Bioacoustics of Brachycephalus actaeus

The dark green general color of Brachycephalus actaeus sp. nov. in life is very distinct from B . boticario , B . coloratus , B . ferruginus , B . fuscolineatus , B . izecksohni , B . leopardus , B . mariaeterezae , B . quiririensis , B . pernix , B . pombali , B . tridactylus , and B . verrucosus , which exhibit a bright yellow or orange general dorsal body color ( Pombal et al . 1998 ; Ribeiro et al . 2005 ; Alves et al . 2006 ; Garey et al . 2012 ; Pie & Ribeiro 2015; Ribeiro et al . 2015; Ribeiro et al . 2017 ). The body color of the new species is similar to B . albolineatus , which has a greenish general coloration ( Bornschein et al . 2016b ), and B . olivaceus , which is predominantly dark green to brown (Ribeiro et al . 2015). The advertisement call of Brachycephalus actaeus sp. nov. differs in structure and frequency from all known advertisement calls within Brachycephalus . It is clearly distinct from B . crispus , B . darkside , B . ephippium , and B . pitanga by having higher frequencies together with a different call structure (Pombal et al . 1994; Araújo et al . 2012 ; Condez et al . 2014 ; Guimarães et al . 2017 ). In these species, the advertisement call is characterized by the regular repetition of one low-frequency note with several pulses, while in B . actaeus sp. nov. the high-frequency notes are composed of just two pulses. In B . crispus , the notes generally last 0.28 seconds and are composed of 10 pulses; the frequency range is 3.5–5.7 kHz, while the dominant frequency is 4.6 kHz ( Condez et al . 2014 ). In B . darkside , the average note duration is 0.11 seconds and notes are composed of six pulses; the frequency range is 2.5–5.8 kHz [considered dominant frequency in Guimarães et al . (2017) ], while the dominant frequency is 3.4 kHz [considered peak frequency in Guimarães et al . (2017) ]. In B . ephippium , notes typically last 0.12 seconds and are also composed of 12 pulses; the minimum and maximum frequencies are 3.4–5.3 kHz (Pombal et al . 1994). In B . pitanga , notes last 0.17 seconds and are composed of 11 pulses; the dominant frequency is 4.9 kHz ( Araújo et al . 2012 ). The high frequency of the advertisement call of the new species is comparable to that described for B . hermogenesi and B . sulfuratus , the latter having the highest dominant frequency known for the genus ( Condez et al . 2016 ). Nevertheless, the advertisement calls of these species differ from the new species in general structure, which is long and composed of a set of high-frequency notes. In B . hermogenesi , the call is composed of 1–5 notes; call lasts 0.2 seconds, with 1–5 pulses ( Verdade et al . 2008 ). The dominant frequency in B . hermogenesi is 6.8 kHz ( Verdade et al . 2008 ). In B. sulfuratus , the call is composed of 4–7 notes, each one lasting 0.19 seconds, with 9 pulses ( Condez et al . 2016 ). The frequency range is 4.9–9.3 kHz and the dominant frequency is 6.7 kHz ( Condez et al . 2016 ). When compared to the advertisement calls of its most closely related species, B . pernix and B . tridactylus , the new species call has shorter notes and higher frequencies. In B . pernix , notes last 0.03–0.06 seconds and are composed of three pulses; the frequency range is 4.5 kHz–6.7 kHz (Wistuba 1998). In B . tridactylus , the frequency range is 3.2–6.4 kHz and dominant frequency is 4.8 kHz ( Garey et al . 2012 ). Description of holotype . Body robust, bufoniform; head wider than long; head length 19% of SVL; snout short, rounded in lateral and dorsal views ( Figures 1A and 1B ); nostrils protuberant; canthus rostralis indistinct; loreal region slightly concave; eyes slightly protruding laterally and dorsally; eye diameter 72% of head length; tympanum absent; lips nearly sigmoid; vocal sac not expanded externally; vocal slits present; tongue longer than wide, with the posterior half not adherent to floor of mouth; vomerine teeth absent; choanae small and ovoid, anterior to eyes. Arm and forearm moderately slender; hands with fingers I and IV reduced; finger II short but distinct; finger III large and robust; fingertips I, II, and IV rounded, fingertip III pointed; finger lengths IV <I <II <III; subarticular tubercles absent; inner and outer metacarpal tubercles absent ( Figure 1C ). Legs relatively short, moderately robust; thigh length 41% of snout-vent length, tibia length 86% of thigh length; foot with toes I and V not visible externally; toe II greatly reduced; toe III short and distinct; toe IV large and robust; toe lengths II <III <IV; toe tips II and III rounded, toe tip IV pointed; subarticular tubercles absent; inner and outer metatarsal tubercles absent ( Figure 1D ). Skin on top of the head smooth; dorsal body slightly rough and without dermal co-ossification. Skin on dorsolateral and dorsal surfaces of legs rough and granular; dorsal surface of arms smooth. Skin on ventral surface of arms, and in pectoral and gular regions smooth; skin on belly and ventral surfaces of legs granular; area around the cloacal opening rough and granular ( Figure 2 ). Measurements of holotype (in mm). SVL 9.4; HL 1.8; HW 3.0; ND 0.4; IND 1.1; ED 1.3; IOD 2.1; END 0.6; AL 2.0; FAL 2.1; HAL 1.4; THL 3.8; TBL 3.3; FL 4.6. Color in life. ( Figure 6 ) Iris black. General body color orange, dorsal surface of body covered by dark green blotches; in dorsal view, a poorly defined dark brown stripe extends from the interorbital region to the posterior end of the vertebral column; arms, legs, fingers III and IV and toe IV dark green, other fingers and toes orange. In lateral view, eye contour, snout tip, lower lip, and corner of the mouth orange. In ventral view, background color orange; dark brown blotches are present on throat, cloacal region, and side of the body, extending toward the dorsum; irregular brownish spots are distributed on belly, arms, legs, fingers, and toes. Color in preservative. General dorsal body color brown; arms, legs, fingers III and IV and toe IV brown, other fingers and toes white ( Figure 2A ). In lateral view, eye contour, snout tip, lower lip, and corner of the mouth white. In ventral view, general background color is white; brown blotches are present on throat, cloacal region, and side of the body, extending toward the dorsum; irregular brown spots are distributed on belly, legs, fingers, and toes ( Figure 2B ). Osteology. ( Figure 3 ) Absence of hyperossification of the skull and skeleton; skull slightly wider than long in dorsal view. Nasals, sphenethmoid, frontoparietals, prootics, and exoccipitals fused; premaxillae broad, not fused medially, odontoids present; alary process of premaxillae distinct and slightly separated from the nasal; maxillae arched in ventral view, odontoids present; vomers not fused medially, vomerine odontophores absent; palatine absent; parasphenoid and sphenethmoid fused and robust; quadratojulgal and pterygoid present; squamosal Tshaped in lateral view, anterior zygomatic ramus short, approximately ½ of the length of the otic ramus; zygomatic ramus oriented to articulate with maxillae; mandible edentate; columella absent; tympanic annulus absent. Arytenoid cartilages not mineralized ( Figure 3D ). Pectoral girdle arciferal and robust; coracoid, clavicle, and scapula fused and completely ossified, except for the epicoracoid region, which is cartilaginous; procoracoid and epicoracoid fused with coracoid but separated from the clavicle by an ovoid fenestra ( Figure 3D ), distant from the epicoracoid; scapula, cleithrum, and suprascapula fused and elongated; suprascapula not expanded; omosternum developed, with distal portion circular and cartilaginous; sternum absent. Vertebral column composed of eight presacral, non-imbricate vertebrae; hyperossification absent in the spinal processes of the vertebrae; transverse processes of the first presacral vertebrae indistinguishable; all others presacral vertebrae with unornamented transverse processes; lengths of transverse processes of presacrals: VIII<II <VIV<II<V<III<IV; sacral diapophyses moderately expanded; urostyle length slightly larger than presacral region ( Figure 3A ). Humerus and forearm of approximately equal length; radius and ulna completely fused ( Figure 3A ); distal carpals of manus (I–IV) fused with centrale; radiale and ulnare about the same size; two prepollical elements present; palmar sesamoid present; phalangeal formula 1–2–3–1; tips of terminal phalangeal elements of fingers I and IV pointed and tips of fingers II and III arrow-shaped ( Figure 3B ). Hindlimbs with tibia and fibula fused, forming the tibiofibula; femur and tibiofibula of approximately equal length; fibulare and tibiale completely fused, but distinguishable; sesamoids present in knees and foot joints; pes with distal tarsal element I present, II–III fused and IV and V absent; centrale present; plantar sesamoid present; one reduced prehallical element; phalangeal formula 1–2–3–4–0; tips of terminal phalangeal elements of toes I and II pointed, III and IV arrow-shaped ( Figures 3 ). Advertisement call. The advertisement call of Brachycephalus actaeus sp. nov. is short, comprised of one high-frequency note and repeated at a rate of 0.13–0.30 calls/second (x̄= 0.20 ± 0.07; n = 110; Figure 4A ). The call lasts 0.03–0.04 seconds (x̄= 0.04 ± 0.01; n = 110; Figure 4B ) and the inter-call interval is 3.27–7.68 seconds (x̄= 5.42 ± 1.83; n = 104; Figure 4A ). Notes are commonly composed of two pulses ( Figure 4B ), but we also registered three pulses, with a repetition rate of 0.26–0.63 pulses/second (x̄= 0.42 ± 0.15; n = 229). The minimum frequency is 3.9–5.9 kHz (x̄=4.8 ± 0.7; n = 110), the maximum frequency is 8.5–9.7 kHz (x̄= 9.1 ± 0.4; n = 110), and the dominant frequency is 6.6–7.3 kHz (x̄= 6.9 ± 0.3; n = 110); most of the energy (90%) is concentrated within 0.7– 1.1 kHz (x̄= 0.8 ± 0.2; n = 110). Spectrograms exhibit a frequency bandwidth that extends to quite high frequencies, (i.e.,> 20 kHz; Figure 4 ). Molecular analysis. Considering DNA sequences of the 16S gene (524 aligned basepairs, 168 terminals), the maximum parsimony analyses retained 680 most parsimonious trees with 519 steps each. The analysis recovered all specimens from Brachycephalus actaeus sp. nov. as a monophyletic group, with 89% of Jackknife support ( Figure 5 ). Additionally, our analysis recovered the two main groups of species within Brachycephalus that are currently recognized; B . ephippium group and B . pernix group. Nonetheless, the position of B . hermogenesi and B . sulfuratus is uncertain. Our analysis supported the individuality of the new species and recovered its position within the B . pernix group ( B . albolineatus , B . auroguttatus , B . boticario , B . brunneus , B . cf. coloratus , B . curupira , B . ferruginus , B . fuscolineatus , B . izecksohni , B . leopardus , B . mariaeterezae , B . olivaceus , B . pernix , B . pombali , B . quiririensis , B . tridactylus , and B . verrucosus ; Figure 5 ). The relationships among several species within this group were not resolved and some were not recovered monophyletic ( B . albolineatus , B . boticario , and B . fuscolineatus ; B . brunneus , B . cf. coloratus , B . curupira , and B . izecksohni ; B . auroguttatus , B . ferruginus , B . mariaeterezae , B . olivaceus , B . pernix , B . pombali , and B . verrucosus ). However, character sampling in our analyses (restricted to 16S) cannot refute relationships inferred with other loci, and call for a more complete analysis including all available molecular evidence. According to the calculated genetic distances, the highest divergence between southern Atlantic Forest species of Brachycephalus is found between the new species and Brachycephalus sulfuratus (9.2%). Brachycephalus actaeus sp. nov. differs from B . brunneus , B . cf. coloratus , B . curupira , B . izecksohni , B . leopardus , and B . tridactylus by 6.6–5.3%; from B . albolineatus , B . boticario , and B . fuscolineatus , by 4.3–4.2%; from B . ferruginus , B . olivaceus , B . pernix , B . quiririensis , and B . verrucosus by 2.4–2.0%; and from B . auroguttatus , B . mariaeterezae , and B . pombali by 1.9–1.7%. The genetic divergence within the populations of Brachycephalus actaeus sp. n. is lower (0.2–1.3%) when compared to the interspecific divergence of species in the same clade (1.7– 6.6%), which additionally supports the new species. The highest genetic divergence (1.3%) within Brachycephalus actaeus sp. nov. was found between mainland populations at Braço do Norte (municipality of Itapoá) and Fazenda Morro Grande on Ilha de São Francisco do Sul (municipality of São Francisco do Sul). The straight-line distance between these two localities is about 20 kilometers. Our data also suggest that genetic divergence within the same population in other Brachycephalus species can be large. For example, we found 0.5% of divergence among the 12 analyzed individuals of B . verrucosus from Morro da Tromba (municipality of Joinville, state of Santa Catarina ). Variation in the type series. Measurements of 32 adult specimens of the type series are given in Table 1 . The sub-adult female (SVL = 10.9 mm ) and juvenile (SVL = 5.0 mm) were not included in the analysis of morphometric variation. The larger SVL of females (average = 11.7 mm ; range 11.1–12.4 mm ) compared to males (average = 10.0 mm; range 9.2–10.8 mm ) revealed sexual dimorphism in size in the new species (Welch’s t-test T = 11.9, DF = 29.6, P <0.01, n = 32). Dorsal skin texture varies from smooth to granular. Color in life varies mainly in extent of green on the dorsum ( Figure 6 ). In some species the dorsal coloration includes green blotches over the orange background ( Figures 6A, 6C, and 6E ), while in others the green color covers the entire dorsum ( Figure 6G ). The dark brown vertebral stripe can be well defined and wide, covering almost all of the dorsum ( Figures 6A and 6C ), or be represented by blotches distributed on the vertebral column and head ( Figures 6E and 6G ). The eye contour is orange and can be well defined, resembling a mask; the snout tip is also orange in most individuals ( Figures 6A, 6C, and 6G ). Some individuals possess a dorsolateral line of evident granules extending from the posterior end of the head until the flanks ( Figure 6A ). In ventral view, irregular dark brown blotches are distributed on the bright orange background; some individuals have dark blotches only in the cloacal region ( Figure 6B ), while others have them concentrated on the throat, belly, cloacal region, and side of the body extending toward the dorsum ( Figures 6D, 6F, and 6H ). The mandible contour is orange and clearly distinguishable ( Figures 6D and 6F ) or with orange shades extending through the gular region ( Figure 6H ). The only known juvenile is mainly brown in color, lacking the orange background of the adults ( Figure 7 ); the dorsum is covered by dark brown and red spots; a dark brown lateral stripe is present from the tip of snout to flanks ( Figure 7A ); the ventral surface of the body is slightly transparent, belly mainly white with a dark brown large blotch; an inverted V-shape mark is present on the chest ( Figure 7B ). The dorsal pattern, lateral stripe, and the inverted V-shape mark on the chest, observed in juvenile are similar to the markings on the sympatric species of flea-toad B. sulfuratus . In preservative, the orange color varies from pale cream to white; brown and green are replaced by dark brown. Males (n = 17) Females (n = 15) Mean SD Range Mean SD Range SVL 10.1 0.4 9.2–10.8 11.7 0.3 11.1–12.4 HL 2.0 0.1 1.8–2.3 2.3 0.2 2.1–2.7 HW 3.4 0.2 3.0–3.9 4.0 0.2 3.7–4.4 ND 0.3 0.0 0.3–0.4 0.3 0.1 0.3–0.4 IND 1.2 0.1 1.1–1.4 1.3 0.1 1.3–1.4 ED 1.3 0.1 1.2–1.4 1.4 0.0 1.3–1.5 IOD 2.1 0.1 1.9–2.3 2.4 0.1 2.2–2.6 END 0.6 0.1 0.6–0.8 0.7 0.1 0.6–0.8 AL 2.1 0.2 1.8–2.3 2.3 0.2 2.0–2.8 FAL 2.1 0.1 1.8–2.3 2.3 0.1 2.1–2.5 HAL 1.7 0.1 1.4–1.8 1.9 0.1 1.5–2.1 THL 4.0 0.2 3.8–4.3 4.5 0.2 3.8–4.8 TBL 3.4 0.1 3.2–3.6 3.8 0.2 3.4–4.1 FL 4.8 0.3 4.2–5.4 5.4 0.2 5.0–5.8 Natural history and ecology. The new species is associated with leaf-litter and lives in the interior of dense sub-montane and/or lowland ombrophilous Atlantic Forest remnants. These forest remnants are well preserved and are represented by secondary forests in medium to advanced stages of regeneration. Individuals of Brachycephalus actaeus sp. nov. were generally found amidst a thin layer of leaf-litter. The species is cryptic and commonly exhibits the behavior of thanatosis when caught. This species is active during the day and its reproduction period appears to be annual because males were found calling from January to December at temperatures between 10– 26°C. Calling males were always found under leaves and females were rarely found exposed. Recently, Goutte et al . (2017) concluded that two species in the genus Brachycephalus are deaf, being incapable of hearing their own vocalizations. For Brachycephalus actaeus sp. nov. we observed, in several instances, that the vocalizations of one male stimulate its neighbors to vocalize, resulting in synchronized alternation in the emission of vocalizations among them. These observations indicate that males of the new species are able to hear and use vocalizations to interact with each other. In November of 2015, at Vila da Glória, we captured a male and a female that were kept in a plastic bag. Some minutes after capture, the pair amplected in an axillary position. Subsequently, in the laboratory, two large yellow eggs were found amidst the leaves ( Figures 8A and 8B ); however, we did not observe the duration of amplexus or the moment of egg deposition. The eggs were covered with a transparent gelatinous capsule that measured 3.3 mm ; without the gelatinous capsule the eggs measured 3.1 mm in diameter ( Figure 8B ). The eggs were deposited in a leaf fold and were visited several times by one or both individuals ( Figure 8A ). We did not observe any physical contact between the parents and the eggs after oviposition. Apparently, the eggs were not fertilized, and a week later their surface was covered by fungi. In its mainland distribution, Brachycephalus actaeus sp. nov. occurs in syntopy with B. sulfuratus . Calling males of both species were found together and the advertisement calls are easily distinguishable by their distinct structures, despite the similarities in their dominant frequencies. Differences between the advertisement calls of these two species are detailed in the section “Comparisons with other species”.

Diagnosis. Brachycephalus actaeus sp. nov. is a new species of the B. pernix group, distinguished from all its congeners by the following combination of characters: (1) “bufoniform” body; (2) general dorsal body color dark green with a dark brown vertebral stripe, and orange background more evident in ventral view; (3) absence of hyperossification of the skull and skeleton; (4) pectoral girdle arciferal and robust, with small ovoid fenestra, distant from the epicoracoid; (5) radius and ulna fused; (6) finger IV greatly reduced, almost not visible externally; (7) manus with two prepollical elements; (8) tips of terminal phalangeal elements of fingers I and IV pointed, II and III arrow-shaped; (9) tibiale and fibulare completely fused; (10) toes I and V present but externally indistinguishable, toe II greatly reduced, toe III short and distinct, and toe IV larger and robust; (11) pes with distal tarsal element I present; (12) pes with phalangeal formula 1–2–3–4–0; (13) tips of terminal phalangeal elements of toes I and II pointed, and of toes III and IV arrow-shaped; (14) arytenoid cartilages not mineralized; (15) body size (SVL of adults: 9.2–10.8 mm for males and 11.1–12.4 mm for females); (16) proportional measurements HL/SVL 18–24% and ED/HL 52–73%; (17) rounded snout in dorsal and lateral views; (18) protuberant nostrils; (19) skin texture rough; and (20) advertisement call short (0.02–0.03 seconds), composed of one high-frequency note (dominant frequency 6.6–7.3 kHz). Comparisons with other species. Brachycephalus actaeus sp. nov. exhibits a “bufoniform” body and an orange background color, both characteristics that clearly differentiate it from the “leptodactyliform” species: B . didactylus , B . hermogenesi , B . pulex , and B . sulfuratus ( Izecksohn 1971 ; Giaretta & Sawaya 1998 ; Napoli et al . 2011 ; Condez et al . 2016 ). These species are generally smaller, exhibit “leptodactyliform” bodies, and always exhibit a brown background color ( Napoli et al . 2011 ; Condez et al . 2016 ). The absence of hyperossification of the skull and skeleton distinguishes Brachycephalus actaeus sp. nov. from B . darkside , B . ephippium , B . garbeanus , and B . margaritatus , which exhibit the extreme condition of hyperossification within Brachycephalus , including a dorsal bony shield ( Clemente-Carvalho et al . 2009 ; Guimarães et al . 2017 ). The absence of hyperossification also distinguishes the new species from B . alipioi , B . atelopoide , B . bufonoides , B . crispus , B . guarani , B . nodoterga , B . pitanga , B . toby , and B . vertebralis , which exhibit the intermediate condition of hyperossification within Brachycephalus ( Clemente-Carvalho et al . 2009 ; Haddad et al . 2010 ; Pombal 2010 ; Clemente-Carvalho et al . 2011 ; Condez et al . 2014 ; Condez et al . 2016 ). Brachycephalus actaeus sp. nov. differs from B . brunneus , B . coloratus , B . ferruginus , B . izecksohni , and B . pombali , which possess pectoral girdles with larger fenestra, disposed closer to the epicoracoid (small fenestra distant from the epicoracoid in the new species). Likewise, B . brunneus , B. ferruginus , B . izecksohni , and B .