A New Species of Hŋlodes (Anura, Hylodidae) from Serra do Mar, Southeastern Brazil: The Fourth with Nuptial Thumb Tubercles Author Malagoli, Leo R. Author de Sá, Fábio P. Author Canedo, Clarissa Author Haddad, Célio F. B. text Herpetologica 2017 2017-06-01 73 2 136 147 http://dx.doi.org/10.1655/herpetologica-d-16-00069 journal article 231396 10.1655/HERPETOLOGICA-D-16-00069 39128a81-7c6c-4fb9-8e48-a23259b36f23 1938-5099 7712269 Hŋlodes caete sp. nov. ( Figs. 1–4 ; Table 3 ) Hŋlodes sp. ( aff. phŋllodes ); Trevine et al. (2014) :130 Holotype . — CFBH 40524 ( Figs. 1–4 ), an adult male , collected by Leo R. Malagoli and Paulo D. P. Pinheiro on 11 March 2015 at Rio Camburi , Núcleo Curucutu , Parque Estadual da Serra do Mar ( 23°59 ′ 56.76 ′ ′ S , 46°44 ′ 17.69 ′ ′ W ; 775 m above sea level [a.s.l.]; in all cases, datum ¼ WGS84), municipality of Itanhaém , State of São Paulo , Southeastern Brazil . Paratypes . — Fifteen adult males : CFBH 12220 , collected on 17 March 2005 by Ricardo J. Sawaya , Fausto Barbo , Fernando Couto , and Leo R. Malagoli ; CFBH 19370–19371 , collected on 6 January 2008 by Leo R. Malagoli ; CFBH 40525 , collected on 11 March 2015 by Leo R. Malagoli and Paulo D. P. Pinheiro ; and CFBH 40528–40529 , collected on 13 February 2016 by Leo R. Malagoli and Alba N. Lozano ; same locality as holotype. CFBH 17667 , collected on 12 October 2007 by Leo R. Malagoli and Januária M. Onça at Núcleo Curucutu , Parque Estadual da Serra do Mar ( 23°59 ′ 50.16 ′ ′ S , 46°44 ′ 41.46 ′ ′ W ; 818 m a.s.l. ), municipality of Itanhaém , State of São Paulo , Southeastern Brazil . CFBH 19351 , collected on 16 November 2007 by Fabio Schunck ; CFBH 25808 , collected on 11 January 2009 by Fabio Schunck ; CFBH 25810–25811 , collected on 12 January 2009 by Fabio Schunck at Rio Mambu , Núcleo Curucutu, Parque Estadual da Serra do Mar ( 24°0 ′ 36.15 ′ ′ S , 46°46 ′ 57.83 ′ ′ W ; 719 m a.s.l. ), municipality of Itanhaém , State of São Paulo , Southeastern Brazil . CFBH 40532–40533 , collected on 2 December 2015 by Leo R. Malagoli and Délio P. Baêta at Núcleo Itutinga-Pilões , Parque Estadual da Serra do Mar ( 23°58 ′ 59.15 ′ ′ S, 46°35 ′ 31.59 ′ ′ W ; 805 m a.s.l. ), municipality of São Vicente , State of São Paulo , Southeastern Brazil . CFBH 40526–40527 , collected on 26 January 2016 by Leo R. Malagoli , Wesley P. Soares , and Kleber E. Rodrigues at Núcleo Curucutu , Parque Estadual da Serra do Mar ( 23°57 ′ 54.62 ′ ′ S , 46°38 ′ 58.24 ′ ′ W ; 718 m a.s.l. ), municipality of São Paulo , State of São Paulo , Southeastern Brazil . Five adult females : CFBH 11172 , collected on 16 December 2005 by Leo R. Malagoli ; CFBH 22131 , collected on 29 November 2008 by Leo R. Malagoli ; same locality as holotype. CFBH 17655 , collected on 12 September 2007 by Leo R. Malagoli at Núcleo Curucutu , Parque Estadual da Serra do Mar ( 23°59 ′ 50.16 ′ ′ S , 46°44 ′ 41.46 ′ ′ W ; 818 m above sea level), municipality of Itanhaém , State of São Paulo , Southeastern Brazil . CFBH 40531 , 40534 , collected on 2 December 2015 by Leo R. Malagoli and Délio P. Baêta at Núcleo Itutinga-Pilões , Parque Estadual da Serra do Mar ( 23°58 ′ 59.15 ′ ′ S , 46°35 ′ 31.59 ′ ′ W ; 805 m a.s.l. ), municipality of São Vicente , State of São Paulo , Southeastern Brazil . FIG. 1.—Live adult male Hŋlodes caete sp. nov. (holotype CFBH 40524; snout–vent length ¼ 31.6 mm). Photograph taken in Parque Estadual da Serra do Mar, municipality of Itanhaém, State of São Paulo, southeastern Brazil. A color version of this figure is available online. FIG. 2.— Hŋlodes caete , CFBH 40524 (holotype), adult male in (A) dorsal and (B) ventral views. Scale bar ¼ 10 mm. Diagnosis. — Hŋlodes caete is a slender species with light, oblique lateral stripes and relatively smooth dorsal surfaces ( Figs. 1 and 2A ). It is diagnosed by the following combination of characters: (1) pointed nuptial tubercles distributed in an elliptical area at the base of the dorsal surface of the thumb of male individuals ( Fig. 4 ); (2) medium-sized (SVL about 31.1–34.0 mm in adult males and 33.2–38.3 mm in adult females; variation reported in Table 3 ); (3) advertisement call with a call duration of 1.22– 2.75 s , 20–45 notes per call, note repetition rate of 14.8–18.3/s, note duration of 0.009 – 0.023 s, and internote duration of 0.036 – 0.058 s ( Table 4 ); and (4) uncorrected p distance of 10.5% ± 0.0003 from H. fredi , 10.2% ± 0.001 from H. phŋllodes , and 9.5% ± 0.001 from H. pipilans for complete 16S gene, and of 6.5% from H. fredi , 6.2% from H. phŋllodes , and 6.2% from H. pipilans for partial 16S gene. Comparisons with other species. — Hŋlodes caete is easily differentiated from all other species of Hŋlodes by the presence of nuptial tubercles on the thumb of males, except for H. fredi , H. phŋllodes , and H. pipilans (nuptial tubercles present on thumb of males in these species; Heyer and Cocroft 1986 ; Canedo and Pombal 2007 ). The males of the four species with nuptial tubercles differ in SVL ( F ¼ 369.6; P <0.0001; n ¼ 120). Males of H. caete (32.3 ± 0.88 mm , 31.1–34.0 mm; n ¼ 16) are distinguishable by their larger size than males of H. phŋllodes (27.8 ± 1.01 mm , 25.4–29.7 mm ; n ¼ 76; personal measurements of type series) and H. pipilans (24.1 ± 0.71 mm , 23.0– 25.1 mm ; n ¼ 9; Canedo and Pombal 2007 ). The mean SVL of male H. caete is larger than the SVL of male H. phŋllodes (Tukey test; Q ¼ 23.85; P <0.01) and H. pipilans ( Q ¼ 28.73; P <0.01). The mean SVL of male H. caete is smaller than that of male H. fredi ( Q ¼ 8.84, P <0.01), although there is overlap in their body size (male SVL 34.4 ± 0.9 mm , 32.8–36.7 mm ; n ¼ 19; Canedo and Pombal 2007 ). Furthermore, H. caete can be distinguished from H. fredi by the distribution of its nuptial tubercles, which are arranged in an elliptical area at the base of the thumb compared to them being distributed all over the dorsum of the thumb in H. fredi ( Canedo and Pombal 2007 ) . In addition to differences in size and nuptial tubercle distribution, H. caete is also distinguished from H. fredi , H. phŋllodes , and H. pipilans by its advertisement call (see call comparisons in Table 4 ). The call notes of H. fredi are emitted at a lower rate (6.06–9.5 notes/s) with a longer interval (0.06– 0.16 s ; Canedo and Pombal 2007 ). The call of H. phŋllodes emits a lower number of notes per call (12–20) at a lower rate (8–11 notes/s) and with a longer duration (0.05– 0.06 s ; Heyer and Cocroft 1986 ). The calls of H. pipilans are shorter (0.06– 0.1 s ), with fewer notes being emitted per call (2) and at a higher rate (20.58–32.79 notes/s; Canedo and Pombal 2007 ). Finally, H. caete can also be differentiated from H. fredi , H. phŋllodes , and H. pipilans by molecular evidence which corroborates our phenotypical analyses (see Table S1 ). FIG. 3.— Hŋlodes caete , CFBH 40524 (holotype), adult male. (A) Dorsal and (B) lateral views of head and ventral views of (C) left hand and (D) left foot. Scale bar ¼ 5 mm . TABLE 3.—Measurements (mm, reported as ¯X ± 1 SD) of males ( n ¼ 16) and females ( n ¼ 5) of the type series of Hŋlodes caete sp. nov.

Males		Females
Trait A	¯X ± SD	Range	¯X ± SD	Range
SVL	32.3 ± 0.88	31.1–34.0	35.24 ± 1.75	33.2–38.3
HL	12.48 ± 0.31	12.06–13.02	12.82 ± 0.72	11.82–13.8
HW	10.63 ± 0.26	10.14–11.01	11.13 ± 0.34	10.74–11.64
ED	5.11 ± 0.25	4.62–5.52	5.23 ± 0.23	4.92–5.53
TD	2.47 ± 0.19	2.04–2.78	2.63 ± 0.18	2.33–2.91
END	2.42 ± 0.10	2.28–2.63	2.48 ± 0.15	2.31–2.78
IOD	3.34 ± 0.14	3.12–3.58	3.32 ± 0.12	3.1–3.46
IND	4.38 ± 0.18	4.1–4.75	4.51 ± 0.16	4.28–4.71
THL	16.02 ± 0.63	15.03–17.53	16.62 ± 0.23	16.45–17.8
TBL	17.83 ± 0.53	16.52–18.73	19.18 ± 0.44	18.5–19.84
TAL	9.14 ± 0.26	8.75–9.65	9.61 ± 0.21	9.29–9.82
FL	17.01 ± 0.70	15.72–18.22	18.34 ± 0.84	17.51–19.53

A SVL ¼ snout–vent length; HL ¼ head length; HW ¼ head width; ED ¼ eye diameter; TD ¼ tympanum diameter; END ¼ eye–nostril distance; IOD ¼ interorbital distance; IND ¼ internostril distance; THL ¼ thigh length; TBL ¼ tibia length; TAL ¼ tarsus length; FL ¼ foot length. Fig. 4.—Distribution of nuptial tubercles on the right thumb of an adult male Hŋlodes caete , CFBH 40524 (holotype). (A) Drawing in lateral view. (B) Picture in dorsal view, with dashed line indicating the elliptical distribution of tubercles. Scale bars ¼ 1 mm. A color version of this figure is available online. Genetic analysis. —Our uncorrected p distance estimates support H. caete being a new species ( Table S1 ). For the new species, and particularly for the others having males with nuptial tubercles, intraspecific distances range from 0 ( H. pipilans ) to 0.3% ± 0.005 ( H. caete ) for the complete 16S gene and from 0 ( H. pipilans ) to 0.5% ( H. phŋllodes ) for partial 16S sequences; interspecific distances range from 9.5% ( H. fredi vs. H. pipilans ) to 11.3% ( H. fredi vs. H. phŋllodes ) for complete 16S sequences and from 6% ( H. phŋllodes vs. H. pipilans ) to 7.2 ( H. fredi vs. H. pipilans ) for partial 16S. Our reconstructed tree from a BI analysis (of the complete mitochondrial gene 16S) distinguishes all included species as independent evolutionary lineages, including Hŋlodes caete ( Fig. 5 ). Monophyly of the genus Hŋlodes , as well as monophyly of the H. lateristrigatus group, are wellsupported. Within the H. lateristrigatus group (represented in our phylogenetic study by H. amnicola , H. caete , H. fredi , H. japi , H. phŋllodes , H. pipilans , and H. ornatus ), the new species and all other Hŋlodes spp. that possess nuptial tubercles form a well-supported clade. Hŋlodes phŋllodes is recovered as the sister taxon of a clade that includes Hŋlodes fredi and H. pipilans (which were recovered as sister species). The new species is found to be the sister taxon of a clade composed of H. fredi , H. phŋllodes , and H. pipilans . Posterior probabilities, particularly within the clade of Hŋlodes with nuptial tubercles, are>0.9. Description of holotype . —Body slender ( Fig. 2 ); head longer than wide; snout nearly rounded in dorsal view and protruding in lateral view ( Fig. 3A,B ); nostrils elliptical, slightly protruding, laterally directed; canthus rostralis distinct, slightly curved; loreal region concave; row of small, light-colored tubercles uniformly distributed on the edge and along the entire upper lip; tympanum visible, nearly rounded, diameter larger than half of eye diameter; supratympanic fold well developed, extending from posterior corner of eye to posterior edge of the shoulder, reaching the dorsal tympanic annulus; oblique lateral fold weak and continuous from eye, above supratympanic fold, to inguinal region; tubercles absent lateral to the oblique lateral fold; paired lateral vocal sacs, widely expanded externally; vocal slits present; tongue large, nearly ovoid, free for its distal 1/3; vomerine teeth in two small series between choanae, bearing three teeth each; choanae small and nearly round, reaching the palatine process of maxilla; maxillary teeth present. TABLE 4.—Advertisement calls of Hŋlodes caete sp. nov. and of the other three Hŋlodes species with males having nuptial tubercles on the thumb: Hŋlodes fredi ( Canedo and Pombal 2007 ) , H. phŋllodes ( Heyer and Cocroft 1986 ) , and H. pipilans ( Canedo and Pombal 2007 ) . Values are reported as ¯X ± 1 SD. (—) in cells indicates unavailable data in cited references.

H. caete (at 20.8–22.8°C)		H. fredi (at 25°C)		H. phŋllodes (at 20.4°C)		H. pipilans (at 22°C)
Call parameters	¯X ± SD	Range	¯X ± SD	Range	¯X ± SD	Range	¯X ± SD	Range
Call duration (s)	1.79 ± 0.38	1.22–2.72	1.35 ± 0.25	0.59–3.36	—	1.05–2.1	0.08 ± 0.43	0.06–0.10
Intercall duration (s)	3.54 ± 1.19	1.85–7.72	—	—	—	—	—	—
Notes per call	30 ± 6.67	20–45	10.49 ± 1.16	5–23	—	12–20	—	2
Rate notes/s	16.6 ± 1.02	14.8–18.3	7.85 ± 0.6	6.06–9.50	—	8–11	26.64 ± 3.67	20.58–32.79
Note duration (s)	0.016 ± 0.002	0.009–0.023	0.03 ± 0.01	0.02–0.04	—	0.05–0.06	0.02 ± 0.01	0.02–0.03
Internote duration (s)	0.045 ± 0.005	0.036–0.058	0.1 ± 0.01	0.06–0.16	—	—	0.03 ± 0.005	0.02–0.04
Dominant frequency (Hz)	4640.63 ± 28.47	3937.5–5062.5	—	3600–4500	—	4100–5700	—	4600–5400

FIG. 5.—Phylogenetic tree reconstructed for representative hylodid anurans from a Bayesian inference analysis of the complete 16S mitochondrial gene. Values adjacent to each node are posterior probabilities. Arms moderately slender and forearms moderately robust; subarticular tubercles single, round ( Fig. 3C ); outer metacarpal tubercle large, round; inner metacarpal tubercle elliptical, smaller than outer metacarpal tubercle; supernumerary tubercles absent from hands; relative lengths of Fingers II <I = IV <III; corneous, pointed whitish nuptial tubercles in an elliptical area at the base of the dorsal surface of thumbs ( Fig. 4 ); thumb slightly fringed on both sides; Finger II fringed on both sides; Finger III fringed laterally from proximal level of proximal subarticular tubercle to disc on both sides; Finger IV fringed along inner margin from distal edge of proximal subarticular tubercle to disc and along outer margin from medial level of proximal subarticular tubercle to disc; finger discs dilated; discs on fingertips nearly oval from ventral view; disc of Finger I small ( Fig. 3C ); paired scutes on dorsal surfaces of finger discs well developed. Legs slender, tibia slightly longer than thigh; foot with elongated oval inner metatarsal tubercle and a smaller, protruding, round outer metatarsal tubercle ( Fig. 3D ); subarticular tubercles single, protruding, slightly elliptical, and slightly larger on Toe I; supernumerary tubercles absent from feet; relative lengths of Toes I <II <V <III <IV; toes exhibit extensive lateral fringes on both sides; the fringe of the outer margin of Toe V extends to slightly beyond the proximal subarticular tubercle; tarsal flap extensive, distally continuous with fringe on the inner side of Toe I, almost reaching heel; toe tips dilated; toe discs oval from ventral view; disc of Toe I small slightly oval ( Fig. 3D ); paired scutes on dorsal surfaces of toe discs well developed. Skin almost smooth on dorsum and smooth on flanks and dorsal surfaces of legs; posterior region of body with few small scattered tubercles; ventral surfaces smooth; ventral surfaces of thighs and nearby areas with granular skin. We observed three protuberances at the end of the dorsal posterior region of body, which were formed by colonies of mites (parasites under the skin). Measurements (mm) of holotype are SVL 31.6, HL 12.1, HW 10.4, ED 4.7, TD 2.8, END 2.4, IOD 3.3, IND 4.3, THL 15.8, TBL 17.6, TAL 9.4, and FL 16.4. Coloration. —In preservative, the coloration of the holotype is as follows: iris gray; dorsum dark brown with slightly clearer blotches; faint whitish line visible in the groin region; slightly darker-brown lateral stripe extends from tip of snout, through nostril, interrupted by the eye, starting again posterior to the eye, passing over the tympanum, and ending near the insertion of the arm; a slightly clearer brown lateral stripe, below the darker-brown stripe, extends from the tip of the snout to the insertion of the arm; lips gray; dorsal surfaces of arm, hand, thigh, and tibia, and hidden surfaces of thighs dark brown; dorsal surfaces of tarsus and foot whitish with many irregular small brown and whitishbrown blotches; few small dark-brown warts on posterior region of the body and near the coccygeal region; venter whitish, but well covered with many irregular, small, darkbrown blotches, which are more concentrated in the gular region ( Fig. 2B ). In life, the colors of the holotype are more vivid and contrasting; however, they are basically the same as described for specimens in preservative, with the following exceptions: iris copper; faint whitish line visible from tip of snout to the anterior corner of the eye; lateral stripe from tip of snout to arm insertion (below the darker brown stripe) whitish-silver; lips pale brown; hidden surfaces of thighs dark brown with black blotches; dark brown transverse bars on thigh, tibia, and tarsus ( Fig. 1 ). Variation among the type series in preservative. — Measurements of paratypes and holotype (totaling 16 males and 5 females ) are presented in Table 3 . Females are larger than males in SVL. Females lack nuptial thumb tubercles, vocal sacs, and vocal slits. Fringes are discreet on fingers and toes of females. Dorsum pale brown to grayish-dark brown, but only males possess darker patterns; dorsum ranging from a uniform pattern to patterns with many slightly clearer brown blotches (some blotched individuals may also exhibit medial longitudinal blotches following the vertebral column); thigh and tarsus with 2–5 transverse bars of variable widths; tibia with 3–5 transverse bars of variable widths; transverse bars are visible in females and can be visible, or not, in males; lateral stripe (below the darker-brown stripe) ranges from dark gray to whitish-cream, but only present in males with darker patterns; lips pale brown to gray, but females always with pale brown lips; posterior region of the body and coccygeal region smooth to warty; ventral surfaces vary from cream or white, with few brownish blotches, to being well covered with many dark-grayish or dark-brownish blotches; gular region varies from possessing a creamy-colored pattern, with a medial longitudinal brownish line, to having a blackish pattern which is only present in males. FIG. 6.—The holotype locality for Hŋlodes caete , a fast-flowing stream of the Rio Camburi at Núcleo Curucutu, Parque Estadual da Serra do Mar, municipality of Itanhaém, State of São Paulo, southeastern Brazil. A color version of this figure is available online. Etymology. —The name of the new species, caete , is a noun in apposition that is derived from the combination of two indigenous Tupi words, ‘‘caa’’ and ‘‘ete.’’ The combination expresses ‘‘true forest’’ with, respectively, ‘‘caa’’ meaning forest and ‘‘ete’’ meaning true. Here, caete refers to the high preserved forests that harbor the fast streams with clear water in which the new species is known to breed. Distribution. — Hŋlodes caete is known from the crests and slopes in high montane, montane, and submontane dense ombrophilous forests of Serra do Mar in the State of São Paulo ( Figs. 6 and 7 ). The new species is known to occur in the municipalities of Pilar do Sul, Ibiúna, Itanhaém, São Vicente , Santos, Cubatão, Santo André (Paranapiacaba), and São Paulo ( Fig. 7 ). The new species is sympatric and syntopic with H. phŋllodes . We recorded these two species together at the type locality and in the municipalities of São Vicente , Santos, Cubatão, and Santo André (Paranapiacaba) of the State of São Paulo. Moreover, we recorded synchronotopic calling activity of the two species in the type locality ( Fig. 8A ; also see audio files associated with the Supplemental Materials available online). Advertisement call. —The advertisement call of H. caete has harmonic structure and the first harmonic is not apparent ( Fig. 8B ). At 20.8–22.8°C air temperature and 19.2–21°C water temperature, call duration ranges from 1.22– 2.72 s (1.79 ± 0.38 s , n ¼ 36 calls from 6 males ); calls occur at intervals ranging from 1.85– 7.72 s (3.54 ± 1.19 s , n ¼ 36 intervals from 6 males ); 20–45 notes per call (30 ± 6.67, n ¼ 36 calls from 6 males ), given at a rate ranging from 14.8–18.3/s (16.6 ± 1.02/s, n ¼ 36 calls from 6 males ); note duration ranges from 0.009 – 0.023 s (0.016 ± 0.002 s , n ¼ 108 notes of 36 calls from 6 males ); notes given at intervals ranging from 0.036 – 0.058 s (0.45 ± 0.005 s , n ¼ 108 intervals of 36 calls from 6 males ); each note consists of a rising frequency–modulated whistle; the dominant frequency occurs in the third harmonic and ranges from 3937.5– 5062.5 Hz (4640.63 ± 28.47 Hz, n ¼ 36 call notes from 6 males ); the first note may be lower, with a dominant frequency range of 3937.5–4687.5 Hz (4283.7 ± 18.01, n ¼ 36 call notes from 6 males ). Natural history and behavior. — Hŋlodes caete was found along medium and large, fast-torrent streams (width ranging from 2–10 m ) within a narrow elevational range, from the middle to the top of the mountains of Serra do Mar (about 450–900 m a.s.l.; not recorded at lower elevations). Hŋlodes caete is predominantly diurnal, with its calling activity ending around 1900 h. During the months of breeding and intensive activity, however, we recorded some males calling until 2000 h ( n ¼ 4). Essentially, males call between September and March, but at lower elevations (~ 450 m a.s.l.) males were recorded calling in July as well. Males called from emergent rocks, from trunks and roots in ravines, from bromeliad leaves in slopes, or from locations concealed among rocks; in all cases, calling males were found in the middle, on the margins, or close to margins of permanent fast streams. Hŋlodes caete called from the ground level up to 2 m when it was perched on vegetation ( n ¼ 26). When disturbed, calling males dove into the water or hid in crevices among rocks on the stream margins, only to return after a few minutes to the same calling site to begin calling again. At night males and females of H. caete were observed resting on stems and leaves of shrubs ( 0.5–1.6 m from the ground; n ¼ 17). We recorded two H. caete males performing visual displays. They displayed foot-flagging ( n ¼ 2 displays from 2 males ), leg lifting ( n ¼ 1 display from 1 male ), arm waving ( n ¼ 7 displays from 1 male ), and mouth gaping ( n ¼ 1 display from 1 male , performed simultaneously with footflagging). See Hartmann et al. (2006) and de Sá et al. (2016) for display definitions. At least three of these four displays had already had been recorded for H. phŋllodes ( Hartmann et al. 2006 ) . On 2 December 2015 (between 1600 and 1610 h) we also observed a partial courtship of H. caete in the municipality of São Vicente , State of São Paulo, before the pair was collected. We noticed the pair and began to observe them when the female (CFBH 40531) jumped on the same rock where the male was calling (CFBH 40533) at the margin of a fast-flowing stream. The male and female stayed side by side with their heads oriented in opposite directions. The male called continuously (inflating vocal sacs on both sides concomitantly), and the female touched the male̕s dorsum with her right arm. The male immediately turned in the direction of the female and emitted a low, short call (this nonrecorded call had fewer notes than the advertisement call) and displayed arm lifting.