**1. Introduction**

The Neotropical family Centrolenidae Taylor 1951 is a monophyletic taxon that contains about 150 species classified into 12 genera [1] (Figure 1). Historical biogeographic evidence in a phylogenetic context suggests that the group originated in South America and subsequently dispersed to Central America [2,3]. Centrolenids share a unique morphology and behavior that makes them readily identifiable; some of the most evident traits include a green dorsum in most species (Figure 2), completely or partially translucent venter (Figure 3), out-of-water deposition of eggs along streams (Figure 4), and forward-directed eyes (Figure 2A). Other interesting features that have evolved within Centrolenidae include parental care [4] (Figure 5), fighting behavior [5] (Figure 6), and humeral spines in males of some species [6] (Figure 7).

**Figure 1.** Relationships among glassfrog genera as inferred herein, using maximum likelihood criterium. Taxonomy sensu Guayasamin et al. [1]. Taxon sampling includes 113 named glassfrog species, 24 putative new species, and 49 outgroup taxa (not shown). The dataset contains complete or partial sequences of 10 genes representing 6513 bp of data (mitochondrial: 12S rRNA, 16S rRNA, ND1; nuclear: BDNF, C-MYC exon 2, CXCR4, NCX1, POMC, RAG1, SLC8A3). Sequences were generated in previous studies, as well as this one (see Table S2). Relationships within each genus are shown in additional figures and follow the same methodology.

**Figure 2.** Dorsal color patterns in glassfrogs. (**A**) Uniform; *Sachatamia ilex*, QCAZ 47193. (**B**) With small and well-defined yellow spots; *Nymphargus humboldti* sp. nov., QCAZ 41084. (**C**) With small and diffuse yellow spots; *Hyalinobatrachium fleischmanni*, QCAZ 45386. (**D**) With large yellow spots; *H*. *aureoguttatum*, QCAZ 45365. (**E**) With small ocelli, *N*. *cochranae*, QCAZ 31113. (**F**) With ocelli and dark flecks; *N*. *anomalus*, QCAZ 47507. (**G**) With irregular light-green marks and small well-defined black spots, *H*. *iaspidiense*, QCAZ 38438. (**H**) With green reticulation; *H*. cf. *valerioi*, ZSFQ 0544 (photo by Jose Vieira). All photographs by Luis A. Coloma, except when noted.

The monophyly of Centrolenidae is well supported by morphological [7–9] and molecular [2,8,10–14] studies. Osteological characters shared by all glassfrogs (Figure 8) include a dilated medial process on Metacarpal III [7], T-shaped terminal phalanges [15], intercalary element between distal and penultimate phalanges [15], and complete or partial fusion of tibiale and fibulare [15,16]. The diversity of glassfrogs is growing and in constant revision (e.g., [1,6,17–21]). Centrolenid species richness is concentrated in mountain chains, where humidity is high, and streams provide suitable

reproductive habitats. Therefore, it is no surprise that the Andes, by far, is the center of diversity and endemism for the group and that Colombia and Ecuador maintain the highest species richness.

In 1973, John D. Lynch and William E. Duellman [22] provided the first review of the glassfrogs of Ecuador, and they reported the presence of 20 species and suggested the occurrence of an additional one. Only 47 years later, the species richness of this family has tripled, reaching the incredible number of 60 species in Ecuador. Since Lynch and Duellman's pioneering work, our understanding of centrolenid biology [23–25], morphology [6,9,17], systematics [1,6,17,26,27], biogeography [3,28], and evolution [2,19,25] has improved substantially. Still, there are novel challenges and conspicuous gaps that need to be filled.

Herein, we provide a new review of Ecuadorian glassfrogs, bringing an update of what is known about the group, highlighting issues pending resolution, and providing a framework that we hope will facilitate further research, particularly with species identification, discovery, and conservation. We employ a candidate species system (e.g., [29]) to identify putative undescribed species to be investigated and to streamline the species discovery process. These tasks are particularly urgen<sup>t</sup> in the current global amphibian extinction phenomenon [30–33].

**Figure 3.** Ventral transparency in glassfrogs. (**A**) Complete transparency of parietal peritoneum and pericardium; *H. aureoguttatum*. (**B**) Partial transparency: parietal peritoneum is transparent only posteriorly; *N. posadae*. (**C**) Venter opaque (no ventral transparency): ventral parietal peritoneum and the urinary bladder peritoneum are opaque (white); *N. gradisonae*. Photos by Martín Bustamante.

**Figure 4.** Egg deposition sites in glassfrogs. (**A**) On upper side of leaves (*C. sanchezi)*. (**B**) On tip of leave (*N. wileyi)*. (**C**) On underside of leaves (*H. cappellei*, photo by C. Barrio-Amorós). (**D**) On the margin of underside of leaves (*Teratohyla spinosa*, photo by R. Puschendorf). (**E**) On rocks (e.g., *Sachatamia albomaculata*, photo by R. Puschendorf). Figure modified from Guayasamin et al. [1].

**Figure 5.** Parental care in glassfrogs. Adult male of *Hyalinobatrachium aureoguttatum*, with egg clutch. Photo by Luis A. Coloma.

**Figure 6.** Combat behavior in glassfrogs. Note diversity of positions. Illustrated species: *Nymphargus grandisonae*. Photos by Carl R. Hutter.

**Figure 7.** Humeral spine in *Cochranella litoralis*. Note that the humeral spine is formed by the prolongation of the *crista ventralis*. Photo by Luis A. Coloma; drawing by Linda Trueb.

**Figure 8.** Synapomorphies of Centrolenidae. (**A**) Partial (*Nymphargus posadae*, QCAZ 25090) and complete ventral transparency (*Hyalinobatrachium aureoguttatum*, QCAZ 32070). (**B**) Partial (*N*. *wileyi*, QCAZ 26029) and complete fusion between tibiale and fibulare (*H*. *munozorum*, KU 155497). (**C**) Presence of medial process on Metacarpal III and intercalary element (*Teratohyla spinosa*, KU 32935). The presence of T or Y-shaped terminal phalanges is a synapomorphy of Allocentroleniae (Allophrynidae + Centrolenidae). Photos in (**A**) by M. Bustamante. Modified from Guayasamin et al. [1].
