Atelopus histrionicus Lötters, Plewnia, Böning, Boistel, Chaparro, Coloma, Ellwein, Orsen, Paluh and, 2025

Atelopus histrionicus Lötters, Plewnia, Böning, Boistel, Chaparro, Coloma, Ellwein, Orsen, Paluh and

Venegas sp. nov.

Species account authors: Stefan Lötters, Amadeus Plewnia, Philipp Böning, Renaud Boistel, Juan C. Chaparro, Luis A. Coloma, Denise J. Ellwein, Ludvig Orsen, Daniel J. Paluh, Pablo Venegas

LSID: urn:lsid:zoobank.org:act:821DEE06-D6BD-44C0-903B-D1016DC7F1C3

Figs. 25–26 View FIGURE 25 View FIGURE 26

Atelopus spumarius View in CoL non-Cope.— Lötters et al. 2011: 48; Herrera-Alva et al. 2020: 420; Jorge et al. 2020a: 5; Jorge et al. 2020b: 7.

Atelopus sp. ( cf. spumarius View in CoL ).— Lötters et al. 2005: 345.

Atelopus aff. spumarius View in CoL .— Lötters et al. 2011: 51.

Atelopus sp. — Rueda-Almonacid et al. 2005: 126.

A. sp. cf. spumarius Tahuayo. View in CoL — Lötters et al. 2023: Supplementary Table 1.

Holotype. PERU: Loreto: Quebrada Blanco, Área de Conservación Regional Comunal Tamshiyacu-Tahuayo (4º 36’02.8” S, 73º17’43.5” W, 130 m asl), MUBI 2449 (field number TAHU006 ; Fig. 25 View FIGURE 25 ), adult male, leg. February 2003 by Rainer Schulte and Dietmar Bernauer. GoogleMaps

Paratopotypes. MUBI 2450–2451 (field numbers TAHU001–002 ) , CORBIDI 26946–26947 View Materials , adult males (field numbers TAHU 003–004 ) , ZFMK 104180 About ZFMK , 104181 About ZFMK (field number TAHU005 , 007 ) , adult males, CORBIDI 26948 View Materials (field number TAHU 008 ) , adult female, same data as holotype GoogleMaps .

Referred material. PERU: Loreto: Genaro Herrera, MUSM 9683; Sierra del Divisor, CORBIDI 2329, 2334, 2381, 2458, 2465–2466, 2469, 2470–2471, 2479, 2482–2483, 3736–3741.

Definition. Atelopus histrionicus sp. nov. is placed in the genus Atelopus based on its mt and nc markers nested within Atelopus (and with the 16S rRNA fragment showing uncorrected p-distance <4% to the genus type species A. flavescens Duméril and Bibron ), and on having presacrals I and II fused. The species is dinstinguished from all other species by the combination of the following characters: (1) A small-sized species with mean SVL of adult males 25.0 ± 0.67, N = 7, and single female 32.0; (2) slender body ( SW / SVL = 0.260 ± 0.008 in 7 males, 0.281 in single female) with (3) long legs ( TIBL / SVL = 0.465 ± 0.014 in 7 males, 0.469 in single female) and (4) blunt acuminate to subacuminate snout, protruding slightly beyond apex of lower jaw; (5) tympanic membrane absent, tympanic annulus not visible, columella present, ostia pharyngea present; (6) phalangeal formula of hand 1-2-3-3, webbing absent on hand; (7) first finger short ( THBL / HAND = 0.462 ± 0.034 in 7 males, 0.515 in single female), in adult males with keratinized dark brown spiculae forming the nuptial pad; (8) phalangeal formula of foot 2-2-3-4-3, webbing formula of toes I 0–0 to 1 II 0–1 to 2 III 1 to 2–3 IV 3–1 to 11 / 2 V; (9) skin smooth, dorsally and laterally covered in almost indistinct dense minute spiculae, resulting in a velvety appearance of the skin in life; ventrally smooth to weakly areolate, cloaca and adjacent thighs most areolate; (10) vertebral column conspicuous, neural processes absent; (11) in life: dorsum black to dark brown with few yellow, whitish-green or green asymmetric circles, dots or lines, which are sometimes becoming brighter towards flanks; dorsolaterally yellow, whitish-green or green foam-like reticulation forming a band from above eye to groin with numerous dark brown rounded irregular spots (smaller than EYDM); laterally a black or dark brown band from tip of snout to groin; limbs and dorsal surfaces of hand and foot with foam-like reticulation with spots (smaller or larger than EYDM), thumb and sometimes tips of fingers and toes dorsally orange to reddish; in males, throat, chest and venter unicolored white to cream, posteriorly with few black dots (smaller than EYDM); in females, throat, chest and venter white to cream with few black rounded to irregular markings (smaller or larger than EYDM) in gular region and on venter, sometimes as a continuous chest band; in both sexes palms, plants, ventral surface of cloaca and thighs and sometimes armpit orange-red to red, in females, extending on posterior parts of vent; iris black with a narrow golden ring surrounding horizontally oriented pupil; (12) in preservative: above brown with cream to light brown pattern; below whitish cream with brown dots; orange to red life markings faded to cream; (13) pulsed call 807 ± 80 ms at dominant frequency 2,929 ± 9 Hz .

Diagnosis. Atelopus histrionicus sp. nov. ( Figs. 25–26 View FIGURE 25 View FIGURE 26 ) can be readily distinguished from all other Atelopus (as far known) by molecular genetics, with an uncorrected p-distance of the 16S rRNA fragment of 2.37% to its closest known described relative, A. franciscus Lescure and the A. hoogmoedi Lescure complex (Appendices 8, 9). It can be morphologically distinguished from all congeners by the combination of small size, dorsal and lateral skin covered with dense, almost indistinct minute spiculae, ventral skin smooth to areolate, presence of a columella and dorsal pattern, except from A. spumarius Cope sensu stricto and A. colomai Plewnia, Terán-Valdez, Culebras, Boistel, Paluh, Quezada Riera, Heine, Reyes-Puig, Salazar-Valenzuela, Guayasamin and Lötters. From both, it differs in call duration (807 ± 80 ms vs. 922 ± 24 ms in A. spumarius sensu stricto; 2,049 ± 220 ms in A. colomai ) and female ventral coloration and pattern in life (whitish to cream with larger rounded to irregular dark brown markings smaller or larger than EYDM vs. throat and chest orange or yellow, venter anteriorly cream yellow with numerous brown or black dots smaller than or equal to EYDM, posteriorly orange or yellow in A. spumarius sensu stricto; yellow with black rounded dots smaller or larger than EYDM in A. colomai ). From A. spumarius sensu stricto it can be distinguished by red palmar and plantar coloration in life (vs. orange). From A. colomai it differs in female snout profile (blunt vs. more projected, sharply pointed in lateral view).

Atelopus histrionicus sp. nov. ( Figs. 25–26 View FIGURE 25 View FIGURE 26 ) is most similar to its congeners of the flavescens-spumarius clade, and to A. andinus Rivero , A. loettersi De la Riva, Castroviejo-Fisher, Chaparro, Boistel and Padial , and the A. tricolor complex (including the junior synonyms A. rugulosus Noble and A. willimani Donoso-Barros ) from the eastern Andean versant of Peru and Bolivia. From A. manauensis Jorge, Ferrão and Lima it differs in larger male SVL (25.0 ± 0.67, N = 7, vs. 21.8 ± 2.4, N = 11; Jorge et al. 2020b). It differs from the nominal A. barbotini Lescure , A. flavescens Duméril and Bibron (including its junior synonym A. vermiculatus McDiarmid ), A. franciscus and the A. hoogmoedi Lescure complex (including the available name A. hoogmoedi nassaui Ouboter and Jairam ) in pattern and coloration in life (dorsally black to dark brown with few yellow, whitish-green or green asymmetric circles, dots or lines; dorsolaterally yellow, whitish-green or green foam-like reticulation forming a band from above eye to groin with numerous dark brown rounded irregular spots; below white to cream, with few black dots and ventral surface of cloaca (plus posterior belly in females) and thighs orange-red to red vs. dorsally black with red to pink markings, ventrally reddish pink in A. barbotini ; dorsally unicolored reddish, brownish or yellow or brown and with olive, brownish reddish and copper-like vermiculation, ventrally reddish pink in males, pink in females in A. flavescens ; dorsally olive to blackish brown, ventrally reddish in A. franciscus ; dorsally dark brown or black with yellow, pink, bluish, white, greenish or orange irregular spots and reticulated dorsolateral bands or solid bars, ventrally yellow, pink, bluish, white, greenish or orange, in some specimens with dark spots or blotches in the A. hoogmoedi complex). All these forms, except some populations of the A. hoogmoedi complex, lack red palmar and plantar surfaces in life (present in the new species). All Guianan harlequin frogs have longer call duration (807 ± 80 ms vs. 1,490 ± 140 ms in A. barbotini ; 1,530 ± 220 ms in A. flavescens ; 1,490 ±150 ms in A. franciscus ; 1,190 ± 10 ms in the A. hoogmoedi complex from French Guiana; 1,060 –1,240 ms in the A. hoogmoedi complex from Pará, Brazil; Lescure 1981; Cocroft et al. 1990), with the exception of the A. hoogmoedi complex from Amapá, Brazil (1,160 ± 390 ms; Costa-Campos & Carvalho 2018). In addition, it can be distinguished from A. franciscus by larger male SVL (25.0 ± 0.67, N = 7, vs. 18.0–21.5, N = 24; Lescure 1974). It can be distinguished from A. pulcher Boulenger by shorter calls (807 ± 80 ms vs. 1,200 ± 100 ms; Lötters et al. 2002a), smaller male SVL (25.0 ± 0.67, N = 7, vs. 27.27 ± 1.07, N = 13) and female coloration in life (white to cream, with few black dots and ventral surface of cloacal region, posterior belly and thighs orange-red to red; dorsally black to dark brown with few yellow, whitish-green or green asymmetric circles, dots or lines; dorsolaterally yellow, whitish-green or green foam-like reticulation forming a band from above eye to groin with numerous dark brown rounded irregular spots vs. bright red ventral surfaces; green dorsolateral bands with few to no reticulation). Atelopus histrionicus sp. nov. differs from A. harlequin sp. nov. in larger male SVL (25.0 ± 0.67, N = 7, vs. 20.6 ± 0.51, N = 4), longer call duration (807 ± 80 ms vs. 680 ± 30 ms; Asquith & Altig 1987) and in dorsal coloration in life (black to dark brown with green, yellowish- or whitish-green reticulation vs. yellowish-gold reticulation on numerous dark brown, rounded marks and irregular shaped blotches). It differs from A. seminiferus Cope in smaller female SVL (32.0, N = 1, vs. 40.0, N = 1), absence of lateral warts (present in A. seminiferus ) and dorsal coloration (dorsally black to dark brown with few yellow, whitish-green or green asymmetric circles, dots or lines; dorsolaterally yellow, whitish-green or green foam-like reticulation forming a band from above eye to groin with numerous dark brown rounded irregular spots vs. dark brown without pattern). From A. andinus , A. loettersi and the A. tricolor complex, A. histrionicus sp. nov. can be distinguished by the presence of a columella (vs. absence; Lötters et al. 2011; authors’ unpubl. data). From A. loettersi , it can be distinguished by female ventral coloration in life (white to cream, with few black dots and ventral surface of cloacal region, posterior belly and thighs orange-red to red vs. mostly red). The new species can be distinguished from A. andinus and the A. tricolor complex by the absence of warts (presence) and absence of dorsal reticulation (presence).

Description of holotype. Adult male. Body slender (SW/SVL = 0.269), head as long as wide (HLSQ/HDWD = 1.010), snout blunt-ended, slightly protruding beyond apex of lower jaw, subacuminate in dorsal view; nostrils directed laterally, moderately protuberant, not visible in dorsal view, about three quarters from eye to tip of snout in lateral view, situated anterior apex of lower jaw; canthus rostralis well defined, slightly concave in dorsal view between eye and nostrils; loreal region concave; lips not flared; top of snout depressed; head plain in lateral view; eyelid flared, covered in minute poorly distinct spiculae; tympanic membrane absent, tympanic annulus not visible; supratympanic crest poorly developed; choanae small, rounded; ostia pharyngea present; tongue three times as long as wide, broadest posteriorly, free for half its length posteriorly; vocal slits present.

Limbs long and slender (TIBL/SVL = 0.463); webbing absent on hand; fingers and toes lack lateral fringes; palmar tubercle rounded, poorly distinct, supernumerary palmar and plantar tubercles absent, thenar and subarticular tubercles indistinct; tips of digits rounded; phalangeal formula of hand 1-2-3-3; first finger short (THBL/HAND = 0.448), dorsally covered with keratinized dark brown spiculae forming the nuptial pad; inner metatarsal tubercle indistinct, outer metatarsal tubercle rounded to oval; relative length of toes I<II<III<V<IV; phalangeal formula of foot 2-2-3-4-3, webbing formula of toes I 0– 1 / 2 II 1 / 2 –2 III 1–3 IV 3–1 V; tarsal fold absent; foot roughly three quarters of tibia; skin smooth on entire body, dorsally and laterally covered in poorly distinct dense minute spiculae resulting in a velvety appearance of the skin; ventrally smooth to slightly areolate; cloacal opening in an inconspicuous tube, directed posteriorly; vertebral column visible through the skin, neural processes absent.

In live, dorsum dark brown with few small whitish-green asymmetric spots; dorsolaterally yellowish-green foam-like reticulation, forming a band from above eye to groin bearing numerous dark brown rounded markings (smaller than EYDM); laterally a blackish-brown band from tip of snout to groin; throat and venter unicolored whitish cream, ventrolaterally with few black dots (smaller than EYDM); lips bordered by thin black line; limbs dorsally dark brown, covered in pale-green bands and reticulation; ventrally, thighs orange-red, distal parts white, bearing dark brown dots or bars; thumb and tips of fingers and toes dorsally orange to reddish orange; palms, plants and ventral surface of cloaca and thighs orange-red; iris black with a golden line surrounding horizontally oriented pupil.

In preservative, above brown with cream pattern; below whitish cream with few brown dots ventrolaterally; orange-red life markings faded to cream.

Measurements: SVL 24.2, TIBL 11.2, FOOT 9.1, HLSQ 7.7, IOD 3.1, HDWD 7.5, EYDM 2.6, EYNO 2.4, ITNA 2.4, FAL 7.8, HAND 5.4, THBL 2.8, SW 6.4.

Variation. For meristic variation of characters see Table 6. Paratypes correspond to the holotype description. Some males present few small dark brown dots on posterior surface of the venter. Dorsal and dorsolateral pattern ranges from green (specimens from Madre Selva and Serrania de Divisor) to yellowish or whitish green (specimens from Tamshiyacu-Tahuayo).

Sexual dimorphism is apparent in A. histrionicus sp. nov. with females being larger than males ( SVL = 32.0 vs. 25.0 ± 0.67). Additional individuals (not collected), measured in the type locality in life corroborate this pattern ( SVL = 31.1 and 31.4 in 2 females; 25.3 ± 1.96 (17.5–29.8) in 45 males). In addition, females lack nuptial pads, differ in coloration by having a whitish to cream venter with larger rounded or oval dark brown markings in the gular region, on chest and venter .

Skull osteology. General osteological features of one male (paratopotype CORBIDI 26947) and one female (paratopotype CORBIDI 26948) are depicted in Figures 5–6 View FIGURE 5 View FIGURE 6 and Appendix 4. The skull of A. histrionicus sp. nov. is triangular in dorsal view. The skull length is 8.2, the skull width is 7.3, and the skull height is 5.1 in CORBIDI 26947. The skull length is 9.4, the skull width is 8.9, and the skull height is 5.7 in CORBIDI 26948. The skull roof is highly rugose. In anterior view, the septomaxilla is U-shaped with medial and lateral rami that extend posteriorly towards the vomer. The lateral ramus is much broader than the medial ramus and bears a nasal process. The ossified sphenethmoid is rugose and underlies the posterior and medial margins of nasals, as well as the anterior margins of the frontoparietals. The posterior limit of the sphenethmoid is about one-half the length of the margin of the orbit. The prootics are rugose and are fused to posterolateral edge of the frontoparietals. The exoccipitals encircle the foramen magnum and are fused to the frontoparietals dorsally, the prootics laterally, and the parasphenoid anteroventrally. The otic capsule is well-ossified. Columellae are present, medially slightly bifurcated. The pars media of the columella is slender and slightly bowed, and the pars interna is broad. The operculum in the fenestra ovalis is not mineralized and therefore not visible in the microCT dataset. The nasals are triangular and bear an acuminate maxillary process that extends ventrolaterally toward the maxilla and contacts the pars facialis of the maxilla. The nasals are in contact with one another medially in CORBIDI 26947 and are narrowly separated medially in CORBIDI 26948. The frontoparietals are rectangular and the orbital edges are straight. Medial articulation is complete in both specimens. No suture line is visible in CORBIDI 26948, whereas an incomplete suture line is visible in the anterior half of the bones in CORBIDI 26947. A supraorbital flange is absent. Occipital groves, which are canals for the carotid artery, are present and partially roofed with bone. In CORBIDI 26948, the left groove is mostly open, while the right groove is covered along the posterior three-fourths of its length. In CORBIDI 26947, the left groove is completely roofed and the right groove is covered along the anterior one fourth of its length. Posteriorly, the frontoparietals are fused to the prootics and exoccipitals. The vomers are small, edentate, medially separated, crescent-shaped, and triradiate with an anterior ramus, prechoanal ramus, and postchoanal ramus. The anterior ramus is directed anterolaterally, pointed at the anterior tip, and is larger than the other rami (nearly reaching the junction of the maxilla and premaxilla). The prechoanal ramus is shorter than the postchoanal ramus; they form the anterior and anteromedial margin of the choana. The prechoanal ramus is directed towards the maxilla, and the postchoanal ramus is directed parallel to the midline. The postchoanal ramus is fused to the overlying sphenethmoid. The neopalatine is elongate and triangular. It underlies, and is partially fused to, the sphenethmoid and extends towards the maxilla, making contact with the preorbital process. The neopalatine is narrow and pointed at its medial tip, widens as it approaches the maxilla, and is cylindrical and rounded at its anterior border. The parasphenoid has an inverted T-shape. The cultriform process underlies the sphenethmoid in CORBIDI 26948 but does not in CORBIDI 26947. The cultriform process extends beyond the midpoint of the orbit. The terminal end of the cultriform process is rounded in shape. The parasphenoid alae are directed posterolateral to the cultriform process and are fused to the overlying otic capsule. The posterior margin of the parasphenoid and a medial posterior process is present. The maxillary arcade is complete and edentate. The paired premaxillae each possess a dorsal alary process that is directed anterolaterally. The lower half of the alary process is the same width as the upper half. The pars palatina is biradiate with medial and lateral processes. The lateral process is more than twice as long as the medial process. The medial process is well developed and triangular. A concave border is present where the lateral and medial processes of the pars palatina come together. The posterior tip of the pars dentalis is pointed and articulates with the maxilla. The external surface of the maxillae is rugose. The pars palatina, which extend along the lingual margin of the maxilla, is narrow, and the posterior half of this shelf articulates with the anterior ramus of the pterygoid. The pars fascialis of the maxilla is directed medially and is a jagged, irregular shape; its dorsomedial margin contacts the neopalatine, sphenethmoid, and the maxillary process of the nasal. The anterior end of the maxilla is truncate and abuts the premaxilla. The posterior end of the maxilla is pointed and contacts the quadratojugal. The quadratojugal is a small, slender, L-shaped bone that underlies the ventral arm of the squamosal and contacts the posterior process of the maxilla at is anterior margin. The paired squamosals possess an otic ramus posterodorsally, a zygomatic ramus anterodorsally, and a ventral ramus. The otic ramus is expanded dorsally and has a broad articulation with the crista parotica of the otic capsule, leaving only the posterior end of the prootic free. The zygomatic ramus is small, triangular, and directly medially. The angle between the dorsal surface of the squamosal and the anterior margin of the ventral ramus is nearly perpendicular. The ventral ramus is flat and blade-like. A squamosal keel is present, extending along the outer surface from the zygomatic ramus to the upper half of the ventral arm. The squamosal surface is rugose in CORBIDI 26947. The pterygoid is triradiate, bearing anterior, medial, and posterior rami. The anterior ramus articulates with the pars palatina of the maxilla. The medial and posterior rami are of equal length although the medial ramus is much wider. The medial ramus nearly contacts the prootic. The posterior ramus is flat and blade-like. The palatoquadrate is cartilaginous and therefore not visible in the microCT dataset. The lower jaw is composed of three ossified elements and Meckel’s cartilage, which is not visible in the microCT dataset. The mentomeckelian is the most anterior element that forms a cartilaginous symphysis at the midline of the jaws. Posterodorsally, the mentomeckelian is fused to the dentary. The dentary is slender, thin, and edentate; this element overlays the anterior half of the angulosplenial. The angulosplenial is the largest mandible element and forms the lingual surface of the lower jaws. The lower jaw length is 7.1 in CORBIDI 26947 and 8.2 in CORBIDI 26948, slightly shorter than the length as the skull from the occipital condyle to the premaxilla.

Atelopus histrionicus sp. nov. is distinguished from the species (re)described herein by having a highly rugose skull roof (vs., in A. spumarius sensu stricto and A. harlequin sp. nov. slightly rugose, in A. colomai , smooth; Fig. 5 View FIGURE 5 ), the lateral margins corresponding to the maxilla more straight, a deeper posteroventral incisure of the sphenethmoid ( Fig. 6 View FIGURE 6 ), and the dorsal end of the dorsal process of the premaxilla (= pars facialis) protruding anteriorly beyond the anterior limit of the nasals (vs., in A. spumarius sensu stricto and A. harlequin sp. nov., remaining at the same level; in A. colomai , remaining slightly recessed; Fig. 6 View FIGURE 6 ).

Further, the new species differs from the neotype of A. spumarius sensu strico by the squamosal laterally extending beyond the posterolateral limit of the maxilla and the jugularis process of the quadratojugal, thus contacting the posterior process of the maxilla anteriorly (vs. the posterior end of the maxilla being free, laterally protruding beyond the lateral limit of the squamosal), the pars glenoidalis of the quadratojugal being triangular with an enlarged but narrow pars jugularis (vs. pars glenoidalis thickened, pars jugularis short) and a less straight orbital margin of the maxilla. However, variation in these characters exists between the two populations of A. spumarius sensu stricto, which might be attributed to imaging conditions or natural variation. Atelopus histrionicus sp. nov. can be distinguished from A. colomai in having the frontoparietals fused only over two posterior thirds of their total length (vs. completely fused), the squamosal laterally extending beyond the posterolateral limit of the maxilla, and the jugularis process of the quadratojugal contacting the posterior process of the maxilla anteriorly (vs. maxilla protruding beyond the lateral limit of the squamosal, posteriorly, contacting the jugularis process of the quadratojugal) and the pars glenoidalis of the quadratojugal being triangular with an enlarged but narrow pars jugularis (vs. pars glenoidalis subrectangular, pars jugularis elongated). Atelopus histrionicus sp. nov. differs from A. harlequin sp. nov. by having the distal end of the medial side of the posterodorsal process of the squamosal free (vs. attached along its entire length to the prootic) and having a reduced posterior process of the parasphenoid (vs. well developed). For details see Figures 5–6 View FIGURE 5 View FIGURE 6 and Appendix 4.

However, the discriminatory power of osteological features needs to be seen with caution as intraspecific variation, sexual dimorphism and taxonomic value remain poorly understood for the species discussed.

Vocalization. Pulsed calls were recorded by LO from an uncollected male on 11. October 2013, between 09:45 am to 03:15 pm, at the type locality with a Sony ICD-UX80 digital voice recorder. Distance to recorder was 0.2 m and ambient temperature varied between 24.8 and 28.1°C. A 20,204 ms recording was available for analysis ( CJ ec.cj.aud.20; Fonozoo Sound Code 14652; Fig. 27 View FIGURE 27 ). It consists of 9 pulsed calls with the first two being interrupted by a pulsed short call (duration 186 ms, intervals between pulsed calls and pulsed short call 1,220 and 379 ms, amplitude peaks in the first two of five pulses, length of pulses 14 ± 2 ms, range 12–17 ms). Pulsed calls have a duration 807 ± 80 ms (688–932 ms). Intervals between calls last 1,458 ± 273 ms (1,105 –1,799 ms). Dominant frequency is 2,929 ± 9 Hz (2,910 –2,941 Hz). Amplitude shows a local peak over the first or second pulse in most calls, followed by a sharp, moderate or no decrease with subsequent increase until it peaks over half to two thirds of the call with a sharp, moderate or no decrease at the end of the call. Frequency shows harmonics and is modulated with a slight increase towards the end of the call. Each call consists of 31 ± 4 pulses (25–38), that show a length of 18 ± 7 ms (7–46 ms) each. Pulses slightly increase in length over the course of each call with longer pulses commonly showing two subsequent amplitude peaks. In two calls, the first two or three pulses are detached by a long interval of 108 and 206 ms respectively from all subsequent pulses GoogleMaps .

Another recording contained pure tone short calls from the same male (same recording data as above; CJ ec.cj. aud.21; Fonozoo Sound Code 14653; Fig. 28 View FIGURE 28 ). It consists of 11 pure tone short calls with an average call duration of 106 ± 51 ms (25–212 ms). Vocalizations are arranged in series of 2–4 pure tones with intervals of 338 ± 44 ms (280–393 ms) within series and intervals of 4,679 ± 710 ms (4,173 –5,491 ms) between them. Pure tone short calls have a dominant frequency of 2,603 ± 99 Hz (2,435 –2,824 Hz). Amplitude peaks at the beginning of the call and sharply descends thereafter, sometimes followed by one or two smaller peaks with a rapid decrease towards the end. Frequency shows harmonics and is modulated with a decrease towards the end of the call GoogleMaps .

Tadpole. The tadpole of A. histrionicus sp. nov. is unknown.

Distribution. Atelopus histrionicus sp. nov. is only known from the Amazonian lowlands of northern Peru, ranging from ca. 85–150 m asl. The species has been reported from eight localities in Loreto, being found right hand side of the Amazon and Ucayali Rivers ( Fig. 2 View FIGURE 2 ). It is likely to be present in more localities in the general area which is poorly studied and difficult to access, perhaps including the species’ presence in adjacent Brasil (Estados Acre, Amazonas).

Natural history. The diurnal species inhabits terra firme lowland rain forest in proximity to slow-moving streams and rivers. In the type locality, A. histrionicus sp. nov. can be found along a stream approximately 4 m wide and 1 m deep with fine white sand and fallen logs about every 30 m on the ground of the stream ( Fig. 29 View FIGURE 29 ). During the day, adults are mostly found actively walking in leaf litter and on fallen trees on the forest floor where they feed on tiny terrestrial termites (Rainer Schulte, unpubl. data). Some individuals could also be found more distant from the closest stream (ca. 40 m away). At the type locality, sex ratio was skewed towards males with only 2 of 51 adult frogs observed in October 2013 being females. Adult males were heard calling throughout the day. Metamorphs and small juveniles were observed in the type locality in late July and October.

Conservation status. We suggest listing Atelopus histrionicus sp. nov. as Vulnerable under criteria B1a, b of the IUCN Red List of Threatened Species as (1) the new species is known only from eight localities, (2) which might be further threatened by ongoing deforestation in the future (currently, at least two localities are affected by small-scale deforestation). Atelopus histrionicus sp. nov. has an EOO of 18,100 km ² and AOO of 32 km ².

It is unknown, if the chytrid fungus is present in A. histrionicus sp. nov.

Atelopus histrionicus sp. nov. is further likely to be threatened by climate change in the near future, which might both impact the species habitat as well as interfer and exacerbate existing threats such as disease ( Lötters et al. 2023). The species from the western Amazon are among the harlequin frogs likely to be most affected by climate change ( Lötters et al. 2023).

The species is known from the National Park Sierra del Divisor, the Comunal Reserve Tamshiyacu-Tahuayo and from Reserva Madre Selva, a private protected area.

Etymology. The specific epithet is derived from the latin histrio meaning “actor”, which refers to the bright coloration and conspicuous diurnal activity pattern of the species with its characteristic foot-flagging behaviour. The name is treated as an adjective in nominative case, singular, masculinum.

3.5 Material of uncertain status

Additional material has become available, which due to the limited data we feel unable to assign it to any of the described species ( Fig. 2 View FIGURE 2 ): (1) Specimens from Parque Nacional Natural Amacayacu, Departamento Amazonas , Colombia ( ICN 45078–9 View Materials ; Fig. 30 View FIGURE 30 ), from Yubineto , region Loreto, Peru ( MNHNP 1979-8416 View Materials ; Fig. 31 View FIGURE 31 ) and from Igará-Paraná , Departamento Amazonas , Colombia ( BMNH 1905.1.31.10–11; Fig. 32 View FIGURE 32 ), resemble A. spumarius sensu stricto (cf. Lescure 1981). Due to the geographic proximity to the distribution of this species, we consider conspecificy likely .

(2) Specimens from Campamento Piedritas, Río Algodón in the Río Putumayo drainage of Peru, region Loreto (CORBIDI 5918–5925; Fig. 33 View FIGURE 33 ), morphologically resemble A. spumarius sensu stricto. Material was included in our molecular phylogeny ( Fig. 1 View FIGURE 1 as A. sp. cf. spumarius from Río Algodón). Given their position in the tree and their genetic distance to the various taxa (see chapter 3.2) they likely represent an undescribed taxon, however.

(3) Specimens from Puerto Leguízamo, Departamento Putumayo, Colombia (GFM 2313, 2317, 2345; Fig. 34 View FIGURE 34 ), from Río Mamansoya, Parque Nacional Natural La Paya, Departamento Putumayo, Colombia (ICN 54651–7; Fig. 35 View FIGURE 35 ) and from Río Puré, Departamento Amazonas, Colombia, (ICN 46833–44; Fig. 36 View FIGURE 36 ) have a color pattern that is intermediate between A. harlequin sp. nov. and A. spumarius sensu stricto. Material from Puerto Leguízamo was included in our molecular phylogeny ( Fig. 1 View FIGURE 1 as A. sp. cf. spumarius from Puerto Leguizamo). Given their position in the tree and their genetic distance to the various taxa (see chapter 3.2.) they likely represent an undescribed taxon, however.

(4) Specimens from Leticia, Departamento Amazonas, Colombia (IAvH-Am 532, 7881; Fig. 37 View FIGURE 37 ), resemble A. harlequin sp. nov. by color pattern and size. As mentioned under the species entry of A. harlequin sp. nov. above, we will not rule out that more than one species is hidden behind this name.

4 Acknowledgements

We are grateful to Sarah Ehl, Diego Ellis Soto, Karin Fischer, Kim Hoke, Sabine Naber, Julia Rautenberg and Alice Schumacher for their help with processing samples. We thank Ana Almendariz, Ronn Altig, Michel Blanc, Dietmar Bernauer, William E Duellman†, Ron Gagliardo, Wilfried Heines, Blake Klocke, William Lamar, Jean Lescure, Jorge Luis Martinez Ruíz, María José Navarrete, Mark Pepper, Manuel Sanchez Rodriguez, Rainer Schulte†, Fredrik Tegner, Evan Twomey, Jorge Valencia, Philine Werner, Katharina C. Wollenberg Valero, Thomas Ziegler, and the staff at Tahuayo Lodge and Orosa Lodge, Peru, for valuable discussions or for providing samples to this study. We are thankful to Steven Guevara Salvador (CJ) and Jose Vieira (www.ex-situphotography.com) for providing photographs. Francesca Angiolani, Ambarka Giehler, Tobias Hildwein, Santiago and Daniel Hualpa, Carlos Iñapi Manuyama, Jos Kielgast, Zane Libke, Jhon and Elmer Macahuachi Serepera, Michael Mayer, Danilo Medina, Christoph Meyer, Maria Alejandra Pinto Erazo, Manuel Sanchez, Raquel Thomas, Philine Werner, Silas Wolf, Freddy and the residents of the Bora village of Colonia, the Iwokrama International Centre for Rain Forest Conservation and Development and the forestry department of the Guyana Forestry Commission (GFCPRDD) kindly supported fieldwork. For providing information on and granting access to material deposited in museum collections, we are thankful to Darrel Frost (AMNH), Ned Gilmore (ANSP), Simon Loader and Jeff Streicher (BMNH), James Aparicio† (CBF), Miguel Urgilez, Gabriela Lagla, Diego Inclán and Francisco Prieto (DHEMCN- INABIO), María Elena Barragán-Paladines (FHGO), Joshua Mata (FMNH), Andrés Aponte, Khristian V. Valencia and Sandra P. Galeano (IAvH), Pedro M. Ruíz-Carranza† and Mauricio Rivera-Correa (ICN), Rafe Brown, William E. Duellman† and Ana Motta (KU), Consuelo Alarcón, Frank P. Condori, Maria I. Diaz, Sergio R. Mallqui, Luis Mamani, Raúl F. Quispe and Jhonatan A. Ttito (MUBI), Rocio Orellana (MHNC), Andreas Schmitz (MHNG),

Pierre-Yves Gagnier and Annemarie Ohler (MNHNP), Silke Schweiger and Georg Gassner (NHMW), Sven O. Kullaner (NHRM), Addison Wynn (USNM), Gregory G. Pandelis (UTA), and Wolfgang Böhme and Morris Flecks (ZFMK). David Dickey (AMNH) kindly provided copies of the 1977 field notes of C.W. Myers for Pebas. For permits related to Peruvian material, we thank the Instituto Nacional de Recursos Naturales (INRENA, Ministerio de Agricultura, Peru), number 010671, Servicio Nacional Forestal y de Fauna Silvestre (SERFOR-Peru), N° 581- 2011-AG-DGFFS-DGEFFS, N° 359-2013-MINAGRI-DGFFS-DGEFFS, RJ Nº001-2014-SERNANP-BPAM- JEF, N° 024-2016-SERFOR-DGGSPFFS, Resolución Directoral Nº 024-2017-SERFOR/DGGSPFFS, Resolución Directoral Nº D000039-2021-MIDAGRI-SERFOR-DGGSPFFS and D000123-2022-MIDAGRI-SERFOR- DGGSPFFS-DGSPFS. In Ecuador, our study was conducted under permits hMAE-DNBCM-2019-012, 001-13

IC-FAU-DNB/MA (collection) and 2018-001-FO-DPAP-MA, 2018-002-FO-DPAP-MA (export), all issued by the Ministerio de Ambiente de Ecuador. For work related to Colombian material, we are grateful to Programas de Investigación INV-2021-128-2323 from the Facultad de Ciencias , Universidad de los Andes , research permit resolution No. 0425 of 2015 from Corpamag and the research permit of the Universidad del Magdalena, resolution No 1293 of 2013 from Autoridad Nacional de Licencias Ambientales de Colombia. For permits related to Guyanan material, we thank the EPA Guyana for research permit number 180609 BR 112 . For permits related to sampling in Venezuela, we thank Ministerio del Poder Popular para El Ambiente , Oficio 3500- 17/08/2009. For imaging, Synchrotron SOLEIL provided beamtime via proposals no. 20220353 and no. 20221502. ANATOMIX is an Equipment of Excellence (EQUIPEX) funded by the Investments for the Future program of the French National Research Agency ( ANR), project NanoimagesX, grant no. ANR-11-EQPX-0031. We further thank the team of CeMIM ( USM 0504 View Materials , Department RDDM, MNHN) for access to their three-dimensional workstation. For funding, we are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/ Universal ), grant number 401120/2016-3, Coordenação de Aperfeiçoamento do Pessoal de Nível Superior ( CAPES), ( Programa de Excelência Acadêmica CAPES / PROEX), grant number 0616/ 2018m, CAPES in partnership with Fundação de Amparo à Pesquisa do Estado do Amazonas ( CAPES / FAPEAM), grant number 24/2014, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro ( FAPERJ), grant number 11/2023 - E-26/200.216 and 200.217/2024 to R. F.J., Scholarship Grant INV- 2018-34-1281 from the Universidad de los Andes, Colombia and Grant 757 of 2016 from Colciencias to L.A. R.S., Universidad San Francisco de Quito USFQ ( HUBI 5467 , 17857 ) to J.M.G., Agence Nationale de la Recherche (Stapes project: ANR-16-CE02-0016; CEBA: ANR-10-LABX-25-01; TULIP: ANR-10-LABX-0041; ANAEE-France: ANR-11-INBS-0001) to R.B. and A.F., Ministry for Foreign Affairs of Finland and Conservation International Foundation (BioCuencas project) to J.C.C. M., German Academic Exchange Service ( DAAD) and Deutsche Forschungsgemeinschaft ( DFG; ER 589 /2-1) to R.E., Mohamed bin Zayed Species Conservation Fund (project number 0925417) to P.J. V., The Field Museum to R. V.M., NSF Postdoctoral Research Fellowship in Biology ( DBI 2109344 ) to D.J.P. An anonymous reviewer, Jörn Köhler and the editor made valuable comments on the original manuscript which helped to improve this paper .

5 References

Asquith, A. & Altig, R. (1987) Atelopus spumarius. Vocalization. Herpetological Review, 18, 32–33.

Ballestas, O., Lampo, M. & Rodríguez, D. (2021) Living with the pathogenic chytrid fungus: Exploring mechanisms of coexistence in the harlequin toad Atelopus cruciger. PLoS ONE, 16, e0254439.

https://doi.org/10.1371/journal.pone.0254439

Barbour, T. (1905) The vertebrata of Gorgona Island, Colombia: Reptilia; Amphibia. Bulletin of the Museum of Comparative Zoology, 46, 98–102.

Benson, D.A., Clark, K., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J. & Sayers, E.W. (2015) GenBank. Nucleic Acids Research, 43, D30.

https://doi.org/10.1093/nar/gku1216

Biton, R., Boistel, R., Rabinovich, S.G., Gafny, S., Brumfeld, V. & Bailon, S. (2016) Osteological observations on the Alytid Anura Latonia nigriventer with comments on functional morphology, biogeography, and evolutionary history. Journal of Morphology, 277, 1131–1145.

https://doi.org/10.1002/jmor.20562

Boistel, R., Grosjean, S. & Lötters, S. (2005) Tadpole of Atelopus franciscus from French Guyana, with comments on other larvae of the genus (Anura: Bufonidae). Journal of Herpetology, 39, 148–153.

https://doi.org/10.1670/0022-1511(2005)039[0147:TOAFFF]2.0.CO;2

Boistel, R., Swoger, J., Kržič, U., Fernandez, V., Gillet, B. & Reynaud, E.G. (2011) The future of three‐dimensional microscopic imaging in marine biology. Marine Ecology, 32, 438–452.

https://doi.org/10.1111/j.1439-0485.2011.00442.x

Boistel, R., Aubin, T., Cloetens, P., Peyrin, F., Scotti, T., Herzog, P., Gerlach, J., Pollet, N. & Aubry, J.-F. (2013) How minute sooglossid frogs hear without a middle ear. Proceedings of the Academy of Natural Sciences of Philadelphia, 110 (38), 15360–15364.

https://doi.org/10.1073/pnas.1302218110

Boulenger, G.A. (1882) Catalogue of the Batrachia Salientia s. Ecaudata in the collection of the British Museum. 2 nd Edition. Taylor and Francis, London, 503 pp.

Böning, P., Lötters, S., Barzaghi, B., Bock, M., Bok, B., Bonato, L., Ficetola, G.F., Glaser, F., Griese, J., Grabher, M., Leroux, C., Gopikrishna, M., Manenti, R., Ludwig, G., Preininger, D., Rödel, M.-O., Seibold, S., Smith, S., Tiemann, L., Thein, J., Veith, M. & Plewnia, A. (2024) Alpine salamanders at risk? The current status of an emerging fungal pathogen. PLoS ONE, 19, e0298591.

https://doi.org/10.1371/journal.pone.0298591

Bravo-Valencia, L. & Rivera-Correa, M. (2011) A new species of harlequin frog (Bufonidae: Atelopus) with an unusual behaviour from Andes of Colombia. Zootaxa, 3045 (1), 57–67.

https://doi.org/10.11646/zootaxa.3045.1.4

Bush, M.B. (1994) Amazonian speciation: a necessarily complex model. Journal of Biogeography, 21, 5–17.

https://doi.org/10.2307/2845600

Byrne, A.Q., Richards-Zawacki, C.L., Voyles, J., Bi, K., Ibáñez, R. & Rosenblum, E.B. (2020) Whole exome sequencing identifies the potential for genetic rescue in iconic and Critically Endangered Panamanian harlequin frogs. Global Change Biology, 27, 50–70.

https://doi.org/10.1111/gcb.15405

Cannatella, D.C. (1981) A new Atelopus from Ecuador and Colombia. Journal of Herpetology, 15, 133–138.

https://doi.org/10.2307/1563371

Carrillo-Bilbao, G. & Martin-Solano, S. (2013) Nuevo registro de distribución de Atelopus spumarius Cope, 1871 (Anura: Bufonidae) para Ecuador. Revista Latinoamericana de Conservación, 3, 48–50.

Cocroft, R.B., McDiarmid, R.W., Jaslow, A.P. & Ruiz-Carranza, P.M. (1990) Vocalizations of eight species of Atelopus (Anura: Bufonidae) with comments on communication in the genus. Copeia, 1990, 631–643.

https://doi.org/10.2307/1446428

Coloma, L.A. (1997) Morphology, Systematics, and Phylogenetic Relationships among Frogs of the Genus Atelopus (Anura: Bufonidae). Doctoral Dissertation, University of Kansas, Lawrence, 267 pp.

Coloma, L.A., Lötters, S. & Salas, A. (2000) Taxonomy of the Atelopus ignescens complex (Anura: Bufonidae): Designation of a neotype of Atelopus ignescens and recognition of Atelopus exiguus. Herpetologica, 56, 303–324.

Coloma, L.A. (2002) Two new species of Atelopus (Anura: Bufonidae) from Ecuador. Herpetologica, 58, 229–252.

https://doi.org/10.1655/0018-0831(2002)058[0229:TNSOAA]2.0.CO;2

Coloma, L.A., Lötters, S., Duellman, W.E. & Miranda-Leiva, A. (2007) A taxonomic revision of Atelopus pachydermus, and description of two new (extinct?) species of Atelopus from Ecuador (Anura: Bufonidae). Zootaxa, 1557 (1), 1–32.

https://doi.org/10.11646/zootaxa.1557.1.1

Coloma, L.A., Duellman, W.E., Almendáriz C., A., Ron, S.R., Terán-Valdez, A. & Guayasamin, J.M. (2010) Five new (extinct?) species of Atelopus (Anura: Bufonidae) from Andean Colombia, Ecuador, and Peru. Zootaxa, 2574 (1), 1–54. https://doi. org/10.11646/zootaxa.2574.1.1

Cope, E.D. (1871) Ninth contribution to the herpetology of Tropical America. Proceedings of the Academy of Natural Sciences of Philadelphia, 23, 200–224.

Costa-Campos, C.E. & Carvalho, T. (2018) The advertisement call of the Hoogmoed’s harlequin toad Atelopus hoogmoedi Lescure, 1974 from northern Brazil (Anura, Bufonidae). Zootaxa, 4521 (1), 141–144.

https://doi.org/10.11646/zootaxa.4521.1.11

Crump, M.L. (1986) Homing and site fidelity in a Neotropical frog, Atelopus varius (Bufonidae). Copeia, 1986, 438–444.

Culebras, J., Novales,A., Quezada Riera,A.B., Medina, D. & Plewnia,A. (2023) New records of the glassfrog Hyalinobatrachium iaspidiense (Ayarzagüena, 1992) in western Amazonia. Herpetology Notes, 16, 179–182.

https://doi.org/10.2307/1445001

Da Silva, G., Cornélio, G.S., de Oliveira, E.A., Trindade, N., França, I. & Hernández Ruz, E. (2020) A candidate species currently classified as Atelopus hoogmoedi (Anura: Bufonidae) in the eastern Amazon, Pará, Brazil. Genetics and Molecular Research, 19, gmr18392.

https://doi.org/10.4238/gmr18392

De la Riva, I., Castroviejo-Fisher, S., Chaparro, J.C., Boistel, R. & Padial, J.M. (2011) A new species of Atelopus (Anura: Bufonidae) from the Amazonian slopes of the Andes in south-eastern Peru. Salamandra, 47, 161–168.

De Magalhães, R.F., Rocha, P.C., Santos, F.R., Strüssmann, C. & Giaretta, A.A. (2018) Integrative taxonomy helps to assess the extinction risk of anuran species. Journal for Nature Conservation, 45, 1–10.

https://doi.org/10.1016/j.jnc.2018.07.001

De Queiroz, K. (1998) The general lineage concept of species, species criteria, and the process of speciation: a conceptual unification and terminological recommendations. In: Raman, S. (Ed.), Endless Forms: Species and Speciation. Oxford University Press, Oxford, pp. 57–75.

Dole, J.W. & Durant, P. (1974) Movements and seasonal activity of Atelopus oxyrhynchus (Anura:Atelopodidae) in a Venezuelan Cloud Forest. Copeia, 1974, 230–235.

https://doi.org/10.2307/1443028

dos Santos Dias, P.H. & Anganoy-Criollo, M. (2024) Harlequin frog tadpoles—comparative buccopharyngeal morphology in the gastromyzophorous tadpoles of the genus Atelopus (Amphibia, Anura, Bufonidae), with discussion on the phylogenetic and evolutionary implication of characters. The Science of Nature, 111 (1), 3.

https://doi.org/10.1007/s00114-024-01889-6

Duellman, W.E. & Lynch, J.D. (1969) Descriptions of Atelopus tadpoles and their relevance to atelopodid classification. Herpetologica, 25, 231–240.

Fabrezi, M. & Alberch, P. (1996) The carpal elements of anurans. Herpetologica, 52, 188–204.

Flechas, S.V., Sarmiento, C. & Amézquita, A. (2012) Bd on the beach: high prevalence of Batrachochytrium dendrobatidis in the lowland forest of Gorgona Island (Colombia, South America). EcoHealth, 9, 298–302.

https://doi.org/10.1007/s10393-012-0771-9

Flechas, S.V., Vredenburg, V.T. & Amézquita, A. (2015) Infection prevalence in three lowland species of harlequin toads from the threatened genus Atelopus. Herpetological Review, 46, 528–532.

Fouquet, A., Recoder, R., Teixeira Jr, M., Cassimiro, J., Amaro, R.C., Camacho, A., Damasceno, R., Carnaval, A.C., Moritz, C. & Rodrigues, M.T. (2012) Molecular phylogeny and morphometric analyses reveal deep divergence between Amazonia and Atlantic Forest species of Dendrophryniscus. Molecular Phylogenetics and Evolution, 62, 826–838.

https://doi.org/0.1016/j.ympev.2011.11.023

Frost, D.R. (2024) Amphibian species of the world: an online reference.Version 6.1.Available from: https://amphibiansoftheworld. amnh.org/ (accessed 21 April 2024)

Frost, D.R. & Kluge, A.G. (1994) A consideration of epistemology in systematic biology, with special reference to species. Cladistics, 10, 259–294. [http://hdl.handle.net/2027.42/31356]

Gascon, C. (1989) The tadpole of Atelopus pulcher Boulenger (Anura, Bufonidae) from Manaus, Amazonas. Revista Brasileira de Zoologia, 6, 235–239.

https://doi.org/10.1590/S0101-81751989000200007

Gosner, K.L. (1960) A simplified table for staging Anura embryos and larvae with notes on identification. Herpetologica, 16, 183–190.

Gray, P. & Cannatella, D.C. (1985) A new species of Atelopus (Anura, Bufonidae) from the Andes of northern Perú. Copeia, 1985, 910–917.

https://doi.org/10.2307/1445241

Guayasamin, J.M., Bonaccorso, E., Duellman, W.E. & Coloma, L.A. (2010) Genetic differentiation in the nearly extinct harlequin frogs (Bufonidae: Atelopus), with emphasis on the Andean Atelopus ignescens and A. bomolochos species complexes. Zootaxa, 2574 (1), 55–68.

https://doi.org/10.11646/zootaxa.2574.1.2

Guindon, S., Dufayard, J.-F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology, 59, 307–321.

https://doi.org/10.1093/sysbio/syq010

Hausdorf, B. (2011) Progress towards a general species concept. Evolution, 64, 923–931.

https://doi.org/10.1111/j.1558-5646.2011.01231.x

Herrera-Alva, V., Díaz, V., Castillo, E., Rodolfo, C. & Catenazzi, A. (2020) A new species of Atelopus (Anura: Bufonidae) from southern Peru. Zootaxa, 4853 (3), 404–420. https://doi.org/10.11646/zootaxa.4853.3.4

Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q. & Vinh L.S. (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution, 35, 518–522.

https://doi.org/10.1093/molbev/msx281

Ibáñez D., R., Jaramillo, C.A. & Solís, F.A. (1995) Una especie nueva de Atelopus (Amphibia: Bufonidae) de Panama. Caribbean Journal of Science, 31, 57–64.

IUCN (2012) IUCN Red List Categories and Criteria. Version 3.1. 2 nd Edition. IUCN, Gland, 32 pp.

IUCN (2023) The IUCN Red List of Threatened Species. Version 2023 -1. Available from: https://www.iucnredlist.org (accessed 22 December 2023)

Jaynes, K.E., Páez-Vacas, M.I., Salazar-Valenzuela, D., Guayasamin, J.M., Terán-Valdez, A., Siavichay, F.R., Fitzpatrick, S.W. & Coloma, L.A. (2022) Harlequin frog rediscoveries provide insights into species persistence in the face of drastic amphibian declines. Biological Conservation, 276, 109784.

https://doi.org/10.1016/j.biocon.2022.109784

Jorge, R.F., Magnusson, W.E., Da Silva, D.A., Polo, É.M. & Lima, A.P. (2020 a) Urban growth threatens the lowland Amazonian Manaus harlequin frog which represents an evolutionarily significant unit within the genus Atelopus (Amphibia: Anura: Bufonidae). Journal of Zoological Systematics and Evolutionary Research, 58, 1195–1205. https://doi.org/10.1111/ jzs.12390

Jorge, R.F., Ferrão, M. & Lima, A.P. (2020 b) Out of bound: A new threatened harlequin toad (Bufonidae, Atelopus) from the outer borders of the Guiana Shield in central Amazonia described through integrative taxonomy. Diversity, 12, 310. https:// doi.org/10.3390/d12080310

Jorge, R.F., Lima, A.P. & Stow, A.J. (2022) Selection and localised genetic structure in the threatened Manauense harlequin frog (Bufonidae: Atelopus manauensis). Conservation Genetics, 23, 559–574.

https://doi.org/10.1007/s10592-022-01436-8

Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haeseler, A. & Jermiin, L.S. (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods, 14, 587–589.

https://doi.org/10.1038/nmeth.4285

Kok, P.J.R., Ratz, S., MacCulloch, R.D., Lathrop, A., Dezfoulian, R., Aubret, F. & Means, D.B. (2018) Historical biogeography of the palaeoendemic toad genus Oreophrynella (Amphibia: Bufonidae) sheds a new light on the origin of the Pantepui endemic terrestrial biota. Journal of Biogeography, 45, 26–36.

https://doi.org/10.1111/jbi.13093

Köhler, J., Jansen, M., Rodríguez, A., Kok, P.J.R., Toledo, L.F., Emmrich, M., Glaw, F., Haddad, C.F.B., Rödel, M.-O. & Vences, M. (2017) The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice. Zootaxa, 4251 (1), 1–124.

https://doi.org/10.11646/zootaxa.4251.1.1

Kimura, M. (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111–120.

https://doi.org/10.1007/BF01731581

Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35, 1547–1549.

https://doi.org/10.1093/molbev/msy096

Langer, M., Boistel, R., Pagot, E., Cloetens, P. & Peyrin, F. (2010) X-ray in-line phase microtomography for biomedical applications. Microscopy: Science, Technology, Applications and Education, 1, 391–402.

La Marca, E. (1983) A new frog of the genus Atelopus (Anura: Bufonidae) from a Venezuelan cloud forest. Contributions to Biology and Geology of the Milwaukee Public Museum, 54, 1–12.

La Marca, E., Lips, K.R., Lötters, S., Puschendorf, R., Ibáñez, R., Rueda-Almonacid, J.V., Schulte, R., Marty, C., Castro, F., Manzanilla-Puppo, J., García-Pérez, J.E., Bolaños, F., Chaves, G., Pounds, J.A., Toral, E. & Young, B.E. (2005) Catastrophic population declines and extinctions in Neotropical harlequin frogs (Bufonidae: Atelopus). Biotropica, 37, 190–201.

https://doi.org/10.1111/j.1744-7429.2005.00026.x

Lecocq, T., Vereecken, N.J., Michez, D., Dellicour, S., Lhomme, P., Valterová, I., Rasplus, J.-Y. & Rasmont, P. (2013) Patterns of genetic and reproductive traits differentiation in mainland vs. Corsican populations of bumblebees. PLoS ONE, 8, e65642.

https://doi.org/10.1371/journal.pone.0065642

Lescure, J. (“ 1973 ” 1974) Présence d’une sous-espéce d’ Atelopus pulcher (Amphibien, Anoure) dans les Guyanes: Atelopus pulcher hoogmoedi. Bulletin du Muséum National d’Histoire Naturelle, 3, 997–1005.

Lescure, J. (“ 1973 ” 1974) Contribution à l’étude des amphibiens de Guyane Française. 1. Notes sur Atelopus flavescens Duméril et Bibron et description d’une nouvelle espèce. Vie et Milieu. Série C, Biologie Terrestre, 23, 125–141.

Lescure, J. (1981) Contribution à l’étude des amphibiens de Guyane française. VIII. Validation d’ Atelopus spumarius Cope, 1871, et désignation d’un néotype. Description d’ Atelopus spumarius barbotini nov. ssp. Données étho-écologiques et biogéographiques sur les Atelopus du groupe flavescens (Anoures, Bufonidés). Bulletin du Muséum National d’Histoire Naturelle, 4, 893–910.

Lescure, J. & Gasc, J.P. (1986) Partage de l’espace forestier par les amphibiens et les reptiles en Amazonie du nord-ouest. Caldasia, 15, 707–723.

Lips, K.R., Diffendorfer, J., Mendelson III, J.R. & Sears, M.W. (2008) Riding the wave: reconciling the roles of disease and climate change in amphibian declines. PLoS Biology, 6, e72.

https://doi.org/10.1371/journal.pbio.0060072

Lötters, S. (1996) The Neotropical Toad Genus Atelopus. Checklist—Biology—Distribution. M. Vences & F. Glaw, Cologne. 143 pp.

Lötters, S. & De la Riva, I. (1998) Redescription of Atelopus tricolor Boulenger from southeastern Peru and adjacent Bolivia, with comments on related forms. Journal of Herpetology, 32, 481–488.

https://doi.org/10.2307/1565201

Lötters, S., Haas, W., Schick, S. & Böhme, W. (2002 a) On the systematics of the harlequin frogs (Amphibia: Bufonidae: Atelopus) from Amazonia. II: Redescription of Atelopus pulcher (Boulenger, 1882) from the eastern Andean versant in Peru. Salamandra, 38, 165–184.

Lötters, S., Haas, W., Schick, S. & Böhme, W. (2002 b) On the systematics of the harlequin frogs (Amphibia: Bufonidae: Atelopus) from Amazonia. I: Description of a new species from the Cordillera Azul, Peru. Salamandra, 38, 95–104.

Lötters, S. (2003) On the systematics of harlequin frogs (Amphibia: Bufonidae: Atelopus) from Amazonia. III:A new, remarkably dimorphic species from the Cordillera Azul, Peru. Salamandra, 39, 169–180.

Lötters, S., La Marca, E. & Vences, M. (2004) Redescriptions of two toad species of the genus Atelopus from coastal Venezuela. Copeia, 2004, 221–233.

Lötters, S., Schulte, R., Córdova, J.H. & Veith, M. (2005) Conservation priorities for harlequin frogs (Atelopus spp.) of Peru. Oryx, 39, 343–346.

Lötters, S., van der Meijden, A., Rödder, D., Koester, T.E., Kraus, T., La Marca, E., Haddad, C.F.B. & Veith, M. (2010) Reinforcing and expanding the predictions of the disturbance vicariance hypothesis in Amazonian harlequin frogs: a molecular phylogenetic and climate envelope modelling approach. Biodiversity and Conservation, 19, 2125–2146.

https://doi.org/10.1007/s10531-010-9869-y

Lötters, S., van der Meijden, A., Coloma, L.A., Boistel, R., Cloetens, P., Ernst, R., Lehr, E. & Veith, M. (2011) Assessing the molecular phylogeny of a near extinct group of vertebrates: the Neotropical harlequin frogs (Bufonidae; Atelopus). Systematics and Biodiversity, 9, 45–57.

https://doi.org/10.1080/14772000.2011.557403

Lötters, S., Mebs, D., Köhler, G., Vargas, J. & La Marca, E. (2019) The voice from the hereafter: vocalisations in three species of Atelopus from the Venezuelan Andes, likely to be extinct. Herpetozoa, 32, 267–275.

https://doi.org/10.3897/herpetozoa.32.e39192

Lötters, S., Plewnia, A., Hönig, A., Jung, A., Laudor, J. & Ziegler, T. (2022) The gastromyzophorous tadpole of the pink harlequin frog from Suriname with comments on the taxonomy of Guianan clade Atelopus (Amphibia, Bufonidae). Zootaxa, 5087 (4), 591–598.

https://doi.org/10.11646/zootaxa.5087.4.7

Lötters, S., Plewnia, A., Böning, P., Jung, A., Pinto Erazo, M.A., Velásquez-Trujillo, D.A., Veith, M., La Marca, E., Catenazzi, A., Shepack, A., Neam, K., Valencia, L.M., Acosta-Galvis, A.R., Alarcon Vela, Y., Allen, J.P., Alfaro Segundo, J.O., Almendáriz Cabezas, A. de L., Alvarado Barboza, G., Alves-Silva, K.R., Anganoy-Criollo, M., Rodrigues, M.T., Arbeláez Ortiz, E., Arpi Lojano, J.D., Siavichay Pesántez, F.R., Arteaga, A., Ballestas, O., García Méndez, C.Z., Lampo, M., Barrera Moscoso, D., Herrera-Alva, V., Torres-Ccasani, G., Barros-Castañeda, J.D., Pérez-González, J.L., Rocha-Usuga, A., Rojas Montaño, S., Rueda Solano, L.A., Villalba-Fuentes, J., Batista, A., Quiroz-Espinoza, M., Bernal, M.H., Betancourt, E., López-Perilla, Y.R., Medina-Rangel, G.F., Rivera-Correa, M., da Cunha Bitar, Y.O., Bravo-Valencia, L., Cáceres Andrade, J.F., Cadenas, D., Chaparro Auza, J.C., Chaves-Portilla, G.A., Chávez, G., Roca-Rey Ross, B., Coloma, L.A., Quezada Riera, A.B., Terán-Valdez, A., Cortez-Fernandez, C.F., Courtois, E.A., Culebras, J., De la Riva, I., Díaz, V., Elizondo Lara, L.C., Ernst, R., Flechas, S.V., Foch, T., Fouquet, A., García-Pérez, J.E., Gómez-Hoyos, D.A., Gomides, S.C., Guerrel, J., Gratwicke, B., Guayasamin, J.M., Griffith, E., Ibáñez, R., Idrovo, C.I., Jiménez Monge, A., Jorge, R.F., Klocke, B., Lehr, E., Lewis, C.H.R., Lindquist, E.D., Mazepa, G., Merino Viteri, A., Mulder, K., Pacheco-Suarez, M., Pereira-Muñoz, A., Tovar-Siso, P.C., Pisso Florez, A.G., Ponce, M., Poole, V., Quiroz, A.J., Ramírez Guerra, A., Ramírez, J.P., Reichle, S., Reizine, H., Rössler, D.C., Señaris, C., Sorokin, A., Venegas, P.J., von May, R. & Webster Bernal, J.F. (2023) Ongoing harlequin toad declines suggest the amphibian extinction crisis is still an emergency. Communications Earth and Environment, 4, 412.

https://doi.org/10.1038/s43247-023-01069-w

Luedtke, J.A., Chanson, J., Neam, K., Hobin L., Maciel, A.O., Catenazzi, A., Borzée, A., Hamidy, A., Aowphol, A., Jean, A., Sosa-Bartuano, A., Fong G., A., de Silva, A., Fouquet, A., Angulo, A., Kidov, A.A., Muñoz Saravia, A., Diesmos, A.C., Tominaga, A., Shrestha, A., Gratwicke, B., Tjaturadi, B., Martínez Rivera, C.C., Vásquez Almazán, C.R., Señaris, C., Chandramouli, S.R., Strüssmann, C., Cortez Fernández, C.F., Azat, C., Hoskin, C.J., Craig Hilton-Taylor, C., Whyte, D.L., Gower, D.J., Olson, D.H., Cisneros-Heredia, D.F., Santana, D.J., Nagombi, E., Najafi-Majd, E., Quah, E.S.H., Bolaños, F., Xie, F., Brusquetti, F., Álvarez, F.S., Andreone, F., Glaw, F., Castañeda, F.E., Kraus, F., Parra-Olea, G., Chaves, G., Medina-Rangel, G.F., González-Durán, G., Ortega-Andrade, H.M., Machado, I.F., Das, I., Ribeiro Dias, I., Urbina-Cardona, J.N., Crnobrnja-Isailović, J., Yang, J.H., Jianping, J., Wangyal, J.T., Rowley, J.J.L., Measey, J., Vasudevan, K., Chan, K.O., Gururaja, K.V., Ovaska, K., Warr, L.C., Canseco-Márquez, L., Toledo, L.F., Díaz, L.M., Khan, M.M.H., Meegaskumbura, M., Acevedo, M.E., Napoli, M.F., Ponce, M.A., Vaira, M., Lampo, M., Yánez-Muñoz, M.H., Scherz, M.D., Rödel, M.O., Matsui, M., Fildor, M., Kusrini, M.D., Ahmed, M.F., Rais, M., Kouamé, N.G., García, N., Gonwouo, N.L., Burrowes, P.A., Imbun, P.Y., Wagner, P., Kok, P.J.R., Joglar, R.L., Auguste, R.J., Albuquerque Brandão, R., Ibáñez, R., von May, R., Hedges, S.B., Biju, S.D., Ganesh, S.R., Wren, S., Das, S., Flechas, S.V., Ashpole, S.L., Robleto-Hernández, S.J., Loader, S.P., Incháustegui, S.J., Garg, S., Phimmachak, S., Richards, S.J., Slimani, T., Osborne-Naikatini, T., Abreu-Jardim, T.P.F., Condez, T.H., De Carvalho, T.R., Cutajar, T.P., Pierson, T.W., Nguyen, T.Q., Kaya, U., Yuan, Z., Long, B., Langhammer, P. & Stuart, S.N. (2023) Ongoing declines for the world’s amphibians in the face of emerging threats. Nature, 622, 308–314.

https://doi.org/10.1038/s41586-023-06578-4

Lynch, J.D. (1986) Notes on the reproductive biology of Atelopus subornatus. Journal of Herpetology, 20, 126–129.

Lynch, J.D. (1993) A new harlequin frog from the Cordillera Oriental of Colombia (Anura, Bufonidae, Atelopus). Alytes, 11, 77–87.

Marcillo-Lara, A., Coloma, L.A., Álvarez-Solas, S. & Terneus, E. (2020) The gastromyzophorous tadpoles of Atelopus elegans and A. palmatus (Anura: Bufonidae), with comments on oral and suction structures. Neotropical Biodiversity, 6, 1–13.

https://doi.org/10.1080/23766808.2019.1709378

McDiarmid, R.W. (1971) Comparative morphology and evolution of frogs of the Neotropical genera Atelopus, Dendrophryniscus, Melanophryniscus and Oreophrynella. Bulletin of the Los Angeles County Museum of Natural History, 12, 1–66.

McDiarmid, R.W. (1973) A new species of Atelopus (Anura, Bufonidae) from northeastern South America. Contributions in Science. Natural History Museum of Los Angeles County, 240, 1–12.

Minh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M. D., van Haeseler, A. & Lanfear R. (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution, 37, 1530–1534.

https://doi.org/10.1093/molbev/msaa015

Miyata, K. (1980) A new species of Atelopus (Anura: Bufonidae) from the cloud forests of northwestern Ecuador. Breviora, 458, 1–10.

Moraes, L.J.C.L., Werneck, F.P., Réjaud, A., Rodrigues, M.T., Prates, I., Glaw, F., Kok, P.J.R., Ron, S.R., Chaparro, J.C., Osorno-Muñoz, M., Dal Vechio, F., Recoder, R.S., Marques-Souza, S., Rojas, R.R., Demay, L., Hrbek, T. & Fouquet, A. (2022) Diversification of tiny toads (Bufonidae: Amazophrynella) sheds light on ancient landscape dynamism in Amazonia. Biological Journal of the Linnean Society, 136, 75–91.

https://doi.org/10.1093/biolinnean/blac006

Noonan, B.P. & Gaucher, P. (2005) Phylogeography and demography of Guianan harlequin toads (Atelopus): diversification within a refuge. Molecular Ecology, 14, 3017–3031. https://doi.org/10.1111/j.1365-294X.2005.02624.x

Osorno-Muñoz, M., Ardila-Robayo, M.C. & Ruíz-Carranza, P. (2001) Tres nuevas especies del género Atelopus A.M.C. Dumeril & Bibron 1841 (Amphibia: Bufonidae) de las partes altas de la Cordillera Oriental Colombiana. Caldasia, 23, 509–522.

Padial, J.M., A. Miralles, I. De la Riva & M. Vences (2010) The integrative future of taxonomy. Frontiers in Zoology, 7, 16.

https://doi.org/10.1186/1742-9994-7-16

Patzelt, E. (1989) Fauna del Ecuador. Banco Central del Ecuador, Quito, 433 pp.

Pearson, K.C. & Tarvin, R.D. (2022) A review of chemical defense in harlequin toads (Bufonidae: Atelopus). Toxicon, 13, 100092.

Pérez-Gonzalez, J.L., Rada, M., Vargas-Salinas, F. & Rueda-Solano, L.A. (2020) The tadpoles of two Atelopus species (Anura: Bufonidae) from the Sierra Nevada de Santa Marta, Colombia, with notes on their ecology and comments on the morphology of Atelopus larvae. South American Journal of Herpetology, 15, 47–62.

Peters, J.A. (1973) The frog genus Atelopus in Ecuador. Smithsonian Contributions to Zoology, 145, 1–49.

Plewnia, A., Lötters, S., Gomides, S., De Agrò, M. & Rössler, D.C. (2024 a) Sex-specific ventral dichromatism and melanization in harlequin toads (Atelopus): a common but overlooked character of unknown function. Evolutionary Ecology, 38, 571– 583.

https://doi.org/10.1007/s10682-024-10288-2

Plewnia, A., Terán-Valdez, A., Culebras, J., Boistel, R., Paluh, D.J., Quezada Riera, A.B., Heine, C.H., Reyes-Puig, J.P., Salazar-Valenzuela, D., Guayasamin, J.M. & Lötters, S. (2024 b) A new species of harlequin toad (Bufonidae: Atelopus) from Amazonian Ecuador. Salamandra, 60, 237–253.

Pons, J., Barraclough, T.G., Gomez-Zurita, J., Cardoso, A., Duran, D. P., Hazell, S., Kamoun S., Sumlin, W.D. & Vogler, A.P. (2006) Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology, 55, 595– 609. https://doi.org/10.1080/10635150600852011

Portik, D.M., Streicher, J. & Wiens, J.J. (2023) Frog phylogeny: A time-calibrated, species-level tree based on hundreds of loci and 5,242 species. Molecular Phylogenetics and Evolution, 188, 107907.

https://doi.org/10.1016/j.ympev.2023.107907

Pramuk, J.B. (2006) Phylogeny of South American Bufo (Anura: Bufonidae) inferred from combined evidence. Zoological Journal of the Linnean Society, 146, 407–452.

https://doi.org/10.1111/j.1096-3642.2006.00212.x

Puillandre, N., Brouillet, S. & Achaz, G. (2020) ASAP: Assemble Species by Automatic Partitioning. Molecular Ecolology Resources, 21, 609–620.

https://doi.org/10.1111/1755-0998.13281

Rambaut, A., Drummond, A.J., Xie, D., Baele, G. & Suchard, M.A. (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67, 901–904.

https://doi.org/10.1093/sysbio/syy032

Ramírez, J., Jaramillo, C., Lindquist, E., Crawford, A. & Ibáñez, R. (2020) Recent and rapid radiation of the highly endangered harlequin frogs (Atelopus) into Central America inferred from mitochondrial DNA sequences. Diversity, 12, 360.

https://doi.org/10.3390/d12090360

Read, M. (2018) Monitoreo biológico Yasuní. Anfibios y reptiles. Vol. 4. Ecuambiente Consulting Group, Quito, 248 pp.

Ringler, M., Himmel, T., Werner, P., Luger, M., Rössler, D.C., Ringler, E. & Lötters, S. (2022) Male life history of a harlequin toad population in French Guiana. Salamandra, 58, 171–186.

Rivero, J.A. (1963) Five new species of Atelopus from Colombia, with notes on other forms from Colombia and Ecuador. Caribbean Journal of Science, 3, 103–124.

Rivero, J.A. (1968) More on the Atelopus (Amphibia, Salientia) from western South America. Caribbean Journal of Science, 8, 19–28.

Rivero, J.A. & Granados Díaz, H. (1993) Nueva especie de Atelopus (Amphibia: Bufonidae) del Departamento del Cauca, Colombia. Caribbean Journal of Science, 29, 12–17.

Roček, Z., Přikryl, T., Baleeva, N., Vazeille, A., Boistel, R., Bravin, A., Nemoz, C., van Dijk, E., Smirina, E.M. & Claessens, L. (2016) Contribution to the head anatomy of the basal frog Barbourula busuangensis and the evolution of the Anura. Russian Journal of Herpetology, 23, 163–194.

Rocha Usuga, A.A., Vargas Salinas, F. & Rueda-Solano, L.A. (2017) Not every drought is bad: Quantifying reproductive effort in the harlequin frog Atelopus laetissimus (Anura: Bufonidae). Journal of Natural History, 51, 1913–1928. https://doi. org/10.1080/00222933.2017.1355075

Rössler, D.C., Lötters, S., Mappes, J., Valkonen, J.K., Menin, M., Lima, A.P. & Pröhl, H. (2019) Sole coloration as an unusual aposematic signal in a Neotropical toad. Scientific Reports, 9, 1128.

https://doi.org/10.1038/s41598-018-37705-1

Ron, S., Bustamante, M., Coloma, L.A. & Mena, B. (2009) Sapos. Museo de Zoología, Pontificia Universidad Católica del Ecuador, Balsa de Sapos, Fundación Otonga, Amphibian Ark, Quito, 256 pp.

Ronquist, F. & Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572–1574.

https://doi.org/10.1093/sysbio/46.1.195

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling,A., Höhna, S., Larget, B., liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539–542.

https://doi.org/10.1093/sysbio/sys029

Rueda-Almonacid, J.V., Rodríguez-Mahecha, J.V., La Marca, E., Lötters, S., Kahn, T. & Angulo, A. (Eds.) (2005) Ranas Arlequines. Serie Libretas de Campo. 5. Conservación Internacional Colombia, Bogotá, 158 pp.

Rueda-Solano, L.A. (2012) Atelopus carrikeri Ruthven, 1916 (Anura: Bufonidae) una especie policromatica. Herpetotropicos, 8, 61–66.

Rueda-Solano, L.A., Vargas Salinas, F., Gonzalez Pérez, J.I., Sanchez A., Ramírez, A., Navas C.A. & Crawford A.J. (2022) Mate-guarding behavior in anurans: intra-sexual selection and the evolution of prolonged amplexus in the harlequin toad Atelopus laetissimus. Animal Behaviour, 185, 127–142.

https://doi.org/10.1016/j.anbehav.2021.12.003

Ruíz-Carranza, P.M. & Hernández-Camacho, J.I. (1978) Una nueva especie Colombiano de Atelopus (Amphibia: Bufonidae). Caldasia, 12, 181–197.

Ruíz-Carranza, P.M. & Osorno-Muñoz, M. (1994) Tres nuevas especies de Atelopus A.M.C. Duméril & Bibron 1841 (Amphibia: Bufonidae) de la Cordillera Central de Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales, 19, 165–179.

Savage, J.M. (1972) The harlequin frogs, genus Atelopus, of Costa Rica and western Panama. Herpetologica, 28, 77–94.

Savage, J.M. & Heyer, R.W. (1997) Digital webbing formulae for anurans: a refinement. Herpetological Review, 28, 131.

Sexton, O. (1958) Observations of the life history of a Venezuelan frog, Atelopus cruciger. Acta Biologica Venezuelica, 2, 235–242.

Standish, I., Leis, E., Schmitz, N., Credico, J., Erickson, S., Bailey, J., Kerby, J., Phillips, K. & Lewis, T. (2018) Optimizing, validating, and field testing a multiplex qPCR for the detection of amphibian pathogens. Diseases of Aquatic Organisms, 129, 1–13.

Sueur, J., Aubin, T. & Simonis, C. (2008) Seewave: a free modular tool for sound analysis and synthesis. Bioacoustics, 18, 213–226.

Tarvin, R.D., Peña, P. & Ron, S.R. (2014) Changes in population size and survival in Atelopus spumarius (Anura: Bufonidae) are not correlated with chytrid prevalence. Journal of Herpetology, 48, 291–197.

Valencia, L.M. & Marin da Fonte, L.F. (2022) Collaborative work brings hope for threatened harlequin toads. Oryx, 56, 12.

Van Bocxlaer, I., Loader, S.P., Roelants, K., Biju, S.D., Menegon, M. & Bossuyt, F. (2010) Gradual adaptation toward a rangeexpansion phenotype initiated the global radiation of toads. Science, 327, 679–682.

https://doi.org/10.1126/science.1181707

Vélez-Rodríguez, C.M. & Ruíz-Carranza, P.M. (1997) Una nueva especie de Atelopus (Amphibia: Anura: Bufonidae) de la Cordillera Central, Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales, 21, 555– 563.

Vences, M., Miralles, A., Brouillet, S., Ducasse, J., Fedosov, A., Kharchev, V., Kostadinov, I., Kumari, S., Patmanidis, S., Scherz, M.D., Puillandre, N. & Renner, S.S. (2021) iTaxoTools 0.1: Kickstarting a specimen-based software toolkit for taxonomists. Megataxa, 6, 77–92.

https://doi.org/10.11646/megataxa.6.2.1

Venegas, P.J., Catenazzi, A., Siu-Ting, K. & Carrillo, J. (2008) Two new harlequin frogs (Anura: Atelopus) from the Andes of northern Peru. Salamandra, 44, 163–176.

Veselý, M. & Batista, A. (2021) A new species of Atelopus (Amphibia: Bufonidae) from eastern Panama. Zoological Research, 42, 272–279.

https://doi.org/10.24272/j.issn.2095-8137.2020.319

Weitkamp, T., Scheel, M., Perrin, J., Daniel, G., King, A., Le Roux, V., Giorgetta, J.-L., Carcy, A., Langlois, F., Desjardins, K., Menneglier, C., Cerato, M., Engblom, C., Cauchon, G., Moreno, T., Rivard, C., Gohon, Y. & Polack, F. (2022) Microtomography on the ANATOMIX beamline at Synchrotron SOLEIL. Journal of Physics: Conference Series, 2380, 012122.

https://doi.org/10.1088/1742-6596/2380/1/012122

Wiley, E.O. (1978) The evolutionary species concept reconsidered. Systematic Biology, 27 (1), 17–26.

https://doi.org/10.2307/2412809

Wiley, E.O. & Liebermann, B.S. (2011) Phylogenetics: The Theory and Practice of Phylogenetic Systematics. 2 nd Edition. Wiley-Blackwell, Hoboken, New Jersey, 406 pp.

Womack, M.C., Christensen-Dalsgaard, J., Coloma, L.A. & Hoke, K.L. (2018) Sensitive high-frequency hearing in earless and partially eared harlequin frogs (Atelopus). Journal of Experimental Biology, 221, jeb169664.

https://doi:10.1242/jeb.169664

Zanette, I., Daghfous, G., Weitkamp, T., Gillet, B., Adriaens, D., Langer, M., Cloetens, P., Helfen, L., Bravin, A., Peyrin, F., Baumbach, T., Dischler, J.-M., Van Loo, D., Praet, T., Poirier-Quinot, M. & Boistel, R. (2014) Looking inside marine organisms with magnetic resonance and X‐ray imaging. In: Reynaud, E.G. (Ed.), Imaging Marine Life: Macrophotography and Microscopy Approaches for Marine Biology. Wiley-Blackwell, Hoboken, New Jersey, pp. 122–184.

6 Appendices

The following appendices can be downloaded from https://figshare.com, available under:

DOI https://doi.org/10.6084/m9.figshare.27610098.v1

Appendix 1. List of samples, primers and models.

Appendix 2. List of Atelopus species, clade allocation and baseline information for a future integrative taxonomic approach.

Appendix 3. Comparative material examined in addition to the taxa described in this paper.

Appendix 4. Additional information on osteological studies.

Appendix 5. Overview of amphibians tested for the presence of the chytrid fungus Batrachochytrium dendrobatidis in this study.

Appendix 6. Additional phylogenetic reconstructions (ML, BI) for single mt markers.

Appendix 7. Haplotype networks for the nc markers POMC and RAG1 created with Hapsolutely (Vences et al. 2021).

Appendix 8. Estimates of evolutionary divergence between 16S sequences: uncorrected p-distances.

Appendix 9. Results of Assemble Species by Automatic Partitioning software (ASAP), based on 16S alignment, suggesting 40 subsets that are potential species.