**2. Methods**

## *2.1. Taxonomy and Species Concept*

Throughout this work, we use the name Centrolenidae as originally intended by Taylor [15]. The taxonomic arrangemen<sup>t</sup> below the family level (i.e., subfamily and genus) follows the proposal by Guayasamin et al. [1]. We use quotations to denote species with uncertain phylogenetic position to di fferentiate them from monophyletic clades (e.g., *"Centrolene"*, *"Cochranella"*), as proposed by Guayasamin et al. [1]. An updated taxonomy for all the species in Centrolenidae is provided in Table S1.

For recognizing species, we adhered to the evolutionary species concept first proposed by Simpson [34,35], modified by Wiley [36] and de Queiroz [37,38]. This concept incorporates important theoretical factors such as lineage independence, identity, and evolutionary tendencies, and provides a flexible framework when reproductive isolation is di fficult to test (e.g., allopatric populations). Evidence supporting the validity of a species can come from di fferent sources (e.g., morphology, DNA, behavior, ecology), and no trait alone can be considered a biological property that a species must have in order to be recognized [37]. In other words, under the evolutionary species concept, the only necessary property for an entity to be a recognized as a species is that it corresponds to a temporal segmen<sup>t</sup> of a metapopulation lineage evolving separately from other lineages [37,38]. Evidence for independent evolution is gathered from di fferent data sources, where integrative taxonomy plays a fundamental role when assessing what represents (or not) a distinctive species (e.g., [39,40]).

## *2.2. Characters and Terminology*

For general terminology and descriptions of morphological characters we follow the proposals by Lynch and Duellman [22] and Cisneros-Heredia and McDiarmid [17]. We illustrate some of the most relevant traits for glassfrog identification, including dorsal color pattern (Figure 2), skin texture (Figure 9), and snout shape (Figure 10). To facilitate comparison with previous literature dealing with anurans, fingers are numbered preaxially to postaxially from I–IV. However, we stress that from an evolutionary perspective, anuran fingers should be numbered from II–V, to reflect the loss of Digit I in anurans [41–43]. Webbing formulae follow the method of Savage and Heyer [44], as modified by Guayasamin et al. [20] (Figure 11). Larval characters follow the terminology recommended by McDiarmid and Altig [45]. The morphology of nuptial excrescences and prepollical spines (Figure 12) follows the types proposed by Flores [46], with the additions and modifications detailed in Cisneros-Heredia and McDiarmid [17] and Guayasamin et al. [1]. Other key traits in centrolenid taxonomy are humeral spines (Figure 7, Figure 13, and Figure 14), ventral transparency (Figure 8), peritoneum and pericardium (with or without iridophores; Figure 13), enlarged subcloacal warts on thighs below vent (Figure 15), and tubercles on the external edge of arm, hand, and foot (Figure 16).

**Figure 9.** Skin texture in glassfrogs. ( **A**) Smooth; *Sachatamia ilex*, QCAZ 47193. (**B**) Shagreen; *Nymphargus humboldti* sp. nov., ZSFQ 0833. ( **C**) Pustular; *Centrolene heloderma*, QCAZ 40200. All photographs by Luis A. Coloma, except (**B**) by Jose Vieira/Tropical Herping.

When discussing parental care, we adopt the terminology by Delia et al. [25]. Egg brooding refers to a specific form of ventral contact where the parent positions its body over the egg clutch; this behavior reduces embryonic mortality by protecting embryos from dehydration and, possibly, by preventing fungal development and predation [25]. Parental care is divided into the following behaviors [25]: (i) Short-term maternal care, where brooding is provided for a few hours just after oviposition; (ii) prolonged male care, where parental care is provided for several weeks; and (iii) prolonged female care, where parental care is provided for several weeks (only observed in *Ikakogi tayrona* [47]).

**Figure 10.** Snout shape in Glassfrogs. (**A**) Round, *Hyalinobatrachium munozorum*, KU 118054. (**B**) Bluntly round, *Centrolene ballux*, KU 164725. (**C**) Truncate, *Nymphargus megacheirus*, KU 143269. (**D**) Slightly protruding, *"Centrolene" medemi*, KU 164493. (**E**) Protruding, *"Cochranella" balionota*, KU 164708. (**F**) Sloping, *Nymphargus grandisonae*, KU 164688. Drawings by Juan M. Guayasamin.

*Morphometrics*—morphological variables were measured with digital callipers to the nearest 0.1 mm, as follows: (1) Snout–vent length (SVL) = distance from tip of snout to posterior margin of vent; (2) tibia length (TL) = length of flexed leg from knee to heel; (3) foot length (FL) = distance from proximal margin of outer metatarsal tubercle to tip of Toe IV; (4) head length (HL) = distance from tip of snout to posterior angle of jaw articulation; (5) head width (HW) = width of head measured at level of jaw articulations; (6) interorbital distance (IOD) = distance between upper eyelids, representing the width of the underlying frontoparietals; (7) upper eyelid width (UE) = greatest transverse width of upper eyelid; (8) internarial distance (IN) = distance between nostrils; (9) eye–nostril distance (EN) = distance from posterior margin of nostril to anterior margin of eye; (10) snout–eye distance (SE) = distance from tip of snout to anterior margin of eye; (11) horizontal eye diameter (ED) = distance between anterior and posterior borders of eye; (12) tympanum diameter (TD) = distance between anterior and posterior margins of tympanic annulus; (13) eye–tympanum distance (ET) = distance from posterior border of eye to anterior margin of tympanic annulus; (14) radio–ulna length (RUL) = length of flexed forearm from elbow to proximal border of palmar tubercle; (15) hand length (HDL) = distance from the proximal margin of palmar tubercle to tip of Finger III; (16) Finger-I length (F1L) = distance from outer margin of palmar tubercle to tip of Finger I; (17) Finger-II length (F2L) = distance from outer margin of palmar tubercle to tip of Finger II; (18) disc of Finger III (3DW) = greatest width of disc of Finger III; and (19) Finger-III width (F3W) = width of Finger III measured at the level of distal subarticular tubercle, including lateral fringes and excluding webbing. For comparing different body sizes (SVL) among glassfrogs, we considered the average size of males and categorized them according to the following criteria: Minute (SVL < 22 mm), small (SVL 22–25 mm), medium (SVL 25–30 mm), large (SVL 30–50 mm), and giant (SVL > 50). Eye diameter was divided into small (eye diameter < 10% SVL), moderate size (eye diameter 10%–15% SVL), and large (eye diameter > 15% SVL). Tympanum was considered to be very small (tympanum < 20% of eye diameter), small (tympanum 20%–30% of eye diameter), moderate (tympanum 31%–40% of eye diameter), large (tympanum diameter 41%–50% of eye diameter), and very large (tympanum diameter > 50% of eye diameter.

**Figure 11.** Webbing in glassfrogs. (**A**) Terminology used for webbing formula in hands and feet (see Guayasamin et al. [20]; modified from Savage and Heyer [44]). Roman numerals (I, II, III, IV, V) represent fingers or toes. Arabic numerals represent the number of phalanges completely or partially free of webbing. We use **0**− to indicate that webbing reaches the distal margin of the disc; **0** indicates that webbing reaches the middle of the disc; **0**<sup>+</sup> indicates that webbing reaches the proximal margin of the disc; **1**− indicates that webbing reaches the distal margin of the intercalary cartilage; **1** indicates that the webbing the middle of the intercalary cartilage; **1**<sup>+</sup> indicates that the webbing the proximal margin of the intercalary cartilage; **2**− indicates that webbing reaches the distal margin of the distal subarticular tubercle; **2** indicates that webbing reaches the middle of the distal subarticular tubercle; **2**− indicates that webbing reaches the proximal margin of the distal subarticular tubercle. For example, webbing formula in the illustrated foot is absent between Toes I and II (lateral fringes are not considered as webbing); **II 12**/**3—3**<sup>+</sup> **III 12**/**3—3**− **IV 3**−**—2 V**. Figure modified from Guayasamin et al. (2006). (**B**) Simplified type of hand webbing. Absent: *Nymphargus cochranae*, QCAZ 31113. Moderate: *Chimerella mariaelenae*, QCAZ 22363. Extensive: *Centrolene geckoidea*, KU 164490. Drawings by Juan M. Guayasamin.

**Figure 12.** Prepollex and nuptial pad morphology in glassfrogs. (**A**) Condition of the prepollex. Concealed, *Nymphargus cochranae*, QCAZ 31113. Distinct, *Teratohyla spinosa*, KU 164668. (**B**) Nuptial pad morphology. **Type I:** Large to medium-size nuptial excrescence present on the dorsal, lateral, and/or ventral sides of the thumb; *Cochranella posadae*, QCAZ 26023. **Type II:** Small circular or squarish nuptial excrescence present on the dorsal or dorsolateral face of the thumb; *Centrolene lynchi*, MCZ 97846 (figure modified from Flores 1985). **Type III:** Medium-size spinous nuptial excrescence extending from the lateral side of the thumb to its dorsomedial surface; *Nymphargus armatus*, UVC 9400 (modified from Lynch and Ruiz-Carranza 1996). **Type IV:** Large nuptial excrescence formed by a granular pad that extends from the side of the thumb to its dorsomedial surface, and on the proximal dorsolateral surface of finger II; *Cochranella litoralis*, ICN 13821. **Type V:** Medium-size diffuse nuptial excrescence formed by glandular clusters and individual glands; a pad as such is absent; *Hyalinobatrachium aureoguttatum*, QCAZ 27429. **Type VI** (not illustrated): Nuptial excrescences formed by a combination of clustered and individual glands that sparse along the flanks of the body (see text).

**Figure 13.** Peritonea and humeral spines in Centrolenidae. (**A**,**Left**): Pericardium lacking iridophores, hepatic, and visceral peritonea with iridophores (*Hyalinobatrachium aureoguttatum*). (**A**,**Right**): Pericardium with iridophores, hepatic, and visceral peritonea lacking iridophores (*Centrolene buckleyi*). (**B**,**Top**): Absence of humeral spine (*H*. *fleischmanni*). (**B**,**Bottom**): Presence of humeral spine in males (*Espadarana callistomma*). Figure modified from Guayasamin et al. [1].

**Figure 14.** Variation of humeral spines, *crista medialis*, and *crista ventralis*, in adult males of Centrolenidae (modified from Guayasamin et al. [1]). Illustrated specimens are: *Centrolene sanchezi*, KU 170116; *C*. *geckoidea*, ICN 5598; *C*. *pipilata*, KU 143286; *Cochranella euknemos*, KU 77534; *Cochranella litoralis*, QCAZ 27693; *Teratohyla spinosa*, KU 32935; *Nymphargus gri*ffi*thsi*, KU 288992, 188148; *N*. *cochranae*, KU 123218; *N*. *megacheirus*, KU 143271; *Sachatamia albomaculata*, KU 65185; *S*. *ilex*, LACM 72910; *Chimerella mariaelenae*, QCAZ 21252; *H*. *valerioi*, KU 178091.

**Figure 15.** Ventral surfaces of tights in glassfrogs. (**A**) With enlarged subcloacal warts. (**B**) Lacking enlarged warts. Illustrations by Juan M. Guayasamin.

**Figure 16.** Tubercles on limbs. (**A**) Tubercles on ventrolateral edge of Finger IV and arm, *Cochranella resplendens*, KU 118053. (**B**) Tubercles on ventrolateral edge of Toe V and tarsus, *C*. *mache*, QCAZ 22412. Photos by Martín Bustamante.

*Vocalizations—*Calls were recorded in the field by different researchers (see Acknowledgments) using an Olympus Linear PCM Recorder LS-10 tape recorder and a Sennheiser K6-ME67 directional microphone. Recordings obtained by us are stored at the Laboratorio de Biología Evolutiva of the Universidad San Francisco de Quito (LBE). Sounds were recorded in PCM format at a sampling frequency of 44.1 kHz. Audiospectograms and oscillograms were generated in the R package

SeeWave [48]. Frequency information was obtained through fast Fourier transformation, with a 50% window overlap (Hanning window size of 1024 and a frequency window of 43.1 Hz). The following call variables were measured: Call duration, duration between calls, number of notes per call, note duration, duration between notes, dominant frequency, first harmonic, second harmonic [49–51] (Table 1). A call is defined as the sound produced in a single exhalation of air. Calls and notes were divided into two categories, tonal and pulsed, based on distinct morphology. Tonal calls/notes have non-peaked amplitude sustained throughout the duration of the note. Pulsed calls/notes are characterized by having at least one clear amplitude peak. Pulses were defined as a visible increase and decrease of amplitude peaks on the oscillogram within a single note, and notes were defined by a single and complete amplitude rise from and return to the base frequency of the background noise. For a review on the use of bioacoustics in taxonomy and specific variable definitions, see Köhler et al. [52].


**Table 1.** Definitions of call variables [51].

*Specimens examined*—We examined ethanol-preserved specimens from the following herpetological collections: Centro de Biodiversidad y Genética, Cochabamba, Bolivia (CBG); Círculo Herpetológico de Panamá (CHP); Centro Jambatu de Investigación y Conservación de Anfibios, San Rafael, Ecuador (CJ); División de Herpetología, Museo Ecuatoriano de Ciencias Naturales, Quito, Ecuador (DHMECN); Field Museum, Division of Amphibians and Reptiles, Chicago, USA (FMNH); Instituto de Ciencias

Naturales, Universidad Nacional de Colombia, Bogotá, Colombia (ICN); University of Kansas, Museum of Natural History, Division of Herpetology, Lawrence, Kansas, USA (KU); Natural History Museum of Los Angeles County, Section of Herpetology, Los Angeles, California, USA (LACM); Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, USA (MCZ); Museo de Historia Natural La Salle, Caracas, Venezuela (MHNLS); Museo de Zoología, Universidad Tecnológica Indoamérica, Quito, Ecuador (MZUTI); Museo de Zoología, Pontificia Universidad Católica del Ecuador, Quito, Ecuador (QCAZ); Colección de Herpetología, Escuela de Biología, Universidad de Costa Rica, San José, Costa Rica (UCR); Museo de Vertebrados, Universidad del Valle, Cali, Colombia (UVC); and Museo de Zoología, Universidad San Francisco de Quito, Ecuador (ZSFQ). When specimens were not available for direct comparison, we relied on descriptions in the literature. Sexual maturity of specimens was determined by the presence of vocal slits and nuptial pads in males and by the presence of eggs or convoluted oviducts in females.
