Two new glassfrogs (Centrolenidae: Hyalinobatrachium) from Ecuador, with comments on the endangered biodiversity of the Andes Author Guayasamin, Juan M. Laboratorio de Biología Evolutiva, Instituto Biósfera, Colegio de Ciencias Biológicas y Ambientales COCIBA, Universidad San Francisco de Quito USFQ, Quito, Ecuador & Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States jmguayasamin@usfq.edu.ec Author Brunner, Rebecca M. Third Millennium Alliance, Quito, Ecuador & Department of Environmental Science, Policy, and Management, University of California, Berkeley, Berkeley, California, United States Author Valencia-Aguilar, Anyelet Division of Behavioral Ecology, Institute of Ecology and Evolution, University of Bern, Bern, Switzerland Author Franco-Mena, Daniela Laboratorio de Biología Evolutiva, Instituto Biósfera, Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito USFQ, Quito, Ecuador Author Ringler, Eva Division of Behavioral Ecology, Institute of Ecology and Evolution, University of Bern, Bern, Switzerland Author Medina Armijos, Anderson Biology & Research Department, Mashpi Lodge, Mashpi, Ecuador Author Morochz, Carlos Biology & Research Department, Mashpi Lodge, Mashpi, Ecuador Author Bustamante, Lucas Tropical Herping, Quito, Ecuador Author Maynard, Ross J. The Biodiversity Group, Tucson, Arizona, United States Author Culebras, Jaime Photo Wildlife Tours, Quito, Ecuador & Fundación Cóndor Andino, Quito, Ecuador text PeerJ 2022 2022-03-18 10 1 34 http://dx.doi.org/10.7717/peerj.13109 journal article 10.7717/peerj.13109 8996732a-01b8-4ffa-8646-33ff5ac5cbaa PMC8935995 35321409 6377348 Hyalinobatrachium mashpi new species LSID: 0815B7E6-33FB-42D9-A367-4FB50885C256 Suggested English name: Mashpi Glassfrog Suggested Spanish name: Rana de Cristal de Mashpi Holotype . CJ11642 , adult male from San Vicente River ( 0.16334 N , 78.86736 W ; 1,040 m a.s.l.), Mashpi Reserve , Pichincha Province , Ecuador , collected by Jaime Culebras and Carlos Morochz on 28 September 2019 . Paratopotypes . CJ11643–44 , adult males with same data as holotype . Paratypes . MZUTI-3921 , adult male from Amagusa River ( 0.15469 N , 78.85322 W ; 1,137 m a.s.l.), Amagusa Reserve , Pichincha Province , Ecuador , collected by Carlos Morochz and Lucas Bustamante on 14 December 2014 . CJ11645, adult male from tributary of the Mashpi River (0.11463 N, 78.88307 W; 1,126 m a.s.l.), Tayra Reserve , Pichincha Province , Ecuador , collected by Jaime Culebras on 28 October 2019 . Generic placement. The new species is placed in the genus Hyalinobatrachium Ruiz-Carranza & Lynch (1991) , as modified by Guayasamin et al. (2009) , on the basis of morphological and molecular data. The molecular phylogeny ( Fig. 2 ) places the new species within the genus Hyalinobatrachium with high confidence. Phenotypically, the main diagnostic traits of Hyalinobatrachium are: (1) completely transparent ventral parietal peritoneum; (2) digestive tract and bulbous liver are covered by iridophores; (3) absent humeral spines; (4) white bones in life; (5) males call from the undersides of leaves; (6) females place the eggs on the undersides of leaves; (7) males provide extended parental care; and (8) tympanum with an orientation that places it almost on a horizontal plane (instead of a more lateral plane as observed in other glassfrog genera). All the aforementioned characteristics are present in Hyalinobatrachium mashpi sp. nov. We note that we have observed males on the same leaves as egg clutches for continuous days, but additional studies are necessary to confirm that these observations actually represent extended paternal care. Diagnosis. Hyalinobatrachium mashpi sp. nov. is distinguished from other species in the genus mainly by its dorsal coloration ( i.e ., head with light yellow spots that may form an interorbital bar; dorsum lime green with small light yellow spots) and by its transparent pericardium ( i.e ., red heart visible in ventral view). Hyalinobatrachium mashpi sp. nov. is most similar to H. aureoguttatum , H. talamancae , H. valerioi , H. vireovittatum , and the new species described below. Differences among these species are indicated in Table 1 and Figs. 3–5 . The new species is morphologically cryptic with Hyalinobatrachium nouns sp. nov. (described below); however, the two new species exhibit a considerable genetic distance (16S; 4.6–4.7%), which is particularly remarkable given that they are found at relatively close geographic proximity (straight distance = 18.9 km), but separated by the Intag-Guayllabamba river valley. Characterization. The following combination of characters are found in Hyalinobatrachium mashpi sp. nov. : (1) dentigerous process of the vomer lacking teeth; (2) snout truncate in dorsal view and slightly protruding in lateral view; (3) tympanum oriented almost horizontally; tympanic annulus barely visible, hidden under skin; tympanic membrane differentiated, with coloration similar to that of surrounding skin; (4) dorsal skin shagreen; (5) ventral skin areolate; cloacal ornamentation absent, paired round tubercles below vent absent; (6) parietal peritoneum and pericardium translucent (in life, red heart visible in ventral view); liver, viscera and testes covered by iridophores; (7) liver bulbous; (8) humeral spines absent; (9) hand webbing formula: I (2 + –2 1/2 )—(3 – –3) II (2–2 – )—(3 + –3 1/4 ) III (2–2 + )—(1 3/4 –2) IV; (10) foot webbing moderate; webbing formula: I 1 + —(2–2 + ) II (1–1 1/3 )—2 1/4 III (1 1/3 –1 1/2 ) —(2 + –2 1/4 ) IV (2 1/2 –2 1/3 )—1 V; (11) fingers and toes with thin lateral fringes; ulnar and tarsal folds absent; (12) nuptial excrescence present as a small pad on Finger I (Type V), prepollex not enlarged; prepollical spine not projecting (spine not exposed); (13) when appressed, Finger I longer than II; Figure 2 Phylogenetic position of Hyalinobatrachium mashpi sp. nov. and H. nouns sp. nov. Phy- logenetic relationships of Hyalinobatrachium inferred from the 16S mitochondrial gene under ML cri- terion. All sequences were downloaded from GenBank, except those in red (Material S2). Genbank codes are listed next to each terminal. Associated locality data is available at Genbank, as well as in Guayasamin et al. (2008 , 2020 ), Castroviejo-Fisher et al. (2014) , and Twomey, Delia & Castroviejo-Fisher (2014) . Full-size DOI: 10.7717/peerj.13109/fig-2 Table 1 Differences between Hyalinobatrachium mashpi sp. nov. , H. nouns sp. nov. , and similar and closely related species.

Species	SVL in mm (adult males)	Dorsal pattern	Hear coloration in life	Interorbital bar	Type of call	Biogeographic distribution/ Country/ Elevation (m a. s.l.)	Sources
H. adespinosai	20.5–22.2	Pale yellowish green with small pale yellow spots and minute gray to black melanophores	Red	Absent	Pulsed	Amazonian slopes of the Andes Ecuador 1,670–1,795	Guayasamin et al. (2019a)
H. anachoretus	20.6–21.4	Apple green with small yellow spots and minute melanophores	Red	Absent	Pulsed	Amazonian slopes of the Andes Peru 2,001–2,050	Twomey, Delia & Castroviejo-Fisher (2014)
H. aureoguttatum	20.4–24.0	Greenish yellow with large, bright yellow spots and, in some populations, dark flecks	Usually red, but also red and white, or white	Usually absent	Tonal	Chocó, Pacific slopes of Andes Colombia, Ecuador, Panama 0–1,340	Barrera-Rodriguez & Ruiz-Carranza (1989) , Guayasamin et al. (2020) , this study
H. bergeri	20.3–22.4	Apple green with small yellow spots and minute melanophores	Mostly white	Absent	Tonal	Amazonian lowlands and Amazonian slopes of the Andes Peru, Bolivia 300–1,980	Castroviejo-Fisher et al. (2009)
H. chirripoi	24–27	Greenish yellow with small yellow spots	Red	Absent	Pulsed	Central America (Costa Rica, Panama); Chocó (Colombia and Ecuador) 0–320	Savage (2002) , Kubicki (2007) , Guayasamin et al. (2020)
H. colymbiphyllum	23–30	Greenish yellow with yellow spots	Red	Absent	Pulsed	Central America (Costa Rica, Honduras, Panama); Chocó (Colombia) 0–1,710	McCranie & Wilson (2002) , Savage (2002) , Kubicki (2007)
H. esmeralda	18.4–22.3	Greenish yellow with yellow spots and some minute dark dots	Red or red and white	Absent	Unknown	Amazonian slopes of the Andes Colombia 1,026–1,700	Ruiz Carranza & Lynch (1998) , Acosta-Galvis (2017) , Twomey, Delia & Castroviejo-Fisher (2014)

(Continued) Table 1 (continued)

Species	SVL in	Dorsal pattern	Hear	Interorbital	Type of	Biogeographic	Sources
mm	coloration	bar	call	distribution/
(adult	in life	Country/
males)	Elevation (m a.
s.l.)
H. mashpi sp.	19.7–20.9	Greenish yellow with small and	Red	Usually	Pulsed	Pacific slopes of	This study
nov.	diffuse yellow spots	present	the Andes
Ecuador
976–1,137
H. nouns sp. nov.	19.1–21.3	Greenish yellow with small and	Red	Usually	Unknown	Pacific slopes of	This study
diffuse yellow spots	present	the Andes
Ecuador
1,177–1,420
H. pellucidum	20.4–21.4	Greenish yellow with small yellow	White	Absent	Tonal	Amazonian	Lynch & Duellman
spots	slopes of the	(1973) , Guayasamin
Andes	et al. (2020)
Ecuador, Peru
523–1,740
H. valerioi	19.5–24.0	Greenish yellow with large and	Red or red	Usually	Tonal	Central	Savage (2002) , Kubicki
diffuse yellow spots	and white	absent	América,	(2007) , Guayasamin
Chocó, Pacific	et al. (2020)
slopes of the
Andes
Costa Rica,
Colombia,
Ecuador
0–1,500
H. vireovittatum	21.5–23.0	Greenish yellow with small yellow	Red	Present or	Tonal	Central America	Savage (2002) , Kubicki
spots. Dark green middorsal	absent	Costa Rica	(2007) , Campos-
stripe outlined by yellow	250–1,957	Villalobos et al.
paravertebral stripes	(2020)
H. talamancae	24–26	Greenish yellow with small yellow	Red	Present or	Tonal	Central America	Kubicki (2006 , 2007 );
spots. Dark green middorsal	absent	Costa Rica	Zamora-Roda,
stripe present.	475–1,600	Herrera-Martínez &
Salazar (2021)
H. yaku	20.8–22.3	Green with small yellow spots and	Red	Absent	Tonal	Amazonian	Guayasamin et al.
minute melanophores; posterior	lowlands	(2017a)
head and anterior half of the	Ecuador
body with few small dark green	300–360
spots placed middorsally

Note: Sources of traits are indicated in the last column. (14) diameter of eye about 2 times wider than disc on Finger III; (15) coloration in life: dorsal surfaces lime green with small light yellow spots; (16) coloration in preservative: dorsal surfaces creamish white, with minute lavender melanophores; (17) eye coloration in life: iris yellow to golden-yellow; pupil surrounded by lavender ring; (18) melanophores absent from fingers and toes, except Toes IV and V; (19) males call from underside of leaves; advertisement call consisting of single pulsed note, with duration of 0.37–0.46 s, peak frequency at 5.25–5.60 kHz, maximum frequency at 5.46–5.81 kHz, and minimum Figure 3 Dorsal and ventral photos of glassfrogs in life. (A) Male of Hyalinobatrachium mashpi sp. nov. , CJ11642 (holotype). (B) Gravid female of H. mashpi sp. nov. , Mashpi Reserve , Ecuador. (C) Male of H. nouns sp. nov. , ZSFQ0537. (D) Male of H . nouns sp. nov. , MZUTI3299 (holotype). (E) Male of H. aureoguttatum , Ecuador. (F) Gravid female of H. aureoguttatum , Ecuador. Photos by Jaime Culebras (A, B, D, E, F) and Ross Maynard (C) . Full-size DOI: 10.7717/peerj.13109/fig-3 frequency at 4.62–4.92 kHz; (20) males attend egg clutches located on the underside of leaves overhanging streams; clutch size of 31 or 32 embryos ( n = 2); (21) SVL in adult males 19.7–20.9 mm (mean = 20.5; n = 5); females unknown; and (22) enameled tubercles absent from sides of head. Description of the holotype . CJ11642, adult male with SVL 20.6 mm. Head wider than long (head width 39% of SVL; head length 78% of head width). Snout truncate in dorsal view and slightly protruding in lateral view. Loreal region concave, nostrils slightly protuberant, elliptical; internarial region concave; canthus rostralis not well defined. Eyes Figure 4 Dorsal and ventral photos of glassfrogs in life. (A, B) Male of Hyalinobatrachium vireovittatum , Costa Rica. (C, D) Male of H. talamancae , Costa Rica. (E, F) Male of H. valerioi , Costa Rica. Photos by Jaime Culebras (A, C, D, E, F) and Josué Alberto Vargas (B) . Full-size DOI: 10.7717/peerj.13109/fig-4 small, directed anterolaterally, eyes about 50 Ǫ relative to midline (where anterior-facing eyes would be 90 Ǫ relative to midline). Tympanum barely visible, oriented almost horizontally; tympanic membrane differentiated and pigmented as surrounding skin. Dentigerous processes on vomers absent; choanae large, oval, separated widely (distance about the same as between nostrils); tongue round, white in preservative, anterior 4/5 attached to mouth; posterior border of tongue widely notched; vocal slits present, extending along floor of mouth lateral to tongue; enameled glands absent from lower part of upper jaw. Ulnar fold absent; humeral spine absent. Relative length of fingers: Figure 5 Dorsal patterns of glassfrogs in life. (A) Hyalinobatrachium mashpi sp. nov. , CJ11642 (holotype). (B) H. nouns sp. nov. , ZSFQ0537. (C) H . aureoguttatum , SC 435. (D) H. talamancae , Costa Rica. (E) H . vireovittatum , Costa Rica. (F) H. valerioi , Costa Rica. Photos by Jaime Culebras (A, D, E, F), Jose Vieira (B) and Luis Coloma (C) . Full-size DOI: 10.7717/peerj.13109/fig-5 II <I <IV <III; finger discs rounded, about the same size as discs on toes, disc on Finger III 42% of eye width; hand webbing reduced between Fingers I–III, moderate between Fingers III and IV, with formula I 2 + —3 – II 2 – —3 1/5 III 2 + —1 3/4 IV. Prepollex concealed; subarticular tubercles round, faint; few small supernumerary tubercles present, palmar tubercle round, of moderate size and difficult to see, thenar tubercle ovoid; nuptial excrescences present as a small pad on external edge of Finger I (Type V). Hind limbs slender, tibia length 55% of SVL; tarsal fold absent; discs of toes round; inner metatarsal tubercle small, outer metatarsal tubercle round, both very difficult to distinguish. Webbing formula of feet: I 1 + —2 II 1—2 1/4 III 1 1/2 —2 + IV 2 1/2 —1 V. In preservative, dorsal skin creamish white, with minute dark lavender melanophores (only visible under the stereomicroscope); dorsal skin shagreen; skin on venter areolate; cloacal opening at level of upper thighs, small and non-enameled cloacal warts present. Parietal peritoneum and pericardium translucent (in life, the red heart is visible ventrally); urinary bladder lacking iridophores; liver, viscera, and tested fully covered by iridophores. Kidneys rounded, approximately bean-shaped; liver bulbous. Coloration in life. Dorsal surfaces apple green to yellowish green with diffuse yellow spots; head with light yellow spots that may form an interorbital bar. Melanophores absent from fingers and toes, except Toes IV and V. Ventrally, parietal peritoneum and pericardium transparent, with a red heart always visible. Gall bladder and urinary bladder covered by translucent peritonea; hepatic and visceral peritonea covered by white iridophores; ventral vein red. Iris yellow, with numerous minute lavender spots. Bones white. Coloration in preservative . Dorsal surfaces creamish white dotted with minute dark lavender melanophores; venter uniform cream, with partial translucence; pericardium translucent; visceral peritoneum covered by iridophores. Iris white with minute lavender melanophores. Melanophores absent from hands and feet, except from some few present on dorsal surfaces of Toes IV and V. Measurements of holotype (in mm) . CJ11642, adult male. SVL = 20.6, femur length = 11.4, tibia length = 11.3, foot length = 9.6, head length = 6.2, head width = 8.0, Figure 6 Visual representation of Hyalinobatrachium mashpi sp. nov. advertisement call, with comparisons of two similar species, H. aureoguttatum and H. valerioi . The call of each species is depicted in three forms: (Top) oscillograms, waveforms representing amplitude changes over time; (Middle) spectrograms, plots of frequency over time, with higher amplitudes represented by brighter colors; and (Bottom) power spectra, representing the relative amplitude of each frequency. Full-size DOI: 10.7717/peerj.13109/fig-6 Figure 7 Discriminant analysis of principal components (DAPC) of glassfrog species’ calls. Variables analyzed include: peak frequency, maximum frequency, minimum frequency, call duration, and inter-call duration. Sample size as follows: H. adespinosai one individual, 10 calls; H. aureoguttatum six individuals, 24 calls; H. mashpi sp. nov. two individuals, 12 calls; H. pellucidum one individual, 41 calls; H. tatayoi four individuals, 26 calls; H. valerioi three individuals, 70 calls. Full-size DOI: 10.7717/peerj.13109/fig-7 interorbital distance = 2.4,eye diameter = 2.6, tympanum diameter = 0.6, arm length = 4.2, hand length = 6.3, Finger I length = 4.6, Finger II length = 4.1, width of Finger III = 1.1. Vocalizations ( Figs. 6 , 7 ). We measured call variables from two individuals, each from a different locality, Mashpi Reserve (CJ11642; call code LBE-C-051) and Tayra Reserve (CJ11645; call code LBE-C-052). The call of Hyalinobatrachium mashpi sp. nov. ( Fig. 6 ) Table 2 Acoustic differences between Hyalinobatrachium mashpi sp. nov. and related species.

Species	Call codes	Call structure	Call duration	Inter-call	Peak	Maximum	Minimum
(s)	interval (s)	frequency	frequency	frequency
(kHz)	(kHz)	(kHz)
H. mashpi	LBE-C-051, LBE-	1 note per call; pulsed;	0.37–0.46	10.07–17.48	5.25–5.6	5.46–5.81 (5.62	4.62–4.92 (4.79
sp. nov.	C-052	8–10 pulses per note	(0.43 ± 0.03)	(12.8 ± 2.17)	(5.38 ±	± 0.11)	± 0.10)
two individuals,	0.12)
12 calls
H. adespinosai	LBE-C-050	1 note per call; pulsed;	0.448–0.646	10.87–30.04	4.87–5.04	5.0–5.25	4.32–4.75
one individual	~12 pulses per note	(0.543 ±	(20.12 ± 8.77)	(4.94 ±	(5.11 ± 0.08)	(4.57 ± 0.15)
10 calls	0.07)	0.07)
H. aureoguttatum	LBE-C-053–057	1 note per call; tonal;	0.078–0.087	1.99–5.20	6.63–7.41	7.78–8.90 (8.11	5.16–5.91
six individuals	harmonics present	(0.082 ±	(3.24 ± 0.79)	(6.87 ±	± 0.27)	(5.62 ± 0.21)
24 calls	0.002)	0.25)
H. chirripoi	LBE-C-010	1 note per call; pulsed;	0.235–0.274	84.3	4.48	4.99–5.77 (5.38	4.16–4.21
one individual	12–13 pulses per note	(0.255 ±	(only 2 notes	(4.48 ± 0)	± 0.05)	(4.19 ± 0.04)
two calls	0.03)	in recording)
H. pellucidum	LBE-C-003	1 note per call; tonal	0.1–0.146	1.67–5.35	5.60–5.86	5.86–6.14	5.05–5.32
1 individual	(0.129 ±	(2.94 ± 0.79)	(5.70 ±	(6.0 ± 0.06)	(5.16 ± 0.07)
41 calls	0.009)	0.06)
H. tatayoi	LBE-C-058	1 note per call; tonal	0.076–0.276	2.05–21.68	4.45–5.11	4.83–5.40 (5.14	3.30–4.61 (4.24
four individuals	(0.143 ±	(7.64 ± 4.92)	(4.82 ±	± 0.17)	± 0.34)
26 calls	0.04)	1.77)
H. valerioi	ML 201469,	1 note per call; tonal;	0.065–0.10	1.76–8.00	6.46–7.24	7.22–7.90	4.09–5.88 (5.12
three individuals	ML201473,	harmonics present	(0.079 ±	(4.27 ± 1.21)	(6.77 ±	(7.53 ± 0.17)	± 0.51)
70 calls	ML201475	0.01)	0.19)

Note: For each variable, data range is followed by the mean and standard deviation in parentheses. consists of a single pulsed (amplitude-modulated) note, which starts with one lower-frequency pulse followed by ~9 more consistent pulses at a slightly higher dominant frequency. We analyzed variables from both individuals: four calls from CJ11645 and eight calls from CJ11642. Calls in our field recordings had a duration of 0.373–0.461 s (mean = 0.425 ± 0.027 SD, n = 12). Time between calls ranged from 10.07–17.48 s (mean = 12.80 ± 2.166 SD, n = 10); intervals between H. mashpi calls were longer when a sympatric glassfrog ( Espadarana prosoblepon ) called in the interim period. Peak frequency was 5.25–5.6 kHz (mean = 5.38 kHz ± 0.12 SD; n = 12), with a maximum frequency of 5.46–5.81 kHz (mean = 5.38 kHz ± 0.11 SD; n = 12) and a minimum frequency of 4.62–4.92 kHz (mean = 4.79 kHz ± 0.10 SD; n = 12). We compared the calls of H. mashpi sp. nov. to those of phenotypically and/or genotypically similar species within the same genus: H. adespinosai , H. aureoguttatum , H. pellucidum , H. tatayoi , and H. valerioi ( Table 2 ). The call of H. adespinosai is a single pulsed (amplitude-modulated) note, consisting of ~12 pulses (mean call duration = 0.54 s ± 0.007 SD, n = 10). Time between calls ranged from 10.87–30.04 s (mean inter-call interval = 20.12 s ± 8.77 SD). Mean peak/fundamental frequency was 4.94 kHz (±0.07 SD; range = 4.87–5.04 kHz), with a mean maximum frequency of 5.11 (±0.08 SD; range = 5.0–5.25 kHz) and a mean minimum frequency of 4.57 (±0.15 SD; range = 4.32–4.75 kHz). The call of H. aureoguttatum ( Fig. 6 ; Table 2 ) consists of a very short, single tonal note (mean call duration = 0.082 s ± 0.002 SD, n = 24). Time between calls ranged from 1.99–5.20 s (mean inter-call interval = 3.24 s ± 0.79 SD, n = 23). Mean peak/fundamental frequency was 6.86 kHz (±0.25 SD; range = 6.55–7.41 kHz; n = 24 calls). Two harmonics are present. We measured call variables from individuals recorded in Canandé (0.5112 N, 79.1343 W; 457 m), Esmeraldas Province, Ecuador, in December 2018 by AVA (LBE- 053–55), and in Mashpi Lodge Reserve (0.17057 N, 78.888 W; 721–723 m) in March 2019 by RMB (LBE-056, 057). The call of H. chirripoi is a single pulsed (amplitude-modulated) note, consisting of ~12 pulses (mean call duration = 0.255 s ± 0.03 SD, n = 2). Since our recording only included two bouts of calling, we were unable to include H. chirripoi in the DAPC analysis. The interval between the two calls was 84.3 s. Peak/fundamental frequency was 4.48 kHz, with a maximum frequency of 4.99–5.77 kHz and a minimum frequency of 4.16–4.21 kHz. We measured call variables from one individual recorded in Reserva Itapoa (0.51307 N, 79.134 W; 321 m), Esmeraldas Province, Ecuador, in July 2016 by JC (LBE-019). The call of H. pellucidum consists of a short, single tonal note (mean call duration = 0.129 s ± 0.009 SD, n = 41). Time between calls ranged from 1.67–5.35 s (mean inter-call interval = 2.94 s ± 0.79 SD). Mean peak/fundamental frequency was 5.70 kHz (±0.06 SD; range = 5.60–5.86 kHz), with a mean maximum frequency of 6.0 (± 0.06 SD, range 5.86–6.14 kHz) and a mean minimum frequency of 5.16 (±0.07 SD, range 5.05–5.32 kHz). We measured call variables from one individual (USNM 286708) recorded at Río Azuela, Napo Province, Ecuador, by Roy McDiarmid on February 23th, 1979. The call of H. tatayoi consists of a short, single tonal note (mean call duration = 0.143 s ± 0.04 SD, n = 26). Time between calls ranged from 2.05–21.68 s (mean inter-call interval = 7.64 s ± 4.92 SD). Mean peak/fundamental frequency was 4.82 kHz (±1.77 SD; range = 4.45–5.11 kHz), with a mean maximum frequency of 5.14 (±0.17 SD, range 4.83–5.40 kHz) and a mean minimum frequency of 4.24 (±0.34 SD, range 3.30–4.61 kHz). We measured call variables from four individuals recorded in Jama Coaque Reserve (0.108264 S, 80.117701 W; 700 m), Manabí Province, Ecuador, in March 2019 by RMB. The call of H. valerioi ( Fig. 6 ) consists of a single tonal note (mean call duration = 0.079 s ± 0.01 SD, n = 70). Time between calls ranged from 1.76–8.00 s (mean inter-call interval = 4.27 s ± 1.2 SD). Mean peak frequency was 6.77 kHz (±0.19 SD; range = 6.46–7.24 kHz). Harmonics are likely present but are difficult to discern in the available recordings. We measured call variables from three individuals recorded in Costa Rica (Limón and Rincón de Oso) by Roy McDiarmid. We used the following recordings from the Macaulay Library at the Cornell Lab of Ornithology: ML212787, ML212788, and ML213430. Results from the discriminant analysis of principal components (DAPC) revealed that the calls of H. mashpi sp. nov. cluster separately, and are thus acoustically distinct from H. adespinosai , H. aureoguttatum , H. pellucidum , H. tatayoi , and H. valerioi ( Fig. 7 ). Overlap occurred between H. aureoguttatum and H. valerioi clusters, as well as between H. tatayoi and H. adespinosai clusters. This suggests that the calls of these pairs may not be adequate for species identification alone; more field recordings with genetic verification of Table 3 Results from the discriminant analysis of principle components (DAPC), comparing the advertisement calls of Hyalinobatrachium mashpi sp. nov. with those of closely related species (see Fig. 7).

PC1	PC2	PC3
PCA Eigenvalues	23.76	1.48	0.16
PCA Loadings
Peak Frequency	0.08	−0.57	−0.09
Call (Note) Duration	−0.02	0.023	0.05
Inter-call Interval	−0.99	−0.14	0.02
Maximum Frequency	0.11	−0.78	−0.19
Minimum Frequency	0.04	−0.21	0.98

Note: Most variation (99.9%) was retained by three principal components. the calling species are thus recommended for future studies. Nearly all (99.9%) of the variance was retained by three principal components. Table 3 lists the eigenvalues and variable loadings of each principal component. Natural history. Most individuals of Hyalinobatrachium mashpi sp. nov. were found on the underside of leaves among riverine vegetation ( Figs. 8 , 9 ). These frogs are difficult to observe because they are found 3–14 m above ground along steep creeks. Males have been observed calling in the months of April, May, June, August, September, October and November. Males that were guarding egg clutches while calling were observed during the rainy season (18 February 2019; 7 May 2021) and dry season (October 2014, June 2015, and August 2021). Examined egg clutches contain 31–34 eggs ( n = 3). A female with mature eggs visible through the skin was observed on 27 May 27 2015. Distribution ( Fig. 10 ). Hyalinobatrachium mashpi sp. nov. is only known from the following localities in the Mashpi river basin, Pichincha Province, Ecuador: (i) Mashpi Lodge Reserve (San Vicente River, 1,040–1,101 m; Laguna River, 1,069 m); (ii) Amagusa Reserve (Amagusa River, 1,137 m; Mashpi Chico River , 1,130 m); and (iii) Tayra Reserve, 976–1,126 m. Unidentified and closely related frogs from Colombia (Departamento de Risaralda, MAR 2147; Departamento de Valle del Cauca; MAR 2222; Fig. 2 ) may prove to be conspecifics of H. mashpi . Evolutionary relationships . Our phylogenetic analyses ( Fig. 2 ) reveal Hyalinobatrachium mashpi sp. nov. as sister to undetermined haplotypes from the Colombian Andes (MAR 2147, 2222) and a new species from the Toisán Mountain Range, described below. Other closely related taxa are endemic to Central America: H. vireovittatum and H. talamancae ( Fig. 2 ). Etymology. The specific epithet mashpi is used as a noun in apposition and refers to the Mashpi area in northwestern Ecuador. There are several conservation efforts to preserve the last patches of forest remaining in Mashpi ( e.g ., Mashpi Lodge Reserve, Tayra Reserve, Amagusa Reserve, Mancomunidad del Chocó Andino, Chocó Andino Biosphere Reserve). Mashpi is a Yumbo word that means ‘friend of water’, an apt description of this glassfrog, which depends on healthy streams for its reproduction. Figure 8 Parental care in Hyalinobatrachium mashpi sp. nov. (A) Male calling at San Vicente River, Mashpi Reserve , Pichincha Province, Ecuador. (B) Male at tributary of the Mashpi River, Tayra Reserve , Pichincha Province, Ecuador. Photos by Carlos Morochz (A) and Jaime Culebras (B). Full-size DOI: 10.7717/peerj.13109/fig-8 Figure 9 Habitat of Hyalinobatrachium mashpi sp. nov. (A) Tributary of the Mashpi River, Tayra Reserve , Pichincha Province, Ecuador. (B) Mashpi Reserve , Pichincha Province, Ecuador. (C) Tayra Reserve, Pichincha Province, Ecuador. (D) Habitat loss in the vicinity of Tayra Reserve, Pichincha Province, Ecuador. Photos by Jaime Culebras. Full-size DOI: 10.7717/peerj.13109/fig-9 Conservation status. We recommend that Hyalinobatrachium mashpi be listed as Endangered, following IUCN Red List criteria B1ab(iii): extent of occurrence estimated to be less than 5,000 km 2 ; known to exist at no more than 5 localities; and continuing decline, observed, inferred or projected, in area, extent, and/or quality of habitat. The main threats for this species are habitat loss and contamination due to cattle ranching, agriculture, and mining activities (see Discussion).