The poverty of adult morphology: Bioacoustics, genetics, and internal tadpole morphology reveal a new species of glassfrog (Anura: Centrolenidae: Ikakogi) from the Sierra Nevada de Santa Marta, Colombia

Ikakogi is a behaviorally and morphologically intriguing genus of glassfrog. Using tadpole morphology, vocalizations, and DNA, a new species is described from the Sierra Nevada de Santa Marta (SNSM), an isolated mountain range in northern Colombia. The new taxon is the second known species of the genus Ikakogi and is morphologically identical to I. tayrona (except for some larval characters) but differs by its genetic distance (14.8% in mitochondrial encoded cytochrome b MT-CYB; ca. 371 bp) and by the dominant frequency of its advertisement call (2928–3273 Hz in contrast to 2650–2870 Hz in I. tayrona). They also differ in the number of lateral buccal floor papillae, and the position of the buccal roof arena papillae. Additionally, the new species is differentiated from all other species of Centrolenidae by the following traits: tympanum visible, vomerine teeth absent, humeral spines present in adult males, bones in life white with pale green in epiphyses, minute punctuations present on green skin dorsum, and flanks with lateral row of small, enameled dots that extend from below eye to just posterior to arm insertion. We describe the external and internal larval morphology of the new species and we redescribe the larval morphology of Ikakogi tayrona on the basis of field collected specimens representing several stages of development from early to late metamorphosis. We discuss the relevance of larval morphology for the taxonomy and systematics of Ikakogi and other centrolenid genera. Finally, we document intraspecific larval variation in meristic characters and ontogenetic changes in eye size, coloration, and labial tooth-rows formulas, and compare tadpoles of related species. Ikakogi tayrona has been proposed as the sister taxon of all other Centrolenidae; our observations and new species description offers insights about the ancestral character-states of adults, egg clutches, and larval features in this lineage of frogs.

Introduction + benzocaine anesthetic and preserved in 5% formalin. Development stages were delimited following [37]. Morphological measurements and terminology for larval characters were noted following [38,39]. Comparative data of related species were taken from tadpole descriptions available in the literature . General descriptions were based on individuals in stage 35 as recommended by [49], but we also measured another 15 tadpoles between stages 25-35 of the undescribed species (CBUMAG: ANF 01015-16) and 26 tadpoles between stages 24-39 of I. tayrona (CBUMAG: ANF 00960, 01017 and ICN 58308). A digital Vernier caliper (model type IP54, accuracy±0.01 mm) in a Zeiss dissecting stereomicroscope was used for measurements in I. tayrona (86 individuals), whereas a Mitutoyo series 500 digital caliper (model type CD-8" CSX accuracy±0.01 mm) was used for specimens of the new species (15 individuals).

Egg clutches and oviposition site features
Reproductive behavior was observed at two localities at Sierra Nevada de Santa Marta, SNMS; see S1 Appendix. Between April 1-2, 2015, and October 1, 2016 we performed visual encounter surveys alongside streams. For each egg clutch of Ikakogi tayrona and Ikakogi sp., we noted the substrate (e.g., leaves, stems), position on the substrate (e.g., upper or lower side of leaves), and height above the ground or water. The clutches were attributed to the species because a female was guarding eggs. For each egg clutch observed we noted number and size (diameter) of eggs, development stage and color of embryos. Quantitative features of oviposition site and egg clutches are reported as mean ± SD (range).

Genetic distances
DNA was extracted from ethanol-preserved thigh muscle, with the DNeasy (QIAGEN, Valencia, CA) isolation kit. Amplification of cytochrome b MT-CYB sequence, ca. 371 bp, was carried out in a 25μl reaction using the primers of [65,66] and a PCR Master Mix (2X) (Thermo Fisher Scientific Inc., USA). For DNA amplifications, the PCR program included an initial denaturing step of 30s at 96˚C, followed by 35 cycles of amplification (96˚C for 30 s; 48-54˚C for 30 s; 60˚C for 60s), with a final extension step at 60˚C for 7 min. PCR amplification products were cleaned using the Agencourt AMPure XP DNA Purification and Cleanup kit (Beckman Coulter Genomics, Brea, CA, USA), and were sequenced in both directions to check for potential errors by a third party using fluorescent dye labeled terminators (ABI Prism Big Dye Terminators v. 1.1 cycle sequencing kits; Applied Biosystems, USA) with an ABI 3730XL DNA Analyzer (Applied Biosystems, USA). Chromatograms obtained were read and contigs made using the sequence editing software Sequencher v5.3 [67]. Sequence variation of the static alignment of mitochondrial Cytochrome b2 (Cytb2) gene were used to calculate the uncorrected pairwise genetic distances in Sequencher v5.3 [67], S3 Appendix. The generated sequences were deposited in GenBank under accession numbers MK809522-MK809523, S4 Appendix.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is urn:lsid:zoobank.org: pub:73CD6D36-B4DB-4120-9A43-69DD0EEE0668. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.
Diagnosis. Ikakogi ispacue sp. nov. can be distinguished from other centrolenids (the only exception is I. tayrona) by having a slightly sloping snout, rounded lateral profile; a tympanum that is not visible; humeral spines in adult males; vomerine teeth absent; parietal peritoneum 1/2 white; green dorsum with black punctuations along the dorsal surfaces and a flanks with a lateral row of small enameled dots that extends from below the eye to just posterior to the insertion of the arm; color of bones in life white, but with a pale green coloration in bone epiphysis; color in preservative uniformly very pale lavender to cream.
Flanks with a lateral row of small enameled dots and white bones are unusual in centrolenids and are otherwise known to occur only in some species of Centrolene [68,69], in Ikakogi tayrona (see [70]), in Nymphargus [4] and Hyalinobatrachium [71]. Species of Centrolene having white dots in an area that extends from below the eye to the insertion of the arm include C. antioquiense [72]; C. bacatum [68]; C. buckleyi [73]; C. daidaleum [74]; C. heloderma [75], C. huilense [69], C. pipilatum [27]; C. peristictum [27]; C. sabini [76], C. savagei [74]; C. scirtetes [77]; C. solitaria [74], C. venezuelense [78] and C. robledoi [69]. However, I. ispacue sp. nov. has a tympanum that is not visible (tympanic membrane and annulus not differentiated) whereas all the species mentioned above have a tympanum that is visible and the tympanic membrane and annulus are clearly differentiated (the exceptions are Centrolene buckleyi and C. venezuelense in which some populations have a tympanum concealed and neither the membrane nor the annulus is evident; (see [4,7]). In addition, preserved I. ispacue sp. nov. have a uniform dorsal coloration, with very pale lavender to cream punctuations (lavender with distinctive marks, clear and/or dark spots). Regarding the color bones in life, of the 35 known species of Nymphargus, only two have white bones: N. anomalus [27] and N. rosada [79]. Ikakogi ispacue sp. nov., is easily distinguished from those taxa by having white bones with pale green coloration in bone epiphyses and the adult males having humeral spines (humeral spine absent in adult males of N. anomalus and N. rosada). Further, the color in life in I. ispacue sp. nov. is uniform green with black punctuations, whereas the dorsum in N. anomalus is tan-brown to very pale green olive with black ocelli surrounding orange spots. Moreover, the dorsum in N. rosada is tan-brown with orange dots [27,79]. Species of Hyalinobatrachium are yellowish green or lime green and have distinctive marks like flecks, dots, and spots on their dorsal skin (see [4]). However, in I. ispacue sp. nov. the dorsum is uniformly green and only has minute star-like punctuations (Fig 3B). The adults of Ikakogi ispacue sp. nov. are morphologically identical to I. tayrona (Fig 5), but differ in their cytochrome B sequence and their advertisement call (Fig 6A and 6B). Genetic distances between both species reveal a noteworthy diversity, uncorrected MT-Cytb2 pairwise distance of 14.8% (identical sites 316 bp = 85.2%; 55 differences; S3 Appendix). This values strongly indicates that the two populations are not conspecific. On the other hand, the call of I. ispacue sp. nov. is a single high-pitched note ("trill") with multiple pulses and a high dominant frequency (2928-3273 Hz); whereas the call of I. tayrona is described as 3-4 well-defined nonpulsed, "beep" notes with a dominant frequency of 2650-2870 Hz ( [80], Table 2). Additionally, the tadpole of I. ispacue sp. nov. is almost indistinguishable from the larvae of I. tayrona externally. However they differ in their internal morphology; specifically, they differ in the number of lateral buccal floor papillae (13 in I. ispacue sp. nov., n = 2, in contrast to 10 in I. tayrona, n = 2) and the position of the buccal roof arena papillae (five papillae distributed laterally in I. tayrona; three papillae latterally and two papillae medially in I. ispacue sp. nov. Additionally, the basihyal was absent in the larvae of Ikakogi ispacue sp. nov., n = 2 but present in I. tayrona (n = 2; but see Discussion, below). New species of glassfrog (Anura: Centrolenidae: Ikakogi) from the Sierra Nevada de Santa Marta, Colombia Description of holotype. A moderate-sized glassfrog, SVL in adult male 29.6 mm (adult male); head slightly wider than body; HW 38% of SLV; head wider than long, HW/HL = 1.3 snout slightly sloping in dorsal profile and truncated in lateral profile; loreal region concave, nostrils elevated, delimiting a slight depression in the internarial area; canthus rostralis round; lips slightly flared; eyes large, directed anterolaterally at ca., 45˚; ED equals 31% of HW; supratympanic fold distinct, tympanic annulus undistinguished; tympanic membrane not differentiated, IN 55% of ED; choanae medium sized, ovoid, separated; vomerine teeth absent; tongue round, with posterior distal margin slightly notched, posterior edge not adherent to floor of mouth; vocal slits elongated, extending posterolaterally from base of tongue to the angle of jaws; large, external, subgular vocal sac.
Humeral spine present; forearm robust; ulnar folds present, low; Finger I slightly longer than Finger II, discs subtruncate; prepollex distinct, forming a elongate "prepollical bulge" at the base of the thumb but not piercing the skin; webbing formula III (2 − 2 − ) − (2 + − 2 + ) IV; subarticular tubercles round, supernumerary tubercles at base of fingers, palmar tubercle slightly longer than wide, thenar tubercle elliptical, longer than palmar tubercle; nuptial excrescences Type I. Hind limbs slender, TL 57.3% of SLV; tarsal fold present, low, tarsal tubercles absent; inner metatarsal tubercle elliptic, outer metatarsal tubercle absent; subarticular tubercles round; formulae on feet I (1 − Color in life. Based on color photographs and MAR´s field notes of holotype and paratypes ICN (56204, 56198-99; 56200-01; 56203-09) and CBUMAG:ANF (00938). Dorsum green with minute black punctuations; margin of upper lip, flanks creamy yellowish; flanks with a very minute lateral row of enameled (white) dots; fingers and toes yellow; ventral surface, including limbs cream; parietal peritoneum white (iridophores) covering 1/2 section of the abdominal region; heart white, not visible; visceral peritonea translucent; iris coloration golden with fine dark brown reticulations and dark colored area toward the midline; bones manly white (diaphysis) but with a very pale green coloration in bone ephiphysis (presumably by accumulation of biliverdin).
Color in preservative (ethanol 70%). Dorsum of head, body and limbs cream to very pale lavender covered with small melanophores (except finger and toes I-IV). Throat, chest, hands, feet, and lower surfaces of legs cream; flanks with a very minute lateral row of white dots ( Fig  2A); venter cream; iris silvery with dark reticulation (Fig 2A).
Variation. Females are about 2.3% larger than males. Measurements of the type series are summarized in Table 1. No other morphological differences are observed between males and Natural history. Males and females were observed in subtropical forest perched on the vegetation at heights of 1-2 m. (Fig 4). Males were observed calling from the lower and upper surfaces of leaves along streams at height of 50-350 cm approx. (Fig 4A). Females deposited and cared for egg clutches (referred to the species by association with their mothers perched on clutches) on either the upper side or lower side of leaves overhanging streams (ca. 50-100 cm; Fig 4B). Clutches contained uniformly pale cream or pale green eggs (n = 4; 55 ± 6.21; 48-62 eggs; Fig 4B). The morphology of the egg mass observed near males of I. ispacue sp. nov. is a monolayer mass lacking eggs and jelly in the center of the clutch, which gives an appearance of a "ring" shape (n = 3; Fig 4C). Embryos exhibit cranial hypervascularization, which turned their color reddish or pink; the heart is translucent but colored reddish by blood. Tadpoles of Ikakogi ispacue sp. nov. were found buried in fallen leaves and sand in small pools (area = 1-2 m 2 ; depth = 30-50 cm) located along the edge of streams.
Advertisement call. The call of the species consisted of a high-pitched note ("trill") of 11-20 pulses with modulated amplitude (Fig 6) (n = 17; Table 3). Mean call duration was 0.122 ± 0.041 s (0.082-0.185 s) with a mean pulse duration of 0.007 ± 0.001 s (0.006-0.007 s); pulses were adjacent or were separated by silent intervals < 0.004 s in duration, although the terminal pulses in two calls were separated by 0.018 s (0.014-0.022 s) (Fig 6)
Spiracle sinistral, tubular, short, posterolaterally located, at 84.1% of body length (BL), directed posterolaterally in dorsal view, dorsad at angle of 30-45˚in lateral view; inner wall present, distally free from the body, longer than external wall; opening elliptical ( Fig 9E). Vent Color in life. High-vascularized skin, which gives the larvae a reddish or pinkish general appearance. Dorsum slightly pigmented but with some minute dark gray scattered punctuations especially concentrated between the eyes, in the posterior section of body and along the tail. Ventral skin of body translucent, intestine translucent, liver dark red, heart and other  parts of the circulatory system are bright red. Tail musculature reddish with conspicuous brownish longitudinal stripe in the midline of myotomes, extending from the tail-body junction until almost 1/ 3 of the tail. Tail fins are transparent with minute melanophores mainly in the distal portion. Color in preservative. The color pattern is similar to that of living tadpoles but loses its red or pink coloration. The dorsum, tail musculature, and venter are light cream. Melanophores on dorsum, tail fins and tail musculature turn pale gray (Fig 9A-9F).
Variation and ontogenetic development. Variation of 13 meristic characters of tadpoles in stages 25-35 are given in Table 4. Labial tooth row formula, 2(2)/3, invariable at different stages (Table 5); nevertheless, at stage 25 the upper and lower tooth rows are less developed, i.e., teeth are weakly keratinized and interrupted in some areas. Extension of middle dark gray stripe of the tail musculature slightly varies between the half and posterior third. In some specimens the tail tip is bluntly pointed or rounded ( Fig 9A). In stages 25 to 28, the eyes of Ikakogi ispacue sp. nov. are small, scarcely pigmented and C-shaped in dorsal view, whereas they are larger and more pigmented and round by stage 35.

Dorsal coloration in preservative
Reference H. orientale tobagoense H. taylori Buccopharyngeal cavity- (Fig 11A and 11B). Buccal roof elongated, elliptical, longer than wide, with most features concentrated in the anterior portion. Prenarial arena semi-circular, with inverted V-shaped dermal crest covered with pustulations. Internal nares elliptical, oriented vertically, longitudinally to body's main axis; anterior and posterior margins with large conical projections; valve projection present, triangular, well developed, positioned laterally. Vacuities present, circumscribed by the inner nares margins. Postnarial papillae present, conical, arranged in series of three; third postnarial papillae is the tallest, bearing pustulations, located laterally to median ridge. Median ridge conical, tall. Lateral ridge papilla present, simple, long, triangular, with small pustulations; three triangular papillae present near the lateral ridge papillae. Buccal roof arena poorly delimited, U-shaped, lacking pustulations; five tall, conical papillae present in the buccal roof arena, the anterior ones laterally located, the posterior two, medially. Dorsal velum arched, devoid of papillae, medially continuous (Fig 11A). Buccal floor wider posteriorly than anteriorly, with most features concentrated in the anterior portion. Infralabial papillae present in two pairs; central pair shorter, conical, bearing postulation; lateral pair large tall, located on Meckel's cartilage, wider on the base, bearing pustulations. Few pustulations on mouth's opening. Lingual bud poorly discernible, bearing four lingual, conical, tall, papillae. Prepocket papillae present. Buccal pocket poorly marked superficially;circularly perforated. Buccal floor arena U-shaped, devoid of pustulations; ten lateral buccal floor arena papillae conical, tall, investing the central arena (one of those papillae is a four branched, large papilla). Glandular zone well marked; spicular support evident. Ventral velum well marked, medially continuous, with marginal projection, and discrete medial notch; ventral surface with glandular porous (Fig 11B). Larval muscles. The musculus interhyodeus posterior and the m. diaphragmatoparaechordalis are present Figs 12-15; S5 Appendix; the former is well developed and continuous medially (Fig 14A). Jaw muscles well developed; the m. levator mandibulae longus profundus et superficialis are massive, occupying the entire area of the fenestra sub-ocular ( Fig 11A). Laterally, the origin of the m. suspensorioangularis occupies the entire lateroventral surface of the palatoquadrate (Fig 11D). Large axial muscles, inserting on the anterior portion of the dorsal otic capsule. The most striking muscle is the well-developed levator arcus branchialium III with two slips: one originating in the dorsal otic capsule and crossing the axial muscles to insert in the ceratobranchial III (Fig 13), and the other on originating on posterior palatoquadrate and ventrolateral otic capsule.
Sinus hyobranchialis. The sinus hyobranchialis lies ventrally to the hyobranchial apparatus [81,51]. In Ikakogi larvae the sinus is reduced and restricted to the basic receptaculum disci oralis (RDO), lacking the receptaculum lateralis et transversalis. The RDO is rounded and small, located at the level of the processus urobranchialis of the ceratohyal; in preserved specimens a mass of blood tissues can still be observed. The vena lateralis hyobranchialis is well developed and large in diameter. Both individuals possess slender guts and lack lungs (Figs 14  and 15).
Skeleton. Chondrocranium completely cartilaginous, almost ovoid, and broad posteriorly (at the level of the posterior palatoquadrate) (Fig 16A and 16B). Rostral region slightly depressed. In lateral view, the highest part of the chondrocranium at the ceratohyal plane and New species of glassfrog (Anura: Centrolenidae: Ikakogi) from the Sierra Nevada de Santa Marta, Colombia muscular process (Fig 16A). Suprarostral cartilage quadripartite, consisting of a central corpus, laterally in contact to the lateral alae ( Fig 16A). Central corpus formed by two robust rectangular plates, articulated medially along most of their extension. Triangular-shaped alae, robust, posterodorsal processes with rounded tip. Suprarostral articulates with the trabecular horns through the corpus and alae regions. Trabecular horns large (45-48% of the chondrocranium length), slightly curved ventrally; anterior margin rounded with a medial projection, inner and outer margins smooth. Lateral trabecular process indistinct. Nasal structures not visible.
In the cranial floor, basicranial fenestra slightly occluded by a thin translucent cartilage, the intertrabeculare planum, pierced by two rounded pairs of foramina. The posterior, the primary carotid and the anterior and smaller, the craniopalatine foramen (Fig 16A and 16B).  Palatoquadrate wider anteriorly than posteriorly, their posterior regions curved to the chondrocranium (Fig 16B). Short articular process, projected anteriorly to articulate with the Meckel's cartilage. Thin quadratocranial commissure, joining the palatoquadrate with the neurocranium. Quadratoethmoid and pseudopterygoid processes of the quadratocranial commissure absent. Muscular process of the quadratocranial commissure very low, its rounded tip oriented vertically, not surpassing the antoorbital process. Hyoquadrate process notable. Outer margin of the palatoquadrate slightly raised, directed laterally in lateral view. Palatoquadrate and neurocranium connected posteriorly through the ascending process, a rod-like cartilage, directed anteromedially and attached posteroventrally to the oculomotor foramen New species of glassfrog (Anura: Centrolenidae: Ikakogi) from the Sierra Nevada de Santa Marta, Colombia [82,63]. The posterior curvature of the palatoquadrate extends to the anterior level of the capsula auditiva.
In the lower jaw (Fig 14B and 14E), sigmoid Meckel's cartilages oriented almost perpendicular to the main axis of the chondrocranium, ventral to the trabecular horns. Meckel's cartilage articulates to the articular process of the palatoquadrate by the posterior surface of the very short retroarticular process and opposite internal margin of Meckel's cartilage ( Fig 16E); dorsomedial and ventromedial processes of the cartilague of Meckel's very low. Infrarostral cartilages rectangular, lateral ends thinner than medial region, joined medially by connective tissue, describing a weak wide V-shape. Small ovoid corpus cartilages between the ceratobranquial and palatoquadrate absent.
In the hyobranchial apparatus (Fig 16C), ceratohyals flat, well extended laterally and projected anteriorly by mean of the sub-acuminate anterior process, and posteriorly by a notable and almost triangular posterior process (see Fig 16C). Anterolateral process notable, tip New species of glassfrog (Anura: Centrolenidae: Ikakogi) from the Sierra Nevada de Santa Marta, Colombia subacuminated. Ceratohyals joined by the partially chondrified pars reuniens. Basihyal (copula I) evident. Basibranchial diamond-shaped, bearing a low and truncate urobranchial process, fused to the hypobranchial plates (two very long thin sheets not articulated medially). Outer margin of hypobranchial plate with notable lateral hypobranchial process, tip sub-truncated. Hypobranchial process at level of the second (CBII) and third (CBIII) ceratobranchial. At level of CBII, Commissura proximalis I and II (CP) joined by a small process arised from hypobranchial I and by the lateral hypobranchial process. Commissura proximalis III absent. Branchial New species of glassfrog (Anura: Centrolenidae: Ikakogi) from the Sierra Nevada de Santa Marta, Colombia basket with four curved ceratobranchials and numerous lateral projections. Ceratobranchial I continuous with the hypobranchial plate by mean of the hypobranchial I, and it bears a notable, medially curved branchial process. Branchial process tip rounded. Spicula I-III long, curved postero-dorsally; spicula IV shorter, straight (Fig 16C). Second and third ceratobranchials with free branchial process, notable on CBIII, not fused between each. Ceratobranchials distally joined by terminal commissures. Posterior process of ceratohyal not connected to the anterior margin of the hypobranchial plate (Fig 16C).
Comparisons. The tadpoles of Ikakogi ispacue sp. nov. differ from others of the family in their slightly pigmented dorsal coloration in preservative (strongly pigmented in Vitreorana helenae, Sachatamia albomaculata, S. ilex, Teratohyla spinosa and T. pulverata;   Table 5). The tadpole of I. ispacue sp. nov. is almost indistinguishable from the larvae of I. tayrona, however they differ in their internal morphology, specifically in the presence of 13 lateral buccal floor papillae in the larvae of the I. ispacue sp. nov. (Fig  11B), 10 lateral buccal floor papillae in I. tayrona sp. nov. (Fig 20A). Additionally, both species have five conical papillae in the buccal roof arena, whereas in the larva of I. ispacue sp. nov. three of them are lateral and two are medial, Fig 11B (all the papillae are laterally located in I. tayrona, Fig 20B) and the basihyal is present in the larvae of Ikakogi tayrona (n = 2) and absent in the larvae of I. ispacue sp. nov. (n = 2; Fig 16). However, given the small sample studied by us, the occurrence of the basihyal should be interpreted with caution (see Discussion section ahead).

Redescription of the tadpole of I. tayrona (Ruiz-Carranza and Lynch, 1991b)
The tadpole of Ikakogi tayrona has been mentioned in two articles. [41] presented a brief description of a series of specimens identified as Geobatrachus walkeri, a species with direct development found in the Cuchilla of San Lorenzo, Santa Marta area in Colombia, and in [49], which cited few external larvae features (i.e. LTRF, spiracle position, snout shape and body coloration). Despite being very valuable, these descriptions lack complete illustrations and were based on a single or small series not taking into account intraspecific variation, thus limiting several characteristics necessary for its identification and species comparisons. Color in life. Highly vascularized skin, which gives the larvae a dark red or pink coloration (body and tail musculature). Dorsum slightly pigmented but with some minute scattered pigmentation especially concentrated between eyes, posterior section of body and on myotomes across tail. Ventral skin of body translucent, intestine translucent but with brown or dark green color because of the intestinal content, liver dark reddish, heart and other parts of the circulatory system red. Tail musculature red with conspicuous medial brownish longitudinal stripe in the midline of myotomes, extending from the tail-body junction until almost three half 1/3 of the tail. Tail fins are transparent but with minute melanophores mainly in distal surface (Fig 19).
Color in preservative. The color pattern is similar to that of living tadpoles, but loses its red or pink coloration. The dorsum, tail musculature and venter light cream. Melanophores in dorsum, tail fins and tail musculature turn pale gray (Fig 17A-17C).
In some specimens the middle dark brown band of the tail musculature are either interrupted or incomplete; its extension slight varies between the half and posterior third of the tail musculature. The tail tip is bluntly pointed (Fig 17A) or rounded. The eyes of Ikakogi are small, scarcely pigmented and C-shaped in dorsal view in stages 25 to 28, whereas they are bigger, pigmented and round when they reach the stage 35 and latter (Fig 17B). Major differences in pigment pattern were observed in the sample: tadpoles from the stage 41 to 43 showed a uniformly greenish color pattern in the dorsal skin and limbs and the eyes are oriented anterolaterally. Also, the snout was rounded or truncate in dorsal and lateral views (Fig 19A-19C).
Internal morphology. Buccopharyngeal cavity. The characteristics of the buccal roof and floor are almost identical in Ikakogi tayrona and I. ispacue sp. nov. with the following exceptions: (1) five tall and conical papillae present in the buccal roof arena, all laterally located and, (2) 13 lateral buccal floor arena papillae in I. tayrona (Figs 20 and 21).
Larval muscles. The origin and insertion of all muscles are identical in I. tayrona and I. ispacue sp. nov. (see S5 Appendix, Fig 22). Sinus hyobranchialis. There are no differences between Ikakogi tayrona larvae and I. ispacue sp. nov (Fig 23).
Skeleton. The internal morphology of Ikakogi tayrona larvae is similar to the descriptions provided before for I. ispacue sp. nov. with the following exceptions: (1) the anterior margin of trabercular horns is rounded without medial projection; (2) the oculomotor foramen is closed totally; (3) the small ovoid corpus cartilage between ceratobranquial I and palatoquedrate is present; (4) the Basihyal (copula I) is absent; (5) in one tadpole in stage 35, the right CBIII is joined to the planun hypobranchiale by a weak and thin cartilage at level of its spicula and, (6) in stage 35, the posterior process of ceratohyal is joined by a connective tissue to the anterior margin of the hypobranchial plate (Fig 24).

Discussion
Anurans of the family Centrolenidae are a diverse clade of arboreal frogs distributed across the Neotropical region [24]. As currently known and including the species here in described, Centrolenidae contains 158 species, 50% of which occur in Colombia [24]. Recently, several species of centrolenids have been described or documented to occur in that country, and more are expected to be discovered as research continues in biologically unexplored and topographically complex regions (e.g. [12,15,16]).
The genus Ikakogi was extracted from Centrolene sensu lato, which was found to be nonmonophyletic [5]. Ikakogi tayrona and its putative sister species I. ispacue sp. nov. are mediumsized centrolenid frogs that are endemic to the Sierra Nevada of Santa Marta [70]. Males of both species emit calls from the upper and lower surfaces of leaves and contrary to most centrolenid frogs, egg care is performed by females rather than males ( [6,80], present study).
Other recent studies have demonstrated that the diversity of the highly endemic herpetofauna of SNSM is underestimated (e.g. [84,85,81,86]). These findings, coupled with the present study, suggest that the diversity of amphibians and reptiles in the SNSM of Colombia is greater than the current estimates and underscore the need to conduct additional surveys of the SNSM fauna.

Comments on the systematics of Ikakogi
Recent molecular-based phylogenetic analyses have greatly improved centrolenid systematics [5,7,10,11]. As a result of these advances, Guayasamin et al. [5] proposed a revised taxonomy composed of 12 genera. However, some disagreement remains regarding the relationships among some genera, especially within the tribe Cochranellini, but also regarding the phylogenetic position of Ikakogi [5,11]. These disagreements are partly attributable to the conflicting phylogenies among different studies that used dissimilar aligment and optimality criteria (e.g. [5,10,11]).
Previous studies found Ikakogi to be the sister of all other glassfrogs [5,7,10,11] or sister taxon of the subfamily Centroleninae [8]. The external morphology of Ikakogi is similar to that of species of Centrolene and Espadarana (i.e., they share some character states like humerus with a conspicuous spine and translucent peritonea); however, they seem to be most similar to species of Hyalinobatrachinae. For example, some morphological traits of adults, such as dorsal coloration in preservative (cream to very pale lavender), bone coloration (white or very pale green concentrated in bone ephiphyses), tympanic membrane and annulus conspicousness (hidden), egg clutch location (under/upper side of leaves), egg clutch (monolayer), and oocyte pigmentation (cream to very pale green) are intriguing and shared mainly with species of Hyalinobatrachium and Celsiella. Because Ikakogi tayrona is considered the sister taxon of all other centrolenids (but with some remaining phylogenetic problems; see 5,7,8,11), our records will offer insights into the ancestral states for egg attendance, egg clutches arrangement and location, and tadpole morphology. Future work incorporating all available molecular and morphological evidence in a phylogenetic analysis is necessary to improve our appreciation of relationships of Ikakogi genus and centrolenids and will further our understanding of the origin and evolution of their morphological characters.

Evidence from vocalizations, egg clutches and larval morphology
Ikakogi tayrona and I. ispacue sp. nov. provide a clear illustration of the relevance of studying multiple sources of evidence to test taxonomic hypotheses. The two species are identical in all   adult external morphological characteristics usually used to identify species of centrolenids. In contrast, comparison of vocalizations, DNA sequences, and internal larval morphology (including the number of lateral buccal floor papillae, the spatial arrangement of the five conical buccal roof papillae, and the basihyal structure in the condochranium) revealed diagnostic differences that support the hypothesis that the two groups are heterospecific. The description of centrolenid advertisement calls facilitates species-level taxonomy and provides phylogenetically and ecologically relevant information [87]. The advertisement calls of Ikakogi tayrona and I. ispacue sp. nov. have several remarkable differences. For example, in addition to differences in dominant frequency, calls are shorter and comprise multi-pulsed notes in I. ispacue sp. nov., whereas they are longer and non-pulsed in I. tayrona (see Table 2, and [80]).

Buccopharyngeal anatomy
Data on centrolenid buccopharyngeal anatomy is extremely restricted, available only for Hyalinobatrachium fleischmanni [64]. Comparison of our data with those of Wassersug [64] shows that both Ikakogi and Hyalinobatrachium share some character states, such as the elongate buccal floor and roof, two pairs of infralabial papillae (one pair adjacent to the midline and the other placed over Meckel's cartilage), four lingual papillae, few papillae and pustulations in the buccopharyngeal cavity, longitudinally oriented internal nares with vacuities, tall conical median ridge, and slit-like buccal pocket. Given that these two genera do not appear to be closely related ( [5,8,10,11] but see comments on the systematics of Ikakogi above), the shared occurrence of those states may represent putative synapomorphies for Centrolenidae. Nevertheless, Ikakogi present projections on the ventral vellum margin, papillae on the arena of the floor and roof, and more (three pairs) postnarial papillae, states absent in Hyalinobatrachium fleischmanni. Our results support the importance of the buccopharyngeal morphology for the systematics of glassfrogs, however, we stress the need for more species descriptions and data.
The elongate body observed in centrolenid larvae might create some difficulties in the interpretation of some characters. For instance, Wassersug [64] interpreted the large, tall, conical papillae near the median ridge as homologous to the lateral ridge papillae and questioned the homology of the "anterolateral compressed flap" papillae observed on each side of the buccal floor. In contrast we interpreted the large papillae near the median ridge as a third pair of postnarial papillae, which occupies this position (near the median ridge) as a consequence of the tadpole's elongation. The lateral ridge papillae, in turn, are the features of unknown homology mentioned by Wassersug [64]. Our choice derives from the parsimony principle; in our hypothesis, rearrangements of some papillae may be the result of body's elongation and the evocation of a new papilla is not needed to explain the observed configurations. We stress, however, that information from other species and developmental series is needed to better test the homology of these characters.

Cranial muscles
Anuran cranial muscles are highly variable [62,63,102] and many different patterns of origin and insertion have evolved independently in several lineages. Frost et al. [2] based on Haas [63] data for Cochranella granulosa. Haas [63] included the m. levator mandibulae externus forming a single muscle and the m. subarcualis rectus II-IV having an anterior insertion on the processus branchialis III as candidate synapomorphies for Centrolenidae. Our data supports [2] hypothesis partially. We also found the m. levator mandibulae externus to be a single muscle; however, we observed the subarcualis rectus II-IV to reach the processus branchialis II, although some fibers also inserted anteriorly in the processus branchialis III. Given that one of the phylogenetic placements of Ikakogi is as a sister to all other centrolenids (the other one is sister of the subfamily Centroleninae, [8]), it is possible that the condition observed by us for Ikakogi tayrona and Ikakogi ispacue sp. nov. is plesiomorphic within Centrolenidae, with the shortening of this muscle being an evolutionary trend in the family.
Nevertheless, the most striking character of the cranial muscles of Ikakogi larvae is the origin of one slip of the m. levator arcus branchialium III on the dorsal surface of the otic capsule; not only is its origin peculiar, but so too is its trajectory until its insertion of the ceratobranchial III passing over the axial muscles (note that the axial musculature also extends further on the otic capsule). As far as we know, there is no record for such condition among anuran larvae; Haas [63] studied the cranial muscles of Cochranella granulosa, however he did not mention this character-state.

Larval skeleton
Published information on the chondrocranium of Centrolenidae is limited to the character states of Cochranella granulosa by Haas [63]. Our observations on Ikakogi show that various character states are shared with C. granulosa; however, some differences were observed, such as the low suspensorium (intermediate in C. granulosa), the presence of the basihyal in Ikakogi ispacue sp. nov. (absent in I. tayrona and C. granulosa), the lack of a medial articulation between the hypobranchial plates (articulated in C. granulosa), and the presence of the commissura proximalis I and II (absent in C. granulosa).
Intraspecific variation in the basihyal has been described in dendrobatoids (see [60]) and interspecific variation in Melanophryniscus (Bufonidae, see [103]), dendrobatids [60] and Centrolenidae (this work). Although, the basihyal is absent in larvae of Ikakogi tayrona and present in I. ispacue sp. nov., given the small sample studied by us, the occurrence of the basihyal should be interpreted with caution. The phylogenetic value of the other differences found between Ikakogi and Cochranella have been showed in other frog families (e.g., [104,105,61,63), which highlights the importance of increasing studies on the chondrocranium of centrolenids.
In addition to the characters proposed by [107], these larvae also present bodies that are compressed laterally and depressed dorsally, strong caudal muscles, shallow tail fins, and depressed and truncated snouts [49,51]. Regarding the internal morphology of fossorial tadpoles, little is known. [110,111] compared the musculoskeletal morphology of Leptobrachella mjobergi and Otophryne robusta and described the crania of Micrixalus herrei, [110] finding that these fossorial taxa differ greatly. Although the larvae of Ikakogi do not appear to share relevant characteristics with those of O. robusta [114], they do share several relevant characteristics with M. herrei and L. mjobergi, including long, laterally compressed crania, robust anterior cartilages (suprarostral and trabecular horns, Meckel's cartilage), a low muscular process, a subocular bar narrowed posteriorly and a thin processus ascendens, and, in Ikakogi and Leptobrachella, weakly chondrified lateral cartilage of the cavum cranii, and thin ceratobranchials. The m. levator mandibulae group is massive and the paraxial musculature reaches far anterior on the otic capsule.
Lastly, we recorded the absence of lungs in both Ikakogi larvae. Several researchers (e.g. [115][116][117]) indicated the importance of the lungs for larval buoyancy. The absence of larval lungs, even in well-developed larvae (Gosner stages 30+) might be related to the fossorial habits. Regarding gas exchanging, the hypervascularization of the skin and branchial area, including the sinus hyobranchialis, probably compensate the absence of lungs and the failure to breath air. areal. considerations on their laboratories we are grateful to J.D. Lynch and M.C. Ardila-Robayo, r.i.