A new species of Amazophrynella (Anura: Bufonidae) from the southwestern part of the Brazilian Guiana Shield Author Rojas, Rommel R. Author Carvalho, Vinícius Tadeu De Author Gordo, Marcelo Author Ávila, Robson W. Author Farias, Izeni Pires Author Hrbek, Tomas text Zootaxa 2014 3753 1 79 95 journal article 36915 10.11646/zootaxa.3753.1.7 23336f49-4d3c-4eb0-87f3-0931764a7237 1175-5326 285395 753637FD-6478-4152-91B1-807250422F2B Amazophrynella manaos sp. nov. ( Figure 1 ) Dendrophryniscus minutus ( McDiarmid, 1971 ; Zimmerman & Rodrigues, 1990 ; Magnusson & Hero, 1991 ; Lima et al. , 2006 ) Amazonella minuta ( Fouquet et al. , 2012a ) Amazophrynella minuta ( Fouquet et al. , 2012b ) Holotype . INPA-H 31866, adult male, collected in forest remnants of the campus of the Universidade Federal do Amazonas—UFAM ( 03° 05' 56"S , 59°58' 09" W ), municipality of Manaus, Amazonas State, by Vinicius Tadeu de Carvalho and Rommel Roberto Rojas, on June 26, 2012 at 16:35 h. Paratypes . INPA-H 29568, INPA-H 29571, INPA-H 29570, INPA-H 29572, INPA-H 29569 (adult males), collected in Mineração Taboca ( 0° 42' 59" S , 60° 10' 24" W ), municipality of Presidente Figueiredo, Amazonas State, by Vinicius Tadeu de Carvalho, on December 17, 2011 ; INPA-H 6983, INPA-H 6984, INPA-H 6987(adult males), collected on the campus of the Universidade Federal do Amazonas—UFAM ( 03° 05' 37" S , 59° 58' 26" W ), Amazonas State, by Ana Cristina de Oliveira Cordeiro, on February 10, 2010 ; INPA-H 20986; INPA-H 21217 (adult females), INPA-H 21028, INPA-H 21170, INPA-H 21060 (adult males), collected in the Reserva Florestal Adolpho Ducke ( 02° 55' 37" S , 59° 58' 14" W ), by Albertina P. Lima, in December 1992 ; INPA-H 30577, INPA-H 30575, INPA-H 30573, INPA-H 30572, INPA-H 30576 (adult females), collected at the municipality of Presidente Figueiredo ( 01° 56' 20" S , 60° 02' 14" W ), Amazonas State, by André Luiz Ferreira da Silva, on January 5, 2012 ; INPA-H 21442, INPA-H 21400 (adult females), INPA-H 21451, INPA-H 21398, INPA-H 21821 (adult males), collected at Reserve ZF-2 ( 02°35' 20"S , 60°06' 55"W ), municipality of Presidente Figueiredo, Amazonas State, by Albertina P. Lima, in December 1997 ; INPA-H 1859 (adult male), collected at Parque Estadual Rio Negro Setor Sul, Cuieiras River ( 02° 43' 27" S , 60° 24' 22" W ), municipality of Manaus, Amazonas State, by Vinicius Tadeu de Carvalho and Lucéia Bonora, on January 10, 2007 . FIGURE 1. Holotype of Amazophrynella manaos sp. nov. (INPA-H 31866): A) Dorsal view, B) Ventral view, C) Dorsal view of head, D) Ventral view of head, E) Ventral view of the right foot, F) Ventral view of right hand. Diagnosis. The new species is assigned to the genus Amazophrynella by the combination of the following generic characters: small size, depressed body, eardrum and parotid glands not visible, presence of granulate bodies, presence of subarticular tubercles on hands, elongated snout, and via phylogenetically nested position within the genus Amazophrynella . The new species is characterized by: 1) triangular snout with a slightly truncate culmination (in ventral view); 2) ventral surface covered with high density fine granules; 3) white abdomen; 4) black patches or stripes on the abdomen; 5) fifteen molecular autapomorphies in the 16S rDNA ( Table 2 ). Divergence from its nominal sister taxon is 9%. TABLE 2. Species level diagnostic characters observed in the 16S rDNA gene of Amazophrynella manaos sp. nov. and other species of Amazophrynella . First line indicates position of the character within the 16S rDNA gene ( Xenopus laevis 16S rDNA gene was used as reference). (-) indicates a deletion.

Position (pb)	308 340	346	370	443	446	452	455	467 478	522	530	537 558 583
A. manaos	C A	T	A	G	C	C	T	C G	T	T	G C A
A. vote	T C	C	C	A	T	A	A	T A	C	A	A A G
A. minuta	T C	C	T	A	T	A	A	T A	C	G	A A G
A. bokermanni	T C	C	G	T	T	-	A	T A	C	G	A T G

Molecular phylogeny and genetic distance. Phylogenetic analysis of nominal and hypothetical taxa of the genus Amazophrynella indicates the existence of six lineages ( Figure 2 ). Basal divergence is between the clade composed of an undescribed Amazophrynella species from western Amazônia and A. minuta specimens sampled from the type locality, and a clade comprising all other species and lineages. The A. minuta + A. aff. minuta “western Amazonia” clade is supported by 97% bootstrap value while the other clade is supported by 80% bootstrap value. The new species, A. manaos is sister to a possibly a new species of Amazophrynella from the Guiana Shield (bootstrap support 70%) and both are sister to A. bokermanni (bootstrap support 75%). The recently described A. vote (italicize) is sister to A. bokemanni + ( A. manaos + A. sp. “Guianas”) with a bootstrap support of 80%. Smallest uncorrected 16S rDNA p -distances (4%) was observed between A. manaos sp. nov. and A. sp. “Guianas”. Greatest intrageneric distance (14%) was observed between A. manaos sp. nov. and A. minuta , and A. sp. “Guianas” and A. minuta (see Table 3 ). TABLE 3. Uncorrected p -distances among Amazophrynella species and the sister genus Dendrophryniscus . Molecular distances are based on the 480 bp fragment the 16S rDNA. Included are A. minuta from its type locality and hypothetical species Amazophrynella sp. “Guianas” and A. aff. minuta “western Amazonia” from Fouquet et al. (2012a); both species pertain to the A. minuta species complex.

16S rDNA 1 2	3	4	5	6	7	8
1 A. sp. "Guianas" - -	-	-	-	-	-	-
2 A. aff. minuta "western Amazonian" 0.13 -	-	-	-	-	-	-
3 D. brevipollicatus 0.21 0.19	-	-	-	-	-	-
4 D. berthalutzae 0.20 0.16	0.0 8	-	-	-	-	-
5 D. krasuae 0.19 0.16	0.0 8	0.0 0	-	-	-	-
6 A. manaos 0.04 0.12	0.20	0.18	0.18	-	-	-
7 A. vote 0.12 0.12	0.20	0.19	0.19	0.11	-	-
8 A. minuta 0.14 0.04	0.20	0.16	0.16	0.14	0.11	-
9 A. bokermanni 0.10 0.13	0.19	0.19	0.19	0.09	0.12	0.13

Characterization of topotypic material of Amazophrynella minuta . Topotypic material of A. minuta was collected in mission Taracuá, right bank of the middle Uaupés River in the last week of August 2013 . Adult males measured 13.5 ± 0.57 mm ( 12.4–14.2 mm ), females 17.5 ± 0.61 mm ( 17.1–18.9 mm ) in snout vent length. The specimens ( Figure 3 ) were characterized by a pointed head (in ventral view); pointed snout (in lateral view); roughly granular dorsal and ventral skin; a scattering of abundant pointed spicules on the body; big warts on the dorso-lateral region; dark gray brown throat and chest; intense yellow-orange coloration of the abdomen; and by irregular black blotches or ocelli on the abdomen. Fingers are unwebbed. Formula of the fingers: I<II<IV<III. Tips of the fingers are unexpanded. Webbing on the base of the foot. Formula of the toe: I<II<III<V<IV. Tips of the toes are unexpanded. FIGURE 2. Phylogenetic relationship of Amazophrynella species inferred using the maximum-likelihood criterion. Analysis was performed with 480 bp of 16S rDNA under the GTR+I+G model of molecular evolution. Branch support values are based on 1000 bootstrap pseudoreplicates.. FIGURE 3. Specimens of Amazophrynella minuta from Taracuá mission, Uaupés River, municipality of São Gabriel da Cachoeira, Amazonas, Brazil (INPA-H 32727 and INPA-H 32735). Comparisons with other species. The new species can be differentiated from the three other nominal species of Amazophrynella by the following characters (characters of compared species in parentheses): a) from A. minuta ( Melin, 1941 ) by the triangular snout with a slightly truncate culmination (snout pointed), absence of spiny tubercles on dorsum (prickly warty skin on dorsum), skin of axillary region and forearm finely granular (rough granular skin on forearm and axillary region, especially on the neck), venter white with black spots and darkbrown chest in life (venter bright reddish-orange, with small black blotches and throat light brown) ( Figure 4 A-C and Figure 5 A, Table 4 ); b) from A. bokermanni ( Izecksohn,1993 ) , by finger I shorter than finger II (finger I longer than finger II), smaller SVL, with males reaching 15.8 mm and females 24.7 mm (maximum SVL of males and females 22 mm and 28 mm , respectively) ( Figure 4 G–I, Figure 5 B); and c) from A. vote Ávila, Carvalho, Gordo, Kawashita & Morais, 2012 , by snout triangular with a slight culmination (rounded), slightly smaller SVL in males ( 14.7–15.8 mm vs. 16.4 mm—Ávila et al. , 2012), and white abdomen with black spots or stripes and blackish to dark-brown chest and throat in life (abdomen reddish-brown with small white dots and brown spots) and subrostral crest converging anteriorly (subrostral crest not converging anteriorly) ( Figure 4 D–F, Figure 5 C). FIGURE 4. Some diagnostically morphological and color characters between the species of Amazophrynella (in life), type of snout (A,D,G,J); texture of the dorsal surface (B,E,H,K) and texture of ventral surface (C,F,I,L) of A–C) A. minuta (INPA-H 312725); D–F) A. manaos sp. nov. (unvoucher specimens); G–I) A. bokermanni (INPA-H 31863); J–L) A. vote (INPA-H 31870). Description of the holotype . Small species; body slender; head longer than wide; head triangular; head length 36% SVL; upper eyelids close to 60% of interorbital distance; snout elongate; snout profile slightly truncated in lateral view; eyes prominent, 31% of head width; nostril is closer to the tip of the snout than to the eyes; tympanums not visible; parotid gland absent. Body covered by abundant rounded granules; texture of dorsal skin granular; abundant granules grouped in the axillar region; texture of the ventral skin granular. Forelimbs slender; upper arm covered by tiny conical tubercles. Fingers unwebbed; formula of the fingers: I<II<IV<III; palmar tubercle rounded; supernumerary tubercle ovoid: one in finger I, two in the II and IV and three in the III; tip of the fingers unexpanded. Hind limbs slender; femur length 52% of SVL; conical tiny granules present on the thigh and tibia; tarsus length approximately 30% of SVL; foot length 68% of thigh length; webbing at the base of toes; formula of the toes: I<II<III<V<IV; elliptical inner metatarsal tubercle present; subarticular tubercles rounded and distinct, nearly the same size as fingertips: two in toe I, II and V, three in the III and IV; tiny triangular spines along rear edge of feet; tips of the toes unexpanded. TABLE 4. Comparison of morphological and color characters of the Amazophrynella (Fouquet et al. , 2012a)species. For all species original descriptions and direct examination of specimens was used. We used adults specimens from all the species.

Species A. manaos	A. bokermanni	A. vote	A. minuta
Source This study	Izecksohn, 1993 + material studied	Ávila et al. , 2012 + material studied	Melin, 1941 + material studied
Males SVL in mm, 15.0 ± 0.6 mean ± SD (range) (14.0–15.8)	22.0 ± 3.7 (16.4–27.7)	16.4 ± 1.3 (15.2–19.3)	13.6 ± 0.8 (12.3–15.8)
Females SVL in mm, 20.6 ± 1.8 mean ± SD (range) (15.9–24.7)	20.1 ± 4.7 (19.4–30.6)	22.7 ± 1.6 (21.4–25.7)	17.9 ± 0.6 (17.9–18.9)

Dorsal skin texture	Fine granules, medium density	Fine granules, medium density	Fine granules, low Rough granules, high density density
Ventral skin texture	Fine granules, high density	Fine granules, medium density	Fine granules, medium Rough granules, high density density
Ventral coloration	White with black blotches	White with black spots	Red-brown with white Yellow-orange with dots brown blotches
Snout profile	Slightly truncated	Pointed	Rounded Pointed
Relative size finger I and II	I<II	I>II	I<II I<II
Gular region	Blackish to dark-brown	Grayish-brown	Brown Light-brown

Measurement of the holotype (in mm). SVL: 13.9; HL: 5.1; HW: 4.1; EW: 1.1; ED: 1.3; SL: 2.3; END : 1.4; IND : 1.0; IOD: 1.8; HAL: 3.0; UAL: 4.1; THL: 7.2; TL: 6.9; TAL: 4.4 and FL: 4.9. Coloration of the holotype . In life, dorsal color pattern of the holotype dark-brown with transversal black and light-brown bars, more evident on limbs; thin cream medial line extending from head to cloaca, a white longitudinal stripe on upper jaw extending from nostril to forearm; arms white ventrally; throat and chest region blackish; belly white with large black spots, thighs and tibiae predominantly grey ventrally with few small black dots; palms and soles reddish ( Figure 5 D). Coloration of preserved specimens is nearly identical to that described in life but black spots on the belly are less evident. Variation. Variation exists among individuals within the same locality as well as between localities. In some individuals the coloration of the throat extends onto the chest and the belly has few black spots; other specimens show a ventral pattern of black stripes that extend dorsally to the upper part of the lateral region. Juveniles have a less pronounced pattern on the belly, with few black spots. Individuals from some localities have a thin clear brown line that extends from the rostrum to the cloaca along the central dorsum ( Figure 6 ). Distribution and natural history . Amazophrynella manaos sp. nov. is distributed throughout the southwestern part of the Brazilian Guiana region. It was collected in eight localities in the state of Amazonas, Brazil : Mineração Taboca, Campus of the Universidade Federal do Amazonas, Reserva Florestal Adolpho Ducke, Presidente Figueiredo, Reserva ZF-2, REBIO Uatumã, RDS Uatumã and Parque Estadual Rio Negro Setor Sul, Cuieiras River. To the south and west its distribution is likely to be delimited by the Amazon and Negro Rivers, respectively. It thus appears to be restricted to the south-central portion of the Guiana Shield ( Figure 7 ). It occurs in primary forests and forest fragments, and is found in leaf litter—often fallen fronds of the buriti palm ( Mauritia flexuosa )—generally close to creeks. The species is diurnal, is clearly sexually dimophic in size ( Figure 9 , Table 5 , online supplement 1) with females being distinctly larger than males. The amplexus is cephalic ( Figure 9 ). Reproductive period is from November to April ( Magnusson & Hero, 1991 ). FIGURE 5. Dorsal and ventral view of male specimens of: A) Topotype of Amazophrynella minuta ,Taracuá , Brazil (INPA-H 32732); B) Amazophrynella bokermanni , Juruti , State of Pará, Brazil (INPA-H 31861); C) Amazophrynella vote , Parque Estadual do Matupiri, State of Amazonas, Brazil (INPA-H 31870); D) Holotype of Amazophrynella manaos sp. nov. campus of the Universidade Federal do Amazonas-UFAM, State of Amazonas, Brazil (INPA-H 31866). FIGURE 6. Patterns of variation in Amazophrynella manaos sp. nov. , dorsal and ventral views. Etymology. We name the species in honor of the Manaos Amerindian tribe that inhabited the region of the present day city of Manaus, Amazonas, Brazil , where the species is distributed.