A new species of brilliant green frog of the genus Tlalocohyla (Anura, Hylidae) hiding between two volcanoes of northern Costa Rica Varela-Soto, Donald Abarca, Juan G. Brenes-Mora, Esteban Aspinall, Valeria Leenders, Twan Shepack, Alex Zootaxa 2022 2022-08-31 5178 6 501 531 Varela-Soto & Abarca & Brenes-Mora & Aspinall & Leenders & Shepack, 2022 Varela-Soto & Abarca & Brenes-Mora & Aspinall & Leenders & Shepack 2022 [151,382,799,826] Amphibia Hylidae Tlalocohyla Animalia Anura 4 505 Chordata species celeste sp. nov.  ( Figures 2–7)     Holotype.UCR 23700, an adult male from Costa Rica, Alajuela, Upala, Bijagua, Tapir Valley Nature Reserve(10.72˚ N, 85.01˚ W;  660 masl), collected on  September 13, 2021, by Juan G. Abarca, Valeria Aspinall, Donald Varela, and Esteban Brenes.  Paratopotypes.Two adult males, ECB-Anf-f50-08-01,01 and YPM13222, same data as holotype; UCR 23701, an adult female collected by Donald Varelaon  September 15, 2021.  Generic placement.Bayesian ( Fig. 1, Suppl. fig. 2) and Maximum likelihood analyses (Suppl. fig. 3) confirm the inclusion of the new species in the genus  Tlalocohyla.   Diagnosis.The new species can be diagnosed by the following combination of traits: (1) small size (adults males: 21 mm, females: 24 mmof SVL; n=4); (2) tympanic membrane not-evident; (3) vocal sac developed, single, subgular, extending to the pectoral region; (4) vomerine teeth absent; (5) dorsal color yellow-green (#103); (6) ventral skin transparent; (7) white peritoneum covering all internal organs; (8) presence of an incomplete sulfur white (#96) dorsolateral stripe that originates at the posterior edge of the orbit and extends posteriorly to a point more than halfway towards the insertion of each hindlimb, bordered above for its entire length by a diffuse mahogany red (34) stripe that extends anteriorly towards the tip of the snout; (9) presence of a small light cyan (#158) axillary membrane. Within its genus,  Tlalocohyla celestecan be differentiated from  T. godmaniand  T. loquaxby the following characteristics (condition for  T. celestein parentheses): maximum SVL 45 mm( 24 mm); venter pale lemon yellow or creamy yellow (transparent ventral skin) and extensive axillary membrane (very small axillary membrane).  Tlalocohyla celesteis similar in overall appearance to  T. smithiiand  T. picta, but can be differentiated based on the following characteristics: from  T. smithiiby its smaller size, maximum SVL 30 mm( 24 mm); pale yellow, tan or brilliant yellow dorsum (bright yellow-green with mahogany red spots); a thin complete white or cream dorsolateral stripe that is usually bordered below by a thin brown line (an incomplete sulphur white dorsolateral stripe bordered above by a diffuse mahogany red stripe); white belly (transparent); vomerine teeth present (absent); and from  T. pictaby the relatively uniform yellowish tan dorsal coloration (bright yellow-green with mahogany red spots); presence of a complete white or cream dorsolateral stripe that may be bordered below by a poorly defined reddish, brown or gray band (an incomplete sulphur white dorsolateral stripe bordered above by a diffuse mahogany red stripe); white bones (green bones) and vocal sac yellow (brilliant light cyan).  Tlalocohyla celestealso differs from these two species in its advertisement call and tadpole morphology (see below).   FIGURE 1.Bayesian phylogenetic tree within the Hylidaefamily based on 12S and 16S mitochondrial DNA genes. Posterior probabilities in nodes, asterisks represent support of>95. Branches are collapsed to illustrate genus-level relationships, see expanded branches on Suppl. material. 2. The scale bar refers to the estimated substitutions per site.   Tlalocohyla celesteis among the smallest hylids in Costa Rica, slightly smaller than  Isthmohyla zeteki, and may be confused with other small hylid species present within its range. Members of the genus  Dendropsophusare small tree frogs with an axillary membrane but none are green in coloration with transparent ventral skin.  Scinaxspecieshave a similar, rounded protruding lateral snout profile but are significantly larger in size and never have light dorsolateral markings.  Tlalocohyla celesteis most likely to be confused with juveniles of the syntopic  Boana rufitela, but the latter has a rounded snout in lateral profile and its white dorsolateral lines are complete, extending from the snout tip backwards to the vent, passing over top of the hindlimbs. These light markings fade and eventually disappear completely as young  B. rufitelamature. Because of its bright green coloration and transparent ventral skin,  T. celestemay be mistaken for a glass frog (family Centrolenidae), however glass frogs invariably lack a pattern of light dorsolateral stripes.   Description of holotype.An adult male with a SVL of 20.10 mm( Fig. 2), in a good state of preservation, head slightly longer than wide, HL 38.3%, and HW 36.5% of SVL. Snout nearly rounded in dorsal view, rounded protruding in profile. Nostrils rounded, dorsolateral, directed laterally, located on slightly raised processes, IND 25.8% of HW. Canthus rostralis well defined and rounded. Loreal region concave. Eyes large and protuberant, ED 39.1% and 10.7% of HW and SVL, respectively. Pupil horizontally elliptical. Tympanum small, TD 23.0% of ED, tympanic membrane inevident, but tympanic annulus visible through the skin ( Fig. 3C), tympanum matches character combination number 2 in Lynch and Duellman (1997). Tongue ovoid, free laterally and posteriorly. Texture of tongue granular. Premaxillary and maxillary teeth present, no vomerine teeth visible between choanae. Choanae rounded anteriorly and widely separated. Vocal apertures are slit-like and elongated, nearly parallel the mandible. Vocal sac single and subgular, extends posteriorly to the pectoral region ( Fig. 2Band Fig. 7).   FIGURE 2.Holotype of  Tlalocohyla celeste  sp. nov.(UCR 23700). (A) Dorsal view. (B) Ventral view. Forelimbs not hypertrophied, upper arms and forearms slender, consistent throughout in its thickness. Hands proportionally large, HAL 33.1% of SVL. Lacks ulnar skin fold. A small axillary membrane present. Fingers robust, relative finger length I<II<IV<III. Discs moderately expanded and round (Fin3DW 25% of ED). Finger discs are comparable in size but finger disc I is slightly smaller than others. Subarticular tubercles single, rounded or globose, large and slightly bifid in fingers III and IV. Supernumerary tubercles large, single and rounded. Accessory palmar tubercles numerous, rounded, large and globular; span the entire palmar area in a tightly packed way. Inner metacarpal tubercle (thenar) elongated, elliptical, globular and large; outer metacarpal tubercle (palmar) not clearly differentiated from the accessory palmar tubercles. White smooth nuptial pads without epidermal projections, at the base of the first finger, formed by a grouping of glandular acini, each circular or ovoid in shape ( Fig. 6C). The interdigital membrane between fingers I–II is vestigial, but it is slightly more developed between fingers II–III–IV. Webbing formula I3+–2- II 2-– 3-III3- –2-IV. Hind limbs slender, THL slightly greater than FL and slightly smaller than TL. TL of about 57.0% of SVL, FL 43.5% of SVL. Toes robust. Relative length of toes I<II<III<V<IV. Discs rounded and expanded. Subarticular tubercles single, rounded and globular. Supernumerary tubercles small, single and rounded. Inner metatarsal tubercle medium-sized, elliptical; outer metatarsal tubercle absent. Webbing formula I 2 -–2 +II 11/2–3 +III 11/2–3 -IV 2 +– 11/2V, free parts of toes fringed. Cloacal dermal fold absent. Body covered in smooth skin throughout, except for the central region of the venter and the undersides of the thighs where skin texture is distinctly granular ( Fig. 6A).   FIGURE 3.Holotype of  Tlalocohyla celeste  sp. nov.(UCR 23700) (A) Dorsal view of head in living specimen. (B) Lateral view of head in preserved specimen. (C) Lateral view in preserved specimen, note the tympanum. Scale bar = 2 mm. (D) Right hand in ventral view. (E) Right foot in ventral view.   FIGURE 4.Variation in dorsal pattern of  Tlalocohyla celeste  sp. nov.(A–B) male holotype of  Tlalocohyla celeste  sp. nov.(UCR 23700) in life. (C) Size differences between females and males. (D) Typical nocturnal pattern of males. (E) Female with the oocytes visible by transparency in the body. (F) Diurnal pattern of adults, males and females, note the white iris chance. (G) Juvenile. (H) Diurnal pattern with red-brown iris.   FIGURE 5.Variation and diagnostic characteristics in  Tlalocohyla celeste  sp. nov.(A-B) pattern change and green tones change of two uncollected males. (C) Ventral view in diurnal pattern. (D) ventral view of female (UCR 23701), note green bones (black arrow) and white peritoneum (blue arrow). (E) Spotted pattern in juvenile. (F) Axillary membrane (red arrow).   FIGURE 6.Ventral view  Tlalocohyla celeste  sp. nov.(A) Ventrals skin texture, and cloacal view. (B) Liver of recently preserved specimen (ECB-Anf-f50-08-01,01.). (C) Nuptial pads of the left hand in  Tlalocohyla celeste  sp. nov.male holotype (UCR 23700), in life, and preserved.  Measurements of the holotype(in mm).SVL 20.10; HL 7.70; HW 7.35; IND 1.89; IOD 3.26; ED 2.14; EN 1.88; TD 0.49; HAL 6.65; FL 8.75; TL 11.45; Fin3DW 0.55; Toe4DW 0.75.  Coloration of the holotypein life (nocturnal coloration).Background color of head, upper back and limbs brilliant yellow-green (#103), with numerous mahogany red (#34) blotches relatively evenly dispersed over the dorsal surfaces of the head, body and lower limbs ( Fig. 4A–B). A narrow sulfur white (#96) dorsolateral stripe starts behind each eye and extends towards the insertion of each hind limb, gradually fading out near a point about halfway down the body. A relatively poorly defined mahogany red stripe extends from the tip of the snout along the canthal region, and continues post-ocularly following the upper border of the sulfur white dorsolateral line; it gradually fades out at the same point as the light stripe. Lateral surfaces of the body and head, below the dorsolateral lines, lack mahogany red spots. Upper surfaces of finger and toe tips light greenish yellow (#87). Upper edge of orbit Pratt’s rufous (#72); remainder of upper eyelid suffused with spectrum yellow (#79). Posterior and anterior edge of orbit bordered by a narrow white line. Iris gold with raw umber (#23) reticulations. Vocal sac yellow-green (#103) when deflated, turquoise green (#147) when inflated. Skin covering ventral surfaces transparent, allowing muscles, veins, bones, and intestines to be visible ( Fig. 5C–D). Internal organs black, but hidden from view by an opaque, white peritoneum ( Fig. 6B).   FIGURE 7.Vocal sac of uncollected males of  Tlalocohyla celeste  sp. nov.(A) Ventral view and vocal sac deflated. (B–D). Calling males perched on vegetation in different positions, note the vocal sac extension.  Coloration of the holotypein preservative.In formalin, yellow-green dorsal coloration becomes pale greenish white (#97), while mahogany red markings (dorsolateral lines and dorsal spots) turn pale vinaceous (#247). The color pattern fades in a similar fashion after ethanol preservation, leaving dorsolateral markings and dorsal spots faded but visible ( Fig. 2). The iris becomes black ( Fig. 3). Hands, feet, and tips of digits fade to a uniform creamywhite. Skin on ventral surfaces retains some transparency in preservative.  Variation.Morphometric variation is detailed in Table 1. Females are larger than males ( Fig. 4C) and lack nuptial pads, vocal slits and a vocal sac. The paratopotypes are comparable in coloration to the holotype. There is a noticeable difference between nocturnal ( Fig. 4D) and diurnal coloration ( Fig. 4F) in this species, especially in the intensity and visibility of the mahogany red dorsal spots and dorsolateral lines. These markings are less prominent during the day, causing the dorsal pattern to appear more uniformly green and less spotted. In addition, the eye color brightens during the daytime to a lighter silver-gray ( Fig. 4F). The presence of blue green bones, lymph, and other tissues ( Fig. 5) indicates that this species has physiological chlorosis, a trait that has been documented in other arboreal frogs ( Taboada et al.2020).   TABLE 2.Measurements (in mm) of the tadpoles of  Tlalocohyla celeste  sp. nov.See Materials and methods section for the abbreviations of measurements.    Measurements Stages 33-34 (N = 3)   1  2  3  TL 26.6 26.1 29.7  BL 10.6 9.4 10.3  TAL 14.9 20.8 21.0  MTH 5.68 4.53 5.61  TMH 2.93 2.92 2.55  TMW 2.63 2.85 1.89  BH 5.50 5.59 5.09  BW 6.54 6.51 5.42  ED 1.44 1.47 1.11  ODW 2.99 2.54 2.31  END 2.07 1.95 1.68  NSD 1.33 1.85 1.34  ND 0.54 0.32 0.43  IND 2.59 2.06 1.95  IOD 4.47 4.11 4.55  DFH 1.77 1.47 2.24  VFH 1.47 1.06 1.44  SL 1.56 1.50 1.36  SS 6.44 6.13 5.15 Metamorphs and small juveniles exhibit a lighter green dorsum and lack the light dorsolateral stripes during early developmental stages ( Fig. 11B–C). The small, light cyan axillary membrane ( Fig. 5F) is not seen in juveniles. Such young individuals generally display clusters of mahogany red blotches on the dorsum, most densely concentrated anteriorly but with considerable variation in their aggregation patterns. A broad, dark canthal stripe and short postocular stripe are usually present in juveniles, while the dorsal surfaces of the head, body, and limbs are marked with bold dark brown to purplish-brown spots. Dark-colored spots and stripes lighten ontogenetically likely to transform into the adult color pattern as the frogs age ( Fig. 4G, 5Eand 11D).  Tadpole description.Tadpoles of  Tlalocohyla celeste( Fig. 11Aand Fig. 12) in Gosner stages 33–44 conform to the following description: body depressed (BH/BW=0.87–0.94), ovoid in dorsal view and ovoid or elliptical in lateral view, from 0.35-0.40 times the TL. Snout rounded in dorsal and lateral aspects. Eyes medium-sized (ED/ BL=0.11–0.16), laterally located, visible in ventral view. The nostrils are situated approximately midway between the eyes and the tip of the snout. Nares rounded, medium-sized (ND/BL=0.03–0.05), dorsally located and dorsally directed. Well-developed fleshy flanges on the marginal rim. Spiracle sinistral, lateral, directed posterodorsally, and located on the posterior third of the body. Spiracle opening small (SL/BL=0.13–0.16). The intestinal mass is positioned perpendicular to the longitudinal body axis and visible through the clear ventral skin. The cloacal tube is short, dextral and has a triangular apex. The small oral disc (ODW/BW=0.39–0.46) is positioned anteroventrally. Oral disc not emarginated; has a single row of short marginal papillae with a small anterior gap. Several submarginal papillae are randomly distributed laterally in the oral disc ( Fig. 12D). LTRF 2(2)/3; A-1 and A-2 of equal size; A-2 with a narrow medial gap; P-1 equal in size to P-2; P-3 approximately one quarter the size of P-1 and P-2. Jaw sheaths small, measuring less than half of the oral disc; finely serrated along their entire margin. Anterior jaw sheath smoothly arched, posterior jaw sheath shallowly V-shaped. Tail of intermediate height (MTH/TAL=0.21-0.38) with moderately robust musculature (TMH/ BH=0.50-0.53); dorsal fin higher than ventral fin (DFH/VFH=1.20-1.56). Dorsal and ventral fins of intermediate height (DFH/TAL=0.07-0.11; VFH/TAL=0.05-0.09); dorsal fin initiates on the body at a point slightly anterior to the base of the tail; maximum fin height is achieved near the midpoint of the tail’s length. Ventral fin originates towards the end of the venter, anterior to the cloacal tube. The lateral line system is indistinct. In life, the general color pattern of the body and tail musculature is pale cream, pale white or pale yellow, with small brown flecks on the dorsal surfaces ( Fig. 11A– 12). Dorsal surfaces of head yellow ochre (#14) to tawny olive (#17); lateral surfaces slightly lighter in hue, marked with concentrations of mahogany red (#34) to warm sepia (#40) spots. A pattern of relatively evenly-spaced, rounded warm sepia (#40) spots is a prominent feature of the dorsal surfaces; dorsal and lateral surfaces also display a fine dusting of irregular white or gold specks. A distinct, but poorly defined warm sepia (#40) stripe extends from a point near the anterior edge of the oral disc towards the eye and continues as an indistinct postocular stripe onto the body. Iris gold, marked on either side of the pupil with a warm sepia (#40) spot; both dark spots align with the dark eye stripe that originates at the snout tip. Ventral skin is transparent, revealing reddish hues anteriorly at the thoracic level and cream white (#52) coloration at the level of the intestines. Throughout the larval development period, the intestinal tract becomes increasingly suffused with black pigment. This ontogenetic change creates a striking black-and-white ventral pattern that contrasts sharply with the dorsal and lateral coloration. Tail fins are transparent, marked with black or raw umber (#23) blotches of irregular shape and size, as well as small white spots. Tail musculature is yellow ochre (#14) with black or warm sepia (#40) spots, occasionally with a fine black line on the medial side of the tail that can be intact or interrupted. Upon preservation in 10% formalin, the larval coloration fades to pale greenish white (#97), whereas the eye stripe, dorsal spots and tail markings become black. Tadpoles of  Tlalocohyla celestediffer most notably from other tadpoles in the genus as follows (Gosner stages in parenthesis): in tadpoles of  T. godmani(38),  T. loquax(26), and  T. picta(38), the dorsal fin originates on the body at the level of, or anterior to the location of the spiracle ( Lee 1996; Duellman 2001), whereas in  T. celestethe dorsal fin originates at the base of the tail. Tadpoles of  T. smithii(37) are most similar to those of  T. celesteas their dorsal tail fin starts close to the tail base. However, tadpoles of  T. smithiidiffer in having the posterior third of the tail black and the height of their dorsal fin is relatively constant throughout until it tapers to a fine tip ( Caldwell 1986). Tadpoles of  T. celestelack a black tail tip and their dorsal fin increases in height towards the middle of the tail before tapering towards the tip.  Advertisement call.The vocal repertoire of  Tlalocohyla celesteis composed of at least three call types. Call typeI ( Fig. 8A–C) is composed of a trill of notes, with average call duration of 0.34 s(0.30– 0.39 s; n=17), emitted at intervals of 2–10 s (n=14) and average call repetition rate of 12 calls/minute. Call typeI is composed of 9–13 notes, each note having average duration of 0.01 s(0.01– 0.02 s; n=17), with a note repetition rate of 33.4 notes/s and inter-note interval of 0.01 s(0.01– 0.02 s; n=17). Notes that comprise the call’s trill-pattern can be modulated to increase amplitude towards the end of the call or display similar amplitude throughout ( Fig. 8A). Call dominant frequency ranges 3.80–4.30 kHz ( Fig. 8D). Bandwidth values range from 1.13 (0.90–1.40) to 13.0 (10.3–23.0) kHz (n=17). This call has several sparse harmonics and each pulsed note (30–50 pulses per note) displays an initial high amplitude pulse but subsequently decreases to lower amplitude pulses ( Fig. 8E). Call type II ( Fig. 8F–I) is composed of one to three squeak-like modulated pulsatile notes, each note having an average duration of 0.08 s(0.07– 0.10 s; n=4), with a note repetition rate of 3.4 notes/s and inter-note interval of 0.02 s(0.01– 0.02 s; n=4). The dominant frequency ranges from 4.00–4.40 kHz, bandwidth values range from 0.85 (0.40–1.5) to 12.1 (11.2–14.6) kHz (n =4). Call typeII usually was emitted 0.17– 0.21 s(n = 4) after call typeI.   FIGURE 8.Calls repertory of  Tlalocohyla celeste  sp. nov.Call type I: (A) oscillogram of a call series type I, first growing in amplitude and the second and third with similar amplitude notes; (B) oscillogram and (C) spectrogram of a call type I with 13 notes; (D) power spectrum and (E) oscillogram of a single note. Call type II:(F) oscillogram and (G) spectrogram of a call group: call type I following call type II; (H) oscillogram and (I) power spectrum of the single squeak-like note in call Type II. Dotted red line indicates the dominant frequency.   FIGURE 9.Call repertoire of  Tlalocohyla celeste  sp. nov.Call type III:(A) oscillogram and (B) spectrogram of a call series: call type I following call type III; (C) oscillogram and (D) power spectrum of notes in call type III. Dotted red line indicates the dominant frequency. (E) Oscillogram of call groups in  Tlalocohyla celeste  sp. nov.Showing a combination of the three call types (type I in orange, type II in green and type III in red) emitted by different males during the chorus. Call type III ( Fig. 9A–D) is a croak of pulsed notes with an average duration of 0.29 s(0.21– 0.34 s; n=7), emitted at intervals of 0.35– 1.61 s(n=7), with an average call repetition rate of 23 calls/s (n=7). Call typeIII is composed of 3–5 pulsed notes of modulated amplitude, with an average call duration of 0.29 s(0.21– 0.34 s; n=7). Each note has an average duration of 0.03 s(0.03– 0.04 s; n=7), an inter-note interval of 0.04 s(0.03- 0.05 s; n=7), and 60–90 pulses per note. The dominant frequency ranges from 3.70–4.10 kHz; bandwidth values range from 1.39 (1.10–1.6) to 11.7 (11.1–13.3) kHz (n=7). Call typeIII can be emitted alone, after, or preceding call typeI. During periods of breeding chorus activity, males have been observed emitting a) only call typeI, b) one to three typeII calls, followed by a single typeI call, c) emit several calls of typeIII followed or preceded by call typeI, or d) produce all three call typessequentially ( Fig. 9E). Call typeI of  T. celesteis more similar to the trills produced by  T. pictaand  T. smithiibut can be differentiated by the following characteristics (condition for  T. celestein parentheses): Call typeI differs primarily in the number of notes per call: the trill speed is faster in  T. picta= 60–70 notes/s and  T. smithii= 43–51 notes/s than in  T. celeste(25–37 notes/s; n=17). In addition,  T. pictaand  T. smithiihave a higher dominant frequency, 4.6–6.30 kHz and 4.29–6.64 kHz respectively, when compared with  T. celeste(3.80–4.30 kHz, n = 28).   FIGURE 10.Oviposition process of  Tlalocohyla celeste  sp. nov.(A) A pair in axillary amplexus with egg deposition. (B-D) Oviposition at the end of different leaves. (E) 5 days of development. (F) 9 days of development.  Reproduction. Males of  Tlalocohyla celestehave been observed calling year-round but display a significant increase in calling activity during periods of heightened precipitation. Vocalizations are generally first observed around 16:00 h and continue throughout the night, until sunrise. Individuals can be heard during the day, especially during periods of heavy rainfall. Most calling activity takes place along the vegetated wetland perimeter, density of calling males decreases towards the center of the wetland where water levels are deeper. Males have been observed calling in a head-down, horizontal, or head-up position, as they perch on vegetation. Individuals occupied a variety of herbaceous plant perches, but most were found on the locally common grass  Rhynchospora corymbosa(Cyperaceae)( Fig. 7). Calling males are positioned 0.1–3 mabove the ground, either calling in isolation or in small groups, with individuals often perched as little as 15–20 cmfrom one another. Satellite male behavior has been observed in this species, a trait also reported for  T. picta( Roble 1985). Females of this species, detected less frequently than males, were observed active in the periphery of the wetland and moved around calling males. On several occasions, females were found in the interior of the forest that surrounds the wetland, some 20 mfrom the water’s edge. Gravid females were first observed on June 12th, 2021( Fig. 4E), followed by subsequent sightings through September.   Tlalocohyla celesteexhibits axillary amplexus ( Duellman & Trueb 1986) ( Fig. 4Cand 10A). The first amplectant pair was discovered on June 13th, 2021at 5:00 h. Amplexus and oviposition have been observed at night and during the pre-dawn morning hours. The amplecting pair separates immediately after oviposition.   FIGURE 11.Metamorphosis of  Tlalocohyla celeste  sp. nov.(A) Tadpole at stage 35. (B) Metamorph of 11 mm of SVL. (C) Recently metamorphosed juvenile of 7 mm. (D) Juvenile.   FIGURE 12.  Tlalocohyla celeste  sp. nov.tadpole at stage 34 (35) according to Gosner (1960). (A) Lateral view, (B) dorsal view, (C) ventral view, (D) oral disc. All egg masses observed to date have been deposited at the tip, or along the drooping edge of leaves located directly above the water ( Fig. 10). This indicates that  T. celesteemploys reproductive mode 24 ( fide Haddad & Prado 2005) where arboreal eggs hatch into exotrophic tadpoles that drop into lentic water. Egg masses contain 20–61 unpigmented eggs (n=8) ( Fig. 10B–D). Measurements taken from three different egg masses indicate that each clutch occupies an area between 12–22 mmin width and 23–28 mmin length; eggs measure an average of 2.0 mm in diameter (without the clear jelly capsules). One gravid female maintained in captivity for two days deposited three clutches containing 23, 45, and 61 eggsrespectively, indicating that females can engage in multiple oviposition events during each breeding cycle. Early embryos, observed five days after oviposition, were uniformly white with visible external gills ( Fig. 10E). Seven days after oviposition, these embryos were light brown and marked with small black spots, particularly on the tail; the ventral region remained white. Nine days after oviposition, the embryos were brown with additional spots present on body and tail ( Fig. 10F). The tadpoles hatched ten days after oviposition. Egg masses were observed from June to November. Metamorphs and small juveniles were observed between November and June. Based on observations derived from tadpoles reared in ex-situ conditions, larval development appears to be slow. Three months after hatching, tadpoles reared under controlled conditions remained at Gosner stages 36–39 ( Gosner 1960). A recent metamorph was measured to have a SVL of 7 mm( Fig. 11C). The following anurans were observed to occur syntopically and likely share resources with  T. celeste:  Agalychnis callidryas, Boana rufitela, Dendropsophus ebraccatus, Dendropsophus microcephalus, Dendropsophus phlebodes, Inciliusvalliceps, Leptodactylus melanonotus, Lithobates forreri, Lithobates vaillanti, Rhinella horribilis, Scinax boulengeri, Scinax elaeochroa, Tlalocohyla loquax.All were confirmed to reproduce in the same wetland.   FIGURE 13.Geographic distribution of the genus  Tlalocohylain Mesoamerica. Inset, map of Costa Rica, showing the distribution of  Tlalocohyla loquax(green circles) and  Tlalocohyla celeste  sp. nov.(red circle). Sources: (GBIF.org 2022).  Geographic distribution and habitat.  Tlalocohyla celeste is only known to inhabit a single wetland system in the privately owned Tapir Valley Nature Reserve(Lat: 10.71 NLong: - 85.01 W; elevation 660 masl). The typelocality is located between Tenorio and Miravallesvolcanoes, in Bijagua de Upala, Alajuela Province, Costa Rica( Fig. 13). The reserve’s life zones are classified as Tropical Premontane Wet Forest and Tropical Moist Forest ( Holdridge 1967). The number of dry months per year ranges from one to two, without a marked dry season. The mean annual precipitation is 3,500 mmand monthly temperatures ranges 20–33 ºC ( IMN 2021). The frogs inhabit an 8-hectare marsh with a seasonally fluctuating, but permanent water level. The wetland is continually fed by a small tributary, by rainwater collected from surrounding hillsides, and likely by groundwater that wells up from bottom springs.A narrow discharge channel facilitates slow, directional water movement through the wetland. In the past, sections of this wetland have been channelized for irrigation purposes but it is now reverting to a more natural state. The surrounding hills and foothills of nearby mountains are largely forested, but areas of cultivated grassland are interspersed throughout the region. The shallow part of the marsh’s littoral zone is dominated by the grass  Rhynchospora corymbosa(Cyperaceae), whereas the riparian zone is occupied by a diverse assemblage of herbaceous vegetation ( Fig. 14). Away from the water’s edge, the vegetation structure changes and herbaceous plants are replaced by woody shrubs and trees, which connect to forest in the upland areas.   Tlalocohyla celesteis an abundant species within the marsh at Tapir Valley Reserve. During periods of increased activity their calls can be heard throughout the littoral zone of the wetland, and one can readily observe up to 50 individuals in a single night. Long-term monitoring is ongoing to determine the population dynamics of this species. In spite of its local abundance, repeated visual and acoustic searches in nearby wetlands within a 2 kmradius that seemingly displayed similar habitat quality and characteristics have thus far not resulted in the discovery of additional  T. celestepopulations.   FIGURE 14.Habitat where  Tlalocohyla celeste  sp. nov.is found. General view of the north (A) and southwest (B) sector of the wetland. The habitat is dominated by the grass  Rhynchospora corymbosa(C), but other species of plants tolerant to flooded environments can be used by the frog for its reproductive activity (D).  Natural history.Individuals of this new species have been observed catching small flies and micro moths, and presumably its diet consists of a variety of small invertebrates. Although no direct observations of predation on adult  T. celestewere made, its wetland habitat harbors abundant snakes that are known predators of anurans. In addition, ctenid spiders, which are common frog predators, abound. During the course of our surveys, opilionid arachnids were observed scavenging a dead frog and small wasps were seen attacking  T. celesteegg masses and preying on early developing larvae   Etymology.The specific epithet “celeste” (light blue, or sky-blue in Spanish), is used as a noun in apposition, and refers to the striking blue coloration seen on this frog’s axillary membranes and groin area, as well as on the inflated vocal sac in adult males. Perhaps more importantly, the authors want to celebrate the river Río Celeste, famous for the distinctive turquoise-blue hue of its water, whose watershed feeds the wetlands that provide critical habitat for its namesake frog. Río Celeste represents a critical natural resource and an important economic driver for local communities, as well as a nexus for sharing the beauty of nature with visitors from outside the region. The fact that this frog was discovered through local wetland restoration efforts is a testament to the critical importance of protecting our aquatic resources. 3897296301 [151,785,835,862] Valley Tree Frog Tapir Valley 4 505 1 Valle 3897296303 UCR 23700 2021-09-13 Juan G. Abarca & Valeria Aspinall & Donald Varela & Esteban Brenes. Costa Rica Upala 660 Tapir Valley Nature Reserve Bijagua 4 505 1 Alajuela holotype 3897296302 UCR 23701 2021-09-15 Donald Varela 4 505 1 3897296304 The & de Upala Costa Rica 660 10.71 Tapir Valley Nature Reserve 778 -85.01 Bijagua de Upala 19 520 1 Alajuela