**1. Introduction**

Neotropical poison frogs, represented by the family Dendrobatidae within Anura, are one of the most charismatic and well-studied groups of amphibians. Popularly known for their powerful skin toxins and extravagant aposematism, dendrobatids have featured in scientific studies for decades in fields as diverse as reproductive behavior [1,2], pharmacology [3–5], color evolution [6–8], and biogeography [9–11], as well as recently fueling important studies in the evolution of monogamy [12] and toxin autoresistance [13,14]. In the context of Anura, Dendrobatidae is moderately diverse, inhabiting a range of habitats and ecological niches throughout Central and South America. Many dendrobatids are unfortunately threatened by a variety of factors including habitat destruction [15,16] and smuggling for the pet trade [17,18], making their conservation an important priority for biologists. Despite heavy popular and scientific interest in dendrobatids, in-depth studies of dendrobatid phylogenetic systematics have become scarce despite the rapid progress of phylogenomics. In this paper, we aim to provide the first evolutionary hypothesis of Dendrobatidae derived from genomic-scale data, to put to rest many of the outstanding questions concerning dendrobatid phylogeny.

The first dendrobatid described was *Rana tinctoria* by Cuvier in 1797 [19], later transferred to *Dendrobates* by Wagler in 1830 [20], where it remains to this day. Since then, described dendrobatid diversity has grown significantly, with roughly 198 species in 16 genera as of 2019 (Table 1). Until the 2006 revision of Dendrobatidae by Grant et al. [21], most dendrobatid species were confined to the genera *Dendrobates, Phyllobates, Colostethus, Epipedobates,* and *Minyobates.* Phylogenetic estimates constructed during this time from molecular data [22–30] generally recovered two main clades of dendrobatids: one composed of mostly aposematic frogs in *Phyllobates* and *Dendrobates*, and the other composed of more cryptic frogs in *Colostethus* and *Epipedobates*. A third group, which would later be established as the subfamily Hyloxalinae, was generally placed as sister to the *Dendrobates* clade ([22,23,27], though see [28] for an exception). During this time, the systematics and taxonomy of Dendrobatidae and its sister family Aromobatidae (then regarded as part of Dendrobatidae; this is still the taxonomy used by AmphibiaWeb) were confused and inconsistent. Phylogenies produced during this period were mostly constructed from alignments of a few mitochondrial loci, making them vulnerable to incomplete lineage sorting [31,32].


**Table 1.** Dendrobatid genera and relevant information. The authority and type species for each genus is given, as well as the number of described species and a very basic description of each genus' geographic range. Species counts and authorities retrieved from Amphibian Species of the World [33].

In 2006, Grant et al. comprehensively revised Dendrobatidae [21], splitting many of the previously paraphyletic genera into a multitude of new, monophyletic ones: *Ranitomeya, Adelphobates*, and *Oophaga* from *Dendrobates; Ameerega* from *Epipedobates;* and *Silverstoneia* and *Hyloxalus* from *Colostethus*. Most species within *Minyobates* were absorbed into *Ranitomeya,* leaving *M. steyermarki* as the sole member of the now-monotypic genus. The dendrobatid tree was becoming clearer now, with the genera previously in *Dendrobates,* along with *Phyllobates* and *Minyobates,* forming the subfamily Dendrobatinae, *Epipedobates, Ameerega, Colostethus,* and *Silverstoneia* forming Colostethinae, and *Hyloxalus* forming its own subfamily Hyloxalinae, which Grant et al. recovered as sister to Dendrobatinae rather than Colostethinae, conflicting with most previous phylogenies [21]. After this seminal study, dendrobatid taxonomy continued to fragment, with Twomey and Brown erecting the genus *Excidobates* in 2008 [35], Brown et al. (2011) splitting *Andinobates* from *Ranitomeya* [43], and Grant et al. (2017) establishing *Leucostethus*, a sister genus to *Ameerega,* as well as *Paruwrobates* and *Ectopoglossus,* both members of Hyloxalinae, in another broad systematic review [41].

Since Grant et al.'s 2006 revision, relatively few large-scale phylogenetic studies of dendrobatids have been undertaken [41,44,45]. Santos et al. published a time-calibrated phylogeny of Dendrobatidae in 2009 constructed from ~2400 bp of mitochondrial data [44], and Pyron and Wiens (2011) published an Amphibia supertree constructed via maximum likelihood containing many representatives of Dendrobatidae [45]. Most recently, Grant et al. (2017) published the most comprehensive dendrobatid tree to date [41], constructed using parsimony, and containing representatives of all genera. They provided evidence for the paraphyly of *Colostethus,* as *C. ruthveni* is nested within Dendrobatinae. The latter two studies were based on approximately a dozen mitochondrial and nuclear loci, with the addition of morphological data in the case of Grant et al. (2017). All of these studies recover Hyloxalinae as the sister group to Dendrobatinae, not Colostethinae, consistent with many pre-2006 studies [22–27,29]. Previous estimates di ffer with respect to the sister genus of *Dendrobates,* which is either *Oophaga* [44] or *Adelphobates* [41,45]. Finally, the problematic taxon *Minyobates steyermarki* is recovered in various places throughout the dendrobatine phylogeny, either as sister to *Adelphobates* [45] or to all other dendrobatines aside from *Phyllobates* and *C. ruthveni* [41].

Many dendrobatid frogs are of conservation concern. The International Union for Conservation of Nature Red List of Threatened Species (also known as the IUCN Red List), is one of the world's most comprehensive inventories of the global conservation status of biological species and has evaluated the status of many dendrobatids. It uses a set of criteria to evaluate the extinction risk of thousands of species globally and is recognized as an authority in the status of biological diversity. Here we present a novel approach for visualizing the relationships between IUCN Red List status, phylogenetic relationships, and spatial distributions. This is a tractable approach for visualizing complex patterns and large quantities of data in relatively simple infographics. These infographics are aimed at summarizing broad patterns, facilitating additional assessment, and complementing more detailed quantitative analyses.

In this study, we attempt to resolve the remaining uncertainties in dendrobatid phylogeny, specifically with regards to relationships between dendrobatid genera and subfamilies. Our primary advance for dendrobatid phylogenetics is the usage of genome-scale molecular markers known as ultraconserved elements (UCEs) [46,47], which provide an order of magnitude more molecular data to work with than previous studies and span the genome across chromosomes [47,48]. UCEs consist of an "ultraconserved" core region with identity or near-identity across the taxon set in question, along with increasingly divergent flanking regions with phylogenetic signal that evidence suggests is greater than in traditional protein-coding loci [49]. UCEs have become popular phylogenomic markers largely thanks to the ease with which thousands of UCE loci can be sequenced from even old museum specimens [50,51]. In recent years, UCEs have been used in many phylogenomic studies of vertebrates, and have been instrumental in resolving di fficult phylogenetic problems at both deep and shallow timescales [52–58]. The use of UCEs in phylogenomic studies is appealing due to UCE loci having little overlap with paralogs [59], being found in genomic regions with few transposons [60], and having low saturation rates that decrease the possibility of homoplasy [58]. A study by Gilbert et al. (2015) showed that UCEs contain considerably more net phylogenetic informativeness than traditional protein-coding nuclear loci [49]. All of these factors led us to use UCE sequence capture as our method of choice for generating a phylogenomic dataset of the dendrobatid poison frogs. Here we provide the first dendrobatid phylogeny constructed from genome-scale data, which we hope will anchor future evolutionary studies of this fascinating amphibian group.

## **2. Materials and Methods**

## *2.1. Data Collection*

We gathered 63 dendrobatoid tissue samples from a combination of museum collections, our own field work, and the collections of collaborators (Table S1). Our sample represents 36 species in 13 dendrobatid genera (the newly erected genera *Paruwrobates, Ectopoglossus,* and *Leucostethus* were not included) and includes the aromobatid *Allobates femoralis* as an outgroup taxon. In many cases, we include multiple representatives of a given species to account for geographic variation.

For each sample, we performed sequence capture of UCEs in the manner of Faircloth et al. [46]. We extracted genomic DNA from each tissue sample with the Qiagen DNeasy Blood and Tissue Kit (Valencia, CA, USA) and performed quality and yield assessment with a Qubit 3 fluorometer (ThermoFisher Scientific). Extracted DNA was sent to RAPiD Genomics (Gainesville, FL, USA), who performed Illumina sequencing of UCEs, enriching the samples with the Tetrapods-UCE-5Kv1 probe set, which contains 5472 probes that target 5060 UCE loci. Raw reads for each sample are available at the NCBI Sequence Read Archive under project number PRJNA547821.
