Uncovering the diversity in the Amazophrynella minuta complex: integrative taxonomy reveals a new species of Amazophrynella (Anura, Bufonidae) from southern Peru.

A new species of the genus Amazophrynella (Anura, Bufonidae) is described from the departments of Madre de Dios, Cusco and Junin in Peru. An integrative taxonomy approach is used. A morphological diagnosis, morphometrics comparisons, description of the advertisement call, and the phylogenetic relationships of the new species are provided. Amazophrynella javierbustamantei sp. n. differs from other species of Amazophrynella by: intermediate body-size (snout-vent length 14.9 mm in males, n = 26 and 19.6 mm in females, n = 20), tuberculate skin texture of body, greatest hand length of the Amazophrynella spp. (3.6 mm in males, n = 26 and 4.6 mm in females, n = 20), venter coloration yellowish, tiny rounded black points covering the venter, and thirteen molecular autapomorphies in the 16S RNA gene. Its distribution varies from 215 to 708 m a.s.l. This discovery highlights the importance of the remnant forest in preserving the biodiversity in Peru, and increase in seven the species formally described in the genus Amazophrynella.

Introduction

Until 2012, two species of were placed in the genus Jimenez de la Espada, 1868. Fouquet et al. (2012a) recognized that species of from the Amazon and Atlantic rainforests represented morphologically and genetically deeply divergent lineages, and thus the authors proposed a new genus: Fouquet, Recorder, Texeira, Cassimiro, Amaro, Camacho, Damaceno, Carnaval, Moritz & Rodrigues, 2012 for the Amazonian species and .

In the following years, an additional four new species of the genus were described: Ávila, Carvalho, Gordo, Ribeiro & Morais, 2012 and Rojas, Carvalho, PageBreakGordo, Ávila, Farias & Hrbek, 2014 based on morphology; and Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2015, based on morphology and genetic data (Ávila et al. 2012; Rojas et al. 2014, 2015). Species of the genus are distributed in nine South American countries: Bolivia, Peru, Ecuador, Colombia, Venezuela, Guiana, French Guiana Brazil, and presumably in Suriname (Frost et al. 2015).

Using a phylogenetic analysis based on mitochondrial and nuclear genes (Fouquet et al. 2007, 2012a), the existence of three independent evolutionary lineages was discovered within the nominal species from Ecuador and French Guianas; subsequently, other independent evolutionary lineages were discovered from Brazil and Peru (Rojas et al. 2014, 2015). The difficulties in delimiting species within the species complex resides in the relatively generalized diagnosis (see Melin 1941) and the poor geographic sampling. For these reasons, historically, the name has been used for individuals distributed throughout the Amazonian biome (e.g. Duellman 1978; Zimmerman and Rodrigues 1990; Magnusson and Hero 1991; Rodrigues and Duellman 1993; Duellman and Mendelson 1995; Fouquet et al. 2012a). Thus, taxonomy and systematics of populations that are currently part of the complex remains largely unresolved (Rojas et al. 2014), in turn limiting the knowledge of the true taxonomic diversity of the genus (Ávila et al. 2012; Rojas et al. 2014, 2015).

Given this scenario, herein is described an additional new species of from the departments of Madre de Dios, Cusco and Junin, Peru, founded on the principles of integrative taxonomy. Morphological, morphometric, bioacoustic and phylogenetic relationships are provided as evidence for the existence of the new taxon.

Material and methods

Morphology

Forty eight specimens previously identified as (Melin, 1941), deposited at the and were analyzed. This material was compared with twenty preserved specimens of from the type locality (Taracuá mission, on the right bank of the Uaupés River, municipality of São Gabriel da Cachoeira, Brazil), deposited in the Collection of Amphibians and Reptiles of the . Further comparisons were made with three syntypes deposited at the , and the original description of the species (Melin 1941).

Museo de Historia Natural del Cusco, Universidad Nacional de San Antonio Abad del Cusco

Museo de Historia Natural de la Universidad Nacional Mayor de San Marcos

Instituto Nacional de Pesquisas da Amazônia–, Brazil

Naturhistoriska Museet, Göteborg, Sweden

Additionally five preserved specimens of (Izecksohn, 1993) from near the type locality (Juruti, 30 Km from type locality), the holotype and paratypes of deposited in the Collection of , the holotype of , deposited in the , seventeen paratypes deposited in the , and the holotype and paratypes of and , deposited at the were analyzed (see Appendix 1 listing all the revised specimens).

Amphibians and Reptiles of the Instituto Nacional de Pesquisas da Amazônia–, Manaus, Amazonas, Brazil

Coleção Zoológica de VerPageBreaktebrados of the Universidade Federal de Mato Grosso–UFMT, Cuiabá, Mato Grosso, Brazil

Collection of Amphibians and Reptiles of the Instituto Nacional de Pesquisas da Amazônia–, Manaus, Amazonas, Brazil

Museo de Zoologia de la Universidad Nacional de la Amazonia Peruana

Morphological character analyses were carried out according to Cruz and Fussinato (2008) and Fouquet et al. (2012a). Sex was determined by gonad analysis.

Measurements were carried out with a digital caliper following Kok and Kalamandeen (2008) and Duellman (1978). SVL from the tip of the snout to the posterior edge of the cloaca; HL from the posterior edge of the jaw to the tip of the snout; HW, the greatest width of the head, usually at the level of the posterior edges of the tympanum; ED; IND, the distance between the edges of the nares; SL from the anterior edge of the eye to the tip of the snout; HAL from the proximal edge of the palmar tubercle to the tip of Finger III; UAL from the edge of the body insertion to the tip of the elbow; THL from the vent to the posterior edge of the knee; TL from the outer edge of the knee to the tip of the heel; TAL from the heel to the proximal edge of the inner metatarsal tubercle; FL from the proximal edge of the inner metatarsal tubercle to the tip of Toe IV. Diagnosis of characters follow Chaparro et al. 2015.

(snout-vent length)

(head length)

(head width)

(eye diameter)

(internarinal distance)

(snout length)

(hand length)

(upper arm length)

(thigh length)

(tibia length)

(tarsal length)

(foot length)

Statistical analysis. We used a total of 80 adult males of the species complex (numbers of individuals and populations of origin in parentheses): sensu stricto (n = 23, from Taracuá), (n = 15, from Puerto Almendras and Fazenda Zamora); (n = 13, from Nuevo Salvador) and the new species of (n = 29, from Tambopata, Nuevo Arequipa, Candamo and Inambari).

All morphometric measures were log10 transformed to conform to requirements of normality (Hayek et al. 2001). The effect of size was removed from all variables by regressing them against SVL and using the residuals of each variable in a . Significance of morphometric differences was tested with with the two first principal components being treated as dependent variables and species as independent variables. The first two principal components were used since they explained the majority of observed variation in shape. A was performed to test classification of individuals in predicted groups. All the statistical analysis were performed in R (R Development Core Team 2011) adopting a 5% significance cut-off. PCA was used to detect groups representing putative cryptic species and DFA was subsequently applied to identify the set of characters that best diagnose those groups (Padial and De la Riva 2009). Additionally we noted large size in the HAL of the new species of , and we used an of the original data (from , , and the new species) to statistically support this hypothesis.

Principal Component Analysis

Multivariate Analysis of Variance

Analysis of Variance

Molecular data

Laboratory procedure. Total DNA was extracted from muscle tissue using standard phenol/chloroform extraction (Sambrook et al. 1989). A 480 bp fragment of the 16S rDNA was PCR amplified using the 16Sar and 16Sbr primers (Palumbi 1996). Amplification was carried out under the following conditions: 60 s hot start at 92 °C followed by 35 cycles of 92 °C (60 sec), 50 °C (50 sec) and 72 °C (1.5 min). Final volume of the PCR reaction was 12 μl and contained 4.4 μL ddH2O, 1.5 μL of 25 mM MgCl2, 1.25 μL of 10 mM dNTPs (2.5mM each dNTP), 1.25 μL of 10x buffer (75 mM Tris HCl, 50 mM KCl, 20 mM (NH4)2SO4), 1 μL of each 2 μM primer, 0.3 μL of 5 U/μL DNA Taq Polymerase (Biotools, Spain) and 1 μL of DNA (about 30 ng/μL). Sequencing reactions were carried out according to the manufacturer’s recommendation for the ABI BigDye Terminator cycle sequencing mix, using 16Sa primer and an annealing temperature of 50 °C. Sequencing reactions were precipitated using standard EDTA/EtOH protocol, and resolved in an ABI 3130xl automatic sequencer.

Phylogenetic analysis. We obtained 16S rDNA sequence data from two specimens of the new species (Accession numbers: KR905184, KR905185), two paratypes of (Accession numbers: KF433970, KF433971), two specimens of (Accession numbers: KF433975, KF433976), two topotypic specimens of (Accession numbers: KF792834, KF792836), two paratopotypes of (Accession number: KP681688, KP681689), the holotype and one paratopotype of (Accession number: KP681868, KP681669) and two paratypes of (Accession number: KF433954, KF433957) deposited in the tissue collection of the Laboratório de Evolução e Genética Animal of the Universidade Federal do Amazonas (CTGA-ICB/UFAM). The dataset also included two sequences of (Accession number: AY326000, DQ158420) from Darst and Canatella (2004), Pramuk (2006) and two sequences of (Accession number: EU201057, JN867570) from Fouquet et al. (2007). As outgroups we used species of the sister taxon (see Table 2 for samples information).

Table 2.

Individuals of the genus (A) and (D) used in the molecular analyses. Information includes samples, collecting locality, GenBank accession number for the 16S rDNA fragment, voucher number and specimen status.

Sample	Locality	Accession Number	Voucher number	Specimen status
Amazophrynella javierbustamantei	Quebrada Guacamayo, Peru	KR905184	MHNC 8331	Holotype
Amazophrynella javierbustamantei	Quebrada Guacamayo, Peru	KR905185	MHNC 8363	Paratype
Amazophrynella matses	Nuevo Salvador, Peru	KF681688	MZUNAP 928	Paratopotype
Amazophrynella matses	Nuevo Salvador, Peru	KF681689	MZUNAP 941	Paratopotype
Amazophrynella minuta sensu stricto	Taracuá, Brazil	KF792834	INPA-H 32729	Topotype
Amazophrynella minuta sensu stricto	Taracuá, Brazil	KF792835	INPA-H 32730	Topotype
Amazophrynella amazonicola	Puerto Almendras, Peru	KF681868	MZUNAP 901	Holotype
Amazophrynella amazonicola	Puerto Almendras, Peru	KF681669	MZUNAP 915	Paratopotype
Amazophrynella vote	Parque Nacional Nascentes do Lago Jari, Brazil	KF433970	INPA-H 28720	Paratype
Amazophrynella vote	Parque Nacional Nascentes do Lago Jari, Brazil	KF433971	INPA-H 28722	Paratype
Amazophrynella bokermanni	Juruti, Pará, Brazil	KF433975	INPA-H 31864
Amazophrynella bokermanni	Juriti, Pará, Brazil	KF433976	INPA-H 31861
Amazophrynella manaos	Mineração taboca, Brazil	KF433954	INPA-H 29566	Paratype
Amazophrynella manaos	Mineração taboca, Brazil	KF433957	INPA-H 29567	Paratype
Amazophrynella sp. aff. manaos	Mitaraka, French Guiana	JN867570	296MC
Amazophrynella sp. aff. manaos	Mitaraka, French Guiana	EU201057	3035T
Amazophrynella sp. aff. minuta	Rio Lagarto Cocha, Peru	AY326000	USNM 520905
Amazophrynella sp. aff. minuta	Equador	DQ158262	QCAZ833
Dendrophryniscus proboscideus	Mata Escura, Brazil	JN867566	MTR17173
Dendrophryniscus proboscideus	Mata Escura, Brazil	JN867564	MTR17171
Dendrophryniscus oreites	Serra das lontras, Brazil	JN867567	MTR16368
Dendrophryniscus carvalhoi	Parna Caparão, Brazil	JN867568	MTR15755
Dendrophryniscus carvalhoi	Parna Caparão, Brazil	JN867569	MTR15757
Dendrophryniscus leucomyxtas	Ilha grande, Brazil	JN867558	MTR15547
Dendrophryniscus leucomyxtas	Ilha grande, Brazil	JN867557	MTR15548
Dendrophryniscus berthalutzae	Treviso, Brazil	JN867551	CFBH10322
Dendrophryniscus brevipollicatus	Estação Biológica de Boracia, Brazil	JN867554	AF1541
Dendrophryniscus brevipollicatus	Estação Biológica de Boracia, Brazil	JN867553	AF1175

Sequences were aligned using the Clustal W algorithm (Thompson et al. 1996) implemented in BioEdit (Hall 1999) and alignment was adjusted as necessary against the secondary structure of the 16S rDNA. The existence of lineages in a phylogenetic tree-based context (Baum and Donoghue 1995) was performed using Maximum Likelihood analysis (Felsenstein 1981) in the program Treefinder (Jobb 2008) using the GTR+I+G model of substitution, selected via Akaike information criterion as implemented in Modeltest 3.7 (Posada 2006). Phylogenetic support was assessed via 10 000 non-parametric bootstrap (Felsenstein 1985). Additionally uncorrected pairwise genetic distances between linages identified by phylogenetic inference of were calculated in MEGA 5.05 (Tamura et al. 2007).

Molecular species delimitation. Evolutionary lineages are diagnosed by discontinuities in character variation among lineages, and correspond to phylogenetic species. The existence of lineages is therefore a necessary and sufficient prerequisite for inferring the existence of a species under the different conceptualizations of the (Cracraft 1983; Baum and Donoghue 1995; De Queiroz 2007). The existence of lineages in a non-tree-based context (Cracraft 1983) was inferred using Population Aggregation Analysis performed at the level of an individual (Davis and Nixon 1992; Rach et al. 2008) using the dataset with the species complex: , , and the new species. The analyses were performed in the program R (R Development Core Team 2011).

Phylogenetic SpePageBreakcies Concept

Bioacoustics

We analyzed one advertisement call obtained from the CD of Frogs of Tambopata, Peru (Macauly Library of Natural Songs and Cornell Laboratory of Ornithology) by the authors Cocroft et al. (2001) from the Natural Reserve of Tambopata, a locality of occurrence of the new species. The call was edit with the software Audacity 1.2.2 for Windows (Free Software Foundation Inc. 1991). The spectral and temporal parameters of the recording were analyzed in the software Raven Pro. 1.3 for Windows (Cornell Laboratory of Ornithology). The advertisement call was obtained from one male in a temperature 25 °C (Crocoft et al. 2001). We measured the following quantitative parameters: call duration (seconds); pulses per call; length of silence between calls (seconds); dominant frequency (kHz); fundamental frequency (kHz) and time to peak at maximum frequency (seconds).

Results

Phylogenetic analysis and systematics

In the resulting phylogeny, the six nominal species of were recognized as monophyletic (Fig. 1). In the genus we can distinguish two monophyletic groups: One clade (bootstrap support = 100) formed by the species: , and and another represented by the species of the “species complex” (bootstrap support = 98): , , and the new species described herein.

Figure 1.

Maximum Likelihood tree of the species based on the GTR+I+G model, analyzing 480 bp of 16S rDNA. Numbers below branches represent bootstrap support for 10 000 pseudoreplications.

In the first clade the species: is sister taxon of the possible new specie from the Guiana Shield: (bootstrap support= 91), and both are sister to (bootstrap support= 98). is sister of + ( + ) with a bootstrap support of 81.

The second clade corresponding to the “species complex”, is sister of + from western Amazonia (bootstrap support= 99). Our analysis further highlighted the occurrence of a new monophyletic lineage ( sp. n.) showing sister relationship with (bootstrap support = 96), both being in turn sister group of + ( + ) with a bootstrap support of 99.

Smallest uncorrected 16S rDNA p-distances estimated between phylogenetic linages was observed between and (= 3%). Greatest interspecific distance (= 14%) was observed between sp. n. and and was comparable to divergence observed between and . Within the “” species complex, the new species shows a high degree PageBreakof genetic divergence from (= 7%), (= 9%) and minor genetic distance with their sister taxon (= 3%) (see all pairwise genetic distance values summarized in Table 3). According to the Population Aggregation Analysis, the newly identified lineage was also diagnosable by thirteen molecular autapomorphic characters (Table 4) leading us to the conclusion that this lineage corresponds to a new species.

Table 3.

Uncorrected p-distances between (A), species and the sister genus (D). Molecular distances are based on the 480-bp fragment of 16S rDNA. We included sensu stricto from its type locality and two candidate species, and mentioned in Fouquet et al. (2012a).

16S rDNA	1	2	3	4	5	6	7	8	9
1 Amazophrynella amazonicola
2 Amazophrynella matses	0.08
3 Amazophrynella sp. aff. minuta	0.06	0.07
4 Amazophrynella minuta	0.05	0.08	0.03
5 Amazophrynella javierbustamantei sp. n.	0.09	0.03	0.06	0.07
6 Amazophrynella vote	0.12	0.12	0.12	0.12	0.13
7 Amazophrynella bokermanni	0.12	0.12	0.11	0.11	0.13	0.10
8 Amazophrynella manaos	0.12	0.12	0.12	0.14	0.12	0.10	0.08
9 Amazophrynella sp. aff. manaos	0.12	0.11	0.12	0.13	0.12	0.10	0.07	0.04
10 Dendrophryniscus leucomystax	0.19	0.21	0.17	0.18	0.20	0.22	0.18	0.20	0.20

Table 4.

Species level diagnostic characters observed in the 16S rDNA gene of sp. n. and other species of genus . First line indicates position of the character within the 16S rDNA gene; (-) indicates a deletion.

Species	213	232	271	276	470	471	473	474	476	477	478	479	480
Amazophrynella manaos	A	C	A	C	A	T	G	T	C	A	A	A	A
Amazophrynella vote	A	T	A	C	C	C	C	T	T	A	A	A	G
Amazophrynella minuta	C	T	A	A	C	C	C	T	T	A	A	A	G
Amazophrynella bokermanni	A	T	A	C	A	T	G	T	C	A	A	A	A
Amazophrynella amazonicola	C	C	A	C	C	C	C	T	T	A	A	T	G
Amazophrynella javierbustamantei sp. n.	T	G	G	T	T	G	T	G	A	G	C	C	-
Amazophrynella matses	C	T	A	C	C	C	C	T	T	A	A	T	T

Morphometric analysis

Comparative analysis of quantitative morphological data allowed us to distinguish sp. n. from the other members of the “species complex”. The first two principal components extracted by the PCA account for 48.56% of the variation found in the dataset. The first component (PC1) explained 24.93% of total variation. In the first principal component axis, is distinguished from the other species due to its larger size (SVL = 14.9 ± 0.7 mm, see Table 1), sharing relative size with sensu stricto (SVL = 13.5 ± 0.6 mm, see Table 1), the species is distinguish by having the smallest size of the genus (SVL range= 12.1 ±0.6 mm, see Table 1), and shares this characteristic with sp. n. (SVL = 14.9 ± 0.9 mm, see Table 1) (Fig. 2). The second component explains 23.63% of the variation. This axis represents a shape variation vector; in this axis sp. n. is well distinguished from the three formally described species, sharing more similarity with (Table 5).

Table 1.

Measurements (mm) of adult male specimens (including the holotype) in the type series spp. Mean ± standard deviation, with ranges in parentheses. Abbreviations are defined in material and methods.

Variable	Amazophrynella minuta sensu stricto (n = 15)	Amazophrynella manaos (n = 29)	Amazophrynella bokermanni (n = 5)	Amazophrynella vote (n = 14)	Amazophrynella amazonicola (n = 15)	Amazophrynella matses (n = 13)	Amazophrynella javierbustamantei sp. n. (n = 26)
SVL	13.5 ± 0.6 (12.5–14.2)	14.2 ± 0.7 (12.3–15.0)	16.8 ± 1.4 (14.6–18.2)	13.1 ± 0.7 (12.0–14.1)	14.5 ± 0.7 (13.3–15.4)	12.1±0.6 (11.5–13.5)	14.9 ± 0.9 (12.7–16.4)
HW	4.2 ± 0.2 (4.0–4.3)	4.2 ± 0.3 (3.7–4.7)	3.2 ± 0.3 (2.5–3.3)	4.0 ± 0.7 (3.3–4.4)	4.4 ± 0.3 (4.2–4.6)	3.6 ±0.2 (3.1–3.8)	4.2 ± 0.2 (3.5–4.7)
HL	4.9 ± 0.2 (4.8–5.3)	5.3 ± 0.3 (4.7–5.6)	3.4 ± 0.4 (2.8–3.8)	4.6 ± 0.3 (4.0–5.2)	5.2 ± 0.3 (5.0–6.2)	4.3 ± 0.3 (3.9–4.8)	5.1 ±0.3 (4.4–5.6)
SL	2.3 ± 0.1 (2.2–2.5)	2.7 ± 0.2 (2.3–2.7)	3.0 ± 0.4 (2.2–3.1)	2.1 ± 0.2 (1.9–2.6)	2.4 ± 0.2 (2.2–2.5)	2.0 ± 0.3 (1.6–2.3)	2.2 ± 0.2 (1.7–2.6)
ED	1.4 ± 0.1 (1.3–1.5)	1.3 ± 0.1 (1.2–1.6)	1.8 ± 0.2 (1.5–2.0)	1.3 ± 0.1 (1.2–1.5)	1.2 ± 0.1 (0.9–1.2)	1.1 ± 0.1 (0.9–1.2)	1.3 ± 0.1 (1.0–1.6)
IND	1.2 ± 0.1 (1–1.3)	1.1 ± 0.1 (1.0–1.4)	1.4 ± 0.2 (1.0–1.5)	1.1 ± 0.1 (1.0 –1.3)	1.2 ± 0.1 (1.0–1.3)	1.0 ± 0.1 (0.8–1.2)	0.9 ± 0.1 (0.8–1.2)
UAL	3.8 ± 0.2 (3.2–4.1)	3.6 ± 0.4 (2.9–4.1)	5.5 ± 0.6 (5.0–5.6)	3.9 ± 0.5 (2.8–3.9)	4.5 ± 0.3 (4.2–5.3)	3.5 ± 0.4 (2.9–4.2)	4.5 ± 0.4 (3.8–5.7)
HAL	2.8 ± 0.2 (2.6–3.0)	2.8 ± 0.6 (1.9–2.9)	3.4 ± 0.6 (2.8–4.2)	2.7 ± 0.3 (2.3–3.2)	3.2 ± 0.2 (2.8–3.3)	2.7 ± 0.2 (2.3–3.1)	3.6 ± 0.4 (2.5–4.5)
THL	6.8 ± 0.2 (6.4–7.2)	6.7 ± 0.3 (2.3–3.1)	8.7 ±1.4 (7.2–8.9)	6.5 ± 0.7 (5.4–7.2)	7.7 ± 0.6 (6.3–8.0)	6.2 ± 0.4 (5.1–6.3)	7.6 ± 0.7 (6.2–9.2)
TAL	6.7 ± 0.3 (6.3–7.1)	6.9 ±0.6 (4.2–7.3)	8.3 ± 1.0 (6.7–9.2)	5.7 ± 0.7 (4.8–7.0)	7.2 ± 0.6 (6.1–7.9)	5.8 ± 0.3 (5.1–6.3)	7.6 ± 0.7 (6.2–8.8)
TL	4.1 ± 0.2 (3.8–4.6)	4.6 ± 0.4 (4.3–6.3)	5.4 ± 1.4 (2.9–6.2)	3.8 ± 1.0 (4.2–7.0)	4.2 ± 0.6 (6.3–8.0)	3.8 ± 0.2 (3.6–4.3)	4.7 ± 0.8 (3.9–8.7)
FL	4.8 ± 0.4 (4.2–5.2)	5.2 ± 0.5 (4.7–6.1)	6.3 ± 1.3 (3.9–7.6)	4.4 ± 0.6 (3.2–5.4)	5.1 ± 0.4 (4.7–6.0)	4.3 ± 0.4 (5.5–3.0)	5.7 ± 0.6 (4.5–7.2)

Figure 2.

Principal Component Analysis (PCA) from: species complex. See Table 5 for character loadings on each component.

Table 5.

Character loadings, eigenvalues, and percentage of explained variance for Principal Components (PC) 1–2. The analysis was based on eleven morphometric variables of adult males: complex ( sensu stricto; ; and sp. n.).

Variables	PC1	PC2
HW	0.462	-0.146
HL	0.455	-0.104
SL	0.374	-0.244
ED	0.261	0.052
IND	0.369	-0.271
UAL	0.139	0.258
HAL	-0.032	0.484
THL	0.311	-0.295
TAL	0.314	0.350
TL	0.116	0.364
FL	0.063	0.433
% of variation	24.93	23.63
%	24.93	48.56

All the species of the group are significantly different in shape (MANOVA, F24.3, Pillae´s trace < 0.001). The found specimens correctly classified in 56.6% of cases and a moderate prior probabilities of groups ( = 28.75%, = 18.75%, = 16.25% and sp. n. = 36.25%). The variables that contributed most to the classification were HAL, SVL and TAL (Table 6). The differences in HAL were significant (ANOVA, F45.27, P < 0.001) among all the species of “species complex” (see Fig. 1), and reveals sp. n. as the species with the largest HAL (Fig. 3).

Table 6.

Character loadings of explained variance for . The analysis was based on twelve morphometric variables of adult males of the complex ( sensu stricto; ; and sp. n.).

Discriminant Function Analysis

Variables	Discriminant Function
SVL	6.343
HW	-7.628
HL	0.146
SL	-5.479
ED	-1.175
IND	-6.015
UAL	1.313
HAL	5.744
THL	-3.871
TAL	13.944
TL	-1.250
FL	1.016

Figure 3.

Measurement comparison of the Hand Length (HAL) between species of complex.

discriminate function analysis

Morphological description

sp. n.

http://zoobank.org/A946B949-1D1F-4FF5-B722-0B33435EE610

Holotype

(Fig. 4). MHNC 8331 (Genbank 16S rRNA: KR905184). Adult male, collected at Quebrada Guacamayo (12°54'24.5"S; 69°59'32.7"W, 215 m a.s.l.) km 105 of the highway Puerto Maldonado–Cusco City, District Inambari, Province Tambopata, Department Madre de Dios, Peru, on 27 October 2009 by Juan C. Chaparro and Oscar Quispe.

Figure 4.

Holotype of sp. n. (MHNC 8331); A dorsal view B ventral view C dorsolateral view D right hand E right foot.

Paratypes

(Fig. 5). Twenty-two specimens (males= 09, females= 13). MHNC 8363, MHNC 8245, MHNC 8238, adult males, MHNC 8316, MHNC 8484, MHNC 8362, MHNC 8354, adult females, collected with the holotype (12°28'25"S, 69°12'36"W, 205 m a.s.l.). MHNC 11001, adult male, MHNC 11002, MHNC 11003, MHNC 11004, adult females collected by E. Aguilar on 17 May 2009, from La Pampa km 107 highway Puerto Maldonado–Cusco City, Department Madre de Dios (12°40'14.14"S, 72°27'30"W, 250 m a.s.l.). MHNSM 17993, adult male collected by A. Angulo in 1999; from Province Manu, locality of Inambari, Department Madre de Dios (13°02'29.28"S, 70°22'46.65"W, 306 m a.s.l.). MHNSM PageBreakPageBreakPageBreakPageBreak25651, adult female, collected by D. Rodriguez on April 2007, from Province La Convención, locality of Camana, Department Cusco (12°05'9.25”S, 73°03'2.61”W, 680 m a.s.l.). MHNC 9939, MHNC 9940, adult females, collected by J. Delgado on 17 January 2010 from Province La Convención, locality of Mapi, Department Cusco (11°31'19.17”S, 73°28'29.83”W, 708 m a.s.l.). MHNC 9387, adult male, collected by G. Estrada on 21 January 2010, from locality of Tambo Poyeni near Quebrada Mayapo, Department Junin (11°19'29.9”S, 73°32'16.7"W, 388 m a.s.l.). MHNC 9754, MHNC 9756, adult males, MHNC 9626 , MHNC 9679, MHNC 9680, MHNC 9757, adult females, collected by A. Pari on January 2010, from locality of Tsoroja, Department Junin (11°18'56.06”S, 73°32'32.11”W, 399 m a.s.l. and 11°23'14.50”S , 73°29'43.00”W, 450 m a.s.l.).

Figure 5.

Dorsal and ventral view of some Paratypes of sp. n. Adult males (MHNC 8245: SVL 13.6 mm; MHNSM 31255: SVL 15.9 mm; MHNSM: 17993 SVL 14.2 mm; Adult females (MHNC 11002: SVL 17.2 mm, MHNC 9739: SVL 21.5, MHNC 8362: SVL 18.0 mm).

Diagnosis.

The new species is part of based on molecular phylogenetic relationships (Fig. 1) and morphological synapomorphies (Fouquet et al. 2012a).

sp. n. is characterized by: (1) skin on dorsum tuberculate, with many subconical tubercles disperse on arms, legs, head and body; ventral skin coarsely areolate, throat and chest aerolate; (2) tympanic membrane and tympanic annulus not apparent through the skin; (3) snout long, subacuminated, protruding in lateral views; (4) upper eyelid with smaller tubercles, cranial crests absent; (5) dentigerous process of vomers absent; (6) vocal sac, vocal slits and nuptial pads absent; (7) finger I shorter than finger II, tips of digits rounded; (8) fingers lacking lateral fringes; (9) ulnar tubercles present; (10) heel bearing eight or more small low tubercles, tarsus with small tubercles and lack of folds; (11) plantar surfaces of feet bearing one metatarsal tubercle, the inner 2.5x larger than the outer, outer subconical; supernumerary plantar tubercles round and low; (12) toes lacking lateral fringes; webbing basal; toe III equal than toe V, tips of digits rounded; (13) dorsally is dark brown to light brown, and gray to black in some, ventrally, cream with yellow to orange marks, with PageBreakPageBreakPageBreakblack to dark brown spots; (14) SVL 16.39–22.25 mm in females, 12.79–16.42mm in males; (15) hand length is the greatest of all species of : 3.6 mm in males (n= 26) and 4.6 mm in females (n=20), see Fig. 3; (16) thirteen molecular autapomorphies in the 16S rDNA gene.

Comparison with other species.

sp. n. (Figs 4, 5, 6) differs in the following character states (states of other species in parentheses). From (Fig. 6A) by having body skin texture tuberculate (roughly granular); relative abundance of spiny granules on the forelimbs (prickly warty skin on axillary region of the forelimbs); absence of large warts on dorsum (presence of large warts); throat and chest cream-grayish (light brown); posterior side of belly color pale orange yellowish with tiny rounded black or dark brown spots (throat and the whole belly intensely orange yellowish); tiny rounded black spots covering the belly (irregular black ocelli or blotches); metatarsal tubercle rounded (oval). From (Fig. 6B) relative size of fingers, with finger I shorter than II (I>II); snout vent length smaller in males (15.8 mm) and females (22.25 mm) ( with maximum 22 mm SVL in males and 28 mm SVL in females, see Izecksohn 1993); smaller snout in males, with 2.2 mm SL, n = 26 (2.7 mm SL, n = 5; see Table 1); posterior side of belly color pale orange yellowish with tiny rounded black or dark brown spots (white coloration with small black dots). From to (Fig. 6C) snout subacuminated in dorsal view (rounded); posterior side of belly color pale orange yellowish with tiny rounded black or dark brown spots (ventral color pattern reddish brown, with presence of small white dots). From (Fig. 6D) snout subacuminated (snout truncate); dorsal skin finely granular (dorsal surfaces granular); throat and chest grayish (dark coloration); posterior side of belly color pale orange yellowish with tiny rounded black spots (venter cream with black spots or stripes). From to (Fig. 6E) snout subacuminated (snout slightly truncate), edges of nasal protrusion not dilated (dilated in ventral view); shape of palmar tubercle rounded (palmar tubercles elliptical); finger tips unexpanded (expanded), rounded tiny black spots covering the belly (medium-sized black ocelli or streaks); coloration of the belly pale yellow (belly completely yellow). From (Fig. 6F) by the absence of small triangular protrusion on the tip of the snout in both dorsal and ventral views (presence); body surface granular (finely granular), dorsum uncovered with medium-sized granules scattered irregularly (covered with medium-sized granules scattered irregularly); posterior side of belly color pale orange yellowish with tiny rounded black or dark brown spots (orange yellowish with dark red and brown blotches).

Figure 6.

Dorsal and ventral morphological comparison between the spp. (Unvoucher specimens): A sp. n. B C D E F G .

Description of the holotype.

Body slender, head triangular, slightly longer than wide; head length 35.5% of SVL, head width 30.9% of SVL. Snout long, subacuminate in dorsal view, protruding in lateral view; canthus rostralis straight and loreal region vertical; without papilla; snout length 39.0% of head length; tympanic membrane and tympanic annulus not apparent through the skin, skin of the tympanic area covered by round sub-conical warts; vocal sac externally not visible, vocal slits absent; eyes prominent 23.8% of head length; upper eyelid covered with small tubercles; those close to the external margin aligned in a more or less distinct row; nostril closer to snout than PageBreakPageBreakto eyes; internarial distance smaller than eye diameter; presence of a line of small spiny granules from the outer edge of the mouth to upper arm, choanas small and circular.

Dorsal skin finely tuberculate with several large tubercles scattered sub-conical tubercles on upper arm; texture of ventral skin granular, covered by rounded granules. Dorsolateral surfaces, granular, with presence of large rounded tubercles. Forelimbs PageBreakslender, upper arm length 29.6% of SVL; edges of lower arm and upper arm finely tuberculate with several large sub-conical and spiny granules; hand length 76.5% of upper arm length; fingers slender, tips not expanded; relative length of fingers Imetatarsal tubercle oval, 2.5× larger than outer; outer metatarsal tubercles small, rounded; subarticular tubercles present, rounded, present one on fingers I, II, and two on fingers III, V and three on finger IV; and tip of toes not expanded.

Measurements of the holotype

(in millimeters). SVL 15.1; HW 4.6; HL 5.3; SL 2.1; ED 1.2; IND 1.0; UAL 4.4; HAL 3.4; THL 8.1; TAL 8.1; TL 4.5; FL 5.3.

Coloration of the holotype.

In life: dorsum of the holotype mostly light brown with dark brown in the dorsum; dorsolaterally creamish-brown with scattered black blotches; dorsal surfaces of hands and feet creamish-brown, and gray on arms and legs; belly creamish-gray with black dots, and the throat gray; fingers, toes and plantar surfaces reddish-black; groin with orange marks; iris with a bronze ring; cloaca with orange flap, black pupil and bronze iris. In alcohol: dorsum brownish-grey; venter cream with black and brown dots; orange surfaces turned cream, with a white longitudinal stripe on upper jaw extending from nostril to forearm.

Variation.

The new species is phenotypically variable. In some individuals (e.g. MHNC 8245 and MHNC 11002, see Fig. 5) patterning on the dorsum varies, with these specimens presenting brown chevrons extending from the head to the vent. Some individuals showed a white line extending from the tip of the nose to the upper arm. Another specimen (MHNC 9739, see Fig. 5) presented a yellow pale coloration in the axillary region (in ventral view). In some individuals, the coloration of the throat extended onto the chest (e.g. MHNC 11002, MHNC 9739 and MHNC 8245, see Fig. 5). The pale yellow coloration of the belly surface may extend from thighs to the chest or just to the middle of the belly (e.g. MHNC 8362, see Fig. 5 and Fig. 7B). In some individuals, the thighs are abundantly covered by rounded tiny spots extending to the shank (Fig. 7B). In preserved specimens the dorsum becomes light brown and the belly coloration vary from white to yellow pale (e.g. MHNSM 31255 and MHNSM 17993, see Fig. 5). The color of the finger becomes pale red and in other individuals the red coloration of the fingers became brown or orange (Fig. 5).

Figure 7.

Dorsal and ventral variation of sp. n. (Unvoucher specimens): A–C Nueva Arequipa, Madre de Dios Department B Basin of Bajo Urubamba, Cusco Department.

Bioacoustics.

The following values are presented as: min-max (average ± SD, number of notes). The call is a trill type call issued during continuous and regular intervals (Fig. 8). Each note had a duration of between 0.03 to 0.08 seconds (0.05 ± 0.01 seconds, n = 20). The number of pulses varied between 8 to 18 pulses per note (10.4 ± 2.6 pulses/note, n = 20). The silence between notes varied from 0.4 to 1.6 seconds (0.8 ± 0.3 seconds, n = 20). The dominant frequency varied from 3962.1 to 3789.8 kHz (3927.6 ± 70.7 kHz, n = 20), and coincides with the fundamental frequency. Time to peak amplitude was around 0.014 to 0.04 seconds (0.02 ± 0.01 seconds, n = 20).

Figure 8.

Advisement call of sp. n. from the Tambopata National Reserve, Madre de Dios, Peru (207 meters a.s.l.) (Macauly Library of Natural Songs and Cornell Laboratory of Ornithology) by the authors Crocoft, Morales and Mc Diarmid (2007). A Oscilogram and spectrogram by one note B Oscilogram and spectrogram of notes from the advisement Call.

Distribution, ecology and conservation.

sp. n. is known from the Department of Cusco, in the lower Urubamba river basin and Department of Madre de Dios (Inambari, Candamo and Nueva Arequipa) in Peru (Fig. 9). Its distribution can vary from 215 m a.s.l. to 708 m a.s.l. Additional specimens were recorded at Los Amigos Biological Station , Tapir Lodge, and Explorers Inn, in Tambopata National Reserve. Individuals were active during the day, jumping on leaf litter, at night they were sleeping on leaves around 30 cm above ground. This species breeds close to the edges of permanent oxbow lakes, males call during the day while perched above streams in tangles (Cocroft et al. 2001). Three of the localities, km 105, 107 and 117 of the highway Puerto Maldonado–Cusco, Department Madre de Dios, show evidence of serious environmental impacts due to illegal gold mining activities, with forest and soil removed, and environmental pollution via organic and inorganic chemicals and heavy metal (specially mercury) poisoning. In addition, the new species is distributed inside of territories where oil companies are operating. On the other hand, the species is present in two protected areas, the Tambopata Natural Reserve and Machiguenga Communal Reserve. The conservation status of this species remains unknow, but was listed in 2008 as Least Concern on the IUCN red list (2015), because it was confused with , and because s.l. had wide distribution at that time, apparent tolerance of a certain degree of habitat modification, presumed large population, and because it is unlikely to be declining, and thus did not qualify for listing in a more threatened category. With recent studies the genus, the species complex of , was split in five species, three of them are now formally described for Peru (, and sp. n.). The recognition of these new species will require the PageBreakPageBreakreevaluation of the conservation status of these species. It should also act as an impetus for additional field and laboratory studies of Peruvian amphibians, in order to understand the real conservation status of this fauna.

Figure 9.

Distribution map of sp. n. in Peru. Holotype locality in square orange, 5 Guacamayo Creek, Department Madre de Dios. Paratypes localities in white circles 1 Tsoroja, Department Junin 2 Mapi, Department Cusco 3 Camana, Department Cusco 4 Inambari, Department Madre de Dios 6 Los Amigos Biological Station, Department Madre de Dios 7 Explorer’s Inn, Department Madre de Dios.

Etymology.

The species is named after Dr. Javier Bustamante, a Peruvian residing in United States, to whom we dedicate this species in recognition of his friendship and support of herpetological taxonomy and systematics research and amphibian conservation in Peru.

Discussion

Taxonomic reviews of Amazonian amphibians suggests that morphological characters are too conservative to permit delimiting species since closely related species share similar morphologies, and amphibians in general are morphologically conservative (e.g., Elmer et al. 2007; Fouquet et al. 2007c; Funk et al. 2011; Padial et al. 2009). Thus, the use of integrative techniques in taxonomy is revolutionizing the identification and delimitation of species based on independent lines of evolutionary evidence (Dayrat 2005; Padial and De la Riva 2009). The use of an integrative approach not only allows for the discovery and delimitation of new species, it also helps us to understand the mechanism of species formation. Thus, integrative taxonomy allows us to have a better understanding of the true scope of anuran diversity in the Amazon, and it allows us to better understand the processes that generated this biodiversity.

The taxonomic ambiguity surrounding the name and to a lesser extent resulted in a severe underestimation of the taxonomic diversity of this genus. Since the descriptions of in 1941 and in 1993, the taxonomy of the genus has not been revised, leading to misdiagnoses of other species as either or due to the relatively generalized descriptions of these taxa. Three publications since 2012 (Ávila et al. 2012; Rojas et al. 2014, 2015) described four new species, increasing the taxonomic diversity of the genus by 200%. All four species were previously classified as populations of a single species with a large distribution ( sensu lato). Although striking, the severe underestimation of taxonomic diversity observed in and the existence of multiple lineages in is nothing particular to this group. Examples of other Amazonian species complexes include and , , , , , and (Fouquet et al. 2007; Elmer and Canatella 2008; Padial et al. 2009; Funk et al. 2011; Caminer and Ron 2014; Gehara et al. 2014, Jungfer et al. 2013).

The descriptions by Rojas et al. (2014, 2015) were based, in part, on diagnostic characters observed in the 16S rDNA. This gene is widely used as a DNA barcode for amphibians, for reliable species identification (Vences et al. 2005, Fouquet et al. 2007), for evaluating monophyly of species and for discovering divergent lineages (PaPageBreakdial et al. 2009, Crawford et al. 2010; Padial et al. 2010 and Padial et al. 2012). Based on 16S rDNA analyses, we also have evidence that and represent species complexes (RRRZ, personal observation). This observation is in addition to the existence of the two candidate species of already observed in previous analyses: one from the Guiana Shield (), sister taxon of , and another from Ecuador (), sister taxon of sensu stricto (Fig. 1). Although the taxonomic status of these candidate species will need to be confirmed using morphological and bioacoustics data, it is clear that even with the recent descriptions, the taxonomic diversity of the genus remains underestimated.

While part of our evidence for the existence of the new species as well as those described previously by Rojas et al. (2014, 2015) comes from the use of molecular data, the descriptions make use of other data types and non-molecular diagnoses. Thus these undiscovered lineages were not truly cryptic (morphologically cryptic), but rather the result of poor taxonomic knowledge of the group. In this respect, the genus again is not the exception, but rather the norm.

The new species sp. n. is clearly differentiated in multivariate morphometric space from the other members of the “species group” (, and ). Together with the description of sp. n. we also provide advertisement call. sp. n. is only the second species of the genus for which an advertisement call is known and recorded (see Duellman 1978). Acoustics can provide evidence of potentially new species with behavioral or premating isolating mechanisms (e.g. De la Riva et al. 1997; Gerhardt 1998; Simões et al. 2008, Padial and De la Riva 2009; Padial et al. 2012), thus providing evidence of evolutionary mechanisms that contributed to the species diversity of the genus .

The threats to the biological conservation of sp. n. are evident, with uncontrolled exploration for gold, illegal mining and the destruction of habitat in the Departments of Madre de Dios and Cusco, probably causing a significant reduction in the population sizes of the species and fragmenting its distribution. For these reasons is necessary to analyze the current population status and trends of this and another amphibian species in this Department of southern Peru.