A new species of Pristimantis (Anura: Strabomantidae) from white-sand forests of central Amazonia, Brazil

Sampling

Twenty-two adults and two juveniles of the new species were manually collected in two localities in the municipality of Iranduba, state of Amazonas, Brazil. Among them, 20 specimens were collected at the Ramal Nova Esperança, Km 20 of the AM-070 Highway (3°09′14.5 ″S, 60°13′59.4″W, 83 m elevation) on 13 December 2020, and four specimens at the RDS Rio Negro (3°03′42.0″S, 60°45′02.1″W, 61 m elevation) on 14 September 2018. Specimens were anaesthetized and killed with topic 5% lidocaine. Muscle or liver tissue was preserved in 100% ethanol for posterior genetic analysis, whereas the specimens were fixed in 10% formalin and subsequently preserved in 70% ethanol. Specimens were sexed by the presence of vocal slits exclusively present in males and internally by the condition of the gonads. All males used in the type series were found calling, which ensures their reproductive status as adults, while gravid females (n = 4) and specimens with large SVL were also considered adults. Vouchers were deposited in the herpetological collection of the Instituto Nacional de Pesquisas da Amazônia—INPA-H (Manaus, Brazil) and Museu Paraense Emílio Goeldi—MPEG (Belém, Brazil). Protocols of collection and animal care follow the Brazilian Federal Council for Biology resolution number 148/2012 (Conselho Federal de Biologia—CFBio, 2012) and the Ethics Committee on the Use of Animals of the Instituto Nacional de Pesquisas da Amazônia—CEUA-INPA (Process n° 35/2020, SEI 01280.001134/2020-63). Specimens were collected under collection permit number 73647-3 issued by the Centro Nacional de Pesquisa e Conservação de Répteis e Anfíbios of the Instituto Chico Mendes de Conservação da Biodiversidade—ICMBio.

Advertisement calls of nine males of the new species were recorded at the Ramal Nova Esperança on 13 December 2020. Recordings were made with a Sennheiser K6/ME66 unidirectional microphone (Sennheiser, Germany) coupled to a digital recorder Marantz PMD660 (Marantz, Japan). Air temperatures (24.6–26.2 °C) and humidity (86–93%) during call recording were measured with a thermohygrometer Incoterm 7663.02.0.00. Each calling male was recorded for three minutes using frequency rate of 44 kHz and 16 bits of resolution in the mono pattern. Recordings were deposited in the Fonoteca Neotropical Jacques Vielliard (FNJV) of the Universidade de Campinas (UNICAMP), Campinas, Brazil under access number FNJV 59105–59115.

Sequencing and phylogenetic analyses

Genomic DNA was extracted from tissues of five specimens from Ramal Nova Esperança and three from RDS Rio Negro using the kit PureLink™ Genomic DNA (Invitrogen by Thermo Fisher Scientific, Carlsbad, CA, USA). Fragments of two mitochondrial (16S rRNA and Cytochrome C Oxidase sub-unit 1—COI) and a nuclear gene (Recombination Activating 1—RAG1) were amplified through polymerase chain reaction (PCR) following the protocol described in Mônico et al. (2022). The 16S was amplified using primers 16Sar (5′-CGCCTGTTTATCAAAAACAT-3′) and 16Sbr (5′-CCGGTCTGAACTCAGATCACGT-3′) (Palumbi, 1996), COI using primers Chmf4f (5′-TYTCWACWAAYCAYAAAGAYATCGG-3′) and Chmr4r (5′-ACYTCRGGRTGRCCRAARAATCA-3′) (Che et al., 2012) and RAG1 using primers R182 (5′-GCCATAACTGCTGGAGCATYAT-3′) and R270 (5′-AGYAGATGTTGCCTGGGTCTTC-3′) (Heinicke, Duellman & Hedges, 2007). Amplicons were sequenced in an ABI PRISMI 3130XL (Thermo Fisher) using the forward and reverse primers of each gene. Sequences were manually edited with Geneious Pro 5.4.6 (Biomatters Ltd.), then subjected to BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul et al., 1997) to compare with sequences of other Pristimantis. Newly generated sequences were deposited in GenBank. Accession numbers are available in Appendix 1.

According to BLAST, sequences of the new species were highly similar to species currently assigned to the Pristimantis unistrigatus species group. To infer the phylogenetic relationships among the new species and its close relatives, newly generated sequences were inserted into a data set retrieved from GenBank containing selected homologous sequences (Appendix 1). Sequence selection in GenBank was focused on specimens of the P. unistrigatus species group from the Andes, Pantepui and Amazonian lowlands. Additionally, sequences of two species of the genus Oreobates were retrieved to root the tree. In total, 265 sequences (16S = 136; COI = 70; RAG1 = 59) corresponding to 137 specimens were selected. We aligned sequences of each gene using MAFFT online server (https://mafft.cbrc.jp/alignment/server/) with default parameters, except by the use of the E-INS-i strategy for the 16S and G-INS-i strategy for protein-coding genes (Katoh & Standley, 2013). The final matrix was concatenated in Mesquite (Maddison & Maddison, 2021) and composed of 137 terminals with 1,827 bp (16S = 561 pb; COI = 636 pb; RAG1 = 630 pb).

Best-fit evolutionary models and partition schemes were determined through ModelFinder (Kalyaanamoorthy et al., 2017) using seven initial partitions: one for the 16S and one for each codon of protein-coding genes. The best evolutionary models for partitions in the concatenated matrix were: TIM2 + F + R5 for 16S, GTR + F + I +G4 for COI 1st and RAG 3rd codons, HKY + F +G4 for COI 2nd, RAG 1st and 2nd codons, and GTR + F +ASC + G4 for COI 3rd position. Phylogenetic relationships were reconstructed using Maximum Likelihood inference (ML). The ML tree was inferred with IQTREE (Nguyen et al., 2015) as implemented in the webserver (http://iqtree.cibiv.univie.ac.at; Trifinopoulos et al., 2016). Clade support was estimated with 10,000 ultrafast bootstrap replications (Hoang et al., 2018), 1,000 maximum iterations, and a minimum correlation coefficient of 0.99. We calculated pairwise genetic distances (p-distance and Kimura-two-parameter distance; Kimura, 1980) among the populations of new species and close relatives using MEGA 11 (Tamura, Stecher & Kumar, 2021). Genetic distances were calculated using pairwise deletion.

Morphology

Twenty five morphometric measurements were taken from 16 adult males and six adult females of the new species following Duellman & Lehr (2009) (eye diameter—ED, eye-nostril distance—EN, foot length—FL, interorbital distance—IOD, internarial distance—IND, head length—HL, head width—HW, snout-vent length—SVL, tibia length—TL, and tympanum diameter—TD), Caldwell, Lima & Keller (2002) (forearm length—FAL, hand length—HAND, snout length—SL, disc width of Finger III—WFD), Heyer et al. (1990) (tarsus length—TAL, thigh length—THL, upper arm length—UAL), Lima, Sanchez & Souza (2007) (hand length from proximal edge of palmar tubercle to tip of Finger I—HANDI, Finger II—HANDII, and Finger IV—HANDIV) and Mônico et al. (2022) (foot length from proximal edge of outer metatarsal tubercle to tip of Toe I—FLI, Toe II—FLII, Toe III—FLIII and Toe V—FLV, and disc width of Toe IV—WTD). Measurements were taken to the nearest 0.01 mm using a Leica stereomicroscope (model S8APO) coupled to a Leica DFC295 camera, except for SVL, measured to the nearest 0.01 mm with a digital caliper. Raw data are provided in Table S1.

Format of the description and terminology of morphological characters follow Kok & Kalamandeen (2008), Duellman & Lehr (2009), and Kok, Means & Bossuyt (2011). Color in life was described based on photographs taken in the field, following the color catalog provided by Köhler (2012).

Bioacoustics

Bioacoustic variables were analyzed with Raven Pro 1.6 (Bioacoustics Research Program, 2014) with the following configuration: window = Blackman, Discrete Fourier Transform = 2,048 samples and 3dB filter bandwidth = 80.0 Hz. The following temporal and spectral traits were measured: call duration—CD, number of notes per call—NN, note duration—ND, inter-note interval—SBN, and minimum—LF, maximum—HF and dominant frequency—DF. Inter-call interval was not measured because it is usually affected by microclimatic conditions at the time of recording (i.e., on rainy days, males call more often in a short period of time than on days without rain) and poorly informative. Dominant frequency was measured using the Peak frequency function; maximum and minimum frequencies were measured 20dB below the peak frequency to avoid background noise interference. Call description follows the call centered approach of Köhler et al. (2017). Spectrogram and oscillogram were generated in R environment (R Core Team, 2019) through the ‘seewave’ package 2.0.5 (Sueur, Aubin & Simonis, 2008) using a Hanning window, 256 points of resolution (Fast Fourier Transform) and an overlap of 85%. Bioacoustic raw data are provided in Tables S2.

Nomenclatural acts

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:F8ED54F6-9E18-49BD-8178-659BDFB79C65. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Phylogenetic relationships and genetic distances

Individuals of the new species show low intraspecific genetic distances between populations (16S p-distance = mean 0.2%, maximum 0.4%). The new species is nested within a strongly supported clade grouping Pristimantis matidiktyo Ortega-Andrade & Valencia, 2012 and P. ockendeni (Boulenger, 1912) (Fig. 1, Appendix 2). This clade is sister to the clade formed by P. delius (Duellman & Mendelson III, 1995) and P. librarius (Flores & Vigle, 1994). Among the species mentioned above, mean interspecific p-distances range from 6.1 to 11.2% (Fig. 2). All these species occur in western Amazonian lowlands.

Taxonomic account

Pristimantis campinarana sp. nov.

LSID: urn:lsid:zoobank.org:act:61E81E96-5309-4FA0-AE8A-B34636CD5616

Figures 3–6, 9.

Pristimantis ockendeni: Lima et al. (2021).

Holotype

INPA-H 44426 (field number APL 23164), adult male, collected at Ramal Nova Esperança, Km 20 of the AM-070 Highway, municipality of Iranduba, state of Amazonas, Brazil (3°09′14.5″S, 60°13′59.4″W, 83 m elevation), on 13 December 2020 by A. T. Mônico and E. D. Koch.

Paratypes

Eighteen adult specimens collected at the same locality as the holotype on 12–19 December 2020 by A. T. Mônico, I. Y. Fernandes and E. D. Koch: twelve males INPA-H 44424, INPA-H 44427–29, INPA-H 44431–35 (field numbers APL 23162, 23165–67, 23169–72 and 23175 respectively) and MPEG 44637, MPEG 44639 and MPEG 44641 (field numbers APL 23181, 23183 and 23185, respectively); and six females INPA-H 44425, INPA-H 44436 and INPA-H 44437 (field numbers APL 23163, 23176 and 23177, respectively) and MPEG 44636, MPEG 44638 and MPEG 44640 (field numbers APL 23179, 23182 and 23184, respectively), and three adult males collected at Reserva do Desenvolvimento Sustentável do Rio Negro, municipality of Iranduba, state of Amazonas, Brazil(3°03′42.0″S, 60°45′02.1″W, 61 m elevation): MPEG 44634–35 and INPA-H 44439 (field numbers APL 22250–52, respectively), on 14 September 2018 by M. Ferrão, A. S. Ferreira and J. Moravec.

Referred material

Four specimens collected in the municipality of Iranduba, state of Amazonas, Brazil. One juvenile (INPA-H 44430, field number APL 23168), same data as the holotype. One juvenile (INPA-H 44440, field number APL 22253) and one adult male (INPA-H 44441, field number APL 22254), same data as the paratype INPA-H 44439. One adult male (INPA-H 44438, field number ATM 013) collected at the surroundings of Reserva do Desenvolvimento Sustentável do Rio Negro (3°06′36.5″S, 60°44′23.5″W), on 15 October 2022 by A. T. Mônico and I. Y. Fernandes. Two females (INPA-H 44698 and INPA-H 44699, field number ATM 49 and ATM 50, respectively) collected at Reserva do Desenvolvimento Sustentável do Rio Negro (2°55′16.6″S, 60°49′18.3″W), on 11 and 12 January 2023 by A. T. Mônico, I. Y. Fernandes, E. D. Koch, B. C. Martins, S. Dantas and A. P. Lima.

Diagnosis

This new species is characterized by the following combination of characters: (1) dorsal skin shagreen; (2) tympanum visible, tympanic membrane poorly differentiated, tympanum diameter 29–40% of eye diameter and annulus partially visible externally; dark supratympanic band; (3) snout moderately long (SL 37–43% of HL), subacuminate in dorsal view and truncated in lateral view, loreal region concave, lips not flared; (4) upper eyelid tubercles present; with or without dark bar between the eyes, and one or two oblique black streaks below the eye; postrictal tubercles absent; cranial crests absent; three scapular tubercles, less visible in specimens having dark dorsal coloration; (5) nostril ovoid, slightly protuberant, directed laterally; internarial distance 73–87% of interorbital distance; (6) tongue ovoid, longer than wide; (7) dentigerous processes of vomers present, small, oblique and positioned posterior to level of choanae, one or two on each side, ill-defined; (8) males with vocal slits; vocal sac small, subgular; nuptial pads absent; (9) Finger II longer than I; finger discs small, rounded (Finger I and II) to expanded (Finger III and IV); (10) fingers without lateral fringes; (11) ulnar tubercles aligned, barely visible in fixed specimens; (12) tibia length 46–54% of SVL; (13) heel tubercle absent; tarsal tubercles aligned, small and barely visible; tarsal fold absent; (14) thenar tubercle ovoid; palmar tubercle bifid, twice the width of the thenar tubercle; (15) inner metatarsal tubercle ovoid; outer metatarsal tubercle small, longer than wide, less than 1/3 of the thenar tubercle; (16) toes III–V with lateral fringes, webbing basal between toes IV–V; Toe I smaller than Toe II, not reaching the edge of disc on Toe II; Toe V longer than Toe III; (17) belly skin weakly areolate, and ventral region of the femur areolate; (18) in life, groin translucent, without brightly colored blotches or marks; posterior surfaces of thighs uniformly brown; (19) in life, iris dichromatic, pale bronze upper and lower parts with dark brown vermiculation and broad median mahogany-red stripe through pupil; (20) SVL in adult males of 17.3–20.1 mm (n = 16) and in females of 23.2–26.5 mm (n = 6); and (21) advertisement call with 5–10 notes and average call duration of 694 ± 115 ms, inter-note interval of 82.7 ± 11 ms, minimum frequency of 2,260–3,176 Hz, maximum frequency of 3,756–5,280 Hz and dominant frequency of 3,295–3,919 Hz.

Comparisons with other species

The new species is compared to all other currently recognized rain frogs of the Pristimantis unistrigatus species group occurring in Amazonian lowlands: P. aaptus (Lynch & Lescure, 1980); P. academicus (Lehr, Moravec & Gagliardi-Urrutia, 2010); P. altamazonicus (Barbour & Dunn, 1921); P. brevicrus (Andersson, 1945); P. carvalhoi (Lutz & Kloss, 1952); P. crepitaculus (Fouquet et al., 2022a; Fouquet et al., 2022b); P. croceoinguinis (Lynch, 1968); P. delius (Duellman & Mendelson III, 1995); P. diadematus (Jiménez de la Espada, 1875); P. divnae (Lehr & Von May, 2009); P. espedeus (Fouquet et al., 2013); P. eurydactylus (Hedges & Schlüter, 1992); P. grandoculis (Van Lidth de Jeude, 1904); P. guianensis (Mônico et al., 2022); P. inguinalis (Parker, 1940); P. kichwarum (Elmer & Cannatella, 2008); P. librarius (Flores & Vigle, 1994); P. luscombei (Duellman & Mendelson III, 1995); P. martiae (Lynch, 1974); P. matidiktyo (Ortega-Andrade & Valencia, 2012); P. miktos (Ortega-Andrade & Venegas, 2014); P. ockendeni (Boulenger, 1912); P. orcus (Lehr, Catenazzi & Rodríguez, 2009); P. variabilis (Lynch, 1968); and P. ventrimarmoratus (Boulenger, 1912).

Pristimantis campinarana sp. nov. differs from P. aaptus, P. diadematus, P. divnae, P. espedeus and P. orcus by having smaller male SVL of 17.3–20.1 mm [SVL 22.9 mm in P. aaptus (Lynch & Lescure, 1980); 20.0–27.4 mm in P. diadematus (Duellman & Mendelson III, 1995); 22.8–23.4 mm in P. divnae (Lehr & Von May, 2009); 20.7–24.8 mm in P. espedeus (Fouquet et al., 2013), and 20.0–25.1 mm in P. orcus (Lehr, Catenazzi & Rodríguez, 2009)] and from P. academicus, P. carvalhoi, P. martiae and P. grandoculis by larger male SVL [SVL 14.9 mm in P. academicus (Lehr, Moravec & Gagliardi-Urrutia, 2010); 13.5–14.8 mm P. carvalhoi (Lynch & Lescure, 1980; Duellman & Mendelson III, 1995); 14.7–17.9 mm in P. grandoculis (Fouquet et al., 2022a); 13.2–16.8 mm in P. martiae (Lynch, 1974)]; from P. aaptus, P. altamazonicus, P. brevicrus, P. delius, P. diadematus, P. eurydactylus, P. miktos, P. ockendeni, P. orcus, and P. ventrimarmoratus by smaller female SVL of 23.2–26.5 mm [SVL 29.9–34.8 mm in P. aaptus (Lynch & Lescure, 1980); 28.4–30.1 mm in P. altamazonicus (Ortega-Andrade et al., 2017); 27.2–35.0 mm in P. brevicrus (Ortega-Andrade et al., 2017); 30.9 mm in P. delius (Duellman & Mendelson III, 1995; Duellman & Lehr, 2009); 35.4–44.5 in P. diadematus (Duellman & Lehr, 2009); 29.4 mm in P. espedeus (Fouquet et al., 2013); 33.5–35.3 mm in P. eurydactylus (Hedges & Schlüter, 1992); 26.7–29.2 mm in P. miktos (Ortega-Andrade & Venegas, 2014); 30.4–30.6 mm in P. ockendeni (Mônico et al., 2022); 32.6–36.5 mm in P. orcus (Lehr, Catenazzi & Rodríguez, 2009), and 33.3–43.8 mm in P. ventrimarmoratus (Duellman & Lehr, 2009)].

Pristimantis campinarana sp. nov. can be easily distinguished from most of the lowland species (n = 16) by presence of vocal slits in males [absent in P. altamazonicus (Ortega-Andrade et al., 2017), P. brevicrus (Ortega-Andrade et al., 2017), P. carvalhoi (Duellman & Lehr, 2009), P. croceoinguinis (Lynch, 1968), P. diadematus (Duellman & Lehr, 2009), P. divnae (Lehr & Von May, 2009), P. eurydactylus (Hedges & Schlüter, 1992), P. grandoculis (Fouquet et al., 2022a), P. miktos (Ortega-Andrade & Venegas, 2014), P. orcus (Lehr, Catenazzi & Rodríguez, 2009), and P. ventrimarmoratus (Duellman & Lehr, 2009)], presence of eyelid tubercles [absent in P. carvalhoi (Duellman & Lehr, 2009), P. delius (Duellman & Mendelson III, 1995; Duellman & Lehr, 2009), P. diadematus (Duellman & Lehr, 2009), P. lythrodes (Lynch & Lescure, 1980), P. variabilis (Duellman & Lehr, 2009) and P. ventrimarmoratus (Duellman & Lehr, 2009)], presence of tympanum [absent in P. brevicrus (Ortega-Andrade et al., 2017), P. carvalhoi (Duellman & Lehr, 2009), P. croceoinguinis (Lynch, 1968; Duellman & Lehr, 2009), P. grandoculis (Fouquet et al., 2022a), P. martiae (Lynch, 1974) and P. ventrimarmoratus (Duellman & Lehr, 2009)], presence of dentigerous processes of vomers [absent in P. delius (Duellman & Mendelson III, 1995; Duellman & Lehr, 2009) and P. guianensis (Mônico et al., 2022)], and by presence of tarsal tubercles in males [absent in P. diadematus (Duellman & Lehr, 2009), P. librarius (Flores & Vigle, 1994), P. lythrodes (Lynch & Lescure, 1980), P. kichwarum (Elmer & Cannatella, 2008), P. martiae (Lynch, 1974; Duellman & Lehr, 2009), P. matidiktyo (Ortega-Andrade & Valencia, 2012) and P. ventrimarmoratus (Duellman & Lehr, 2009)].

Furthermore, the absence of brightly colored blotches or marks in the groin distinguishes the new species from P. aaptus (black groin; Lynch & Lescure, 1980), P. academicus (yellow groin; Lehr, Moravec & Gagliardi-Urrutia, 2010), P. altamazonicus (red to bright orange groin with black mottling; Ortega-Andrade et al., 2017), P. brevicrus (bluish white to yellowish white groin with black mottling; Ortega-Andrade et al., 2017), P. carvalhoi (yellow to yellowish white groin; Duellman & Lehr, 2009), P. crepitaculus (dark grey groin; Fouquet et al., 2022a), P. croceoinguinis (yellow or orange groin; Lynch, 1968; Elmer & Cannatella, 2008; Duellman & Lehr, 2009), P. diadematus (bluish white, yellowish tan or pink groin; Duellman & Lehr, 2009), P. divnae (yellow groin with brown marks; Lehr & Von May, 2009), P. espedeus (red-orange groin; Fouquet et al., 2013), P. eurydactylus (pale tan groin with dark brown vertical or diagonal bars; Hedges & Schlüter, 1992), P. grandoculis (dark grey groin; Fouquet et al., 2022a), P. inguinalis (bright yellow groin; Fouquet et al., 2013), P. librarius (reddish orange paler groin; Flores & Vigle, 1994; Elmer & Cannatella, 2008), P. lythrodes (black with yellowish white groin; Lynch & Lescure, 1980), P. martiae (pale brown groin; Lynch, 1974), P. matidiktyo (pale yellowish white groin; Ortega-Andrade & Valencia, 2012), P. miktos (yellowish-tan groin; Ortega-Andrade & Venegas, 2014), and P. orcus (black groin with white or whitish blue blotches; Lehr, Catenazzi & Rodríguez, 2009). Moreover, dichromatic iris differs P. campinarana sp. nov. from the species having monochromatic iris: P. academicus (golden to bronze iris; Lehr, Moravec & Gagliardi-Urrutia, 2010); P. altamazonicus (coppery red iris; Ortega-Andrade et al., 2017); P. brevicrus (coopery red to silver iris; Ortega-Andrade et al., 2017); P. carvalhoi (pale gray iris; Duellman & Lehr, 2009); P. croceoinguinis (gray to dull bronze iris; Lynch, 1968; Duellman & Lehr, 2009); P. divnae (silver iris; Lehr & Von May, 2009; P. lythrodes (grayish brown iris; Duellman & Lehr, 2009); P. miktos (deep orange iris; Ortega-Andrade & Venegas, 2014); P. orcus (dark gray to gold with cupper tint; Lehr, Catenazzi & Rodríguez, 2009); in P. ventrimarmoratus (pale bronze iris; Duellman & Lehr, 2009).

The advertisement call of Pristimantis campinarana sp. nov. is relatively similar with the calls of P. espedeus, P. guianensis and P. ockendeni. Nevertheless, calls of the new species differ in temporal and spectral characteristics [P. campinarana sp. nov.: call duration 694 ± 115 ms (550–1061 ms), inter-note interval 82.7 ± 11.9 ms (64–109 ms), and dominant frequency 3,587 ± 204 Hz (3,295–3,919 Hz) from P. espedeus (call duration 330 ms (240–500 ms), dominant frequency 2,700 Hz (2,680–2,840 Hz); Fouquet et al., 2013), P. guianensis (call duration 232 ± 42 ms (158–371 ms), inter-note interval 44 ± 5 ms (14–56 ms); Mônico et al., 2022) and P. ockendeni (dominant frequency 2,864 ± 202 Hz (2,519–3,143 Hz); Mônico et al., 2022)]. Furthermore, the advertisement call of P. campinarana sp. nov. easily differs from P. inguinalis and P. orcus by having multi-noted calls (single note in both mentioned species: Fouquet et al., 2013; López-Rojas et al., 2013).

Description of holotype

INPA-H 44426 (field number APL 23164). Morphometric measurements are presented in Table 1. An adult male (Fig. 3), SVL 19.1 mm; head slightly longer than wide (HL 103% of HW); head width 35.9% of SVL; head length 36.9% of SVL; cranial crest absent. Snout moderately long(SL 131% and 106% of EN and ED, respectively), subacuminate in dorsal view (Fig. 3A) and moderately truncated in lateral view (Fig. 3C); nostril ovoid, slightly protuberant, directed dorsolaterally; IND 87.3% of IOD; internarial region almost straight; canthus rostralis almost straight in dorsal view, slightly angular in profile; loreal region concave; lips not flared; one small tubercle on upper eyelid; interorbital region straight, IOD 32.1% of HW; eyes large (ED/TD = 2.7), pupil horizontally elliptical; supratympanic fold slightly distinct, extending from posterior margin of eyelid angling posteroventrally behind tympanic annulus; tympanum visible and rounded, TD 36.5% of ED; tympanic membrane poorly prominent, directed laterally; tympanic annulus poorly distinct, obscured dorsally by the supratympanic fold; one small postrictal tubercle, poorly visible; choanae of moderate sized, rounded to ovoid, not concealed by palatal sheath of maxilla; dentigerous processes of vomers present, with two ill-defined teeth, small, oblique and positioned posterior to level of choanae; tongue ovoid, longer than wide; short vocal slits, located in posterior half of mouth floor between tongue and margin of jaw; vocal sac small, simple and subgular.

Forearm shorter than hand (FAL 81.2% of HAND), notched posteriorly, nearly 78% free; three small ulnar tubercles ill-defined and aligned, almost indistinct after preservation; relative length of fingers I <II <IV <III (Fig. 3D); discs small and rounded on fingers I and II, expanded on fingers III and IV, with circumferential grooves; thenar tubercle poorly distinct, ovoid; palmar tubercle distinct, bifid; subarticular tubercles ill-defined, most prominent on fingers III and IV, rounded in dorsal and lateral view; small supernumerary tubercles present, but poorly visible; ventral pads well-defined on fingers III and IV.

Hindlimbs slender; tibia length 49% of SVL; heel without tubercles; tarsus with a row of small, poorly defined tubercles; tarsal fold absent; foot length 41% of SVL; relative length of toes I <II <III <V <IV (Toe III reaches the second subarticular tubercle of the Toe IV; Fig. 3E); toes with lateral fringes, more developed on toes III–V, webbing basal between toes IV–V; discs small and rounded on toes I and II, expanded on toes III–V; inner metatarsal tubercle large and ovoid, more than two times the size of ovoid outer metatarsal tubercle; subarticular tubercles large, protuberant, single, round on toes I–III and elliptical on toes IV and V; small supernumerary tubercles more visible on toes II-IV; ventral pads well-defined on toes III–V.

Dorsal skin shagreen (Fig. 3A), with longitudinal stripes and an irregular dorsolateral fold composed of spaced tubercles; small tubercles on scapular region; upper eyelid shagreen, with small tubercles; skin on flanks and chest smooth; skin on belly slightly areolate; upper and posterior surfaces of hindlimbs smooth, with small flat tubercles on thigh; dorsolateral folds absent; pectoral and discoidal folds not visible; cloaca protuberant, cloacal region without tubercles.

In life, dorsum yellow ocher (color 14 by Köhler, 2012) with dark brown mid-dorsal stripe running from snout to cloaca. Canthal stripe present, black, running from the tip of snout to anterior margin of eyelid. Dorsolateral stripe present, irregular, formed by a series of small, dark brown dots and blotches, running from posterior eye to cloacal region (Fig. 4A). Upper lip with dark brown subocular and dark brown supratympanic bar. Dark brown transversal bars on forearms; light brown transversal bars on thigh and tibia. Posterior surfaces of thigs uniformly brown. Groin white, translucent, with sparse brown melanophores. Throat, chest, belly and ventral surfaces of legs white, translucent, densely covered by tiny brown melanophores (Fig. 4B). Iris pale bronze with dark brown vermiculation and broad horizontal median mahogany-red (color 34 by Köhler, 2012) stripe through pupil.

In alcohol, color pattern faded (Fig. 3), upper lip with five dark bars (Fig. 3C), bars on dorsal surfaces of forearms, thighs and tibias dark brown; venter whitish with dense dark brown melanophores.

Variation in the type series

SVL ranges from 17.3 to 20.1 mm in males (n = 16) and from 23.2 to 26.5 mm in females (n = 6) (Table 1). Canthus rostralis almost straight in dorsal view (e.g., Fig. 3B, holotype) to slightly curved in some individuals (e.g., Figs. 5E and 5F). Three to four ulnar tubercles are present in males (barely visible in 63% of them), ulnar tubercles absent in females. Dorsal skin texture varies from shagreen to granular, small tubercles are present or absent (character of skin texture probably depends on individual activity at the moment of sampling; see Guayasamin et al., 2015; Kok et al., 2018).

In preservative, three types of basic dorsal color patterns can be detected in the type series of Pristimantis campinarana sp. nov.: dorsum with irregular dark brown markings (68%; Figs. 5A, 5D and 5E), dorsal coloration sharply outlined against the flanks (9%; Fig. 5B), dorsum with dark brown stripes as in the holotype (23%; Figs. 5C and 5F). In addition, a dark brown interorbital bar is present in 73% of the specimens (Figs. 5A, 5D and 5E); a dark brown W-shaped mark is presented on the scapular region of 18% of the individuals, in some of them more conspicuous (Figs. 5A and 5D) than in others (Figs. 5D and 5E). Dark brown bars and blotches are present on the upper lip of all type specimens, dark and distinct in 73% of the specimens, faded or less conspicuous in the others. Obvious transversal dark brown bars are present on the arms and hands of 63% of the individuals, less conspicuous or absent in 23% and 14% of the specimens, respectively. Distinct transversal dark bars are present on the thigh and tibia of 32% of the specimens (Figs. 5C and 5F), poorly conspicuous in 54% (Fig. 5A) and absent in 14% of the individuals (Fig. 5B). Dark supratympanic stripe present in all specimens. Ventral surface is whitish cream to yellowish white, with a moderate amount of melanophores in 59% of individuals (Fig. 5I), small and large amount in 32% (Fig. 5G) and 9% (Fig. 5H) of individuals, respectively.

In life, dorsal background coloration is widely variable, from yellow ocher (color 14 by Köhler, 2012; Figs. 6A, 6D and 6J) and light chrome orange (color 76 by Köhler, 2012; Figs. 6D, 6G and 6J) to antique brown (color 24 by Köhler, 2012; Figs. 6C, 6G, 6I and 6L). Dorsolateral stripe present in some individuals (Figs. 6H and 6I, 6L), but absent in others (Figs. 6A–6G, 6J–6K). Canthal stripe present in some individuals (Figs. 6D, 6H, 6I and 6L), but absent in others (Figs. 6A–6C, 6E–6G, 6J–6K). The iris of most specimens is similar to that of the holotype, but some specimens show a small portion of the lower iris pale bronze to gold (Figs. 6A, 6C, 6I and 6K). At the day, the groin and ventral surface are platt’s-payne’s-gray (color 293 by Köhler, 2012) with brown melanophores (Fig. 5B); at night, when melanophores are less expanded, the groin (Fig. 7A) and ventral surface (Fig. 7B) becomes lighter, almost white.

Advertisement call

The advertisement call of Pristimantis campinarana sp. nov. (n = 9 males) is composed of 5–10 notes (n = 26 calls)—most commonly of 6–8 notes (n = 21)—and has a call duration of 694 ± 115 ms (550–1,061 ms). Notes are tonal, have duration of 26.5 ± 7.1 ms (13.2–39.8 ms) and an inter-note interval of 82.7 ± 11.9 ms (64.4–109.4 ms). Calls are emitted with a minimum frequency of 2,853 ± 141 Hz (2,260–3,176 Hz), a maximum frequency of 4,490 ± 413 Hz (3,756–5,280 Hz) and a dominant frequency of 3,587 ± 204 Hz (3,295–3,919 Hz) (Fig. 8). Temporal and spectral traits summarized according to individual call arrangement are presented in Table 2.

Etymology

The specific epithet ‘campinarana’ is used as a noun in apposition and refers to the word in Portuguese that defines the type of forest that the new species occupies: the white-sand forest campinarana.

Distribution, natural history and conservation

Currently, Pristimantis campinarana sp. nov. is known only from primary and slightly anthropized forests at two localities in the municipality of Iranduba, state of Amazonas, Brazil (Fig. 9). All individuals were recorded in WSE characterized as campinarana (a forest with canopy height below 20 m; Fig. 10A), where the species is locally abundant. However, none was found in campina, a white-sand forest with canopy height below 10 m and large patches of exposed white sandy soils.

Pristimantis campinarana sp. nov. is a crepuscular and nocturnal species, with peak activity at crepuscule. Its breeding activity takes place in the rainy season (November to February). In the dry season, we found males sheltering among the leaves of terrestrial bromeliads of the genus Guzmania (Fig. 10B). In the rainy season, males start calling at dusk (∼18:00 h) and are very active until ∼20:00 h. Then their activity decrease and end usually around 22:00 h. In rainy days, the vocalization is sustained throughout the night. Males were observed calling perched on the vegetation (Fig. 10C) usually 1 m above the ground, rarely above 3 m. Calling males aggregate in groups of up to ten individuals, separated from each other by ∼4–5 m, but it is not uncommon to observe smaller groups of three to four calling individuals that are spatially more spaced. The amplexus (n = 3) is axillary (Fig. 10D). Clutches were not found in situ, but females produce about 14–17 large oocytes (n = 4). The new species occurs in sympatry with a candidate species closely related to Pristimantis orcus (AT Mônico, 2022, unpublished data). We do not have sufficient data to categorize the new species following the criteria of the International Union for Conservation of Nature (IUCN), it should be thus considered Data Deficient (DD).