (KU). We thank to Camila Plata for the help in the early stages of the development of this project and to Maria José Gomez for the help with the mitochondrial genome ensemble. We thank Andres M. Cuervo, Luisa Castellanos, Luisa Dueñas, Santiago

Herrera, Andrea Paz, and Camila Plata for providing valuable comments on earlier versions of this manuscript and to all of the Biomi|cs lab members.

DISCLOSURE STATEMENT

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the Colciencias doctoral fellowship 567 and the Colciencias grant Purdue grant 655, Proyecto semilla Universidad de los Andes, FAPA grant AJC, and The University of Arizona and U.S. National Science

Foundation Grant DEB 1655690 to JJW.

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10 Table 1. Included species, museum voucher numbers, and results of the ensembled data set of mitochondrial genomes. Voucher information and amount of DNA data produced for each sample, including number of contigs assembled using the software Velvet (v. 1.2.10), and the resulting number of mitochondrial contigs assembled, the read coverage of each contig, and the Sequence Read Archive (SRA) accession numbers provide access to all reads obtained for each individual.

Mitochondrial Total number Contig length Coverage SRA accession Species Id number assembled of contigs (bp) (X) number contigs

Allophryne 8,483 10.49 MAD 1852 13,011 2 SAMN05559872 ruthveni 2,189 11.49 Calytocephalla CM 68336 29,581 1 12,768 16.54 SAMN05559935 gayi Espadarana MVZ 149741 25,601 1 13,871 20.43 SAMN05559886 prosoblepon Ceratophrys KU 215537 40,359 1 14,086 292.55 SAMN05559887 cornuta Craugastor JWS 296 7,375 1 15,018 73.55 augusti Cyclorampus MTR-H-526 14,662 1 15,511 87.23 SAMN05559891 boraceiensis Hemiphractus 10,890 38 KU 217513 10,460 2 SAMN05559908 proboscideus 2,169 24.58 Hypodactylus KU 178258 23,432 1 13,128 24.2 brunneus Lepidobatrachus UWSP 3599 20,065 1 7,198 15.39 laevis Lynchius 2,223 14.42 ERW 86 19,056 2 SAMN05559921 nebulanastes 13,575 17.2

Odontophrynus 15,718 43.33 ZVCB 10097 23,145 2 SAMN05559924 americanus 301 15.12

Pristimantis 5,945 36.84 WED 56667 7,763 2 simonsii 7,846 44.49 Scinax USNM 208986 5,950 1 12,375 22.63 SAMN0555993 catharinae Strabomantis ANDES-A 2,224 22.93 257,299 2 anomalus 1416 6,914 37.211

Telmatobius 5,358 21.26 KU 214828 7,643 2 SAMN05559932 carrillae 10,157 23.09 Thoropa MTR17674 9,829 1 15,634 63.9 SAMN05559934 miliaris

11 1 Figure 1. Partial mitochondrial genomes obtained from Illumina sequencing of the UCEs genomic libraries, ensembled using Velvet

2 software version 1.2.10, the annotation was performed using MITOS 2 webserver http://mitos2.bioinf.uni-leipzig.de/index.py. The

3 standard vertebrate mitochondria modified from Irisarri et al., 2012, in gray color the tRNAs and in white the protein coding genes and

4 the two ribosomal RNAs. For the ensembled mtDNA genomes the translocated protein-coding genes (orange) and transfer RNA genes

5 (blue), and indicated with red arrows the loses of locus. Abbreviations of mitochondrial genes follow (Boore, 1999).

6

7

8

12 L1 L1 L1 L1 L1 L1 L1 L1 L1 L1 L1 L1 Hypodactylus brunneus Allophryne ruthveni gayi Calyptocephalella prosoblepon Espadarana cornuta Ceratophrys augusti Craugastor boraceiensis Cyclorampus proboscidus Hemiphractus laevis Lepidobatrachus nebulanastes Lynchius Odontophrynus americanus simonsii Pristimantis Scinax catharinae anomalus Strabomantis carrillae Telmatobius miliaris Thoropa A Standard vertebrate mitochondria mitochondria vertebrate Standard M M M Q rrnS, V rrnS,

9

10

13 11 Table S1. Characteristics of the partial mitochondrial genomes recovered for the 16-species

12 assembled using Velvet 1.2.10 and annotated using MITOS 2. The negative symbol shows

13 when the loci is localized in the light strand and the positive symbol when the loci is localized

14 in the heavy strand. The tRNAs follows Boore, (1999) abbreviations.

Allophryne ruthveni

Position Length Contig Gene Start Stop Strand (bp) cob 105 1226 1122 - trnE(gaa) 1238 1305 68 + nad6 1309 1797 489 + nad5 1796 3553 1758 - trnY(tat) 3548 3606 59 + trnS1(agc) 3623 3690 68 - trnH(cac) 3691 3759 69 - nad4 3756 5123 1368 - nad4l 5120 5389 270 - trnR(cga) 5417 5485 69 - 1 nad3 5487 5828 342 - trnG(gga) 5826 5894 69 - cox3 5896 6678 783 - atp6 6684 7361 678 - atp8 7358 7516 159 - trnK(aaa) 7517 7589 73 - cox2 7606 8277 672 - trnD(gac) 8279 8347 69 - trnS2(tca) 8349 8419 71 + cox1 8428 8535 108 -

Ceratophrys cornuta

Position Length Contig Gene Start Stop Strand (bp) nad6 8 385 378 + nad5 384 2159 1776 - 1 trnS1(agc) 2212 2278 67 - trnH(cac) 2279 2347 69 -

14 nad4-0 2349 3710 1362 - nad4l 3707 3997 291 - trnR(cga) 4005 4074 70 - nad3 4076 4417 342 - trnG(gga) 4415 4483 69 - cox3 4485 5267 783 - atp6 5273 5950 678 - atp8 5947 6105 159 - trnK(aaa) 6106 6177 72 - cox2 6194 6865 672 - trnD(gac) 6867 6935 69 - trnS2(tca) 6937 7007 71 + cox1 7018 8547 1530 - trnY(tac) 8555 8624 70 + trnC(tgc) 8625 8690 66 + trnN(aac) 8715 8787 73 + trnA(gca) 8788 8856 69 + trnW(tga) 8857 8926 70 - nad2 8931 9956 1026 - trnM(atg) 9957 10025 69 - trnQ(caa) 10025 10095 71 + trnI(atc) 10095 10165 71 - nad1 10173 11108 936 - trnL2(tta) 11127 11199 73 - rrnL 11199 12798 1600 - trnV(gta) 12799 12867 69 - rrnS 12865 13800 936 - trnF(ttc) 13801 13868 68 - trnP(cca) 13870 13938 69 + trnT(aca) 13938 14008 71 - trnL1(cta) 14009 14080 72 -

Craugastor augusti

Position Length Contig Gene Start Stop Strand (bp) trnL1(cta) 141 212 72 + 1 trnT(aca) 213 282 70 + trnP(cca) 283 351 69 -

15 trnF(ttc) 351 418 68 + rrnS 419 1348 930 + trnV(gta) 1346 1413 68 + rrnL 1414 2999 1586 + trnL2(tta) 2997 3069 73 + nad1 3082 4023 942 + trnI(atc) 4031 4100 70 + trnQ(caa) 4099 4169 71 - trnM(atg) 4169 4239 71 + nad2 4240 5262 1023 + trnW(tga) 5273 5341 69 + trnA(gca) 5342 5410 69 - trnN(aac) 5411 5483 73 - trnC(tgc) 5510 5572 63 - trnY(tac) 5573 5642 70 - cox1 5650 7179 1530 + trnS2(tca) 7189 7259 71 - trnD(gac) 7261 7329 69 + cox2 7330 8001 672 + trnK(aaa) 8012 8080 69 + atp8-0 8086 8244 159 + atp6 8241 8912 672 + cox3 8927 9709 783 + trnG(gga) 9711 9777 67 + nad3 9775 10116 342 + trnR(cga) 10118 10186 69 + nad4l 10250 10504 255 + nad4 10501 11859 1359 + trnH(cac) 11866 11933 68 + trnS1(---) 11934 12000 67 + nad5 12044 13819 1776 + nad6 13818 14303 486 - trnE(gaa) 14310 14377 68 - cob 14389 15066 678 +

Cyclorampus boraceiensis

Position Length Contig Gene Start Stop Strand (bp)

16 cob 96 1217 1122 - trnE(gaa) 1229 1296 68 + nad6 1297 1788 492 + nad5 1793 3544 1752 - trnS1(agc) 3614 3681 68 - trnH(cac) 3682 3749 68 - nad4 3755 5116 1362 - nad4l 5113 5409 297 - trnR(cga) 5416 5484 69 - nad3 5486 5824 339 - trnG(gga) 5828 5896 69 - cox3 5898 6680 783 - atp6 6686 7363 678 - atp8 7360 7518 159 - trnK(aaa) 7519 7590 72 - cox2 7604 8278 675 - trnD(gac) 8282 8350 69 - trnS2(tca) 8352 8422 71 + 1 cox1 8436 9962 1527 - trnY(tac) 9970 10039 70 + trnC(tgc) 10040 10103 64 + trnN(aac) 10129 10201 73 + trnA(gca) 10202 10270 69 + trnW(tga) 10271 10340 70 - nad2 10345 11373 1029 - trnM(atg) 11374 11442 69 - trnQ(caa) 11442 11512 71 + trnI(atc) 11512 11582 71 - nad1 11590 12528 939 - trnL2(tta) 12544 12616 73 - rrnL 12616 14216 1601 - trnV(gta) 14217 14287 71 - rrnS 14286 15222 937 - trnF(ttc) 15223 15291 69 - trnP(cca) 15291 15359 69 + trnT(aca) 15359 15432 74 - trnL1(cta) 15433 15504 72 -

Hypodactylus brunneus

17 Position Length Contig Gene Start Stop Strand (bp) trnS1(agc) 522 588 67 + trnL1(cta) 613 684 72 + trnT(aca) 685 755 71 + trnP(cca) 755 823 69 - trnF(ttc) 823 890 68 + rrnS 891 1823 933 + trnV(gta) 1821 1888 68 + rrnL 1889 3458 1570 + trnL2(tta) 3458 3529 72 + nad1 3557 4483 927 + trnI(atc) 4491 4561 71 + nad5 4573 4668 96 + trnM(atg) 4635 4703 69 + nad2 4704 5723 1020 + trnW(tga) 5737 5805 69 + trnA(gca) 5804 5872 69 - 1 trnN(aac) 5873 5945 73 - trnC(tgc) 5973 6035 63 - trnY(tac) 6039 6109 71 - cox1 6111 7640 1530 + trnS2(tca) 7656 7726 71 - trnD(gac) 7728 7795 68 + cox2 7796 8461 666 + trnK(aaa) 8481 8552 72 + atp8 8554 8715 162 + atp6 8709 9386 678 + cox3 9392 10174 783 + trnG(gga) 10176 10244 69 + nad3 10251 10583 333 + trnR(cga) 10585 10653 69 + nad4l 10680 10952 273 + nad4 10949 12307 1359 + trnH(cac) 12364 12431 68 +

Lepidobatrachus laevis

Position

18 Length Contig Gene Start Stop Strand (bp) cox3 3 461 459 - atp6 467 1144 678 - atp8 1141 1299 159 - trnK(aaa) 1300 1371 72 - cox2 1388 2059 672 - trnD(gac) 2061 2129 69 - trnS2(tca) 2131 2201 71 + cox1 2218 3741 1524 - trnY(tac) 3749 3818 70 + trnC(tgc) 3819 3883 65 + 1 trnN(aac) 3909 3981 73 + trnA(gca) 3982 4050 69 + trnW(tga) 4051 4120 70 - nad2 4125 5153 1029 - trnM(atg) 5154 5222 69 - trnQ(caa) 5222 5292 71 + trnI(atc) 5292 5362 71 - nad1 5370 6308 939 - trnL2(tta) 6324 6396 73 - rrnL 6398 7252 855 -

Lynchius nebulanastes

Position Length Contig Gene Start Stop Strand (bp) cob 269 1387 1119 - trnE(gaa) 1402 1469 68 + nad6 1470 1961 492 + nad5 1966 3711 1746 - trnS1(agc) 3784 3850 67 - trnH(cac) 3851 3917 67 - 1 nad4 3920 5278 1359 - nad4l 5275 5544 270 - trnR(cga) 5574 5641 68 - nad3 5643 5957 315 - trnG(gga) 5982 6050 69 - cox3 6052 6834 783 - atp6 6840 7514 675 -

19 atp8 7508 7669 162 - trnK(aaa) 7670 7740 71 - cox2 7754 8422 669 - trnD(gac) 8423 8490 68 - trnS2(tca) 8492 8562 71 + cox1 8572 10101 1530 - trnY(tac) 10109 10175 67 + trnC(tgc) 10176 10239 64 + trnN(aac) 10277 10346 70 + trnA(gca) 10347 10415 69 + trnW(tga) 10416 10485 70 - nad2 10497 11516 1020 - trnM(atg) 11517 11585 69 - trnQ(caa) 11585 11655 71 + trnI(atc) 11655 11725 71 - nad1 11733 12665 933 - trnL2(tta) 12682 12754 73 - rrnL 12756 13607 852 -

Odontophrynus americanus

Position Length Contig Gene Start Stop Strand (bp) trnL1(cta) 374 445 72 + trnA(gca) 448 517 70 + trnF(ttc) 522 589 68 + rrnS 590 1525 936 + trnV(gta) 1523 1591 69 + rrnL 1592 3192 1601 + trnL2(tta) 3192 3264 73 + nad1 3280 4218 939 + 1 trnI(atc) 4226 4297 72 + trnQ(caa) 4297 4367 71 - trnM(atg) 4367 4435 69 + nad2 4436 5470 1035 + trnW(tga) 5475 5544 70 + trnN(aac) 5553 5625 73 - trnC(tgc) 5652 5715 64 - trnY(tac) 5716 5785 70 -

20 cox1 5793 7319 1527 + trnS2(tca) 7333 7403 71 - trnD(gac) 7404 7472 69 + cox2 7474 8142 669 + trnK(aaa) 8162 8233 72 + atp8 8235 8393 159 + atp6 8390 9067 678 + cox3 9073 9855 783 + trnG(gga) 9857 9925 69 + nad3 9923 10264 342 + trnR(cga) 10266 10334 69 + nad4l 10371 10634 264 + nad4 10631 11989 1359 + trnH(cac) 11998 12065 68 + trnS1(agc) 12066 12132 67 + nad5 12180 13952 1773 + nad6 13951 14442 492 - trnE(gaa) 14444 14511 68 - cob 14526 15647 1122 +

Pristimantis simonsii

Position Length Contig Gene Start Stop Strand (bp) cox1 1 1143 1143 + trnS2(tca) 1163 1233 71 - trnD(gac) 1234 1302 69 + cox2 1304 1972 669 + trnK(aaa) 1989 2058 70 + atp8 2059 2217 159 + atp6 2214 2888 675 + 1 cox3 2893 3675 783 + trnG(gga) 3677 3745 69 + nad3 3743 4084 342 + trnR(cga) 4086 4154 69 + nad4l 4177 4452 276 + nad4 4449 5810 1362 + trnH(cac) 5813 5880 68 + trnS1(agc) 5881 5947 67 +

21 nad5 6006 7772 1767 + cox1 2 388 387 - trnY(tac) 396 465 70 + trnC(tgc) 466 524 59 + trnN(aac) 550 622 73 + trnA(gca) 623 691 69 + trnW(tga) 692 760 69 - nad2 775 1791 1017 - trnM(atg) 1792 1860 69 - trnQ(caa) 1860 1930 71 + 2 trnI(atc) 1930 2000 71 - nad1 2008 2943 936 - trnL2(tta) 2962 3034 73 - rrnL 3034 4611 1578 - trnV(gta) 4612 4680 69 - rrnS 4678 5609 932 - trnF(ttc) 5610 5678 69 - trnP(cca) 5678 5745 68 + trnT(aca) 5745 5816 72 - trnL1(cta) 5817 5888 72 -

Strabomantis anomalus

Position Length Contig Gene Start Stop Strand (bp) cox3 23 805 783 - atp6 811 1485 675 - atp8 1482 1640 159 - trnK(aaa) 1642 1710 69 - cox2 1733 2398 666 - trnD(gac) 2400 2468 69 - trnS2(tca) 2470 2540 71 + 1 cox1 2556 4082 1527 - trnY(tac) 4087 4156 70 + trnC(tgc) 4163 4220 58 + trnN(aac) 4247 4319 73 + trnA(gca) 4320 4388 69 + trnW(tga) 4389 4459 71 - nad2 4465 5490 1026 -

22 trnM(atg) 5491 5560 70 - trnQ(caa) 5560 5630 71 + trnI(atc) 5630 5700 71 - nad1 5708 6643 936 - trnL2(tta) 6662 6734 73 - rrnL 6736 6917 182 - rrnL 6918 6934 17 - rrnL 43 437 395 - trnS1(agc) 686 761 76 - trnK(aaa) 823 871 49 + atp8 1076 1186 111 - 2 trnS2(tcg) 1208 1274 67 + trnL2(tta) 1719 1779 61 + trnF(ttc) 2078 2146 69 - trnP(cca) 2146 2214 69 +

Telmatobius carrillae

Position Length Contig Gene Start Stop Strand (bp) trnN(aac) 11 83 73 - trnC(tgc) 110 174 65 - trnY(tac) 175 244 70 - cox1 252 1778 1527 + trnS2(tca) 1792 1862 71 - trnD(gac) 1864 1932 69 + cox2 1934 2605 672 + trnK(aaa) 2622 2693 72 + atp8 2694 2852 159 + 1 atp6 2849 3526 678 + cox3 3532 4314 783 + trnG(gga) 4316 4384 69 + nad3 4382 4723 342 + trnR(cga) 4725 4794 70 + nad4l 4824 5096 273 + nad4 5093 6451 1359 + trnH(cac) 6458 6525 68 + trnS1(agc) 6526 6592 67 + nad5 6645 8420 1776 +

23 nad6 8419 8904 486 - trnE(gaa) 8911 8978 68 - cob 8993 10111 1119 + trnA(gca) 9 77 69 + trnW(tga) 78 147 70 - nad2 154 1182 1029 - nad4 1188 1220 33 - trnQ(caa) 1251 1321 71 + trnI(atc) 1321 1391 71 - nad1 1399 2337 939 - 2 trnL2(tta) 2353 2425 73 - rrnL 2425 4026 1602 - trnV(gta) 4027 4095 69 - rrnS 4093 5026 934 - trnF(ttc) 5027 5095 69 - trnP(cca) 5095 5163 69 + trnT(aca) 5163 5234 72 - trnL1(cta) 5235 5306 72 -

Thoropa miliaris

Position Length Contig Gene Start Stop Strand (bp) trnL1(cta) 139 210 72 + trnT(aca) 211 281 71 + trnP(cca) 281 349 69 - trnF(ttc) 349 418 70 + rrnS 419 1359 941 + trnV(gta) 1360 1428 69 + rrnL 1429 3018 1590 + trnL2(tta) 3018 3090 73 + 1 nad1 3109 4044 936 + trnI(atc) 4052 4122 71 + trnQ(caa) 4122 4192 71 - trnM(atg) 4192 4260 69 + nad2 4261 5289 1029 + trnW(tga) 5295 5365 71 + trnA(gca) 5364 5432 69 - trnN(aac) 5433 5505 73 -

24 trnC(tgc) 5531 5594 64 - trnY(tac) 5595 5664 70 - cox1 5672 7195 1524 + trnS2(tca) 7212 7282 71 - trnD(gac) 7284 7352 69 + cox2 7354 8037 684 + trnK(aaa) 8042 8113 72 + atp8 8114 8272 159 + atp6 8269 8946 678 + cox3 8952 9734 783 + trnG(gga) 9736 9804 69 + nad3 9805 10143 339 + trnR(cga) 10145 10214 70 + nad4l 10220 10516 297 + nad4 10513 11874 1362 + trnH(cac) 11880 11948 69 + trnS1(agc) 11949 12016 68 + nad5 12065 13840 1776 + nad6 13842 14333 492 - trnE(gaa) 14334 14402 69 - cob 14417 15538 1122 +

Scinax catharinae

Position Length Contig Gene Start Stop Strand (bp) nad5 15 227 213 + nad6 226 714 489 - 1 trnE(gaa) 721 788 68 - cob 803 1921 1119 + nad5 1 228 228 - trnS1(gct) 331 397 67 - trnH(gtg) 398 465 68 - nad4 471 1830 1360 - 2 nad4l 1824 2123 300 - trnR(tcg) 2125 2193 69 - nad3 2164 2509 346 - trnG(tcc) 2534 2602 69 - cox3 2602 3386 785 -

25 atp6 3386 4069 684 - atp8 4060 4224 165 - trnK(ttt) 4225 4296 72 - cox2 4297 4984 688 - trnD(gtc) 4986 5054 69 - trnS2(tga) 5056 5126 71 + cox1 5122 6672 1551 - trnY(gta) 6674 6743 70 + trnC(gca) 6744 6807 64 + OL 6807 6831 25 + trnN(gtt) 6834 6906 73 + trnA(tgc) 6907 6975 69 + trnW(tca) 6976 7045 70 - nad2 7092 8133 1042 - trnQ(ttg) 8148 8218 71 + trnI(gat) 8218 8288 71 - nad1 8310 9273 964 - trnL2(taa) 9247 9319 73 - rrnL 9319 10920 1602 - trnV(tac) 10923 10993 71 - rrnS 10991 11921 931 - trnF(gaa) 11922 11989 68 - trnP(tgg) 11989 12057 69 + trnT(tgt) 12057 12127 71 - trnL1(tag) 12128 12199 72 -

Calyptocephalella gayi

Position Length Contig Gene Start Stop Strand (bp) OH 16 60 45 + cob 82 1225 1144 - trnE(ttc) 1229 1297 69 + nad6 1299 1793 495 + 1 nad5 1777 3564 1788 - trnS1(gct) 3624 3690 67 - trnH(gtg) 3691 3758 68 - nad4 3765 5129 1365 - nad4l 5123 5419 297 -

26 trnR(tcg) 5421 5488 68 - nad3 5489 5828 340 - trnG(tcc) 5829 5897 69 - cox3 5897 6681 785 - atp6 6681 7364 684 - atp8 7355 7519 165 - trnK(ttt) 7520 7591 72 - cox2 7592 8279 688 - trnD(gtc) 8282 8350 69 - trnS2(tga) 8352 8422 71 + cox1 8418 9962 1545 - trnY(gta) 9964 10033 70 + trnC(gca) 10034 10097 64 + OL 10097 10125 29 - trnN(gtt) 10128 10200 73 + trnA(tgc) 10201 10269 69 + trnW(tca) 10270 10339 70 - nad2 10388 11432 1045 - trnQ(ttg) 11446 11516 71 + trnI(gat) 11516 11586 71 - nad1 11598 12524 927 - trnL2(taa) 12548 12620 73 - rrnL 12622 12822 201 - OH 1 49 49 + trnL1(tag) 100 171 72 + trnT(tgt) 172 243 72 + 2 trnP(tgg) 245 313 69 - trnF(gaa) 315 384 70 + rrnS 385 1263 879 +

Espadarana prosoblepon

Position Length Contig Gene Start Stop Strand (bp) trnL1(tag) 119 190 72 + trnT(tgt) 199 268 70 + 1 trnP(tgg) 268 336 69 - trnF(gaa) 336 403 68 + rrnS 404 1340 937 +

27 trnV(tac) 1338 1406 69 + rrnL 1409 3010 1602 + trnL2(taa) 3010 3082 73 + nad1 3098 4066 969 + trnI(gat) 4044 4114 71 + trnQ(ttg) 4117 4187 71 - trnM(cat) 4187 4255 69 + nad2 4256 5290 1035 + trnW(tca) 5290 5359 70 + trnA(tgc) 5360 5428 69 - trnN(gtt) 5429 5501 73 - OL 5504 5528 25 - trnC(gca) 5528 5591 64 - trnY(gta) 5592 5661 70 - cox1 5663 7207 1545 + trnS2(tga) 7209 7279 71 - trnD(gtc) 7281 7349 69 + cox2 7351 8038 688 + trnK(ttt) 8039 8112 74 + atp8 8113 8277 165 + atp6 8268 8951 684 + cox3 8951 9735 785 + trnG(tcc) 9735 9803 69 + nad3 9804 10145 342 + trnR(tcg) 10144 10212 69 + nad4l 10213 10515 303 + nad4 10509 11868 1360 + trnH(gtg) 11874 11942 69 + trnS1(gct) 11943 12009 67 + nad5 12044 13846 1803 + nad6 13833 1 114 - OH 13945 37 38 +

Hemiphractus proboscidus

Position Length Contig Gene Start Stop Strand (bp) trnL2(taa) 25 97 73 + 1 nad1 92 1060 969 +

28 trnI(gat) 1059 1126 68 + trnQ(ttg) 1126 1196 71 - trnM(cat) 1196 1264 69 + nad2 1238 2282 1045 + trnW(tca) 2293 2361 69 + trnA(tgc) 2360 2428 69 - trnN(gtt) 2428 2500 73 - OL 2503 2526 24 + trnC(gca) 2526 2589 64 - trnY(gta) 2591 2656 66 - cox1 2658 4211 1554 + trnS2(tga) 4207 4277 71 - trnD(gtc) 4279 4347 69 + cox2 4349 5036 688 + trnK(ttt) 5034 5103 70 + atp8 5104 5268 165 + atp6 5259 5942 684 + cox3 5942 6726 785 + trnG(tcc) 6726 6795 70 + nad3 6751 7097 347 + trnR(tcg) 7133 7200 68 + nad4l 7201 7499 299 + nad4 7494 8868 1375 + trnH(gtg) 8857 8923 67 + trnS1(gct) 8924 8991 68 + nad5 9026 10828 1803 + nad6 10815 10940 126 - rrnL 20 1527 1508 - trnV(tac) 1530 1599 70 - 2 rrnS 1597 2223 627 - OH 2214 13 23 +

29 15

30 GENERAL CONCLUSIONS

Many previous studies on the phylogenetic relationships within terraranan frogs were in disagreement, uor results provide a generally well-supported estimate of relationships among most terraranan families and subfamilies based on concatenated and species-tree methods, including the largest number of genetic loci. At the family level, our results show that

Brachycephalidae is the sister taxon of Eleutherodactylidae + “Craugastoridae” +

Strabomantidae. Within these families, we suggest that Strabomantidae sensu (Heinicke et al.,

2018) should be placed in the synonymy of Craugastoridae. Within Craugastoridae, we find that

Pristimantinae should be placed in the synonymy of Holoadeninae. We also found that including more UCE loci with more missing data generally increased mean bootstrap values for concatenate ML and coalescent based methods. These results add additional support to the idea that the benefits of including more loci can potentially overcome any negative consequences of including more missing data in phylogenomic analyses.

With our study, we promote the combination of massively parallel sequencing (MPS) techniques with mitochondrial data to estimate phylogenetic relationships to expand the impact of these sources of information. Whit this kind of data (mtDNA + UCEs) we explore the phylogenetic relationships among eleutherodactylid frogs and found that of the distribution of this family must be attributed to an ancient overseas dispersal from South America towards

Middle America and the proto Caribbean, over the vicariant hypothesis. In our results we found that the most important process to explain the biogeographic patterns of the family are dispersal events, as suggested by the DIVALike model.

1 In the local scale exploring the cryptic diversity of Diasporus, we found at least 26 genetic clusters. Nine of that genetic groups are clearly assignable a previous described species, six require a morphological and molecular corroboration to assign the available names, and finally we found at least 11 genetic groups that could be undescribed species. Also, our analysis of environmental niche models (ENM) on four pairs of sister lineages, give to us weak evidence for divergence that would support a role environmental diversification. On the other hand, at least for one pair of sister lineages we have evidence that niche characteristics are conserved across evolutionary time.

Finally, we show the utility of using off-target sequences data from MPS give to us the chance to obtain mitochondrial information as a byproduct of the library construction. Here we report the capture of 16 partial mitochondrial genomes of neobatrachian frogs. For 11 frogs, we get genomes ranged from approximately 12 to 16 Kb in length (lacking only the control region), plus 5 long fragments of over 5 kilobases (Kb) for 5 additional neobatrachian frogs.

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6 Supplemental material Chapter 1. Fig S1 A. RAxML 80% missing taxa/locus 8 3 Stefania coxi Dendropsophus leali 100 Espadarana prosoblepon 7 2 Hyloxalus nexipus 100 Leptodactylus dydimus Brachycephalidae Brachycephalus quiririensis Phyzelaphryne miriame 100 Phyzelaphryninae 100 Adelophryne adiastola Eleutherodactylus longipes Eleutherodactylidae 100 100 Eleutherodactylus johnstonei Eleutherodactylinae 100 Diasporus gularis 100 Diasporus vocator 7 3 100 Strabomantis anomalus Craugastor daryi Craugastorinae 9 8 Craugastor augusti 9 3 Craugastor longirostris 100 Lynchius nebulanastes Craugastoridae 100 Oreobates quixensis 100 Barycholos pulcheus Holoadeninae 100 Pristimantis miyatai 100 Pristimantis simonsii

substitutions / site 0.005

B. NJst 80% missing taxa/locus

6 0 Dendropsophus leali 100 Stefania coxi Espadarana prosoblepon 3 6 Hyloxalus nexipus 9 1 Leptodactylus didymus Brachycephalidae Brachycephalus quiririensis 9 6 Adelophryne adiastola Phyzelaphryninae 9 8 Phyzelaphryne miriame Eleutherodactylidae Eleutherodactylus johnstonei 100 100 Eleutherodactylus longipes Eleutherodactylinae 9 9 Diasporus gularis 9 9 Diasporus vocator 9 2 Craugastor augusti 9 9 Craugastor longirostris Craugastorinae 8 9 Craugastor daryi 9 2 Strabomantis anomalus 8 5 Lynchius nebulanastes Craugastoridae 9 9 Oreobates quixensis 9 6 Barycholos pulcheus Holoadeninae 9 7 Pristimantis miyatai 100 Pristimantis simonsii 0.4

C. ASTRAL-II 80% missing taxa/locus

Stefania coxi Dendropsophus leali 100 2 9 Espadarana prosoblepon 6 5 Leptodactylus dydimus 8 5 Hyloxalus nexipus Brachycephalidae Brachycephalus quiririensis 100 Adelophryne adiastola Phyzelaphryninae 100 Phyzelaphryne miriame Eleutherodactylus longipes Eleutherodactylidae 100 100 Eleutherodactylus johnstonei Eleutherodactylinae 100 Diasporus gularis 100 Diasporus vocator

8 5 7 0 Craugastor longirostris 9 3 Craugastor augusti Craugastorinae Strabomantis anomalus 7 3 Craugastor daryi 9 6 Oreobates quixensis Craugastoridae 100 Lynchius nebulanastes 9 4 Barycholos pulcheus Holoadeninae 100 Pristimantis miyatai 100 Pristimantis simonsii

0.4 Supplemental material Chapter 1. Fig S2 A. 90% missing taxa/locus Brachycephalus quiririensis Brachycephalidae Phyzelaphryne miriame Phyzelaphryninae 100 Adelophryne adiastola

100 Diasporus vocator 100 100 Eleutherodactylidae Diasporus gularis Eleutherodactylinae 100 Eleutherodactylus johnstonei 100 Eleutherodactylus longipes 5 8 100 Strabomantis anomalus Craugastortor daryi Craugastorinae 100 Craugastor augusti 100 Craugastor longirostris 100 Lynchius nebulanastes Craugastoridae 100 Oreobates quixensis 100 Barycholos pulcheus Holoadeninae 100 Pristimantis simonsii 100 0.005 Pristimantis miyatai

B. 70% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae Phyzelaphryne miriame Phyzelaphryninae 100 Adelophryne adiastola Diasporus vocator Eleutherodactylidae 100 100 100 Diasporus gularis Eleutherodactylinae 100 Eleutherodactylus johnstonei 100 Eleutherodactylus longipes 6 7 100 Strabomantis anomalus Craugastortor daryi Craugastorinae 100 Craugastor augusti 9 6 Craugastor longirostris 100 Lynchius nebulanastes Craugastoridae 100 Oreobates quixensis

100 Barycholos pulcheus Holoadeninae

100 Pristimantis simonsii 100 Pristimantis miyatai 0.004

C. 60% missing taxa/locus Brachycephalus quiririensis Brachycephalidae Adelophryne adiastola 100 Phyzelaphryninae 100 Phyzelaphryne miriame Diasporus gularis 100 100 Diasporus vocator Eleutherodactylinae Eleutherodactylidae 100 Eleutherodactylus longipes 100 Eleutherodactylus johnstonei 7 4 9 9 Strabomantis anomalus Craugastortor daryi Craugastorinae 9 9 Craugastor longirostris 9 6 Craugastor augusti 100 Oreobates quixensis Craugastoridae 100 Lynchius nebulanastes Holoadeninae 100 Barycholos pulcheus

100 Pristimantis simonsii 100 0.005 Pristimantis miyatai

D. 50% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae Phyzelaphryne miriame Phyzelaphryninae 9 4 Adelophryne adiastola 9 4 Eleutherodactylus johnstonei 100 100 Eleutherodactylidae Eleutherodactylus longipes Eleutherodactylinae 100 Diasporus gularis 100 8 5 Diasporus vocator 8 8 Strabomantis anomalus Craugastortor daryi Craugastorinae 9 2 Craugastor longirostris 100 Craugastor augusti 8 7 Craugastoridae 100 Oreobates quixensis Lynchius nebulanastes 9 6 Barycholos pulcheus Holoadeninae 100 Pristimantis miyatai 100 0.003 Pristimantis simonsii E. 40% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae Phyzelaphryne miriame Phyzelaphryninae 9 4 9 4 Adelophryne adiastola Eleutherodactylus johnstonei Eleutherodactylidae 100 100 Eleutherodactylus longipes Eleutherodactylinae 100 Diasporus gularis 100 Diasporus vocator 8 4 8 9 Strabomantis anomalus Craugastortor daryi Craugastorinae 9 2 Craugastor augusti 100 Craugastor longirostris 9 0 100 Craugastoridae Lynchius nebulanastes Oreobates quixensis 9 4 Barycholos pulcheus Holoadeninae

100 Pristimantis simonsii 100 Pristimantis miyatai 0.003 F. 30% missing taxa/locus

Brachycephalus quiririensis 3 2 Adelophryne adiastola Craugastortor daryi 3 9 Phyzelaphrynee miriame 8 9 2 5 Diasporus vocator 6 8 Diasporus gularis 5 2 Eleutherodactylus johnstonei 8 4 3 4 Eleutherodactylus longipes 4 5 Craugastor augusti Strabomantis anomalus 3 7 Craugastor longirostris 1 8 Barycholos pulcheus Pristimantis miyatai 4 0 100 Pristimantis simonsii 5 6 Lynchius nebulanastes 4 8 Oreobates quixensis

0.003

G. 20% missing taxa/locus Dendropsophus leali Stefania coxi Eleutherodactylus longipes 4 5 Eleutherodactylus johnstonei 7 5 Pristimantis miyatai 5 4 Espadarana prosoblepon 8 0 Diasporus gularis 3 0 Craugastor augusti Barycholos pulcheus 7 2

1 0 Adelophryne adiastola 7 2 Diasporus vocator 9 0 Hyloxalus nexipus 1 0 Leptodactylus dydimus 1 2 Phyzelaphryne miriame 2 6 Brachycephalus quiririensis 3 6 2 Craugastor longirostris Craugastortor daryi 8 Lynchius nebulanastes 1 6 Pristimantis simonsii

2 2 Strabomantis anomalus 6 7 Oreobates quixensis

0.007 H. 10% missing taxa/locus

Adelophryne adiastola Brachycephalus quiririensis Barycholos pulcheus Lynchius nebulanastes 5 8 Pristimantis simonsii 6 0 1 5 Oreobates quixensis 6 3 Pristimantis miyatai

3 Dendropsophus leali 2 Diasporus gularis 0 Eleutherodactylus longipes 8 Espadarana prosoblepon 5 6

0 Craugastor longirostris Craugastor augusti 1 1 Eleutherodactylus johnstonei 0 Phyzelaphryne miriame 3 9 Stefania coxi 1 Hyloxalus nexipus 8 Leptodactylus dydimus 1 8 Diasporus vocator 0.01 Supplemental material Chapter 1. Fig S3 A. 90% missing taxa/locus Brachycephalus quiririensis Brachycephalidae Adelophryne adiastola 100 Phyzelaphryninae Phyzelaphryne miriame 9 7 100 100 Eleutherodactylus johnstonei Eleutherodactylus longipes 9 8 Eleutherodactylinae Eleutherodactylidae 9 9 Diasporus gularis Diasporus vocator

8 8 9 7 Craugastor augusti 9 4 Craugastor longirostris Craugastorinae 9 3 Craugastor daryi

9 1 Strabomantis anomalus Craugastoridae

100 Lynchius nebulanastes

100 Oreobates quixensis Barycholos pulcheus 9 6 Holoadeninae

100 Pristimantis miyatai 0.3 Pristimantis simonsii B. 70% missing taxa/locus Brachycephalus quiririensis Brachycephalidae

9 8 Adelophryne adiastola Phyzelaphryninae Phyzelaphryne miriame 9 6 100 100 Eleutherodactylus johnstonei Eleutherodactylidae

9 9 Eleutherodactylus longipes

9 9 Diasporus vocator Eleutherodactylinae Diasporus gularis

8 9 9 9 Craugastor augusti

8 5 Craugastor longirostris

9 2 Craugastor daryi Craugastorinae

8 4 Strabomantis anomalus

9 9 Lynchius nebulanastes Craugastoridae

9 6 Oreobates quixensis Barycholos pulcheus 9 8 Holoadeninae

100 Pristimantis miyatai

0.4 Pristimantis simonsii C. 60% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae Adelophryne adiastola 100 Phyzelaphryninae Phyzelaphryne miriame 9 7 100 100 Eleutherodactylus johnstonei Eleutherodactylidae 9 8 Eleutherodactylus longipes Eleutherodactylinae 9 9 Diasporus vocator Diasporus gularis

8 4 9 7 Craugastor augusti 8 5 Craugastor longirostris Craugastorinae 9 3 Craugastor daryi

8 5 Strabomantis anomalus

100 Lynchius nebulanastes Craugastoridae 100 Oreobates quixensis Barycholos pulcheus 9 6 Holoadeninae

100 Pristimantis miyatai 0.3 Pristimantis simonsii D. 50% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae 9 9 Adelophryne adiastola Phyzelaphryninae Phyzelaphryne miriame 9 0 100 9 9 Diasporus vocator Eleutherodactylidae 9 9 Diasporus gularis Eleutherodactylinae

100 Eleutherodactylus johnstonei Eleutherodactylus longipes

7 6 9 6 Craugastor augusti 7 4 Craugastor longirostris Craugastorinae 8 8 Craugastor daryi

7 6 Strabomantis anomalus Lynchius nebulanastes 100 Craugastoridae 100 Oreobates quixensis Barycholos pulcheus Holoadeninae 100

100 Pristimantis miyatai

0.4 Pristimantis simonsii E. 40% missing taxa/locus Brachycephalidae Brachycephalus quiririensis

9 8 Adelophryne adiastola Phyzelaphryninae Phyzelaphryne miriame 8 3 Eleutherodactylidae 9 9 100 Eleutherodactylus johnstonei Eleutherodactylinae 9 9 Eleutherodactylus longipes

9 9 Diasporus vocator Diasporus gularis 6 6

9 3 Craugastor augusti Craugastor longirostris 6 3 Craugastorinae Craugastoridae 8 8 Craugastor daryi

5 6 Strabomantis anomalus

100 Lynchius nebulanastes

9 3 Oreobates quixensis Barycholos pulcheus Holoadeninae 9 4

100 Pristimantis miyatai 0.4 Pristimantis simonsii F. 30% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae 6 4 Adelophryne adiastola Phyzelaphryninae Phyzelaphryne miriame 2 0 9 8 Eleutherodactylus johnstonei 8 0 Eleutherodactylidae 8 6 Eleutherodactylus longipes Eleutherodactylinae 9 4 Diasporus vocator Diasporus gularis 1 8

7 8 Craugastor augusti Craugastor longirostris 1 1 Craugastorinae 6 6 Craugastor daryi Strabomantis anomalus

9 6 7 Lynchius nebulanastes Craugastoridae Oreobates quixensis 2 5 Barycholos pulcheus 6 4 Holoadeninae

100 Pristimantis miyatai 0.4 Pristimantis simonsii G. 20% missing taxa/locus

8 1 Strabomantis anomalus Brachycephalus quiririensis Oreobates quixensis

8 1 Pristimantis simonsii

7 Adelophryne_adiastola 2 1 Pristimantis miyatai Eleutherodactylus longipes 1 6 3 Eleutherodactylus johnstonei Diasporus vocator 1 0 Hyloxalus_nexipus 3 Dendropsophus leali 6 Leptodactylus dydimus Barycholos pulcheus 4 1 Stefania coxi Craugastor augusti 1 Centrolene prosoblepon 4 Diasporus gularis Lynchius nebulanastes 3 Phyzelaphryne miriame 8

3 7 Craugastor longirostris 50.0 Craugastor daryi H. 10% missing taxa/locus

2 7 Hyloxalus nexipus

Diasporus vocator

Leptodactylus dydimus

2 0 Dendropsophus leali

6 Espadarana prosoblepon

2 7 2 2 Diasporus gularis

Craugastor augusti

8 Barycholos pulcheus

Pristimantis simonsii 2 9 Stefania coxi 1 4

6 8 Eleutherodactylus johnstonei

Eleutherodactylus longipes

4 0 Adelophryne adiastola

Pristimantis miyatai 1 Craugastor longirostris 6 Phyzelaphryne miriame 4 Lynchius nebulanastes 2 0

3 8 Brachycephalus quiririensis 0.7 Oreobates quixensis Supplemental material Chapter 1. Fig. S4 A. 90% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae Adelophryne adiastola Phyzelaphryninae 9 7 Phyzelaphryne miriame Eleutherodactylus longipes 9 9 100 100 Eleutherodactylus johnstonei Eleutherodactylidae Eleutherodactylinae 100 Diasporus gularis 100 Diasporus vocator

8 2 Craugastor longirostris 7 1 8 9 Craugastor augusti Craugastorinae Strabomantis anomalus 7 1 Craugastor daryi 9 4 Oreobates quixensis Craugastoridae 100 Lynchius nebulanastes 9 3 Barycholos pulcheus Holoadeninae 9 9 Pristimantis miyatai 100 Pristimantis simonsii 0.4

B. 70% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae Adelophryne adiastola Phyzelaphryninae 100 Phyzelaphryne miriame Eleutherodactylus longipes 9 9 Eleutherodactylidae 100 100 Eleutherodactylus johnstonei Eleutherodactylinae 100 Diasporus gularis 100 Diasporus vocator Craugastor longirostris 8 6 7 3 9 1 Craugastor augusti Craugastorinae Strabomantis anomalus 7 3 Craugastor daryi 9 5 Oreobates quixensis Craugastoridae 9 8 Lynchius nebulanastes 9 2 Barycholos pulcheus Holoadeninae

100 Pristimantis miyatai 100 Pristimantis simonsii 0.4 C. 60% missing taxa/locus

Brachycephalidae Brachycephalus quiririensis Adelophryne adiastola Phyzelaphryninae 9 7 Phyzelaphryne miriame

9 9 Eleutherodactylus longipes 100 100 Eleutherodactylus johnstonei Eleutherodactylinae 100 Diasporus gularis Eleutherodactylidae 100 Diasporus vocator

8 2 7 1 Craugastor longirostris 8 9 Craugastor augusti Craugastorinae Strabomantis anomalus 7 1 Craugastor daryi 9 4 Oreobates quixensis 100 Lynchius nebulanastes Craugastoridae 9 3 Barycholos pulcheus Holoadeninae

9 9 Pristimantis miyatai 100 Pristimantis simonsii 0.4

D. 50% missing taxa/locus

Brachycephalus quiririensis Brachycephalidae Adelophryne adiastola Phyzelaphryninae 9 9 Phyzelaphryne miriame Eleutherodactylus longipes Eleutherodactylidae 9 8 100 100 Eleutherodactylus johnstonei Eleutherodactylinae 9 9 Diasporus gularis 100 Diasporus vocator 7 6 7 6 Craugastor longirostris 9 4 Craugastor augusti Strabomantis anomalus Craugastorinae 8 1 Craugastor daryi 9 2 Oreobates quixensis Craugastoridae 100 Lynchius nebulanastes

9 0 Barycholos pulcheus Holoadeninae 9 8 Pristimantis miyatai 100 Pristimantis simonsii 0.4

E. 40% missing taxa/locus

Adelophryne adiastola Phyzelaphryninae 9 5 Phyzelaphryne miriame 9 6 Eleutherodactylus longipes Eleutherodactylidae 100 Eleutherodactylus johnstonei Eleutherodactylinae 9 8 Diasporus gularis 100 Diasporus vocator Brachycephalidae Brachycephalus quiririensis 9 9 7 Craugastor longirostris 8 9 Craugastor augusti Craugastorinae 7 2 Strabomantis anomalus 7 7 Craugastor daryi 2 5 Oreobates quixensis 9 9 Craugastoridae Lynchius nebulanastes 9 5 Barycholos pulcheus Holoadeninae 9 9 Pristimantis miyatai 100 Pristimantis simonsii

0.4 F. 30% missing taxa/locus

Dendropsophus leali Leptodactylus dydimus Espadarana prosoblepon 1 1 Stefania coxi Hyloxalus nexipus 3 4 Brachycephalus quiririensis 1 6 3 6 Strabomantis anomalus 6 5 Craugastor daryi Craugastor longirostris 8 3 8 4 Craugastor augusti Oreobates quixensis 8 4 Lynchius nebulanastes 6 5 6 Barycholos pulcheus

6 4 Pristimantis simonsii 100 Pristimantis miyatai 5 Phyzelaphryne miriame 6 6 Adelophryne adiastola Diasporus gularis 3 0 9 1 Diasporus vocator 7 8 Eleutherodactylus johnstonei 0.3 9 9 Eleutherodactylus longipes

G. 20% missing taxa/locus

1 8 Dendropsophus leali Leptodactylus mystaceus Phyzelaphryne miriame Craugastor augusti

6 Strabomantis anomalus 1 8 2 3 Brachycephalus quiririensis Oreobates quixensis 0 1 9 Lynchius nebulanastes Pristimantis simonsii 2 0 Pristimantis miyatai 0 2 5 Adelophryne adiastola Craugastor longirostris

0 5 Craugastor daryi 4 Eleutherodactylus longipes 5 8 Eleutherodactylus johnstonei 0 Hyloxalus nexipus

7 Diasporus gularis 2 9 Diasporus vocator 0 Stefania coxi

2 Espadarana prosoblepon 0.4 1 6 Barycholos pulcheus

H. 10% missing taxa/locus

Dendropsophus leali

Diasporus gularis

Espadarana prosoblepon

Craugastor augusti

1 9 Hyloxalus nexipus 0 Brachycephalus quiririensis 3 2 Leptodactylus didymus

9 Diasporus vocator

1 4 1 Lynchius nebulanastes

2 8 Pristimantis simonsii 1 5 Pristimantis miyatai 1 3 1 Oreobates quixensis Adelophryne adiastola

3 Phyzelaphryne miriame 2 8 Stefania coxi 0 Eleutherodactylus longipes

6 Barycholos pulcheus

0 Craugastor longirostris

5 Eleutherodactylus johnstonei 0.3 Supplemental material Chapter 2. Fig S1

100 Phyzelaphryne miriamae Phyzelaphryne sp.1b Adelophryne sp.7 9 8 100 Adelophryne patamona 6 4

100 Adelophryne gutturosa 100 9 7 Adelophryne adiastola

Adelophryne pachydactyla 100

7 2 Adelophryne sp.6 8 3 Adelophryne sp.5 8 8 Adelophryne sp.4 4 0 Adelophryne maranguapensis 100 Adelophryne sp.1 4 5 Adelophryne baturitensis 100 Adelophryne sp.2 Diasporus diastema 9 9 4 4 Diasporus gularis

100 Diasporus sp CH4717 “tinker” Diasporus anthrax 4 7 Diasporus vocator 5 7 Diasporus aff. hylaeformis 5 6

8 0 Diasporus quidditus 100 Diasporus tigrillo Diasporus citrinobapheus Eleutherodactylus inoptatus 100 100 Eleutherodactylus nortoni Pelorius 8 6 Eleutherodactylus chlorophenax 100 Eleutherodactylus ruthae 100 Eleutherodactylus bothroboans 9 2 Eleutherodactylus hypostenor 100 Eleutherodactylus parapelates Eleutherodactylus counouspeus Schwartzius 9 8 Eleutherodactylus unicolor Eleutherodactylus richmondi 7 5 100 Eleutherodactylus barlagnei

100 Eleutherodactylus pinchoni Eleutherodactylus johnstonei 9 4 100 Eleutherodactylus martinicensis 5 1 Eleutherodactylus amplinympha 100 Eleutherodactylus auriculatoides Eleutherodactylus patriciae 100 Eleutherodactylus fowleri 9 8 4 7 Eleutherodactylus lamprotes

9 9 100 5 5 Eleutherodactylus wetmorei 8 6 Eleutherodactylus sommeri 100 Eleutherodactylus leberi

100 Eleutherodactylus melacara

100 Eleutherodactylus varians 9 9 Eleutherodactylus guantanamera Eleutherodactylus ionthus

9 2 Eleutherodactylus flavescens

100 Eleutherodactylus cooki 100 100 Eleutherodactylus eneidae Eleutherodactylus locustus 9 7 Eleutherodactylus antillensis 8 8

9 5 Eleutherodactylus brittoni 7 5 Eleutherodactylus hedricki Eleutherodactylus 8 1 Eleutherodactylus cochranae Eleutherodactylus gryllus 7 4 6 7 8 2 9 4 Eleutherodactylus schwartzi 7 7 Eleutherodactylus coqui Eleutherodactylus portoricensis 7 0 Eleutherodactylus wightmanae 8 1 Eleutherodactylus poolei Eleutherodactylus minutus Eleutherodactylus pituinus 100 6 9 1 8 Eleutherodactylus haitianus 6 9 Eleutherodactylus abbotti

3 4 3 8 Eleutherodactylus parabates Eleutherodactylus audanti Eleutherodactylus eileenae 7 1 100 Eleutherodactylus ronaldi

100 Eleutherodactylus mariposa Eleutherodactylus glamyrus 5 2 9 5 Eleutherodactylus auriculatus Eleutherodactylus bartonsmithi Eleutherodactylus longipes 100 Eleutherodactylus klinikowskii Eleutherodactylus zugi 100 Eleutherodactylus zeus

9 9 Eleutherodactylus symingtoni

100 Eleutherodactylus marnockii 100 6 9 Eleutherodactylus nitidus 9 9 Eleutherodactylus pipilans Eleutherodactylus schmidti 100 Eleutherodactylus albipes 100 Eleutherodactylus maestrensis

6 6 6 8 100 Eleutherodactylus dimidiatus Eleutherodactylus emiliae 100 Eleutherodactylus glandulifer 9 2 9 4 Eleutherodactylus sciagraphus Eleutherodactylus brevirostris 6 3 100 Eleutherodactylus ventrilineatus Eleutherodactylus paulsoni 100 Eleutherodactylus furcyensis 7 4 Eleutherodactylus rufifemoralis 100 Eleutherodactylus apostates 7 7 Eleutherodactylus oxyrhyncus 8 9 Eleutherodactylus jugans Eleutherodactylus glanduliferoides 4 6 8 6 Eleutherodactylus glaphycompus 7 4 3 1 Eleutherodactylus dolomedes 5 9 Eleutherodactylus thorectes

9 Eleutherodactylus bakeri

1 1 Eleutherodactylus heminota

1 9 Eleutherodactylus amadeus

3 8 Eleutherodactylus corona 2 5 Eleutherodactylus caribe 9 5 Eleutherodactylus eunaster Eleutherodactylus greyi 3 1 Eleutherodactylus probolaeus Eleutherodactylus monensis 100 Eleutherodactylus pictissimus 100 Eleutherodactylus grahami

8 0 Eleutherodactylus lentus

7 3 9 2 Eleutherodactylus weinlandi 8 1 Eleutherodactylus rhodesi Eleutherodactylus atkinsi

9 6 Eleutherodactylus gundlachi 100 Eleutherodactylus intermedius 6 7 Eleutherodactylus varleyi 9 0 8 2 Eleutherodactylus cubanus 6 8 Eleutherodactylus orientalis

4 4 Eleutherodactylus etheridgei 7 8

100 Eleutherodactylus iberia 8 3 100 Eleutherodactylus limbatus Eleutherodactylus jaumei

100 Eleutherodactylus guanahacabibes 100 Eleutherodactylus casparii Eleutherodactylus planirostris 8 8 4 3 Eleutherodactylus goini

9 3 Eleutherodactylus rogersi 6 8 4 4 Eleutherodactylus tonyi Eleutherodactylus pezopetrus

2 8 8 8 Eleutherodactylus pinarensis 100 Eleutherodactylus blairhedgesi Eleutherodactylus thomasi

100 Eleutherodactylus acmonis Eleutherodactylus ricordii 7 5 Eleutherodactylus bresslerae 9 7 Eleutherodactylus alcoae 8 5 Euhyas 7 9 Eleutherodactylus armstrongi + 100 Eleutherodactylus leoncei Syrrhopus Eleutherodactylus darlingtoni 3 4 100 Eleutherodactylus turquinensis Eleutherodactylus cuneatus Eleutherodactylus toa 6 8 4 4 5 6 Eleutherodactylus rivularis Eleutherodactylus riparius

4 6 7 4 Eleutherodactylus sisyphodemus 7 8 Eleutherodactylus cavernicola

6 4 Eleutherodactylus grabhami 4 4 Eleutherodactylus jamaicensis

4 5 Eleutherodactylus nubicola Eleutherodactylus andrewsi

2 7 Eleutherodactylus orcutti Eleutherodactylus fuscus 3 3 4 2 Eleutherodactylus luteolus

1 5 Eleutherodactylus alticola

2 8 Eleutherodactylus junori

6 5 Eleutherodactylus gossei

3 9 Eleutherodactylus griphus Eleutherodactylus pantoni 3 2 7 8 Eleutherodactylus pentasyringos 2 3 0.04 9 6 Eleutherodactylus cundalli Eleutherodactylus glaucoreius Supplemental material Chapter 2. Fig S2 DIVALIKE+J d=0.004 South America e=0 A j=0.0177 B Middle America LnL= -34.31 C Antilles AB South America + Middle America AC South America + Antilles BC Middle America + Antilles

B Diasporus diastema

A Diasporus anthrax

AB Diasporus vocator

B Diasporus aff. hylaeformis

B Diasporus citrinobapheus

B Diasporus tigrillo

AB Diasporus quidditus

AB Diasporus sp CH4717

A Diasporus cf gularis

C Eleutherodactylus johnstonei

C Eleutherodactylus amplinympha

C Eleutherodactylus martinicensi

C Eleutherodactylus pinchoni

C Eleutherodactylus barlagnei

C Eleutherodactylus patriciae

C Eleutherodactylus auriculatoid

C Eleutherodactylus sommeri

C Eleutherodactylus wetmorei

C Eleutherodactylus lamprotes

C Eleutherodactylus fowleri

C Eleutherodactylus melacara

C Eleutherodactylus leberi

C Eleutherodactylus varians

C Eleutherodactylus ionthus

C Eleutherodactylus guantanamera

C Eleutherodactylus minutus

C Eleutherodactylus poolei

C Eleutherodactylus pituinus

C Eleutherodactylus audanti

C Eleutherodactylus parabates

C Eleutherodactylus abbotti

C Eleutherodactylus haitianus

C Eleutherodactylus glamyrus

C Eleutherodactylus bartonsmithi

C Eleutherodactylus auriculatus

C Eleutherodactylus mariposa

C Eleutherodactylus ronaldi

C Eleutherodactylus eileenae

C Eleutherodactylus wightmanae

C Eleutherodactylus portoricensi

C Eleutherodactylus coqui

C Eleutherodactylus schwartzi

C Eleutherodactylus gryllus

C Eleutherodactylus brittoni

C Eleutherodactylus cochranae

C Eleutherodactylus hedricki

C Eleutherodactylus antillensis

C Eleutherodactylus locustus

C Eleutherodactylus eneidae

C Eleutherodactylus cooki

C Eleutherodactylus flavescens

C Eleutherodactylus richmondi

C Eleutherodactylus unicolor

C Eleutherodactylus counouspeus

C Eleutherodactylus monensis

C Eleutherodactylus probolaeus

C Eleutherodactylus pictissimus

C Eleutherodactylus rhodesi

C Eleutherodactylus weinlandi

C Eleutherodactylus lentus

C Eleutherodactylus grahami

C Eleutherodactylus toa

C Eleutherodactylus luteolus

C Eleutherodactylus griphus

C Eleutherodactylus glaucoreius

C Eleutherodactylus cundalli

C Eleutherodactylus pentasyringo

C Eleutherodactylus pantoni

C Eleutherodactylus gossei

C Eleutherodactylus junori

C Eleutherodactylus alticola

C Eleutherodactylus fuscus

C Eleutherodactylus orcutti

C Eleutherodactylus andrewsi

C Eleutherodactylus nubicola

C Eleutherodactylus jamaicensis

C Eleutherodactylus sisyphodemus

C Eleutherodactylus grabhami

C Eleutherodactylus cavernicola

C Eleutherodactylus riparius

C Eleutherodactylus rivularis

C Eleutherodactylus cuneatus

C Eleutherodactylus turquinensis

C Eleutherodactylus darlingtoni

C Eleutherodactylus leoncei

C Eleutherodactylus armstrongi

C Eleutherodactylus alcoae

C Eleutherodactylus bresslerae

C Eleutherodactylus ricordii

C Eleutherodactylus acmonis

C Eleutherodactylus orientalis

C Eleutherodactylus jaumei

C Eleutherodactylus limbatus

C Eleutherodactylus iberia

C Eleutherodactylus etheridgei

C Eleutherodactylus cubanus

C Eleutherodactylus varleyi

C Eleutherodactylus intermedius

C Eleutherodactylus gundlachi

C Eleutherodactylus planirostris

C Eleutherodactylus casparii

C Eleutherodactylus guanahacabib

C Eleutherodactylus pezopetrus

C Eleutherodactylus pinarensis

C Eleutherodactylus thomasi

C Eleutherodactylus blairhedgesi

C Eleutherodactylus tonyi

C Eleutherodactylus rogersi

C Eleutherodactylus goini

C Eleutherodactylus atkinsi

C Eleutherodactylus greyi

C Eleutherodactylus albipes

C Eleutherodactylus maestrensis

C Eleutherodactylus emiliae

C Eleutherodactylus dimidiatus

C Eleutherodactylus schmidti

B Eleutherodactylus marnockii

B Eleutherodactylus pipilans

B Eleutherodactylus nitidus

C Eleutherodactylus symingtoni

C Eleutherodactylus zeus

C Eleutherodactylus rufifemorali

C Eleutherodactylus furcyensis

C Eleutherodactylus jugans

C Eleutherodactylus thorectes

C Eleutherodactylus bakeri

C Eleutherodactylus eunaster

C Eleutherodactylus caribe

C Eleutherodactylus corona

C Eleutherodactylus amadeus

C Eleutherodactylus heminota

C Eleutherodactylus dolomedes

C Eleutherodactylus glaphycompus

C Eleutherodactylus glandulifero

C Eleutherodactylus oxyrhyncus

C Eleutherodactylus apostates

C Eleutherodactylus paulsoni

C Eleutherodactylus ventrilineat

C Eleutherodactylus brevirostris

C Eleutherodactylus sciagraphus

C Eleutherodactylus glandulifer

C Eleutherodactylus zugi

C Eleutherodactylus klinikowskii

B Eleutherodactylus longipes

C Eleutherodactylus inoptatus

C Eleutherodactylus chlorophenax

C Eleutherodactylus nortoni

C Eleutherodactylus parapelates

C Eleutherodactylus hypostenor

C Eleutherodactylus bothroboans

C Eleutherodactylus ruthae

A Adelophryne sp.2

A Adelophryne baturitensis

A Adelophryne sp.1

A Adelophryne maranguapensis

A Adelophryne sp.6

A Adelophryne sp.4

A Adelophryne sp.5

A Adelophryne pachydactyla

A Adelophryne patamona

A Adelophryne gutturosa

A Adelophryne adiastola 2

A Adelophryne adiastola 1

A Adelophryne patamona

A Adelophryne sp. 7

A Phyzelaphryne sp.1b

A Phyzelaphryne miriame

Paleocene Eocene Oligocene Miocene 70 60 50 40 30 20 10 0 Millions of years ago Supplemental material Chapter 3. Fig S1. ML tree mtDNA + UCEs Adelophryne_adiastola 7 1 Phyzelaphryne sp.1b AF-2012 9 3 Phyzalephryne sp. AJC 3606 Eleutherodactylus johnstonei 2 4 Diasporus vocator AB 564 Diasporus vocator CH 4786

9 9 Diasporus sp. UCR 21844 Diasporus vocator UCR 21966

4 1 Diasporus vocator UCR 21857 Diaiasporus vocator Pichi 248 Diasporus vocator UCR 20479 3 6 Diasporus vocator AJC 0127 Diasporus vocator CH 4791

3 6 Diasporus diastema CH 4794 Diasporus sp. AB 476 100 Diasporus sp.6 MHCH 1678 100 3 0 Diasporus diastema Pichi 216 6 6 Diasporus diastema UCR 22968 Diasporus sp. UCR 21676

7 2 Diasporus hylaeformis Pichi 229 Diasporus aff. diastema USNM 572456 Diasporus sp. KRL 0782 100 Diasporus sp. KRL 0831 Diasporus aff. diastema MVUP 1826 Diasporus sp. CH 5900 8 6 9 8 Diasporus diastema CH 5370 5 8 Diasporus diastema CH 5031 Diasporus diastema CH 5004 50 Diasporus hylaeformis KRL 0831

8 3 Diasporus aff. diastema AAB 3745 Diasporus hylaeformis KRL 0782 3 3 Diasporus diastema CH 5712

6 6 Diasporus vocator UCR 21946 100 Diasporus sp. UCR 21843

Diasporus sp. UCR 21953 Clade America Middle Diasporus sp. MHCH 1298 8 6 Diasporus aff. hylaeformis SMF 89875 Diasporus aff. hylaeformis SMF 89872 Diasporus hylaeformis Pichi 240 5 0 Diasporus hylaeformis Pichi 241 9 1 Diasporus aff. hylaeformis SMF 89868 Diasporus aff. hylaeformis SMF 89869 Diasporus diastema CH 5695 Diasporus sp. CH 5694 6 4 Diasporus hylaeformis AJC 0468 7 9 Diasporus hylaeformis Pichi 234 Diasporus sp. DL 685 Diasporus hylaeformis AJC 468 6 2 Diasporus diastema Pichi 218 Diasporus hylaeformis UCR 21935 6 4 Diasporus diastema Pichi 224 Diasporus diastema MVZ 203844 3 1 Diasporus sp. AJC 414 Diasporus sp. AJC 369 5 4 Diasporus diastema Pichi 223 Diasporus sp. AB 032 8 3 Diasporus sp. CH 6525 6 1 Diasporus sp. CH 6121 Diasporus sp. CH 6578 Diasporus hylaeformis CH 6520 Diasporus hylaeformis CH 6134 Diasporus cf. hylaeformis AJC 1732 Diasporus sp. CH 6577 Diasporus sp. CH 6583 Diasporus sp. CH 6617 Diasporus vocator CH 4773 Diasporus vocator CH 4772 Diasporus sp. CH 5918 Diasporus sp. MHCH 1288 Diasporus sp.5 AB032 Diasporus hylaeformis CH 4774 Diasporus sp. CH 6505 Diasporus hylaeformis CH 6124 Diasporus sp. CH 6251 100 Diasporus antrax MHUA 7237 Diasporus sp. MHUA 7305 9 7 Diasporus antrax MHUA 7374 4 7 Diasporus aff. quidditus AB 1030

8 Diasporus sp. AB 1065 100 Diasporus sp. CH 6297 Diasporus sapo AB 429

9 4 Diasporus sapo AB 435 9 9 Diasporus sapo AB 430 Diasporus sp.4 AB 439 Diasporus sp.4 AB 431 3 2 Diasporus sp. CH 9142 100 Diasporus sp. AB 1268

5 8 Diasporus darienensis AB 329 Diasporus sp. AJC 595 9 7 Diasporus sp. CH 6425 Diasporus sp. CH 6431 5 6 Diasporus darienensis AB 1185

4 0 Diasporus sp.3 AB 159

9 6 Diasporus darienensis AB 1134 Diasporus darienensis AB 1144 Diasporus darienensis AB 151 Diasporus sp. JJS 065 5 2 Diasporus sp. AJC 2119/MAR1486 9 3 Diasporus quidditus AJC 1194 Diasporus gularis MAA 300 6 4 Diasporus gularis MAA 299 Diasporus pequeno AB 860 Diasporus pequeno AB 822 9 7 Diasporus pequeno AB 857 Diasporus pequeno AB 856 Diasporus pequeno AB 861

100 Diasporus sp.2 AB 823 Diasporus sp. CH 4718 Diasporus sp. CH 4721 Diasporus quidditus CH 5807 Diasporus quidditus CH 4760 Diasporus sp. CH 4741 Diasporus vocator CH 4780 Diasporus quidditus CH 5340 Diasporus quidditus CH 5338 Diasporus quidditus CH 5792 Diasporus quidditus CH 5337 Diasporus quidditus CH 5341 Diasporus quidditus CH 5339 Diasporus quidditus CH 5802 Diasporus quidditus CH 5543 1 4 Diasporus quidditus CH 5540 8 3 6 7 Diasporus quidditus AB 689 4 4 Diasporus quidditus KRL 0856 9 6 Diasporus quidditus KRL 0647 9 0 3 9 Diasporus aff. quidditus AB 931 Diasporus quidditus AB 1130 Diasporus quidditus AB 138 Diasporus quidditus AB 131 Diasporus quidditus AB 158 Diasporus quidditus CH 4748 Diasporus sp. CH 4966 100 Diasporus quidditus CH 4717 Diasporus vocator CH 5392 Diasporus vocator CH 5391 Diasporus vocator CH 5390 Diasporus quidditus CH 6804 3 0 Diasporus diastema CH 6648 Diasporus quidditus AJC 1789 Diasporus diastema CH 4830 Diasporus quidditus CH 6803 Diasporus sp. AJC 600 Diasporus quidditus CH 5624 100 Diasporus quidditus CH 5544 Diasporus quidditus CH 5557 100 4 3 Diasporus quidditus CH 5556 Diasporus sp. AJC 1866

9 9 Diasporus sp. CH 6439 Diasporus sp. CH 9147 Diasporus sp. AJC 1866 7 8 Diasporus tinker AB 308 Diasporus sp. CH 4751

100 Diasporus tinker AB 1270 South and Middle America Clade Diasporus tinker AB 1269 Diasporus tinker AB 1271 Diasporus tinker AB 1272 Diasporus quidditus CH 5373 Diasporus quidditus CH 5371 100 Diasporus quidditus CH 5372 Diasporus sp. CH 5214 Diasporus vocator AB 028

2 6 Diasporus quidditus CH 5314 Diasporus quidditus CH 5213 Diasporus vocator CH 5400 Diasporus vocator CH 5401 Diasporus sp. AA2178 9 9 Diasporus gularis MHUA 7264 7 9 Diasporus sp. JJS 074 100 Diasporus gularis LEP 264 Diasporus gularis VCK13 100 Diasporus gularis VCK14 9 2 Diasporus quidditus MHUA 6789 Diasporus gularis MHUA 5379 Diasporus sp. MHUA 7405 Diasporus gularis MHUA 7414 2 1 Diasporus sp. MHUA 7412 6 1 Diasporus aff. gularis AJC 3417 3 7 Diasporus gularis MAR 777 Diasporus sp. MHUA 5129

6 3 Diasporus gularis AJC 3419 Diasporus gularis AJC 1183

5 5 Diasporus sp. AJC 2287 Diasporus sp. AJC 1192 Diasporus gularis Toe 1 8 5 Diasporus gularis Toe 2 Diasporus gularis Toe 3 Diasporus sp. AJC 2291 Diasporus diastema AB 073

100 Diasporus diastema AB 084 Diasporus diastema AB 637 Diasporus gularis MAR 689 Diasporus diastema AB 818 2 0 9 5 Diasporus diastema AJC 1536 9 6 Diasporus aff. diastema AB 035 9 7 Diasporus diastema AB 675

8 100 Diasporus aff. diastema AB 218

7 7 Diasporus sp. CH 6503 100 Diasporus sp. CH 6556 Diasporus tigrillo UCR 22364 Diasporus tigrillo UCR 22365 1 8 100 Diasporus tigrillo UCR 22366 Diasporus tigrillo UCR 22367 Diasporus tigrillo UCR 22368 Diasporus diastema CH 5799 Diasporus diastema AJC 1940 Diasporus sp. CH 4764 100 Diasporus diastema CH 5806 Diasporus diastema CH 6676 3 6 Diasporus diastema AB 979

100 Diasporus diastema CH 6786 Diasporus quidditus CH 4726 Diasporus diastema AB 602 Diasporus diastema CH 4834 9 9 Diasporus quidditus CH 5745 Diasporus diastema CH 4746 Diasporus quidditus CH 6792 Diasporus diastema CH 4719 4 8 6 7 Diasporus diastema CH 6800 Diasporus diastema CH 6802

6 6 Diasporus citrinobapheus SMF 89820 Diasporus citrinobapheus SMF 89814

8 5 Diasporus citrinobapheus MHCH 2370 Diasporus citrinobapheus MHCH 2371 Diasporus diastema CH 5302 5 0 9 5 Diasporus diastema CH 5270 Diasporus diastema CH 5225 Diasporus diastema CH 4992 Diasporus diastema CH 5226 9 7 Diasporus diastema CH 5221 Diasporus diastema CH 5211 Diasporus citrinobapheus KRL 0694 Diasporus citrinobapheus KRL 1181

0.03 Diasporus citrinobapheus KRL 0902 Diasporus citrinobapheus KRL 0900 Diasporus citrinobapheus KRL 0901 Diasporus citrinobapheus KRL 0840 Supplemental material Chapter 3. Fig. S2

OTU 4 vs. OTU 5

Identity test: Identity test: OTU4 vs. OTU5 OTU4 vs. OTU5 15 25

20

10 15 count count 10 5

5

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 D D, Environmental Space Identity test: Identity test: OTU4 vs. OTU5 OTU4 vs. OTU5

20 15

15

10 count count 10

5 5

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 I I, Environmental Space Identity test: Identity test: OTU4 vs. OTU5 OTU4 vs. OTU5 15

30

10 20 count count

5 10

0 0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 Rank Correlation Rank Correlation, Environmental Space

OTU 8 vs. OTU 9

Identity test: Identity test: OTU8 vs. OTU9 OTU8 vs. OTU9

15 15

10 10 count count

5 5

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 D D, Environmental Space Identity test: Identity test: OTU8 vs. OTU9 OTU8 vs. OTU9

40 10

30 count count 20 5

10

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 I I, Environmental Space Identity test: Identity test: OTU8 vs. OTU9 OTU8 vs. OTU9

60 30

40 20 count count

20 10

0 0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 Rank Correlation Rank Correlation, Environmental Space

OTU 11 vs. OTU 12

Identity test: Identity test: OTU11 vs. OTU12 OTU11 vs. OTU12 20 15

15

10

10 count count

5 5

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 D D, Environmental Space Identity test: Identity test: OTU11 vs. OTU12 OTU11 vs. OTU12 10.0 40

7.5 30

5.0 20 count count

10 2.5

0 0.0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 I I, Environmental Space Identity test: Identity test: OTU11 vs. OTU12 OTU11 vs. OTU12

60

10

40 count count

5 20

0 0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 Rank Correlation Rank Correlation, Environmental Space

OTU 21a vs. OTU 21b

Identity test: Identity test: OTU21a vs. OTU21b OTU21a vs. OTU21b 25

20 15

15 10 count count 10

5 5

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 D D, Environmental Space Identity test: Identity test: OTU21a vs. OTU21b OTU21a vs. OTU21b 30

20 20 count count 10 10

0 0 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 I I, Environmental Space Identity test: Identity test: OTU21a vs. OTU21b OTU21a vs. OTU21b 60 60

40 40 count count

20 20

0 0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 Rank Correlation Rank Correlation, Environmental Space