A new diploid species belonging to the Odontophrynus americanus species group (Anura: Odontophrynidae) from the Espinhaço range, Brazil Author Garcia, Paulo Christiano De Anchietta text Zootaxa 2017 2017-10-06 4329 4 327 350 journal article 31876 10.11646/zootaxa.4329.4.2 dda6f3e2-723b-40b6-a098-75ac90b036c1 1175-5326 1002988 8A5Ed9Ea-Dad8-46Ee-A7Af-F9C4Ac164838 Odontophrynus juquinha sp. nov. ( Figs. 1–4 ; Table 1 ) Odontophrynus americanus ; Eterovick & Sazima (2000) : 443 ; Eterovick & Fernandes (2001) : 687 ; Eterovick & Fernandes (2002) : 34 ; Eterovick & Barros (2003) : 443 ; Eterovick (2003): 222; Eterovick & Sazima (2004) : 99 ; Nascimento et al. (2005) : 146 ; Soma et al. (2006) : 23 ; Leite et al. (2008) : 171 ; Eterovick et al. (2010) : 8 ; Pimenta & Camara (2015) : 218 . Odontophrynus sp.; Rosset et al. (2006) : 472 . Holotype . UFMG 5516 ( Figs. 2–3 ), an adult male, collected by P. C. A. Garcia and F. Natali on 1 November 2010 at Serra do Cipó , Municipality of Santana do Riacho ( 19°15’29”S , 43°32'5”W , 1360 m a.s.l. ), Minas Gerais State, Brazil . Paratypes . Sixteen adult males: Municipality of Jaboticatubas , Minas Gerais State: UFMG 417 collected by F. T. M. C. Gomes on 3 November 1996 ; Municipality of Santana de Pirapama , Minas Gerais State: UFMG 11118 collected by H. Thomassen on 14 November 2011 ; Municipality of Santana do Riacho , Minas Gerais State: UFMG 419 collected by P. C. Eterovick on November 1996 , UFMG 420–1 collected by J. Cassimiro on January 2000 , UFMG 5511–2 and 5517 collected by P. C. A. Garcia and F. Natali on 1 November 2010 (same as the holotype), UFMG 5597–8 collected by P. P. G. Taucce and F. Leal on 2 November 2010 , UFMG 11171–2 collected by H. Thomassen , F. Leal and P. C. Rocha on 30 March 2012 , UFMG 13901–4 ( Fig. 1 , used for karotype) collected by P. C. Rocha and P. H. V. B. P. Silva on 14 October 2012 . Two adult females collected at Municipality Santana do Riacho , Minas Gerais State: UFMG 5518 collected by P. C. A. Garcia and F. Natali on 1 November 2010 (same as the holotype ) , UFMG 5596 collected by P. P. G. Taucce and F. Leal on 2 November 2010 . All paratypes were collected at Serra do Cipó , Minas Gerais State. FIGURE 1. Odontophrynus juquinha sp. nov. (paratype UFMG 13903; SVL = 46.2 mm); live adult male. Parque Nacional da Serra do Cipó, Municipality of Santana do Riacho, Minas Gerais State, Brazil. Photo by F. Leal. Referred specimens. Pico do Itobira, Municipality of Rio de Contas, Bahia State: one adult female ( UFMG 7824) collected by T. L. Pezzuti, L. O. Drummond, B. Imai and L. Rodrigues on 11 January 2010 . Pico das Almas, Municipality of Rio de Contas, Bahia State: two adult males ( UFMG 4470 and 4476) and three adult females ( UFMG 4469, 4471, and 4488) collected by F. S. F. Leite, M. R. Lindemann and R. B. Mourão on 11 January 2010 . Serra Nova, Municipality of Rio Pardo de Minas, Minas Gerais State: three adult males ( UFMG 6831, 6833, and 6875) and three adult females ( UFMG 6019–20, and 6832) collected by F. S. F. Leite on 16 and 17 December 2007 . Serra de Botumirim, Municipality of Botumirim, Minas Gerais State: one adult female ( UFMG 12250) collected by F. Leal and H. Thomassen on 8 January 2012 . Serra do Cabral, Municipality of Joaquim Felício, Minas Gerais State: four adult males ( UFMG 7277–80) and one adult female ( UFMG 7275) collected by T. L. Pezzuti and L. O. Drummond on 15 December 2007 ; four adult males ( UFMG 13945–8) used for karyotype; one adult male ( UFMG 13949) and one adult female ( UFMG 13950) collected by F. Leal and T. L. Pezzuti on 28 November 2012 . Serra do Cabral, Municipality of Buenópolis, Minas Gerais State: one adult male ( UFMG 7318) collected by T. L. Pezzuti and L. O. Drummond on 15 December 2007 ; four adult males ( UFMG 13927–30) and one adult female ( UFMG 13931) collected by F. Leal and T. L. Pezzuti on 27 November 2012 . Municipality of Diamantina, Minas Gerais State: three adult males ( UFMG 3957–9) collected by F. S. F. Leite and R. B. Mourão on 20 October 2008 . Alto Palácio, Municipality of Santana do Riacho, Minas Gerais State: five adult males ( CFBH 40116–20) and one adult female ( CFBH 40121) collected by C. F. B. Haddad, A. M. Haddad, K. Zamudio, H. Greene, C. P. A. Prado and F. Zara on 10 February 2016 ; one adult male ( CFBH 39947) collected by C. F. B. Haddad, A. Haddad, V. Mallerba, no collection date. Kilometer 113 (Serrotes), Serra do Cipó, Municipality of Santana do Riacho, Minas Gerais State: one adult female ( CFBH 00794) collected by C. F. B. Haddad and L. Castanho on 0 5 October 1989 . Serra do Cipó. Kilometer 132, Municipality of Santana do Riacho, Minas Gerais State: one adult male ( CFBH 35055) collected by J.P. Pombal Jr, O. C. Oliveira. Crossroad between MG10 and the road to Morro do Pilar, Serra do Cipó, Municipality of Santana do Riacho, Minas Gerais State: one adult male ( CFBH 39816) and one adult female ( CFBH 39822) C. F. B. Haddad, P. G. Taucce and A. Valencia on 0 3 February 2015 . TABLE 1. Measurements (in mm, reported as mean ± SD, range) of males and females of Odontophrynus juquinha sp. nov. type series. See Abbreviations List for definitions of trait acronyms.

Males (n=16)	Females (n=2)
Trait	Mean ± SD	Range	Mean ± SD	Range
SVL	44.27 ± 3.83	38.45–52.09	46.05 ± 1.46	45.01–47.08
HL	13.49 ± 1.33	11.50–16.00	13.73 ± 0.26	13.54–13.91
HW	17.46 ± 1.73	14.41–21.16	17.81 ± 0.04	17.78–17.84
IND	2.97 ± 0.35	2.04–3.40	2.65 ± 0.25	2.47–2.82
AMD	6.76 ± 0.55	5.90–8.32	6.41 ± 0.16	6.30–6.52
END	3.13 ± 0.20	2.55–3.37	3.37 ± 0.07	3.32–3.42
ED	4.77 ± 0.46	4.30–5.56	5.31 ± 0.30	5.09–5.52
AL	22.50 ± 2.65	18.47–29.89	22.98 ± 0.12	22.89–23.06
THL	16.40 ± 12.81	12.81–20.19	16.11 ± 0.30	15.89–16.32
TL	14.34 ± 1.19	12.66–16.66	15.33 ± 0.28	15.13–15.52
FL	26.74 ± 2.26	22.59–31.34	27.56 ± 0.27	27.37–27.75
MTL	4.33 ± 0.39	3.80–5.29	4.44 ± 0.11	4.36–4.52

Diagnosis. Odontophrynus juquinha sp. nov. is a medium-sized species belonging to the genus Odontophrynus based on a combination of morphological characters (i.e. head wider than long, snout truncate in profile, tympanum hidden, first subarticular tubercle on toe I enlarged, inner metatarsal tubercle large, tarsal fold short, granular skin on the dorsum and venter) ( Savage & Cei 1965 ; Cei 1987 ; Caramaschi 1996 ; Caramaschi & Napoli 2012 ). The new species belong to the Odontophrynus americanus species group based on the absence of large dorsal, tibial and forearm glandular warts, with postorbital, temporal, and parotoid glandular warts not distinctly developed but with a series of small glandular warts of irregular size and shape, forming glandular ridges longitudinally oriented, on postorbital-parotoid regions ( Fig. 2A , 5 ). Odontophrynus juquinha sp. nov. is distinguished from the remaining species belonging to the O. americanus species group by the following combination of characters: (1) medium sized (SVL = 38.45–52.09 mm in males in O . juquinha ); (2) head wider than long (HL/HW = 0.7–0.8); (3) dorsal dark brown blotches of small to medium-size with low contrast on a light brown background ( Fig. 5 ); (4) light mid-dorsal stripe absent or greatly interrupted in most of the specimens; (5) yellowish coloration of mid-dorsal stripe and background of both the head and the flanks of the body; (6) moderate number of glandular warts on dorsum; (7) diploid complement of 2n = 2x = 22 chromosomes with fundamental number FN = 44; (8) secondary constriction on the interstitial region of the short arm of the pair 4; (9) advertisement call with dominant frequency of 840–1080 Hz; (10) pulse rate of 90.5–106.7 pulses/s; (11) small tadpoles (mean TL = 24.30–35.69 mm ); (12) single lateral emargination on each side of oral disc; (13) caudal end acute; (14) 4–8 submarginal papillae in both sides of lip; and (15) internal wall of spiracle with a small free portion. FIGURE 2. Odontophrynus juquinha sp. nov. , UFMG 5516 (holotype), adult male in (A) dorsal and (B) ventral view. Comparison with other species. Odontophrynus juquinha sp. nov. is clearly distinguished from the species of O. cultripes group ( O . cultripes , O . carvalhoi , and O . monachus ) and O. occidentalis group ( O . occidentalis , O . barrioi , and O . achalensis ) by the absence of distinctly enlarged postorbital, temporal, and parotoid glandular warts (three or more pairs of enlarged rounded-oval postorbital, temporal, and parotoid glandular warts in O. cultripes and O. occidentalis species groups) (See Appendix 1 for complete list of examined material). It is distinguished from O . salvatori by its larger size (SVL> 38 mm ), supernumerary tubercles on hands and feet absent, dorsal surface of fingers and toes smooth, and nuptial pads present (SVL < 32 mm , supernumerary tubercles on hands and feet present, dorsal surface of fingers and toes rugose, and nuptial pads absent in O . salvatori ( Caramaschi 1996; present work ). Within the Odontophrynus americanus species group, O. juquinha sp. nov. is distinguished from O. americanus ( Fig. 5A ) by having a diploid complement of 2N = 2X = 22 chromosomes (tetraploid complement of 44 chromosomes in O. americanus ); and a secondary constriction (SC) on the interstitial region of the short arm of pair 4 (SCs on the proximal region of the short arm of chromosomes group 4 and/or on the interstitial region of the long arm of chromosomes group 11). Moreover, the advertisement call of O. juquinha sp. nov. distinguishes it from O. americanus by the higher pulse rate ( O. juquinha PR = 90.5–106.7 pulses/s, air temperature = 19.2°C; O. americanus PR = 73–77 pulses/s, air temperature = 20°C, Martino & Sinsch, 2002 ), and its tadpoles distinguish it by being smaller ( O. juquinha TL = 24.30–35.69 mm , stages 30–38, n = 6; O. americanus TL = 42 mm , stage 38– 39; Nascimento et al . 2013 and literature cited therein). Odontophrynus juquinha sp. nov. is distinguished from O . lavillai ( Fig. 5B ) by having dorsal dark brown blotches of small to medium-size with low contrast on a light brown background (two or three large dorsal dark brown blotches, transversally fused, with high contrast in all examined specimens of O. lavillai ) ( Fig. 1 , 4 and 5B ), by having a yellowish coloration in the mid-dorsal stripe and in the background of both the head and the flanks of the body (whitish or greyish coloration in O. lavillai ). Moreover, the advertisement call of O. juquinha sp. nov. distinguishes it from O. lavillai by the higher dominant frequency ( O. juquinha DF = 840–1080 Hz, SVL = 44.3 ± 3.8; O. lavillai DF = 637.1–790.2 Hz, SVL = 58.3 ± 4.3) and by the lower pulse rate ( O. juquinha PR = 90.5–106.7 pulses/s, air temperature = 19.2°C; O. lavillai PR = 107.2–132.5 pulses/s, air temperature = 25°C) (see Table 2 for call comparisons, see also Discussion section for comments on the possible influence of temperature). The tadpoles of the new species can be distinguished from O. lavillai by being smaller ( O. juquinha TL = 24.30–35.69 mm , stages 30–38; O. lavillai TL = 55.8 mm , stage 37; Lavilla & Scrocchi 1991 ), by having a single lateral emargination on each side of the oral disc (ventral and lateral emarginations in O. lavillai ), and by having 4–8 submarginal papillae (one in O. lavillai ). FIGURE 3. Odontophrynus juquinha sp. nov. , UFMG 5516 (holotype), adult male. (A) Left hand, (B) left foot, (C) lateral and (D) dorsal views of head. Odontophrynus juquinha sp. nov. is distinguished from O. cordobae ( Fig. 5C ) by having dorsal dark brown blotches of small to medium-size with low contrast on a light brown background (dorsal dark brown blotches with high contrast in all examined specimens of O. cordobae ) ( Fig. 1 , 4 , and 5C). It is also distinguished by the lack of a light mid-dorsal stripe, or greatly interrupted in most of the specimens (light mid-dorsal stripe slightly interrupted in O . cordobae ) ( Fig. 5C ). Whenever the mid-dorsal stripe is present, its yellowish coloration distinguishes the new species from O. cordobae (whitish or greyish coloration in O. cordobae ). Additionally, the new species is distinguished by the yellowish coloration in the background of both the head and the flanks of the body (whitish or greyish coloration in O. cordobae ). Moreover, the advertisement call of O. juquinha sp. nov. distinguishes it from O. cordobae by the lower pulse rate ( O. juquinha PR = 90.5–106.7 pulses/s, air temperature = 19.2°C; O. cordobae PR = 111–116 pulses/s, air temperature = 20°C) (see Table 2 for call comparisons, see also Discussion section for comments on the possible influence of temperature). The tadpoles of the new species are distinguished by being smaller than those of O. cordobae ( O. juquinha TL = 24.30–35.69 mm , stages 30–38, n = 6; O . cordobae TL = 40 mm , stage 37; Nascimento et al . 2013 and literature cited therein) and by having 4–8 submarginal papillae (single or paired in O. cordobae ). TABLE 2. Advertisement calls of species belonging to the Odontophrynus americanus group. Parameters presented as range.

Species	Note duration (ms)	Internote interval (s)	Pulses/ Note	Pulse duration (ms)	Interval between pulses (ms)	Pulse rate (pulses/s)	Dominant frequency (Hz)	Reference
O. juquinha sp. nov.	322–610	–	32–62	5–8	3–6	90.5–106.7	840–1080	This work
O. americanus	524–558	–	39–41	5.1–5.5	8.0–8.6	73–77	1025–1075	Martino & Sinsch (2002)
O. cordobae	421–475	–	48–53	3.3–3.7	5.1–5.7	111–116	990–1040	Martino & Sinsch (2002)
O. lavillai	301.5–583.0	1.8–8.4	40.0–62.5	4.5–7.0	1.8–5.0	107.2–132.5	637.1–790.2	Rosset & Baldo (2014)
O. maisuma	570–785	1.6–18.6	43–57	–	–	71.1–77.2	1124–1211	Borteiro et al. (2010)

Odontophrynus juquinha sp. nov. is distinguished from O. maisuma ( Fig. 5D ) by the head wider than long (HL/HW = 0.7–0.8, n = 16 males of O. juquinha ; HL/HW = 0.8–0.9, n = 12 males of O. maisuma , sensu Rosset 2008 ), by the high density of glandular warts on dorsum (few glandular warts in all examined specimens of O . maisuma ) ( Fig. 5D ) (See Appendix 1 for complete list of examined material), by having dorsal dark brown blotches of small to medium-size, with low contrast on a light brown background (one or two pairs of large, nearly symmetrical, longitudinal dorsal dark brown blotches with high contrast in O. maisuma ) ( Fig. 1 , 4 and 5D ), and by the lack of a light mid-dorsal stripe, or greatly interrupted in most of the specimens (light mid-dorsal stripe uninterrupted in all examined specimens of O . maisuma ) ( Fig. 5D ). Whenever the mid-dorsal stripe is present, its yellowish coloration distinguishes the new species from O. maisuma (whitish or greyish coloration in O. maisuma ). Furthermore, O. juquinha sp. nov. is distinguished by having yellowish coloration in the background of both the head and the flanks of the body (whitish or greyish coloration in O. maisuma ). The advertisement call of Odontophrynus juquinha sp. nov. distinguishes it from O. maisuma by its lower dominant frequency ( O. juquinha DF = 840–1080 Hz, SVL = 44.3 ± 3.8; O. maisuma DF = 1124–1211 Hz, SVL = 38.4 ± 2.2), and by the higher pulse rate ( O. juquinha PR = 90.5–106.7 pulses/s, air temperature = 19.2°C; O. maisuma PR = 71.1–77.2 pulses/s, air temperature = 6°C) (see Table 2 for call comparisons; see also Discussion section for comments on the possible influence of temperature). Tadpoles of the new species are smaller than those of O. maisuma ( O. juquinha TL = 24.30–35.69 mm , stages 30–38, n = 6; O. maisuma TL = 47 mm , stages 33–36; Borteiro et al. 2010 ) and have the caudal end acute (rounded in O. maisuma ). Additionally, the tadpoles of O. juquinha sp. nov. are distinguished from those of O. maisuma by having 4–8 submarginal papillae (one or few in O. maisuma ) and by having the internal wall of spiracle with a small free portion (entirely fused to the body in O. maisuma ). Description of the holotype . Body stout; head wider than longer, its length 77% of its width, 33% of SVL ( Fig. 2 ). Snout rounded in dorsal profile, between sloping and truncate in lateral view ( Fig. 3C, D ). Canthus rostralis slightly distinct, concave in dorsal view; loreal region slightly concave. Nostrils elliptic elongated, directed dorsolaterally, and situated at the tip of the snout in lateral profile; internarial distance 44% of distance between the anterior margins of eyes, 77% of eye-nostril distance. Eye large, prominent, 32% of head length, its diameter 40% longer than eye-nostril distance. Upper eyelid with distinct warts varying from rounded to elongated, external border of the upper eyelid with a glandular ridge. Tympanum hidden, not visible externally ( Fig. 3C, D ). Premaxillary and maxillary teeth present; tongue round, approximately half free and notched posteriorly; vomerine teeth in two patches slightly posteromedian to choanae. Vocal sac median, subgular; vocal slits present, longitudinal. Skin of dorsum granular, consisting of glandular warts with variable size, distributed scattered throughout the dorsum, and a series of distinct glandular warts, with irregular size and shape, forming small ridges longitudinally oriented, on postorbital-parotoid region. No distinct temporal glandular wart. Venter uniformly granular. Forelimbs relatively short, stout; glandular longitudinal ridge in the external part of the forearm ( Fig. 3C ). Lengths of fingers: III <V <II <IV, with a deformation in the tip of left finger III; fringes absent, webbing absent. Inner and outer metacarpal tubercles well developed; inner oval; outer divided, with the inner oval and the outer elongated; subarticular tubercles well developed, oval, slightly bilobated on fingers III and IV; supernumerary tubercles rounded, scattered distributed in the palm. Nuptial pad present on thumb. Hind limbs relatively short, stout; lengths of toes: I <II <V <III <IV; tarsal fold present; subarticular tubercles present, rounded, oval and enlarged on finger I. Inner metatarsal tubercle strong, shovel-like, nonkeratinized; outer metatarsal tubercle slightly distinct, rounded; supernumerary tubercles rounded, scattered distributed in the foot palm. Webbing formula: I 1– 2 II 1 1/2– 3 III 2 1/2– 4- IV 4–2+ V, with dermal fringes developed ( Fig. 3B ). Measurements of the holotype (mm). SVL 44.3, HL 14.6, HW 18.9, IND 2.6, AMD 6.3, END 3.4, ED 4.8, AL 23.15, THL 17.2, TL 15.0, FL 27.9, MTL 5.1. Coloration of the holotype . Unknown in life. In preservative, dorsal color predominantly dark brown ( Fig. 2 ). Light brown stripe between the eyes, resembling a “U” shape. Head and extremities dark brown in dorsal view. Light brown stripe dorsolaterally, starting at the medial third of the body toward the hind limb. Upper lip light brown; two dark brown botches connecting each nostril to the upper lip; two dark brown blotches connecting the eyes to the upper lip; four dark brown small blotches in the upper lip. Light brown stripe from the posterior part of the eye towards the forelimb insertion. Venter yellowish-colored, with scattered dark brown blotches gradually increasing on the sides of the body. Gular area with dark grey pigmentation. Coloration in life. Description based on adult male paratypes UFMG 13901, 13903–4, Municipality of Santana do Riacho, Minas Gerais State, Brazil ( Fig. 1 ). Dorsal surface of head, body and extremities dark brown. Yellowish stripe between the eyes, resembling a “U” shape. Yellowish stripe dorsolaterally, starting at the medial third of the body toward the hindlimb. Nut-brown blotches distributed below the dorsolateral yellowish stripe. Upper lip yellowish; two dark brown blotches connecting each nostril to the upper lip; two dark brown blotches connecting the eyes to the upper lip; four dark brown small blotches in the upper lip. Yellowish stripe from the posterior part of the eye towards the forelimb insertion. Venter whitish, with scattered grey blotches gradually increasing on the side of the body. Gular area whitish, crowded with dark grey blotches. Pupil horizontally elliptical; iris golden marbled with dark stripes; dark edges of eyes. The size and shape of the yellowish blotches varied in the analyzed individuals. Dark brown blotch bellow the eye well defined in UFMG 13901 and 13903, whereas blurred in UFMG 13904. A yellow mid-dorsal stripe well defined in UFMG 13904, interrupted in UFMG 13901 and 13903. Dorsum predominantly light brown in UFMG 13901, dark brown blotches surrounding the longitudinal glandular ridges. Variation. Adult males can be distinguished from adult females by the presence of clearly developed nuptial pads on thumb, and a dark grey-pigmented gular region at the vocal sac. Adult females are usually about the same size as males (SVL = 44.3 ± 3.8, 38.4–52.1 mm in males, n = 16; SVL = 46.1 ± 1.5, 45.0– 47.1 mm in females, n = 2). Full mid-dorsal stripe was present in only one individual (i.e. UFMG 13904; Fig. 4L ); an interrupted mid-dorsal stripe was present in some individuals (e.g. UFMG 417, 5511–2, 5596, 5598, and 13901) whilst others presented the mid-dorsal stripe only in the supra-cloacal region (e.g. UFMG 419, 420–1, 5518, 5597, 11118, 11171, and 13902–3) (see Fig. 4 for dorsal color variation). Some individuals presented the dorsum light brown in preservative, with dark brown blotches surrounding the longitudinal glandular ridges (e.g. UFMG 417, 5511–2, 5518, 11171, 13901). Some variation was observed in the outer metacarpal tubercle division, with individuals presenting it only partially divided (e.g. UFMG 5512, 5517), and others with a merely distinguishable division (e.g. UFMG 5518, 11171). Although the level of keratinization varied, all paratypes presented the inner metatarsal tubercle keratinized. FIGURE 4. Odontophrynus juquinha sp. nov. dorsal color variation. Paratypes UFMG (A) 5518 (female), (B) 419, (C) 420, (D) 11118, (E) 5598, (F) 5597, (G) 5512, (H) 5511, (I) 11172, (J) 13901, (K) 5596 (female), (L) 13904. FIGURE 5. Live specimens of (A) Odontophrynus americanus , (B) O . lavillai , (C) O . cordobae , and (D) O . maisuma . Karyotype. All examined specimens of Odontophrynus juquinha sp. nov. presented a diploid number of 2n = 2x = 22 chromosomes. All chromosomes were biarmed (fundamental number FN = 44) and the karyotype consisted of four large (1–4), three medium (5–7), and four small pairs (8–11) ( Fig. 6A ). Chromosome pairs 1, 5–7, and 11 were metacentric and chromosome pairs 2–4 and 8–10 were submetacentric. Pair 4 had an evident secondary constriction (SC) in the interstitial region of its short arm, which probably bears the nucleolus organizer region (NOR) as it has been observed in the other diploid species of the genus Odontophrynus (Barrio & Pistol de Rubel 1972 ; Ruiz et al. 1981 ; Rosset et al. 2006 ; Borteiro et al. 2010 ). Meiotic analysis revealed 11 ring bivalents with terminal chiasmata in diplotene, diakinesis, and metaphase I; and 11 dyads in metaphase II ( Fig. 6B–D ). FIGURE 6 . (A) Karyotype of a male Odontophrynus juquinha sp. nov. (2n = 2x = 22; FN = 44) after conventional Giemsa staining. Meiotic stages: (B) Diakinesis, (C) Metaphase I, (D) Metaphase II. Vocalizations. Advertisement call of Odontophrynus juquinha sp. nov. is characterized by a single pulsed note (n = 10 individuals), without frequency modulation and with variable duration (ND = 446.8 ± 61.0, 322–610 ms, n = 61 calls) and number of pulses (PN = 44.5 ± 5.7, 32–62 pulses/note, n = 61 calls) ( Fig. 7A , Table 3 ). Pulses had mean duration of 6 ± 0.8 ms (PD = 5–8 ms, n = 92 pulses), at regular intervals and constant rate (IPD = 4.3 ± 0.9, 3–6 ms, n = 85 intervals; PR = 99.4 ± 4.1, 90.5–106.7 pulses/s, n = 58 notes). FIGURE 7. (A) Advertisement call of Odontophrynus juquinha sp. nov. and (B) call including a suffix, unvouchered male from Serra do Cipó, municipality of Morro do Pilar, MG, 1 November 2010, air temperature 19.2°C. (C) Call including a prefix, unvouchered male from Serra do Cabral, municipality of Buenópolis, MG, 27 November 2012, air temperature 19.2°C. The calls from Serra do Cabral and Diamantina had a slightly higher dominant frequency then the one from Serra do Cipó. Other variables presented small variations that overlapped. We observed an affix in 18% of the analyzed notes (n = 11: six calls from two individuals recorded at Serra do Cipó, and five calls from one individual recorded at Serra do Cabral) ( Fig. 7B, C ). This affix was always part of the same note described as the advertisement call. Males displayed it either after, as a suffix (45.5%, n = 5; Fig. 6B ), or before, as prefix (54.5%, n = 6; Fig. 7C ). The affix presented short duration (Affix Duration = 99.8 ± 29.7, 58–149 ms, n = 11; Table 3 ) and less pulses than the advertisement call (PN = 10.1 ± 3.0, 6–16 pulses/note, n = 11). Affix’ pulses presented the same characteristics of the advertisement call (PD = 6.4 ± 0.9, 5–8 ms, n = 63; IPD = 4.3 ± 0.9, 3–6 ms, n = 55; PR = 101.7 ± 3.6, 97.3–107.3 pulses/s, n = 11). Dominant frequency is slightly lower than the advertisement call (DF = 890 ± 94, 796–984 Hz, n = 11). Despite the similarity between the affix and the advertisement call, they are clearly distinguishable in the spectrogram ( Fig. 7B, C ). Moreover, calls with an affix are audibly distinct than the common advertisement call. Although the affix seems to be related to the presence of other calling males, we did not test its social context. TABLE 3. Call parameters of Odontophrynus juquinha sp. nov. based on 61 calls from Serra do Cipó, Serra do Cabral, and from the municipality of Diamantina. Data are presented as mean ± SD, range. Note duration (ms) Pulses per note Dominant frequency (Hz) Mean ± SD Range Mean ± SD Range Mean ± SD Range All localities 446.8 ± 61.0 322–610 44.5 ± 5.7 32–62 941.1 ± 71.8 843.8–1078.1 Type locality (n = 35 calls; 19.2°C) 460.6 ± 50 360–610 45.5 ± 5.8 34–62 887.8 ± 38.7 843.8–937.5 Serra do Cabral (n = 20 calls) 396.5 ± 39.1 322–443 41.3 ± 4.5 32–47 1007.8 ± 35 984.4–1078.1 Diamantina (n = 6 calls) 533.5 ± 28.7 492–567 49.3 ± 2.5 46–52 1012.1 ± 52.7 947.5–1076.7 Affix (all localities) 99.8 ± 29.7 58–149 10.1 ± 3.0 6–16 890 ± 94 796–984 Pulse duration (ms) Interpulse interval (ms) Pulse rate Mean ± SD Range Mean ± SD Range Mean ± SD Range All localities 6.0 ± 0.8 5–8 4.3 ± 0.9 3–6 99.4 ± 4.1 90.5–106.7 Type locality (n = 35 calls; 19.2°C) 6.0 ± 0.7 5–8 4.3 ± 0.8 3–6 98.6 ± 2.8 92.1–102.5 Serra do Cabral (n = 20 calls) 5.8 ± 0.7 5–8 4.1 ± 0.9 3–6 103.5 ± 1.9 99.3–106.7 Diamantina (n = 6 calls) 6.5 ± 0.9 5–8 4.3 ± 0.9 3–6 92.5 ± 1.6 90.5–94.9 Affix (all localities) 6.4 ± 0.9 5–8 4.3 ± 0.9 3–6 101.7 ± 3.6 97.3–107.3 Tadpole description. Tadpoles in stage 30–38 have 24.3–35.7 mm of TL (n = 6 individuals). Body depressed (BH/BW = 0.74–0.78) ( Fig. 8A, B ), longer than one third of total length (BL/TL = 0.40–0.46); oval in dorsal view and globular depressed in lateral view. Snout ovoid in dorsal view (BWN/BWE = 0.69–0.75) and rounded in lateral view. Nostrils elliptic (only one individual has shown rounded nostrils), dorsally located (IND/BWN = 0.28–0.32), with small medial apophysis; closer to snout than to eyes (NSD/END = 0.55–0.65). Eyes large (ED/BWE = 0.25– 0.29), dorsally located (IOD/BWE = 0.55–0.61), dorsolaterally directed. Spiracle sinistral, and lateroventral (SDEH/BH = 0.47–0.51); posterodorsally directed, visible in lateral and dorsal view; its inner wall fused to the body wall, with posterior portion free and longer than external wall (one individual has the inner and external edge of spiracle of same size); opening located approximately at half of the body (SED/BL = 0.51–0.57). Intestinal switchback point located at the center of the abdominal region ( Fig. 8B ). Vent tube long, dextral, with ventral wall fused to bottom edge of ventral fin. Tail and body with approximately same height (MTH/BH = 0.94–1.02); tail musculature slightly developed (TMH/MTH = 0.36–0.38); caudal end acute. Dorsal fin low (DFH/TMH = 0.39– 0.43), with margin slightly convex and maximum height nearly half of the tail; emerges on posterior third of the body. Ventral fin with margin slightly convex; origin concealed by vent tube; lower than dorsal fin (DFH/VFH = 1.35–1.55). Oral disc ( Fig. 8C ) anteroventrally positioned, small (ODW/BW = 0.28–0.32), laterally emarginated; one row of marginal papillae in alternate disposition throughout the oral disc; wide anterior gap present; four to eight submarginal papillae present in both sides of oral disc. Labial tooth row formula (LTRF) 2(2)/3(1); A-1 and A-2 of the same length; P-1 and P-2 of the same length, longer than P-3. Jaw sheaths wide, finely serrated on the margins; upper jaw sheath M-shaped and lower V-shaped. Natural History. Adult males of Odontophrynus juquinha sp. nov. can be found calling with the body partially submersed in the shallow water of temporary ponds or streamlets within campo rupestre (rupestrian grasslands) vegetation. They often call at the beginning of the raining season (i.e. from September to January) after heavy rains. Males usually prefer to call hidden beneath grasses and bushes, and promptly cease calling when approached. Chorus can be heard from long distances. We also witnessed O. juquinha sp. nov. calling during daylight after heavy rains. Tadpoles are exotrophic, benthic, inhabiting mainly lentic environments such as streamlets backwater, swamps and temporary ponds (ecomorphological guild II: A:1; sensu McDiarmid & Altig 1999). In addition, species such as Scinax curicica Pugliese et al., 2004 , S . pinima ( Bokermann and Sazima, 1973 ) , S. squalirostris ( Lutz, 1925 ) , Boana cipoensis ( Lutz, 1968 ) , Leptodactylus camaquara Sazima and Bokermann, 1978 , L. cunicularius Sazima and Bokermann, 1978 , L. furnarius Sazima and Bokermann, 1978 , L. fuscus ( Schneider, 1799 ) , L. jolyi Sazima and Bokermann, 1978 , L. syphax Bokermann, 1969 , and Physalaemus cuvieri Fitzinger, 1826 , can also be found calling at the same streamlets and ponds that the new species inhabit ( Eterovick & Fernandes, 2002 ; Eterovick & Barros 2003 ). FIGURE 8. Tadpole of Odontophrynus juquinha sp. nov. collected at Serra do Cabral, lot UFMG 1581, stage 34: (A) Lateral view, (B) dorsal and ventral view of body, (C) oral disc. FIGURE 9. Geographic distribution in Brazil of Odontophrynus juquinha sp. nov. related to the altitude. MG = Minas Gerais State, ES = Espírito Santo State, BA = Bahia State, GO = Goiás State. TABLE 4. Tadpole measurements (in mm) of Odontophrynus juquinha sp. nov. from Serra do Cabral, municipality of Buenópolis, Minas Gerais State, Brazil. Developmental stages 30–38 (Gosner 1960) (n = 6). See Abbreviations List for definitions of trait acronyms.

Characters	Serra do Cabral (UFMG 1581; n = 6) Mean ± SD	Range
TL BL TAL	30.6 ± 3.9 12.9 ± 0.9 17.6 ± 3.1	24.3–35.7 11.4–14.3 12.8–21.4
MTH DFH VFH	7.7 ± 0.5 2.9 ± 0.3 2.0 ± 0.1	6.2–7.6 2.5–3.4 1.9–2.0
TMH BH SL	2.6 ± 0.2 7.3 ± 0.7 1.1 ± 0.1	2.3–2.9 6.2–8.2 1.0–1.2
SDEH SED ED	3.6 ± 0.3 7.0 ± 0.5 2.0 ± 0.1	3.1–3.9 6.5–7.6 1.8–2.2
BW BWN BWE	9.6 ± 0.9 5.4 ± 0.4 7.5 ± 0.4	8.5–10.8 4.8–5.8 6.8–7.9
TMW END ESD	2.4 ± 0.1 2.6 ± 0.1 4.1 ± 0.2	2.3–2.5 2.3–2.7 3.8–4.4
NSD ND IND	1.5 ± 0.2 0.5 ± 0.05 1.6 ± 0.15	1.4–1.8 0.4–0.5 1.4–1.8
IOD ODW	4.3 ± 0.2 2.8 ± 0.2	3.9–4.6 2.4– 3.0

Distribution and conservation. Odontophrynus juquinha sp. nov. occurs in the Espinhaço range, Brazil ( Fig. 9 ), in altitudes ranging from 900 to 1540 m a.s.l. The southernmost record is at Serra do Cipó, municipality of Santana do Riacho, Minas Gerais State; the northernmost record is at Serra do Itobira, municipality of Rio de Contas, Bahia State. The species extent of occurrence measured by a minimum convex polygon (EO, sensu IUCN 2001 ) has 30,117 km 2. Within its EO, there are two Federal and six State strictly protected areas (equivalent to IUCN category II , IUCN 1994 ), namely from south to north Parque Nacional ( PARNA ) Serra do Cipó * ( 338 km 2) , PARNA Sempre- Vivas ( 1241.5 km 2), Parque Estadual (PE) Serra do Intendente * ( 135.1 km 2), PE Pico do Itambé ( 46.9 km 2), PE Biribiri ( 169.9 km 2), PE Rio Preto ( 121.8 km 2), PE Serra do Cabral * ( 224.9 km 2), and PE Serra Nova * ( 126.5 km 2), all in the state of Minas Gerais . PE Grão Mogol ( 284 km 2) and PE Caminho das Gerais ( 562.3 km 2), at the northern state of Minas Gerais , are really close to the species EO border but not within it. There is no Federal or State strictly protected areas within its EO in the state of Bahia . Nonetheless, Odontophrynus juquinha sp. nov. has vouchered records only for the four reserves above highlighted by an asterisk. The Espinhaço range is known for its extremely high species richness and large number of microendemisms, both for amphibian ( Leite et al. 2011 , 2012 ; Barata et al. 2013 ; Carvalho et al. 2013 ; Araujo-Vieira et al. 2015 ) and plant species ( Silveira et al. 2015 ). Nevertheless, it has currently been subject of many antropogenic threats. Among major disturbances frequently associated to the Espinhaço, opencast mining, uncontrolled urbanization, especially linked to tourism expansion, and eucalypt and pine plantations are probably the most likely negatives impacts to Odontophrynus juquinha sp. nov. populations. However, due to the relatively large distribution of the new species along the Espinhaço, and the fact it occurs in some reserves, these impacts may be geographically restricted. At Serra do Cipó ( type locality), where authors usually make field trips at least once every two years, on average, since 1999, it is unlikely that Odontophrynus juquinha sp. nov. populations are declining. At the beginning of the rainy season, the species can be found (or heard) easily, in suitable habitats, being not a rare one. Therefore, we suggest that the new species does not meet the criteria and does not qualify for listing in a threatened category, been evaluated as Least Concern (LC), according to IUCN (2001). Etymology. The specific epithet is a noun in apposition and honors “Juquinha das Flores ”, a hermit whose simplicity and gentleness made him a folkloric figure at Serra do Cipó, type locality of the new species. According to the legend, Juquinha nursed from a wolf, ate scorpions, was bitten by over a hundred snakes and his age exceeded a hundred years. He used to collect flowers and roots and trade them for general items or food. His love for the mountains and the joy he provided to those who met him made him a very popular and loved figure. Juquinha lived and died twice. The first time, he was found dead by his brother and woke up during his own funeral, strengthening the belief that he was immortal. In fact, he suffered from catalepsy, a rare disease that causes the heart to stop, as if the person had died. The second and actual death happened in 1983, at unknown age. In 1987, a homage statue to Juquinha was erected at the Serra do Cipó and became a tourist spot.