A third species of glassfrog in the genus Chimerella (Anura, Centrolenidae) from central Peru, discovered by an integrative taxonomic approach Author Koehler, Joern https://orcid.org/0000-0002-5250-2542 Hessisches Landesmuseum Darmstadt (HLMD), Friedensplatz 1, 64283 Darmstadt, Germany joern.koehler@hlmd.de Author Venegas, Pablo J. https://orcid.org/0000-0002-6501-4492 Rainforest Partnership, 4005 Guadalupe St., Austin, TX 78751, USA & Instituto Peruano de Herpetologia (IPH), Augusto Salazar Bondy 136, Urb. Higuereta, Surco, Lima, Peru Author Castillo-Urbina, Ernesto https://orcid.org/0000-0002-8293-758X Universidad Nacional Mayor de San Marcos, Museo de Historia Natural (MUSM), Departamento de Herpetologia, Av. Arenales 1256, Lima 11, Peru Author Glaw, Frank https://orcid.org/0000-0003-4072-8111 Zoologische Staatssammlung Muenchen (ZSM-SNSB), Muenchhausenstr. 21, 81247 Muenchen, Germany Author Aguilar-Puntriano, Cesar https://orcid.org/0000-0001-6372-7926 Universidad Nacional Mayor de San Marcos, Museo de Historia Natural (MUSM), Departamento de Herpetologia, Av. Arenales 1256, Lima 11, Peru Author Vences, Miguel https://orcid.org/0000-0003-0747-0817 Zoological Institute, Technische Universitaet Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany text Evolutionary Systematics 2023 2023-05-16 7 2 195 209 http://dx.doi.org/10.3897/evolsyst.7.102950 journal article http://dx.doi.org/10.3897/evolsyst.7.102950 2535-0730-2-195 FCC50241B78C4EDB8991B7410EBF186A 5603DEC1CC7F53E0BB3028EA9604ECF0 Chimerella mira sp. nov. Type material. Holotype . MUSM 40278 (FGZC 6233), adult male (Fig. 3a-c ), from a point approximately 16 km airline west of Tingo Maria ( 09°18.09'S , 76°08.71'W , 798 m above sea level), close to the settlement "Corvina Colorada", at the bank of the Rio Patay Rondos, Provincia Leoncio Prado, Departamento Huanuco , Peru, collected on 6 November 2019 by Ernesto Castillo-Urbina, Frank Glaw and Joern Koehler . Figure 3. Chimerella mira sp. nov. from west of Tingo Maria in life: male holotype (MUSM 40278, FGZC 6233) in a frontal, b dorsolateral, and c ventral views; male paratype (MUSM 40264, FGZC 6215) in d lateral and e ventral views. Paratype . MUSM 40264 (FGZC 6215), adult male (Fig. 3 ), same data as holotype, but collected on 5 November 2019. Etymology. The specific epithet is a Latin adjective (feminine form) meaning 'surprising' . It refers to the fact that this species surprisingly turned out to be undescribed, after at first impression in the field having been tentatively identified as C. corleone . Definition. A species in the genus Chimerella , based on molecular relationships and shared morphological traits, characterized by the following combination of characters: (1) dentigerous processes of vomer and vomerine teeth absent; (2) snout truncate in dorsal view, truncate in lateral profile; canthus rostralis straight in dorsal view, rounded in cross-section; nostrils flush with surrounding skin; (3) tympanum and tympanic annulus evident, round, its diameter about 25% of eye diameter; supratympanic fold weakly defined, not concealing upper tympanum; (4) dorsal skin finely shagreened, with few minute scattered dorsal tubercles; skin on venter and ventral surfaces of thighs granular; (5) a pair of enlarged subcloacal warts; (6) ventral parietal peritoneum transparent (condition P0 sensu Cisneros-Heredia and McDiarmid 2007 ); iridophores in pericardium and peritonea covering digestive tract; kidneys and urinary bladder lacking iridophores (condition V5); (7) liver with two broadly rounded right/left lobes, not forming free flaps, covered by iridophores (condition H1); (8) humeral spine and single subgular vocal sac present in adult males; (9) webbing absent between fingers I and II, basal webbing between fingers II and III; webbing formula II2--3-III2+-2+IV; (10) webbing between toes extensive; webbing formula I1+-2+II1+-2.5III1+-3-IV3--1+V; (11) enamelled fringe present on postaxial edge of finger IV; ulnar fold diffuse; tarsal fold absent; enlarged tubercles on ventrolateral edges of arm and tarsus absent; (12) concealed prepollex, not enlarged, prepollical spine not projecting; nuptial pad absent, but diffuse nuptial excrescence formed by glandular clusters (Type V); (13) finger I slightly longer than finger II; (14) diameter of eye three times wider than width of disc on finger III; (15) in life, dorsum yellow-green with small round scattered pale-yellowish flecks; venter transparent white; bones green; (16) in preservative, dorsum lavender with small scattered round cream flecks; dorsal surfaces of limbs yellowish cream, with scattered melanophores; ventral surfaces yellowish cream; (17) in life, iris silvery white with fine black spotting, and a dark brown median streak formed by fine spots; circumpupillary ring absent; (18) dorsal surfaces of fingers and toes lacking melanophores, except for toes IV and V; (19) males call from the upper surface of leaves; calls consist of 2-3 high-pitched pulsed notes ( 'Trii' calls sensu Duarte-Marin et al. 2022 ), 42-85 ms note duration, 160-239 ms inter-note interval duration within calls; dominant frequency 5543-6135 Hz; (20) fighting behavior unknown (but probably present in males; see below); (21) egg clutches unknown; (22) tadpoles unknown; (23) minute body size (sensu Guayasamin et al. 2020 ), SVL in adult males 18.1-19.6 mm (n = 2); females unknown. Diagnosis. The new species is morphologically most similar to C. corleone . However, it differs from C. corleone by fine dark spots in the iris in life (versus dark reticulation; Figs 4 , 5 ), a truncate snout in lateral profile (versus slightly rounded; Fig. 6 ), tarsal fold absent (versus present as a line of low white warts on the lateral edge of tarsus), greyish-lavender dorsal colour in preservative (versus greyish-green), a dispersed network of melanophores on dorsal surfaces resulting in a light yellow-green colour in life (versus a very dense network of melanophores on dorsal surfaces, resulting in dark green life colouration; Figs 3 , 4 ), a call consisting of pulsed 'Trii' notes (sensu Duarte-Marin et al. 2022 ) with 42-85 ms duration (versus simple 'Tic' notes of 10-15 ms duration), and substantial differentiation in certain molecular markers. The new species mainly differs from C. mariaelenae by a yellow-green dorsum with small round scattered yellowish flecks (versus green dorsum with black flecks and punctuation; Fig. 7 ), greyish-lavender dorsum with small round cream flecks in preservative (versus pale lavender with dark lavender flecks), silvery white iris in life (versus orange to reddish iris), an advertisement call consisting of pulsed 'Trii' notes with 42-85 ms duration (versus simple 'Tic' notes of 4-7 ms duration; Guayasamin et al. 2020 ), and substantial differentiation in certain molecular markers. Figure 4. Chimerella corleone in life: a dorsolateral view of amplectant couple from the type locality photographed at night (note the striking dark reticulation of the iris); b dorsolateral and c ventral views of the male holotype (CORBIDI 10467). Courtesy of J. Delia and E. Twomey. Figure 5. Comparison of eye colouration in life: a male holotype (MUSM 40278) and b male paratype (MUSM 40264) of Chimerella mira sp. nov.; c male holotype (CORBIDI 10467) and d female paratype (CORBIDI 10465) of Chimerella corleone (courtesy of J. Delia and E. Twomey). Note the fine dark spotting versus dark reticulation in the iris. Figure 6. Lateral views of heads of preserved holotypes of a Chimerella mira sp. nov. (MUSM 40278) and b Chimerella corleone (CORBIDI 10467; courtesy of E. Twomey). Orange lines indicate outline of snout shape in lateral profile. Not to scale. Figure 7. Amplectant couple of Chimerella mariaelenae from the Cordillera de Kampankis, 1100 m a.s.l., Departamento Amazonas, Peru, in life. Description of the holotype. Adult male, SVL 19.6 mm, in good state of preservation (Fig. 8 ). HW about 1/5 wider than body; HW 29% of SVL; HW 1.15 times HL. Snout truncate in dorsal view, truncate in lateral profile (Fig. 6a ); END/ED 0.65; END/IOD 0.55. Loreal region concave, nostrils flush with surrounding skin, round; internarial region concave anterodorsally; canthus rostralis well defined, straight in dorsal view, rounded in cross-section. Eyes directed anterolaterally, angled 51° relative to midline of body (where anteriorly facing eyes would be 90° relative to midline); ED 3.0 times wider than width of disc on finger III; ED 41% of HL and 100% of IOD. Tympanum noticeable with tympanic annulus visible, more evidently ventrally than dorsally, annulus and membrane coloured as dorsum; supratympanic fold weakly defined leaving entire tympanum visible, tympanum round with slight dorsal inclination. Dentigerous processes on vomers absent; dentigerous process on premaxillae and maxillae present; choanae large, circular, separated more widely than nostrils; tongue removed for tissue sample; vocal slits present, wide, oblique and lateral to the tongue. Forelimbs moderately robust, with forearm flattened and roughly 1.4 times as wide as arm; ulnar fold present, low diffuse, white; tubercles on ventrolateral edge of arm absent; humeral spine externally visible as an elongated bump, slightly less defined in preservative than in life. Relative length of fingers: II <I <IV <III; finger discs distinctly expanded, those on fingers I and II rounded, on fingers III and IV slightly truncate, larger than toe discs; width of disc on finger III 32% of ED; webbing absent between fingers I and II, basal webbing between fingers II and III, webbing formula II2--3-III2+-2+IV. Prepollex concealed; subarticular tubercles round, evident; supernumerary tubercles absent, palmar tubercle round and small, thenar tubercle barely distinct, minute, ovoid; nuptial pads absent, but diffuse nuptial excrescence formed by glandular clusters (Type V sensu Guayasamin et al. 2020 ). Hind limbs slender, TL 51% of SVL; tarsal fold absent; tubercles on ventrolateral edge of tarsus absent. Relative length of toes: I <II <III <V <IV; toe discs slightly expanded, round; inner metatarsal tubercle narrow, elongated, ovoid, slightly protruding; outer metatarsal tubercle not visible. Webbing formula of feet: I1+-2+II1+-2.5III1+-3-IV3--1+V. Dorsal skin finely shagreened, with few small scattered cream coloured tubercles on dorsum and dorsal surfaces of limbs; skin on venter and ventral sides of thighs granular, skin on throat smooth; cloacal opening at level of upper thighs, concealed by faint superior dermal fold; a pair of round, low, unpigmented subcloacal warts present on ventral side; crenulated flaps absent. Figure 8. Preserved male holotype of Chimerella mira sp. nov. (MUSM 40278, FGZC 6233) in a dorsal and b ventral views. Measurements (in mm). SVL 19.6, HL 5.8, HW 6.7, TD 0.7, IND 1.5, IOD 2.7, ED 2.7, EW 1.2, END 1.5, HaL 5.4, TL 9.9, THL 10.8, FL 7.9. In life (Fig. 3 ), dorsal surfaces translucent yellow-green, with small, round, widely scattered yellowish cream flecks on dorsum and dorsal surfaces of thighs; dorsal surfaces of hands and feet yellow-green; dorsal surfaces of finger and toe discs orange-yellow; area around nostrils, dorsal surfaces of arms and thighs dusted with minute dark melanophores; upper lip with a narrow tan line anteriorly that vanishes posteriorly; venter transparent, whitish; throat transparent with a turquoise tint; ventral surfaces of limbs lemon green; ventral surfaces of finger and toe discs orange-yellow; greyish-white diffuse line along lateral edge of proximal ulna; diffuse white spots in cloacal area; subcloacal wart ornamentation transparent; parietal peritoneum transparent; pericardium, hepatic peritonea and visceral peritonea white, urinary bladder transparent; iris silvery white with fine black spotting, median brown streak formed by densely spaced fine spots, circumpupillary ring absent, posterior iris periphery white. After three years in preservative, dorsum greyish-lavender with scattered small cream flecks; dorsal surfaces of limbs yellowish cream with minute scattered melanophores; dorsal surfaces of hand and fingers yellowish cream; dorsal surfaces of feet and toes yellowish cream, with scattered melanophores extending on dorsal surfaces of toes IV and V; posterior surfaces of thighs yellowish cream; venter and ventral surfaces of arms and legs yellowish cream, throat cream (Fig. 8 ). Variation. Overall, the male paratype MUSM 40264 is rather similar to the holotype. In life, it had a slightly paler green dorsal colouration, with fewer and less distinct minute yellowish flecks on dorsum. The throat lacked the turquoise tint and the brown median streak in the iris was less distinctly expressed when compared to the holotype (Fig. 3 ). Measurements (in mm) of the paratype are as follows: SVL 18.1, HL 5.5, HW 6.7, TD 0.6, IND 1.7, IOD 2.4, ED 2.4, EW 1.3, END 1.4, HaL 5.2, TL 10.7, THL 10.7, FL 7.6. The male paratype was dissected for inspection of internal organs which appear as follows: liver with two broadly rounded right/left lobes sagittally divided, not forming free flaps, completely covered in iridophores (white), corresponding to state H1 sensu Cisneros-Heredia and McDiarmid (2007) . Gall bladder, pericardium, liver and gastrointestinal peritoneum covered in iridophores (white). Kidneys and urinal bladder are tan in colour and thus not covered by iridophores. Testes ovoid and partially covered in a white iridophore reticulum. Distribution of iridophores in visceral peritonea falls into state V5 sensu Cisneros-Heredia and McDiarmid (2007) . Natural history, distribution, and threat status. Both males were collected at the stream bank of the Rio Patay Rondos, a medium-sized tributary of the Rio Monzon, which itself is part of the Huallaga river system. The habitat consisted of a swampy area, apparently temporarily flooded by the river, with small lentic waterbodies, emerging shrub vegetation and younger trees (Fig. 9 ). Shortly after dusk, male individuals were sitting on upper surfaces of leaves approximately 0.5 to 2.5 m above the ground while calling during light rainfall. Some fine transverse scratches were visible on the anterior dorsum of the paratype MUSM 40264, arguing for the occurrence of male-male fighting behaviour (e.g., Hutter et al. 2013b ). Egg clutches and larvae are unknown. Anuran species found in sympatry were Boana lanciformis , Leptodactylus griseigularis , and Adenomera sp. The glassfrog Hyalinobatrachium carlesvilai occurred at nearby sites within a few hundred metres distance. So far, the species is only known from the type locality at an elevation of 798 m, but might be more widespread in the Huallaga River basin at similar elevations. Because population size, actual range, and thus potential threats are unknown, we propose the IUCN Red List status 'Data Deficient' for C. mira (see also Scherz et al. 2019 ). Figure 9. Type locality and habitat of Chimerella mira sp. nov. on the bank of the Rio Patay Rondos, a tributary of the Rio Monzon: a view to the east along the river bed. The yellow arrow indicates the area where both reported specimens were collected; b night view of the swampy habitat at the edge of the river showing shrub vegetation from which males were calling. Advertisement call. Calls were emitted at somewhat irregular intervals and occurred in 'waves' with several males calling nearly synchronously. The advertisement calls recorded on 5 November 2019 at the type locality (estimated air temperature ca. 25 °C; recording distance approximately 1.5 m) consist of 2 to 3 high-pitched, pulsed notes of short duration (Fig. 10a ). Notes exhibit considerable amplitude modulation, with maximum call energy present at the beginning of the note, continuously decreasing towards its end. Pulse structure is rather irregular within notes, with pulses being partly fused and of differing amplitude. As a consequence, the total number of pulses per note is not reliably countable, but in most cases 3 to 4 distinctly separated pulses are evident at the beginning of each note, followed by about 10-12 less distinctly separated pulses. We observed pulse rate within notes to range approximately around 200 pulses/second. Other numerical parameters of 12 analysed calls from 4 individuals are as follows: number of notes per call 2-3 (2.8 +/- 0.5); call duration 322-707 ms (552.1 +/- 141.8 ms); note duration 42-85 ms (64.6 +/- 11.7 ms); inter-note interval within calls 160-239 ms (197.0 +/- 26.4 ms); call repetition rate approximately 1.5-1.9 calls/minute; dominant frequency 5543-6135 Hz (5897 +/- 148 Hz); prevalent bandwidth 4500-7500 Hz, with weak call energy present up to 21 kHz. Among the notes within a call, dominant frequency is highest in the first note and slightly decreases in subsequent notes. The character of this call would qualify as a 'Trii' call according to the definition of Duarte-Marin et al. (2022) . Figure 10. Audiospectrograms and corresponding oscillograms of calls of Chimerella : a Chimerella mira sp. nov. (call voucher MUSM 40264) from west of Tingo Maria, Departamento Huanuco , Peru. Below an expanded oscillogram depicting the first note of the call; b Chimerella corleone from the type locality, Departamento San Martin , Peru. Below an expanded oscillogram depicting the first note of the call. Both recordings high-pass filtered at 1000 Hz. Comparative call data. The only available call recording of Chimerella corleone is that described by Twomey et al. (2014) recorded from a topotypic male in captivity after dislodging it from the female with which it has been in amplexus. These conditions argue for the call representing a mating call, not an advertisement call (J. Delia, pers. comm.) and thus comparison should be regarded with some reservation. However, as mentioned by Twomey et al. (2014) , advertisement calls heard in the field were rather similar in character. We re-analysed the available recording of the single call using the methodology described above (for recording equipment used see Twomey et al. 2014 ). The call (Fig. 10b ) has the following numerical parameters: number of notes per call 2; call duration 521 ms; note duration 10 and 15 ms; inter-note interval 493 ms; dominant frequency of the first note 6485 Hz, and 6526 Hz in the second note; prevalent bandwidth difficult to determine due to oversaturated recording level, but call energy is apparently present up to 20 kHz. The character of this call would qualify as a 'Tic' call according to the definition of Duarte-Marin et al. (2022) . Calls of C. mariaelenae from Pangayaku Creek (929 m a.s.l.), Provincia Napo, Ecuador, have been described by Guayasamin et al. (2020) . The calls (call duration 231-1761 ms) contain 2-10 unpulsed high-pitched notes of very short duration (4-7 ms), repeated at comparatively short intervals. Dominant frequency ranged from 6718-8010 Hz ( Guayasamin et al. 2020 ). Also, Batallas and Brito (2016) described the call of C. mariaelenae , from Sangay National Park (1750 m a.s.l.), Provincia Morona Santiago, Ecuador. Their analysis described call parameters quite different from those reported by Guayasamin et al. (2020) , with calls always containing 3 notes (call duration 668-808 ms) and much longer note durations of 54-116 ms. The differences among these two call descriptions for calls of C. mariaelenae are beyond those usually considered to represent inter-specific call variation (see Koehler et al. 2017 ) and would argue for the calls belonging to different species. However, although apparently rare in centrolenids (see Duarte-Marin et al. 2022 ), the calls described could also refer to two different call types of C. mariaelenae . Batallas and Brito (2016) described the males calling in dense choruses containing numerous males, thus territorial and/or aggressive function of the calls recorded could be an explanation. With the data at hand, we are unable to clarify the reason for these call differences described for C. mariaelenae , but for our diagnosis above we here relied on the description provided by Guayasamin et al. (2020) . However, if considering the calls described by Batallas and Brito (2016) to represent the advertisement call of C. mariaelenae , there would be some overlap in numerical parameters with calls of C. mira , but the distinct pulsed amplitude structure present in C. mira has not been observed in calls described by the mentioned authors.