Brachycephalus actaeus, Monteiro & Condez & Garcia & Comitti & Amaral & Haddad, 2018

Monteiro, Juliane Petry De Carli, Condez, Thais Helena, Garcia, Paulo Christiano De Anchietta, Comitti, Estevão Jasper, Amaral, Ivan Borel & Haddad, Célio Fernando Baptista, 2018, A new species of Brachycephalus (Anura, Brachycephalidae) from the coast of Santa Catarina State, southern Atlantic Forest, Brazil, Zootaxa 4407 (4), pp. 483-505 : 486-500

Brachycephalus actaeus sp. nov. Figures 1 , 2 , 6 , 7 , and 8

Holotype. CFBH 39850 , adult male, collected at Serra da Palha , Laranjeiras , Ilha de São Francisco do Sul, municipality of São Francisco do Sul, state of Santa Catarina, Brazil (26°17'50"S; 48 ° 40'28"W, Datum WGS 84, ca 60 meters above sea level), on 19 May 2015, by C.F.B. Haddad, J.P.C. Monteiro, and E.C. Nardin ( Figures 1 and 2 ).

Paratopotypes. CFBH 39851, adult female, collected with the holotype; CFBH 39872, 39873, and UFMG 18973, adult males, collected on 25 November 2015, by J.P.C. Monteiro, T.H. Condez, and E.C. Nardin; CFBH 39876, adult male, and CFBH 39877, adult female, collected on 0 2 December 2015, by J.P.C. Monteiro and E.C. Nardin.

Paratypes. CFBH 39846 , adult male, cleared and double-stained, collected at Fazenda Morro Grande, Morro Grande, Ilha de São Francisco do Sul , municipality of São Francisco do Sul, state of Santa Catarina, Brazil (26 ° 17'47"S; 48°37'10"W, Datum WGS 84, ca 60 meters above sea level), on 14 November 2014, by E.J. Comitti . UFMG 18970, adult female, collected on 15 November 2014, by J.P.C. Monteiro, T.H. Condez, and E.J. Comitti; CFBH 39849, sub-adult female, collected on 21 November 2014, by J.P.C. Monteiro and E.C. Nardin; CFBH 39848, adult male, cleared and double-stained, collected on 21 January 2015, by C.F.B. Haddad, J.P.C. Monteiro, T.H. Condez, and E.J. Comitti; UFMG 18971, adult male, collected on 0 1 August 2015, by J.P.C. Monteiro and E.C. Nardin; CFBH 39855–39858 and 39861, adult males, CFBH 39860, adult female, CFBH 39859 , juvenile, collected on 23 November 2015, by J.P.C. Monteiro, T.H. Condez, and E.C. Nardin; all collected at Centro de Estudos e Pesquisas Ambientais da Univille ( CEPA), Vila da Glória , Distrito do Saí, municipality of São Francisco do Sul, state of Santa Catarina, Brazil (26 ° 13'39"S; 48 ° 41'31"W, Datum WGS 84, ca 120 meters above sea level) . CFBH 39853 and 39854, adult males, collected on 27 August 2015, by J.P.C. Monteiro and E.C. Nardin; CFBH 39862 , adult female, collected on 23 November 2015, by J.P.C. Monteiro, T.H. Condez, and E.C. Nardin; at Estrada do Saí, Distrito do Saí, municipality of São Francisco do Sul , state of Santa Catarina, Brazil (26°12'06"S; 48 ° 41'37"W, Datum WGS 84, ca 80 meters above sea level) . CFBH 39863, 39864, 39867, 39868, and UFMG 18972 adult females, CFBH 39865 and 39870, adult males, collected on 24 November 2015, by J.P.C. Monteiro, T.H. Condez, and E.C. Nardin; CFBH 39875 , adult female, collected on 30 November 2015, by J.P.C. Monteiro and E.C. Nardin; at Braço do Norte , municipality of Itapoá, state of Santa Catarina, Brazil (26°07'29"S; 48 ° 43'48"W, Datum WGS 84, ca 220 meters above sea level) . CFBH 42005–42008 , adult females, collected on 17 September 2016, by J.P.C. Monteiro and E.C. Nardin; Fazenda Palmito Juriti, municipality of São Francisco do Sul , state of Santa Catarina, Brazil (26°08'09"S; 48 ° 43'54"W, Datum WGS 84, 125–170 meters above sea level).

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.

pombali differ in having the radius and ulna not fused, pes with distal tarsal element I absent, and toe V reduced ( Ribeiro et al. 2005; Alves et al. 2006); the new species has the radius and ulna fused, pes with distal tarsal element I present, and toe V externally not visible. Also, B. albolineatus , B. coloratus , B. curupira , B. ferruginus , and B. pombali have just one prepollical element, and B. izecksohni has no prepollical element ( Ribeiro et al. 2005; Alves et al. 2006; Bornschein et al. 2016b; Ribeiro et al. 2017); the new species has two prepollical elements. The phalangeal formula for the pes of B. brunneus , B. izecksohni , and B. pombali is 0–2–3–4–0 ( Ribeiro et al. 2005; Alves et al. 2006), and for B. curupira it is 0–1–3–4–0 ( Ribeiro et al. 2017); the phalangeal formula of the pes of the new species is 1–2–3–4–0. In B. albolineatus , B. coloratus , and B. curupira the fibulare and tibiale are not completely fused; tips of terminal phalangeal elements of toes II–IV are arrow-shaped (fibulare and tibiale completely fused; tips of terminal phalangeal elements of toes I and II pointed, III and IV arrow-shaped in the new species). Finally, the arytenoid cartilages are mineralized in B. albolineatus and B. coloratus (arytenoid cartilages not mineralized in the new species). Although the osteology of B. pernix was not studied in detail ( Pombal et al. 1998), on the basis of the available information its osteology is quite similar to that of the new species. See Figure 3 for osteological details of the new species.

Body size (males SVL = 9.2–10.8 mm; females SVL = 11.1–12.4 mm) distinguishes the new species from Brachycephalus ferruginus (males SVL = 11.6–12.5 mm; females SVL = 13.0– 14.5 mm; Alves et al. 2006), B. pernix (males SVL = 12.0– 13.3 mm; females SVL = 14.1–15.8 mm; Pombal et al. 1998), and B. pombali (males SVL = 12.6–13.9 mm; females SVL= 14.6–15.3 mm; Alves et al. 2006). Also, in B. coloratus (males SVL = 10.3– 10.6 mm; females SVL = 12.2–13.3 mm; Ribeiro et al. 2017), B. izecksohni (males SVL = 10.3–12.1 mm; females SVL = 12.5–13.1 mm; Ribeiro et al. 2005), and B. tridactylus (males SVL = 10.6–11.6; females SVL = 13.5–13.8 mm; Garey et al. 2012) males and/or females are slightly larger than in the new species. Brachycephalus actaeus sp. nov. is distinguishable from B. albolineatus , B. auroguttatus , B. boticario , B. fuscolineatus , B. leopardus , B. mariaeterezae , B. olivaceus , B. quiririensis , and B. verrucosus by a proportionally shorter head relative to body length (HL/SVL) and by a proportionally larger eye diameter related to head length (ED/HL). In B. actaeus sp. nov., HL/ SVL is 18–24% (x̄= 20, SD = 1) and ED/HL is 52–73% (x̄= 63, SD = 4), for the 32 adult specimens of the type series, without sexual distinction. In B. albolineatus , HL/SVL is 28–34% (x̄= 31, SD = 4) and ED/HL is 36–42% (x̄= 38, SD = 4); in B. auroguttatus , HL/ SVL is 29–38% (x̄= 33, SD = 2) and ED/HL is 30–44% (x̄= 33, SD = 3); in B. boticario , HL/ SVL is 31–36% (x̄= 34, SD = 2) and ED/HL is 30–34% (x̄= 32, SD = 1); in B. fuscolineatus , HL/ SVL is 29–34% (x̄= 31, SD = 1) and ED/HL is 36–41% (x̄= 39, SD = 1); in B. leopardus , HL/ SVL is 31–35% (x̄= 33, SD = 1) and ED/HL is 34–43% (x̄= 38, SD = 3); in B. mariaeterezae , HL/ SVL is 29–36% (x̄= 33, SD = 2) and ED/HL is 36–42% (x̄= 39, SD = 2); in B. olivaceus , HL/ SVL is 32–36% (x̄= 34, SD = 1) and ED/HL is 26–36% (x̄= 32, SD = 3); in B. quiririensis , HL/ SVL is 31–36% (x̄= 34, SD = 1) and ED/HL is 28–34% (x̄= 32, SD = 2); and in B. verrucosus , HL/ SVL is 30–36% (x̄= 33, SD = 2) and ED/HL is 30–40% (x̄= 34, SD = 3) (Pie & Ribeiro 2015; Ribeiro et al. 2015; Bornschein et al. 2016b).

Additionally, Brachycephalus actaeus sp. nov. exhibits a rounded snout in dorsal and lateral views, which distinguishes it from B. brunneus , which has a slightly mucronate snout in dorsal view ( Ribeiro et al. 2005); from B. leopardus , which has a slightly truncate snout in dorsal and lateral views (Ribeiro et al. 2015); and from B. quiririensis , which has a mucronate snout in dorsal view (Pie & Ribeiro 2015). Nostrils are not protuberant in B. auroguttatus , B. pernix , and B. fuscolineatus ( Pombal et al. 1998; Ribeiro et al. 2015), which differ from the protuberant nostrils of the new species.

In Brachycephalus tridactylus , finger IV is not externally visible ( Garey et al. 2012), while in the new species it is reduced but externally distinct. In B. actaeus sp. nov., fingertips I, II, and IV are rounded, differing from B. pernix in which these fingertips are pointed ( Pombal et al. 1998) and from B. brunneus , B. izecksohni , and B. leopardus , which have the tip of finger II pointed ( Ribeiro et al. 2005; Ribeiro et al. 2015).

The texture of the skin on the dorsum of Brachycephalus actaeus sp. nov. is rough; this characteristic distinguishes the new species from B. albolineatus , B. brunneus , B. coloratus , B. curupira , B. ferruginus , B. izecksohni , B. leopardus , B. pernix , B. pombali , and B. tridactylus , which have a smooth dorsum ( Pombal et al. 1998; Ribeiro et al. 2005; Alves et al. 2006; Garey et al. 2012; Ribeiro et al. 2015; Bornschein et al. 2016b; Ribeiro et al. 2017).

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.

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”.

Distribution. Brachycephalus actaeus sp. nov. is known from six nearby localities along the coast in the state of Santa Catarina, Brazil ( Figure 9 ). The new species occurs at two localities on Ilha de São Francisco do Sul (Serra da Palha and Fazenda Morro Grande), and three mainland localities in Distrito do Saí (Centro de Estudos e Pesquisas Ambientais da Univille, Estrada do Saí, and Fazenda Palmito Juriti), all located in the municipality of São Francisco do Sul. On the mainland, the new species also occurs at Braço do Norte, municipality of Itapoá, in the same mountain range (Serra do Saí). The distribution of the new species seems to be restricted to the coastal region in northeastern Santa Catarina, including lowlands near sea level and coastal mountains up to 250 meters. We recorded Brachycephalus actaeus sp. nov. at 20 meters above sea level in Serra da Palha, Ilha de São Francisco do Sul. Despite its occurrence in lowlands, the distribution is restricted to the foothills of the mountains and it is not associated with the flat relief of the coastal plains.

Etymology. The specific epithet actaeus is a Latin adjective that means situated on the edge. It is derived from the Latin word acta, which was incorporated from Greek, and means shore, coast, or beach. The name is used in allusion to the typical habitat of the new species, the coastal lowlands of the Atlantic Forest.

Remarks. Most species of Brachycephalus are considered microendemic, occurring at one or a few high elevation sites along the mountains of the Atlantic Forest ( Pie et al. 2013; Bornschein et al. 2016a). However, in contrast to this pattern, the expanding knowledge for the genus is revealing that even species with small geographic ranges can be found beyond their type localities and even over broad elevational ranges. At least nine species of Brachycephalus are known to have broad geographic distributions: B. brunneus ( Pie et al. 2013) , B. didactylus ( Oliveira et al. 2012) , B. ephippium ( Clemente-Carvalho et al. 2008) , B. hermogenesi ( Pimenta et al. 2007; Verdade et al. 2008), B. leopardus ( Bornschein et al. 2016a) , B. margaritatus ( Pombal & Izecksohn 2011) , B. nodoterga ( Clemente-Carvalho et al. 2015; Abegg et al. 2015), B. olivaceus ( Bornschein et al. 2016a) , B. quiririensis ( Bornschein et al. 2016a) , and B. sulfuratus ( Condez et al. 2016) . Of these, B. didactylus , B. hermogenesi , and B. sulfuratus also exhibit broad elevational ranges, occurring from sea level up to 1000 meters ( Condez et al. 2016). Broad elevational ranges for species of Brachycephalus have been associated with those species that possess a “leptodactyliform” body and cryptic coloration (sometimes defined as “flea-toads”), while those with a “bufoniform” body and conspicuous coloration (sometimes defined as “pumpkin-toadlets”) have been associated with restricted distributions at higher elevations ( Pie et al. 2013; Bornschein et al. 2016a; Guimarães et al. 2017). However, the new species described herein, Brachycephalus actaeus sp. nov., represents the first record of a “pumpkin-toadlet” occurring in the lowlands of the Atlantic Forest, and thus is of particular significance to understanding the evolution of the genus.

Local adaptation to distinct climatic conditions notwithstanding, it seems likely that the current conditions of the lowland and sub-montane forests of the southern Atlantic Forest are similar to those required for the persistence of species of Brachycephalus in the highlands along the southeastern part of the Atlantic Forest. Furthermore, the development of suitable climatic conditions for Brachycephalus occurrence on the emerged continental shelf during the Quaternary (Leite et al. 2015) may explain the expansion of its distribution and colonization of coastal lowlands by Brachycephalus .

Climate change has been demonstrated to be one of the main threats to the conservation of species of Brachycephalus , especially considering their close association with montane forests, which are particularly vulnerable to changes in climate and will probably shrink or disappear with a warmer future climate (Haddad et al. 2008). Moreover, with the addition of new lowland records for the genus, alteration in sea level represents another potential source of negative effects to these species as a result of global climate change. Presently, the main threats faced by Brachycephalus actaeus sp. nov. are habitat loss and modification associated with the intense rapid urban expansion and seaport construction in the coastal region of northeastern Santa Catarina. These changes in the land use are particularly detrimental to the new species, which is an exclusive forest leaf-litter dweller with a limited area of occurrence. Despite occurring at six localities, the distribution of the new species is restricted to areas near this coastal region where there are few established conservation units. The new species could potentially be present inside a protected area called Reserva Particular do Patrimônio Natural (RPPN) Morro da Palha, located near the type locality. However, its occurrence in Parque Estadual Acaraí (Ilha de São Francisco do Sul) and RPPN Volta Velha (Itapoá) is not expected because of the flat relief formations present there, such as sandy coastal plains and/or lowland forests, where populations of Brachycephalus actaeus sp. nov. are not known to occur.

Figures

FIGURE 1. Brachycephalus actaeus sp. nov. CFBH 39850 (holotype), adult male. (A) Dorsal and (B) lateral views of head, (C) ventral view of left hand, and (D) ventral view of left foot. Scale bar = 1.0 mm.

FIGURE 4. Advertisement calls of Brachycephalus actaeus sp. nov., voucher specimen CFBH 39848 (paratype), recorded at Centro de Estudos e Pesquisas Ambientais da Univille, Vila da Glória, municipality of São Francisco do Sul, state of Santa Catarina, Brazil, on 21 January 2015, 10:50 a.m., air temperature 25°C, air relative humidity 100%. (A) oscillogram of three consecutivecalls, a single call is highlighted in orange; and (B) spectrogram and oscillogram of a single call.

FIGURE 6. Color variation in Brachycephalus actaeus sp. nov. (A) Dorsolateral and (B) ventral views of CFBH 39864 (paratype), adult female, from municipality of Itapoá, state of Santa Catarina.(C) Dorsolateral and (D) ventral views of CFBH 39863 (paratype), adult female, from municipality of Itapoá, state of Santa Catarina. (E) Dorsolateral and (F) ventral views of CFBH 39876 (paratopotype), adult male, from municipality of São Francisco do Sul, state of Santa Catarina. (G) Dorsolateral and (H) ventral views of CFBH 39846 (paratype), adult male, from municipality of São Francisco do Sul, state of Santa Catarina, Brazil.

FIGURE 8. Eggs of Brachycephalus actaeus sp. nov. (A) Male left, CFBH 39861 (paratype) and female right, CFBH 39860 (paratype), after oviposition. (B) Detail of the eggs of Brachycephalus actaeus sp. nov.

FIGURE 9. Distribution of Brachycephalus actaeus sp. nov. in northeastern state of Santa Catarina, Brazil. (1) Type locality, Serra da Palha, Laranjeiras and (2) Fazenda Morro Grande, both at Ilha de São Franciscodo Sul, municipality of São Francisco do Sul; (3) Centro de Estudos e Pesquisas Ambientais da Univille, Vila da Glória, (4) Estrada do Saí, and (5) Fazenda Palmito Juriti,at mainland region, municipality of São Francisco do Sul; and (6) Braço do Norte, municipality of Itapoá.Abbreviations to Brazilian states: PR = Paraná, SC = Santa Catarina,and RS = Rio Grande do Sul.

FIGURE2. Brachycephalus actaeus sp. nov. CFBH39850 (holotype), adult male.(A) Dorsal and (B) ventral views.Scale bar = 2.0 mm.

FIGURE7. Juvenile of Brachycephalus actaeus sp.nov. in life (snout-vent length = 5.0 mm). (A) Dorsolateral and (B) ventral views of the specimen CFBH 39859 (paratype), from Centro de Estudos e Pesquisas Ambientais da Univille, Vila da Glória, Distrito do Saí, municipality of São Francisco do Sul, state of Santa Catarina, Brazil.

FIGURE3. Detailsof a cleared and double-stained specimenof Brachycephalus actaeus sp.nov. (paratype CFBH 39848).(A) Dorsal view of skull and skeleton, arrow indicates radio and ulna fused; (B) right hand in palmar view, arrows indicates the prepollical elements; (C)rightfoot inplantar view; (D)ventral view of pectoral girdle, arrowsindicate arytenoidscartilage;and (E)right leg in ventral view,arrow indicates fibulare and tibiale fused.Scale bar = 2.0 mm in A and 0.5mm in B, C, D, and E.

FIGURE5. Phylogenetic relationships of the genus Brachycephalus based on a fragment of the mitochondrial 16SrRNA (519 base pairs). Topology represents the strict consensus of the 680 most parsimonious trees (of 519 steps). Branch lengths correspond to the number of unambiguous parsimony transformations. Node values represent> 70% support of Jackknife absolute frequencies.Sequencesof the new species are highlighted in bold.See Appendix 2 for details of sequencesincluded in this analysis and complete locality names.