Zootaxa 5100 (4): 521–540 ISSN 1175-5326 (print edition)

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Copyright © 2022 Magnolia Press
Article ZOOTAXA
ISSN 1175-5334 (online edition)
https://doi.org/10.11646/zootaxa.5100.4.4
http://zoobank.org/urn:lsid:zoobank.org:pub:570D580F-CDA3-42A1-A9A2-E023282A3F4A

A new species of Pristimantis Jiménez de la Espada, 1870 (Anura: Strabomantidae)

from the “Brejos de Altitude” in Northeast Brazil
IGOR JOVENTINO ROBERTO1,*, DANIEL LOEBMANN2, MARIANA L. LYRA3,4,
CÉLIOF.B HADDAD3,5 & ROBSON WALDEMAR ÁVILA1,6
1
Departamento de Biologia, Centro de Ciências, Campus do Pici, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
2
Laboratório de Vertebrados, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Av. Itália, Km 8, 96203-900, Rio
Grande, Rio Grande do Sul, Brazil
� pinguimfiel@yahoo.com.br; https://orcid.org/0000-0003-0106-7197
3
Departamento de Biodiversidade e Centro de Aquicultura (CAUNESP), Instituto de Biociências, Universidade Estadual Paulista
(UNESP), Av. 24A, 1515, Rio Claro, São Paulo, CEP 13506-900, Brazil
4�
marillyra@gmail.com; https://orcid.org/0000-0002-7863-4965
5�
haddad1000@gmail.com; https://orcid.org/0000-0002-7044-5764
6�
robsonavila@gmail.com; https://orcid.org/0000-0003-3641-8321
*
Corresponding author. � igorjoventino@yahoo.com.br; https://orcid.org/0000-0003-3268-9597

Abstract

Pristimantis is the most diverse Neotropical genus of terrestrial vertebrates and is distributed from Central America to
Argentina. The last few years have seen the description of several new species of the genus, suggesting that its diversity
is still underestimated. After decades of uncertainties about the taxonomic status of populations of Pristimantis from
the “Brejos de Altitude” of the state of Ceará, Northeast Brazil, we finally found morphological, acoustic and molecular
evidence confirming their distinctiveness from other Atlantic Forest species. The new species is characterized by the
following: shagreen dorsal skin with small-scattered tubercles, absence of dorsolateral fold, presence of tarsal fold,
advertisement call composed of 1–8 pulsed notes, (2–5 pulses per note), and dominant frequency located in the second
energy band, ranging from 3617–4220 Hz. Phylogenetic analysis placed the new species in the Pristimantis conspicillatus
species group and the sister lineage of the Atlantic Forest clade comprising P. ramagii, P. paulodutrai and the P. vinhai
species complex.

Key words: Amphibia, Ceará State, Neotropics, Systematics, Taxonomy

Introduction

Pristimantis (Anura: Strabomantidae) is one of the most diverse genera of terrestrial vertebrates in the world (Ttito &
Catenazzi, 2021), with 590 described species (Frost 2021) distributed in 13 species groups (sensu Padial et al. 2014;
Gonzalez-Durn et al., 2017; Zumel et al. 2021). Several species complexes with morphologically cryptic lineages have
been identified by different authors in the last few years, demonstrating that the number of species of Pristimantis is
underestimated (Padial et al. 2009; Oliveira et al. 2017; 2020; Trevisan et al. 2020; Taucce et al. 2020). Some species
complexes have been found within the Pristimantis conspicillatus species group (sensu Padial et al. 2014). Although
P. conspicillatus species group still lacks morphological synapomorphies, it forms a well-supported monophyletic
clade based on molecular phylogenetic analysis. Additionally, the species within the P. conspicillatus species group
have morphological and acoustic similarities, such as the presence of a tarsal fold, a smooth to slightly granular belly, a
distinct tympanic membrane, length of Finger I equal to or longer than Finger II, and an advertisement call consisting
of pulsatile and amplitude-modulated notes (Padial et al. 2009, 2014, 2016; Taucce et al. 2020).
The Pristimantis conspicillatus species group is currently composed of 41 species distributed from Costa Rica
to eastern Amazon with an isolated clade in the northern Atlantic Forest in Brazil. The latter, named hereafter as the
“Atlantic Forest clade”, comprises the species P. ramagii (Boulenger, 1888), P. paulodutrai (Bokermann, 1975) and
P. vinhai (Bokermann, 1975), (Hedges et al., 2008; Canedo & Haddad 2012; Padial et al. 2014; Taucce et al. 2020).

Accepted by M. Rivera-Correa: 5 Jan. 2022; published: 22 Feb. 2022 521

Pristimantis rupicola was recently described Taucce, Nascimento, Trevisan, Leite, Haddad & Napoli, 2020, from
the highlands of Chapada da Diamantina in the state of Bahia in Northeast Brazil, however, this species does not
form a monophyletic clade within the Atlantic Forest clade, being more related to P. gaigei (Dunn, 1931) from Cen-
tral America and Colombia. Another species that was described recently from Northeast Brazil is P. moa Oliveira,
Silva, Silva, Guimarães, Penhacek, Martínez, Rodrigues, Santana & Hernádez-Ruz, 2020, however, its distribution
is restricted to a transitional zone between the Amazon and the Cerrado in the states of Maranhão and Tocantins
(Oliveira et al. 2020). This species forms a monophyletic clade with P. giorgi, P. pluvian, P. pictus Oliveira, Silva,
Silva, Guimarães, Penhacek, Martínez, Rodrigues, Santana & Hernádez-Ruz, 2020 and P. latro Oliveira, Rodrigues,
Kaefer, Pinto & Hernádez-Ruz, 2017, all from the Amazon domain (Oliveira et al. 2017; 2020).
Canedo & Haddad (2012) identified another distinct genetic lineage of Pristimantis in Northeast Brazil, not
recovered within the Atlantic Forest clade, but instead as the sister lineage to P. gaigei, although with low support.
This lineage occurs in the humid mountain forest of Ceará, a region called the “Brejos de Altitude” (sensu Silva
& Castelleti 2003). These enclaves of mountain forest are isolated and surrounded by the xeric vegetation of the
Caatinga and harbor an endemic fauna evidencing a preterit connection between the Amazon and the Atlantic Forest
(Hoogmoed et al. 1994; Fouquet et al. 2012; Roberto & Loebmann 2016).
In this study we gathered morphological, acoustic, and molecular data that corroborate the distinctiveness of
this taxa and herein described it as a new species, endemic to the state of Ceará in Northeast Brazil.

Material and methods

Taxon sampling
Around 15 field expeditions were carried out throughout 2007 and 2019 in 13 municipalities in the “Brejos de Al-
titude” of Ceará, Brazil: Planalto da Ibiapaba mountain range (municipalities of Ipu, Tianguá, Ubajara, Granja, and
Viçosa do Ceará), Serra de Maranguape mountain range (municipality of Maranguape), Serra da Aratanha moun-
tain range (municipality of Pacatuba), Serra de Baturité mountain range (municipalities of Guaramiranga, Pacoti,
Aratuba, Mulungu), Serra da Uruburetama mountain range (municipality of Uruburetama), and Serra da Meruoca
mountain range (municipality of Meruoca). The collected Pristimantis specimens were euthanized by applying 2%
lidocaine to their skin. Muscle tissue samples were obtained and stored in 99% ethanol for molecular analyses. Indi-
viduals were fixed in 10% formalin and stored in 70% ethanol. Specimens were deposited in the Célio F. B. Haddad
Amphibian collection (CFBH), Universidade Estadual Paulista (UNESP), Rio Claro, state of São Paulo; Coleção
de Herpetologia da Universidade de Rio Grande (CHFURG), Rio Grande, state of Rio Grande do Sul; Museu Na-
cional, Universidade Federal do Rio de Janeiro (MNRJ), state of Rio de Janeiro; and Coleção de Herpetologia da
Universidade Regional do Cariri, Crato, state of Ceará (URCA-H), all in Brazil.

Molecular data acquisition and analysis

Total genomic DNA was extracted using a standard ammonium precipitation method adapted for microcentrifuge
tubes (Lyra et al. 2017). Two overlapping fragments of the 16S ribosomal RNA gene (16S rRNA) were ampli-
fied using primers 12sL13 (Feller & Hedges 1998) and H10 (Hedges 1994), and 16SAR and 16SBR (Palumbi
et al. 1991) and PCR cycling schemes optimized in Lyra et al. (2017). PCR products were purified by enzymatic
reaction and sent to Macrogen Inc., Seoul, Republic of Korea, for sequencing. The new DNA sequences were
quality trimmed and assembled using Geneious R11 (Biomatter) and submitted to GenBank (accession numbers:
MW263916-MW263919). The access of genetic heritage was registered at the National System for the Management
of Genetic Heritage and Associated Traditional Knowledge (SISGen # AD85091).
For phylogenetic inferences, the new sequences were combined with sequences available in GenBank for al-
most all named species within the P. conspicillatus species group (appendix 1), as in Taucce et al. (2020). The final
matrix includes one to four individuals per named species of the group, Pristimantis species from other groups, and
some species from other genera of Strabomantidae, totaling 97 terminals (Appendix 1). Sequence alignment was
performed with the software MAFFT version 7.273 (Katoh & Standley 2013) using E-INS-i and the final length of
the alignment was 1490 bp, including gaps. Alignments indicated that few hypervariable regions of the 16S rRNA
fragment available for four species sequenced by Oliveira et al. (2020; P. giorgii, P. moa, P. pictus, and P. pluvian)
are missing, and probably was accidentally deleted before submitted to the GenBank. To be able to include these
sequences in our analyses we filled the missing region with ‘question marks’ based on alignments, denoting missing
data. External gaps were also coded as question marks for subsequent analyses.

522 · Zootaxa 5100 (4) © 2022 Magnolia Press Roberto et al.

 Phylogenetic trees were obtained using maximum likelihood (ML) and Bayesian inference (BI) as optimality
criteria. The ML analysis was performed with RAxML 8.2.12 (Stamatakis 2014) using the GTRCAT substitution
model with 100 searches for the most likely tree. Node support was estimated with 1000 non-parametric bootstrap
replicates under the same model. Bayesian inference was performed with MrBayes 3.2.6 (Ronquist et al. 2012),
using two independent runs of 10 million generations and four Markov chains (one cold). Parameters were sampled
every 1,000 generations and burn-in was 10%. To assess run convergence of BI analyses, we used standard devia-
tion of split frequencies (<0.01) and estimated sample size (ESS >100) using the software Tracer v.1.7 (Rambaut et
al. 2018). The most likely tree of the ML analyses and the 50% majority rule consensus tree from BI were visual-
ized and edited in FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree). Both ML and BI analyses were run in
CIPRES Science Gateway (Miller et al. 2010).
The minimum uncorrected pairwise distances were estimated between species of the P. conspicillatus species
group that are present in northeast Brazil, except P. moa (see below). For that, we used only a fragment of the 16S
rRNA gene, flanked by primers 16SAR and 16SBR, with the package SPIDER in R ver. 3.6.1 (Brown et al. 2012; R
Core Team 2019), since this fragment was available for all individuals. The alignment length was 546 bp, including
gaps. Gaps and missing data were treated as pairwise deletions. The species P. moa were excluded from this analysis
do avoid bias in the distance’s estimates since variable regions as missing, as explained before.

Morphology and species description

A total of 131 specimens were directed analyzed for morphological comparisons, including 68 specimens for the
new species and other species of Pristimantis housed in the following collections: Natural History Museum, Lon-
don, England (BMNH); Célio F. B. Haddad Amphibian Collection, Universidade Estadual Paulista, Rio Claro, São
Paulo, Brazil (CFBH); Coleção Herpetológica da Universidade de Rio Grande, Rio Grande, Rio Grande do Sul, Bra-
zil (CHFURG); Coleção de Herpetologia da Universidade Federal da Paraíba, João Pessoa, Paraíba, Brazil (CHUF-
PB); Coleção de Herpetologia da Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil (CHUFPE),
Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (MNRJ); Coleção Zoológica da
Universidade Federal do Mato Grosso, Cuiabá, Mato Grosso, Brazil (UFMT-A); United States National Museum,
Smithsonian Institution, Washington, USA (USNM); Coleção de Herpetologia da Universidade Regional do Cariri,
Crato, Ceará, Brazil (URCA-H); Naturhistorisches Museum, Wien, Austria (NHMW) (Appendix 2). Sex was deter-
mined by the distinctive gular color and the presence of vocal slits and vocal sac in males, and the presence of eggs
in reproductive females. Terminology for diagnostic characters follows Heyer et al. (1990) and Lynch & Duellman
(1997). Measurements were taken with digital calipers to the nearest 0.1 mm and follow Taucce et al. (2020). The
following measurements were taken: SVL (snout-vent length), HL (head length), HW (head width), ED (eye diam-
eter), SL (snout length), IND (internarial distance), END (eye to nostril distance), IOD (interorbital distance), UEW
(upper eyelid width), TD (tympanum diameter), FAL (forearm length), HAL (hand length), FIII (width of disc of
Finger III), FIV(width of disc of Finger IV), THL (Thigh length), TL (tibia length), TAL (tarsal length), FL (foot
length), and TIV (width of disc of Toe IV).

Recordings and acoustic analysis

We recorded three males from three distinct localities in Ceará. Audio files deposited in Fonoteca Neotropical Ja-
cques Vielliard (FNJV 0050599–601). The first individual, MNRJ 55582, was recorded at the Reserva Particular
do Patrimônio Natural Monte Alegre in the Serra da Aratanha mountain range, municipality of Pacatuba, on 26
December of 2006. The second individual (not vouchered) was recorded at Sítio Monte Belo in the Serra de Batu-
rité mountain range, municipality of Pacoti, on 11 February of 2007, both individuals were recording using a Sony
TCM 5000 EV tape recorder coupled to a Sennheiser ME66/K6 directional microphone. The third individual (not
vouchered) was recorded at Ubajara National Park, municipality of Ubajara, on 2 February of 2007 using a Sony
cassette tape recorder (TCM-150) equipped with an external directional microphone Yoga (HT 81 Boom) positioned
at least 3 m from the calling males. Recordings were digitalized at 44.1 kHz, with a resolution of 16 bits. Calls were
analyzed in Raven Pro v1.6.1 (Center for Conservation Bioacoustics 2019), and Spectrograms were produced with
Fast Fourier Transform (FFT) length of 1,024 samples, overlap 89.8 %, window Hanning, 3-dB filter bandwidth of
496 Hz, and window size of 256 (Taucce et al. 2020). Spectrogram and oscillogram figures were produced with the
Seewave package version 1.6.4 (Sueur et al. 2008) in the R platform version 3.3.3 (R Core Team 2019). Terminol-
ogy of call descriptions follows the note centered approach Köhler et al. (2017), while that for note rate parameters
follow Cocroft & Ryan (1995).

New species of Pristimantis Zootaxa 5100 (4) © 2022 Magnolia Press · 523
Results

Phylogenetic relationships and genetic distance

Maximum likelihood analyses and Bayesian inference recovered similar topologies in which the Pristimantis con-
spicillatus species group was recovered as monophyletic (BI posterior probability = 1; ML bootstrap = 93%);
however, the deepest relationships among species within the group lack support (Figure 1). The new specimens
sequenced here, from the “Brejos de Altitude” of the state of Ceará, were recovered in both analyses together with
Pristimantis sp. 2 sensu Canedo & Haddad (2012) as sister of the Atlantic Forest clade composed of the species
complex formed by P. paulodutrai, P. ramagii and P. vinhai, but with low support (BI posterior probability = 0.53
and ML bootstrap = 41%). This Atlantic Forest clade was recovered with high support in both analyses (BI posterior
probability = 1 and ML bootstrap = 100%).
The clade composed of the specimens from the Brejos de Altitude and the Atlantic forest clade was consistently
recovered as the sister clade to [(P. gaigei + P. rupicola) + (P. incertus + P. gutturalis) + P. ardilae], but with low
support in both analyses (BI posterior probability = 0.61 and ML bootstrap = 28%).
Intraspecific genetic distances of the 16S rRNA gene for the clade containing specimens from the “Brejos de
Altitude ranged from 0% to 1.8%, and minimum distances between this clade and other species of the P. conspicil-
latus group present in Northeast Brazil ranged from 14.8% (with P. rupicola) to 16.3% (with P. ramagii) (Appendix
3).

Pristimantis relictus sp. nov.

Hylodes ramagii Rocha, 1948

Eleutherodactylus sp Lima-Verde & Cascon (1990)
Eleutherodactylus cf. ramagii Borges-Nojosa & Lima (2001)
Ischnocnema gr. ramagii Carnaval & Bates (2007)
Pristimantis sp Loebmann & Haddad (2010)
Pristimantis sp 2. Canedo & Haddad (2012)
Pristimantis sp Roberto & Loebmann (2016)
Pristimantis sp (cf. ramagii)—Castro et al. (2019)
Pristimantis sp (Northern Ceará clade)—Trevisan et al. (2020)

Holotype. URCA-H 13344 (adult male) collected at Parque Nacional de Ubajara, municipality of Ubajara, state of
Ceará, Brazil (03°50’18.5”S, 40°54’37.9” W; 833 m above sea level), on April 10, 2013 by Igor J. Roberto (Figure 2).
Paratypes. 17 males, 33 females and 3 juveniles of undetermined sex: URCA-H 13343 (adult male) and 13342
(adult female) collected with the holotype. CFBH 20304 (adult male), CFBH 20306–08, 20310–11, 20313 (adult
females), collected at Parque Nacional de Ubajara, municipality of Ubajara, state of Ceará, Brazil (03°50’31.57” S,
40°53’56” W, 850 m a.s.l.), on June 29, 2008 by Daniel Loebmann. CFBH 15899–00, 15904 (adult males), collected
at Mata das Bromélias, Parque Nacional de Ubajara, municipality of Ubajara, state of Ceará, Brazil (03°51’16”
S, 40°55’16” W, 820 m a.s.l), on March 24, 2007 by Daniel Loebmann. URCA-H 1546 (adult male), 1543–45,
1547 (adult females) collected at municipality of Guaramiranga, state of Ceará, Brazil (04°15’21”S, 38°58’17”
W, 830 m a.s.l), on February 12, 2012 by Robson W. Ávila. CFBH 20316–17, 25415–16, 25419 (adult females),
CFBH 25417–18, 25420–21 (adult males), collected at Parque das Trilhas, municipality of Guaramiranga, state of
Ceará, Brazil (04°15’48”S, 38°55’59” W, 853 m asl), on January 23, 2009 by Daniel Loebmann. URCA-H 6988
(adult male), 6979–80, 6984, 6987 (adult females) collected on February 3–5, 2013. URCA-H 9214 (adult male)
collected on April 7, 2014 at Área de Proteção Ambiental Bica do Ipu, municipality of Ipu, state of Ceará, Brazil
(41°7’21.78”S, 40°42’47.69”W), by Robson W. Ávila. URCA-H 2373 (adult female) collected on March 23, 2011
by Igor J. Roberto, 9828–30 (adult females) and 9633, 9837–38 (juveniles) collected by Herivelto F. Oliveira on
August 14–20, 2014 at Serra de Maranguape, municipality of Maranguape, state of Ceará, Brazil (35°3’59.23”S,
38°43’0.32”W, 800 m a.s.l). CFBH 24531 (adult female), collected at Serra de Maranguape, municipality of Ma-
ranguape, state of Ceará, Brazil (35°3’59.23”S, 38°43’0.32”W, 770 m a.s.l), on December 11, 2008 by Daniel

524 · Zootaxa 5100 (4) © 2022 Magnolia Press Roberto et al.

Figure 1. The 50% majority rule consensus tree from Bayesian inference showing the position of Pristimantis relictus sp.
nov. (in bold) and its relationships within the P. conspicillatus species group. Numbers above branches indicate posterior prob-
abilities while numbers below branches indicate maximum likelihood nonparametric bootstrap values. Support values within
species are not shown. Asterisk indicate terminal previously identified as Pristimantis sp. 2 in Canedo & Haddad (2012).

Loebmann. CFBH 25887 (adult female), CFBH 25888 (adult male) collected at Serra da Aratanha, municipality of Pa-
catuba, state of Ceará, Brazil (03°58’60”S, 38°38’00” W, 700 m a.s.l), on March 29, 2010 by Daniel Loebmann. CFBH
25406–12 (adult females) collected at Fazenda Gameleira, municipality of Tianguá, state of Ceará, Brazil (03°43’16”S,

New species of Pristimantis Zootaxa 5100 (4) © 2022 Magnolia Press · 525
40°55’46” W 800 m a.s.l), on October 28, 2008 by Daniel Loebmann. CFBH 25413–14 (adult males) collected at
Estrada do Retiro, municipality of Uruburetama, state of Ceará, Brazil (03°35’53.4”S, 39°34’50” W), on January 23,
2009 by Daniel Loebmann. URCA-H 16276–77 (adult males) collected at Serra da Meruoca, municipality of Meruoca,
state of Ceará, Brazil (03°37’03.3”S, 40°21’46.7” W, 700 m a.s.l), on February 20, 2019 by Kassio C. Araújo.

Figure 2. Holotype of Pristimantis relictus sp. nov. (URCA-H 13344): Above—dorsal and ventral views; Below—head in
lateral view; right hand and right foot in ventral view. Scale bars = 10mm.

Referred specimens: URCA-H 9835-9836 (juveniles) collected at Serra de Maranguape, municipality of Ma-
ranguape, state of Ceará, Brazil; URCA-H 6981-6983, 12287 collected at municipality of Ipu, state of Ceará; MNRJ
55882-55883 collected at Serra da Aratanha, municipality of Pacatuba, state of Ceará, Brazil; MNRJ 55884 col-
lected at Serra da Ubatuba, municipality of Granja, state of Ceará, Brazil; CHFURG 2119-23 collected at Sítio São
José, municipality of Guaramiranga, state of Ceará, Brazil.
Definition. Pristimantis relictus sp. nov. is diagnosed by the following combination of characters: (1) dorsal
skin shagreen with small scattered tubercles; (2) ventral skin areolate; (3) dorsolateral fold absent; (4) discoidal fold
present; (5) tarsal fold present; (6) lateral fringes absent on fingers, narrow on toes; (7) vocal slits present in males;
(8) advertisement call composed of 1–8 pulsed notes (2–5 pulses per note), note rate of 0.5–2.2 notes per second,
call duration 20–660 ms, fundamental frequency located at the first energy band between 1895–2153 Hz and domi-
nant frequency located at the second energy band between 3617–4220 Hz; (9) in life dorsum coloration dark brown
to yellowish-brown, with presence of dark brown chevrons and black spots in some individuals, transversal brown
bars on legs and arms; (10) immaculate cream-colored pattern of concealed surfaces of thighs; (11) presence of a

526 · Zootaxa 5100 (4) © 2022 Magnolia Press Roberto et al.

dark brown stripe from tip of snout to eye, including inter-nostril area, and a dark brown interorbital bar; (12) supra-
tympanic fold bold, black; (13) gular region yellowish; (14) iris color cooper with brown reticulations.
Morphological comparisons with other species within the Pristimantis conspicillatus species group. The
shagreen texture of the dorsal skin differentiates Pristimantis relictus sp. nov. from P. avicuporum (Duellman &
Pramuk, 1999), P. metabates (Duellman & Pramuk, 1999), P. johannesdei (Rivero & Serna, 1988), P. phalaroin-
guinis (Duellman & Lehr, 2007), and P. pictus (all have smooth dorsal skin). Nineteen species have smooth ventral
skin, differing from the areolate ventral skin of P. relictus sp. nov.: P. achatinus (Boulenger, 1898), P. buccinator
(Rodriguez, 1994), P. charlottevillensis (Kaiser, Dwyer, Feichtinger & Schmid, 1995), P. chiastonotus (Lynch &
Hoogmoed 1977), P. citriogaster (Duellman, 1992), P. condor (Lynch & Duellman, 1980), P. conspicillatus (Gün-
ther, 1858) , P. fenestratus (Steindachner 1864), P. gaigei, P. gutturalis, P. johannesdei P. latro, P. lymani (Bar-
bour & Noble, 1920), P. malkini (Lynch, 1980), P. metabates , P. peruvianus (Melin, 1941), P. phalaroinguinis, P.
samaipatae (Köhler & Jungfer 1995), and P. vilarsi (Melin, 1941). The areolate ventral skin also distinguishes P.
relictus sp. nov. from P. dundeei (Heyer & Muñoz, 1999), P. giorgii, P. iiap Padial, Gagliardi-Urrutia, Chaparro
& Gutiérrez, 2016, P. incertus, and P. ventrigranulosus Maciel, Vaz-Silva, Oliveira & Padial, 2012 (ventral skin
granular or slightly granular). The absence of dorsolateral folds distinguishes P. relictus sp. nov from Pristimantis
adiastolus (Duellman & Hedges, 2007), P. ardilae, P. avicuporum, P. bipunctatus (Duellman & Hedges, 2005), P.
buccinator, P. chiastonotus, P. conspicillatus, P. iiap, P. latro, P. malkini, P. meridionalis (Lehr & Duellman, 2007),
P. peruvianus, P. pluvian, P. skydmainos, and P. zeuctotylus (presence of dorsolateral folds) The subovoid snout in
dorsal view of P. relictus sp. nov. distinguishes it from P. carranguerorum (Lynch, 1994) (obtuse) and P. medemi
(Lynch, 1994), P. latro, and P. zeuctotylus (subacuminate). The immaculate cream-colored pattern of concealed sur-
faces of thighs differentiates P. relictus sp. nov. from P. pluvian (red), P. bipunctatus, P. condor, P. conspicillatus, P.
malkini, P. peruvianus, and P. pictus (with well-defined spots).
In addition to the new species, five other species of Pristimantis are distributed in Northeast Brazil: P. moa, P.
paulodutrai, P. ramagii, P. rupicola and P. vinhai (see Table 1). While Pristimantis relictus sp. nov. is distributed in
highland marshes of Ceará, P. moa is known from transition areas between the Cerrado and Amazon Forest (Oliveira
et al. 2020), P. rupicola occurs in Campos Rupestres of Chapada Diamantina (Taucce et al. 2020), and the remaining
three species inhabit the Atlantic Forest domain (Trevisan et al. 2020). Besides geographic distribution, P. relictus
sp. nov. can be distinguished from P. moa by areolate ventral skin (smooth in P. moa), snout subovoid in dorsal view
(truncate in P. moa), immaculate cream-colored pattern of concealed surfaces of thighs (strongly stained yellow on
a dark background in P. moa), finger fringes absent (present in P. moa), and webbing on toes absent (present in P.
moa) (Oliveira et al. 2020). The new species differs from P. rupicola by possessing shagreen dorsal skin (granular
in P. rupicola), and snout subovoid in dorsal view (rounded or truncate in P. rupicola) (Taucce et al. 2020). The
new species can be distinguished from the Atlantic Forest species as follows: from P. paulodutrai by the presence
of nuptial pads (absent in P. paulodutrai), ventral skin areolate (granular in P. paulodutrai), and immaculate cream-
colored pattern of concealed surfaces of thighs (with red blotches in P. paulodutrai) (Bokermann, 1975); from P.
ramagii by snout subovoid in dorsal view (subacuminate in P. ramagii), presence of nuptial pads and toe fringes
(absent in P. ramagii) (Boulenger, 1888); and from P. vinhai by ventral skin areolate (slightly granular in P. vinhai),
snout subovoid in dorsal view (acuminate in P. vinhai) (Bokermann, 1975).
Bioacustical comparisons with other species within the Pristimantis conspicillatus species group. The
shorter note duration (20–50 ms) differentiates P. relictus sp. nov. from P. samaipatae (59–141 ms) and P. fenestra-
tus (50–91 ms) (Padial & De La Riva 2009). The lower note rate (0.5–2.2 notes/s) distinguishes P. relictus from P.
fenestratus (7.7–12.7), P. koehleri (11.8–17.3), P. samaipatae (2.7–14.9), P. dundeei (13.7–20.7) (Heyer & Muñoz
1999; Giaretta et al. 2018), and P. vilarsi (14) (Heyer & Barrio-Amorós 2009). The lower number of pulses per note
of P. relictus sp. nov. (2–5) also differentiates the new species from P. fenestratus (9–17), P. samaipatae (11–23), P.
latro (6–9), and P. pluvian (8–16). The higher fundamental frequency of P. relictus sp. nov. (1894.9–2153.3 Hz) dis-
tinguishes the new species from P. samaipatae (1535–1834 Hz), P. latro (1342–1448.6 Hz), P. moa (1320.8–1660.2
Hz), and P. giorgii (663.2–1872.3 Hz). The higher peak frequency of P. relictus sp. nov. (3617.6–4220.5 Hz) dif-
ferentiates the new species from P. fenestratus (1710–3591 Hz), P. latro (2635.9–3272 Hz), P. moa (2657.1–3400
Hz), and P. pictus (2487.4–3272.2 Hz) (Padial & De La Riva 2009; Oliveira et al. 2017, 2020). Also, advertisement
call differentiates P. relictus from P. rupicola. The new species has a higher peak of frequency (3617.6–4220.5 Hz),
located in the second band of energy, [peak frequency (2410–3490 Hz) in the first band of energy of the call; Taucce
et al. (2020)]. The advertisement call of P. relictus is very similar to that of P. ramagii but the species can be distin-
guished by the number of pulses per note of 2–5 (7–53 in P. ramagii; Octaven et al. 2017).

New species of Pristimantis Zootaxa 5100 (4) © 2022 Magnolia Press · 527
 Table 1. Comparison of diagnostic characters between Northeastern Brazilian species of the Pristimantis conspicillatus Group.
P. relictus sp. nov P. ramagii P. paulodutrai P. vinhai P. rupicola P. moa
Male (SVL–mm) 23.1–29.7 19.3–26.2 25–27 16–19 18.1–28.2 30.6–34.4
Female (SVL–mm) 26.8–32.8 26.9–30.5 34–36 23–25 30.7–33.4 30.4–43.4
Snout shape—dorsal view subovoid subacuminate subacuminate acuminate truncate or ovoid truncate
Dorsal skin texture shagreen granular granular shagreen granular shagreen
Ventral skin texture areolate granular granular granular granular Smooth
Finger fringes absent absent absent absent absent Present
Basal toe webbing absent absent absent absent absent Present
Nuptial pad present absent absent present present Absent
Color pattern of posterior sur- cream-colored cream-colored/red- reddish carmine cream-colored dark yellow
faces of thighs dish
Peak Frequency (Hz) 3617.6–4220.5 2217–4898 3540–3970 ? 2410–3490 2657.1–3400

528 · Zootaxa 5100 (4) © 2022 Magnolia Press

Notes/call 1–8 1–4 1–5 ? 1 3–5
Call duration (s) 0.02–0.66 0.07–0.24 0.02–1.6 ? 0.01–0.09 0.21–0.38
Pulses/note 2–5 7–53 3–5 ? 2–23 4–18
Source This study Boulenger, 1888; Bokermann, 1975; Bokermann, 1975 Taucce et al. 2020 Oliveira et al. 2020
Oitaven et al. 2017; Heyer & Carvalho,
this study 2000

Roberto et al.
 Description of the holotype. Adult male (URCA-H 13344; Figure 2). Head longer than wide; head width 37%
of SVL; head length 40% of SVL; snout subovoid in dorsal view, rounded in lateral view; nostrils not protuberant,
directed anterolaterally; internarial distance less than eye-nostril distance; canthus rostralis angular, weakly con-
cave, loreal region slightly concave; eye large, with horizontally elliptical pupil; eye diameter slightly larger than
interorbital distance; cranial crests absent; upper eyelid smooth, 74% of eye diameter; tympanum large, with distinct
rounded tympanic annulus; tympanic membrane present, visible; supratympanic fold present; labial bars absent; vo-
cal sac subgular, small; tongue ovoid covering the entire floor of mouth, free and notched behind; vocal slits present,
lateral to tongue; choana small, rounded; odontophores oblique and widely separated posterior to the choanae, each
one bearing six teeth; skin on dorsum shagreen with scattered low tubercles; dorsolateral fold absent; skin on venter
areolate, weakly spotted, discoidal fold present; hands large, 29% of SVL; relative lengths of fingers II < IV < I <
III; finger fringes absent; finger discs expanded, truncate; disc of Finger III wider than the others, discs of fingers II
and IV wider than disc of Finger I; nuptial pad present, single; outer metacarpal tubercle divided inner metacarpal
tubercle large, in contact with nuptial pad; subarticular tubercles large, rounded; one subarticular tubercle on fingers
I and II, and two on fingers II and IV; supernumerary tubercles present; three horizontal bars present on thigh and
tibia; tibia length greater than thigh length, tibia length 51% of SVL; tarsal fold present; inner metatarsal tubercle
present, larger than outer metatarsal tubercle, both rounded; toes bearing narrow lateral fringes; webbing absent;
relative lengths of toes I < II < III < V < IV; toe discs expanded, smaller on Toe I; subarticular tubercles rounded, one
on toes I and II; two on toes III and V, and three on Toe IV; supernumerary tubercles present, barely distinct.
Measurements of the holotype (mm). SVL 24.4, HL 9.7, HW 8.9, ED 3.4, SL 4.4, IND 2.0, END 2.8, IOD 3.3,
UEW 2.5, TD 2.0, FAL 5.4, HAL 7.0, FIII 1.4, FIV 1.3, THL 12.4, TL 13.1, TAL 7.1, FL 10.7, and TIV 1.4.
Color in preservative. Dorsal and lateral surfaces pale cream, snout light brown, brown interocular band in
dorsal view, distinct dark brown stripe from tip of snout to posterior region of supratympanic fold. Irregular brown
marks in lower orbital region. Two brown chevrons on dorsum with irregular brown dots in inguinal region, light
brown stripes on dorsal surfaces of limbs. Ventral surface background pale cream in color.
Advertisement call. The call of Pristimantis relictus sp. nov. is described based on three individuals (MNRJ
55582 and two unvouchered individuals) (see Table 2;). The vocalization is composed of 1–8 multi pulsed notes
(2–5 pulses per note), with an ascendant amplitude modulation. The call duration ranges 0.02–0.66 s (0.06 ± 0.08;
n = 181), with intercall interval of 0.8–9.6 s (2.12 ± 1.5; n = 74), note duration ranges 0.02–0.05 s (0.03 ± 0.01; n
= 150), with a note rate of 0.52–2.20 notes per second (Figure 3. The call has three visible bands. The fundamental
frequency is in the first band, ranging 1894.9–2153.3 Hz, followed by a second band located in the dominant fre-
quency, ranging 3617.6–4220.5 Hz, and a third weak band with a higher frequency, ranging 4565–6287.7 Hz.

Table 2. Analyzed acoustic parameters for the advertisement calls of three individuals of Pristimantis relictus sp.
nov: MNRJ 55582 from Serra da Aratanha, municipality of Pacatuba (FNJV 0050599); one unvouchered individual
from Serra de Baturité (FNJV 0050600), municipality of Pacoti; and one unvouchered individual other from Natio-
nal Park of Ubajara (FNJV 0050601), municipality of Ubajara, Ceará, Brazil.
Acoustic parameters Pristimantis relictus sp. nov.
Peak Frequency (Hz) 3922.8 ± 125.6 (3617.6–4220.5, n =149)
Fund. Frequency (Hz) 2032.5 ± 54.7 (1894.9–2153.3, n =149)
Third band Frequency (Hz) 5692.3 ± 341.7 (4565–6287.7, n =149)
Note duration (s) 0.03 ± 0.01 (0.02–0.05, n =150)
Call duration (s) 0.06 ± 0.08 (0.02–0.66, n =181)
Notes/call 1.94 ± 1.24 (1–8, n = 151)
Note rate(s) 1.39 ± 0.8 (0.52–2.20, n =3)
Pulses/call 2.6 ± 1.0 (2–5, 386)
Intercall interval (s) 2.12 ± 1.5 (0.8–9.6, n =74)

New species of Pristimantis Zootaxa 5100 (4) © 2022 Magnolia Press · 529
Figure 3. Advertisement call of Pristimantis relictus sp. nov: A—individual, MNRJ 55582, recorded on the Serra da Ara-
tanha mountain range, municipality of Pacatuba, state of Ceará, Brazil, on December 26, 2006 by Igor J. Roberto, showing one
call composed of two notes, each with two distinct pulses. B–unvouchered individual from the Serra de Baturité mountain range,
municipality of Pacoti, state of Ceará, recorded on February 11, 2007, by Igor J. Roberto, showing a call composed of eight
pulsed notes. Spectrogram (above) and oscillogram (below).

Table 3. Measurements (mm) of males and females of the type series of Pristimantis relictus sp. nov. including the
holotype. Mean standard deviation (range).
Males (n = 18) Females (n = 33)
SVL 25.4 ± 1.7 (23.1–29.7) 30.5 ± 1.5 (26.8–32.8)
HL 9.7 ± 0.6 (9.0–11.3) 12.4 ± 1.1 (10.8–15.9)
HW 8.9 ± 0.7 (7.9–10.9) 11.6 ± 0.6 (9.9–12.3)
ED 3.2 ± 0.4 (2.6–4.3) 3.7 ± 4.2 (3.1–4.2)
SL 4.5 ± 0.3 (3.8–5.4) 5.5 ± 0.4 (4.6–6.4)
IND 2.1 ± 0.2 (1.7–2.6) 2.6 ± 0.3 (1.9–3.3)
END 3.2 ± 0.4 (2.7–4.2) 3.9 ± 0.4 (3.1–4.7)
IOD 3.1 ± 0.4 (2.4–3.7) 4.0 ± 0.4 (3.3–4.8)
UEW 3.3 ± 0.8 (2.2–4.3) 4.0 ± 0.8 (2.6–5.3)
TD 1.9 ± 0.2 (1.6–2.5) 2.2 ± 0.2 (1.8–2.5)
FAL 5.8 ± 0.5 (5.1–6.8) 7.3 ± 0.5 (6.4–8.4)
HAL 6.8 ± 0.4 (6.1–7.7) 8.4 ± 0.6 (6.8–9.9)
FIII* 1.5 ± 0.1 (1.4–1.6) 1.5 ± 0.1 (1.3–1.6)
FIV* 1.4 ± 0.1 (1.3–1.5) 1.4 ± 0.1 (1.2–1.7)
THL 12.3 ± 0.9 (10.2–13.8) 15.2 ± 1.3 (12.8–18.4)
TL 13.6 ± 0.6 (12.5–15.5) 17.0 ± 1.2 (14.9–19.8)
TAL 7.3 ± 0.5 (6.4–8.3) 9.2 ± 1.1 (7.4–12.3)
FL 10.7 ± 0.6 (9.9–12.6) 13.4 ± 0.8 (11.8–14.9)
TIV* 1.3 ± 0.1 (1.2–1.4) 1.4 ± 0.1 (1.2–1.6)

Variation among paratypes. Adult females (SVL 26.8–32.8 mm) are slightly larger than adult males (SVL
23.1–29.7 mm) (Table 3). Variation in coloration of fixed specimens is mostly related to the presence of chevrons
on the dorsum (URCA-H 6980, 2373, 1544, 13342, 6988, and 9828). In life, the dorsal coloration is highly poly-
morphic: dark brown, reddish to yellowish-brown, or grey (Figure 4), with the presence of dark brown chevrons
and black spots in some individuals. Other variations in coloration among paratypes are: brown bars on the leg and
arm; dark brown stripe from the tip of the nose to the eye, including the internarial area; dark brown bar in the in-

530 · Zootaxa 5100 (4) © 2022 Magnolia Press Roberto et al.

terorbital region; labial region yellowish to white; supratympanic fold bold black; gular region yellowish; and iris
cooper-colored with brown reticulations.
Geographic distribution. Pristimantis relictus is endemic to the “Brejos de Altitude” of the state of Ceará,
Northeast Brazil. This species occurs in the Planalto da Ibiapaba (municipalities of Ipu, Tianguá, Ubajara, Granja,
Viçosa do Ceará), Serra de Maranguape (municipality of Maranguape), Serra da Aratanha (municipality of Pacatu-
ba), Serra de Baturité (municipalities of Guaramiranga, Pacoti, Aratuba, Mulungu), Serra da Uruburetama (Roberto
& Loebmann 2016), and Serra da Meruoca (municipality of Meruoca) (Figure 5).
Natural history. Pristimantis relictus occurs in dry forests and humid forests (Loebmann & Haddad 2010;
Roberto & Loebmann 2016; Castro et al. 2019), from 100 to 900 m.a.s.l. The species has a prolonged breeding
period throughout the raining season from December to April. Vocalization occurs from 5 pm throughout the night,
with a peak of individuals vocalizing during 6–9 pm. Individuals were found vocalizing on leaves, tree trunks and
twigs, mostly facing downwards. Amplexus is axillary, and the eggs are deposited on humid surfaces of rock and
wooden debris. Mature females range 28–33 mm and 1.45–3.27 g (n = 13), and clutches have 21–53 eggs (n = 13).
We observed territorial behavior involving agonistic combat and the emission of territorial and release calls; un-
fortunately, these calls were not recorded. At Serra de Baturité mountain range we recorded the following species
occurring in sympatry with P. relictus: Boana raniceps, Dendropsophus minusculus, D. minutus, D. tapacuarensis,
Pithecopus gonzagai, Scinax tropicalia, Trachycephalus typhonius, Adelophryne baturitensis, Odontophrynus car-
valhoi, Rhinella casconi, R. dapsilis, R. diptycha, R. granulosa, Adenomera juikitam, Leptodactylus macrosternum,
L. natalensis, L. pustulatus, L. mystaceus and Physalaemus cuvieri. See Loebmann & Haddad (2010), and Castro
et al. (2019) for the anuran species that occur in sympatry with Pristimantis relictus at the Planalto da Ibiapaba
mountain range.

Figure 4. Adult specimens of Pristimantis relictus sp. nov. in life: A—Adult male, B–D adult female, municipality of Uba-
jara; E–F adult female municipality of Guaramiranga, G—amplectant pair, municipality of Pacatuba; H–I—adult calling males,
municipality of Guaramiranga, state of Ceará, Brazil.

New species of Pristimantis Zootaxa 5100 (4) © 2022 Magnolia Press · 531
Figure 5. Geographic distribution map of Pristimantis relictus: yellow triangle, type locality in the Ubajara National Park,
Serra da Ibiapaba mountain range; green circles, localities of paratypes: 1-municipality of Tianguá, 2-Serra da Ubatuba moun-
tain range, municipality of Granja, 3-Serra da Meruoca mountain range, 4-municipality of Ipu, 5-Serra da Uruburetama moun-
tain range, 6-Serra de Baturité mountain range, 7-Serra de Maranguape mountain range, 8-Serra da Aratanha mountain range.

Etymology. The specific epithet is derived from the Latin noun word relictus, meaning left behind, abandoned,
in reference to the isolated geographic distribution of the species between the Amazon and Atlantic Forest, where it
only occurs in mountain forests of highland marshes of the state of Ceará, Brazil.

Discussion

The taxonomy of the populations of Pristimantis from the “Brejos de altitude” of Ceará have been questioned since
the 1990’s and some authors have referred to the species as Eleutherodactylus sp., Ischnocnema gr. ramagii, Pris-
timantis sp. and Pristimantis gr. ramagii (Lima-Verde & Cascon 1990; Borges-Nojosa & Lima, 2001; Carnaval &
Bates 2007; Canedo & Haddad 2012; Roberto & Loebmann 2016; Castro et al. 2020), especially because its con-
servative external morphology is very similar to that of P. ramagii. Using several lines of evidence, we were finally
able to describe this endemic species, which had already been considered genetically distinct in recent decades (see
Carnaval & Bates 2007; Canedo & Haddad 2012; Trevisan et al. 2020). Pristimantis relictus is the fifth endemic
species of anuran described for the “Brejos de Altitude” of Ceará; the others being Adelophryne baturitensis, A.
maranguapensis, Proceratophrys ararype, and Rhinella casconi (Roberto & Loebmann 2016; Mângia et al. 2018).
Carnaval & Bates (2007) argued that the “Brejos de Altitude” are not a single biogeographical unit, but with distinct
entities across the northern and southern enclaves. The higher level of endemism of amphibians, reptiles and plants
in thee Brejos de Altitude of Ceará (e.g. Borges-Nojosa & Caramaschi 2003; Santos et al. 2007; Roberto & Loeb-
mann, 2016) confirms this and reinforces the need for conservation measures to protect these forest enclaves, espe-
cially in the Serra da Uruburetama, Serra da Meruoca, Serra de Maranguape and Serra da Aratanha mountain ranges,
where deforestation pressure has increased in recent last decades and there are no restrictive protected areas .
We recovered Pristimantis relictus as the sister species to the Atlantic Forest clade of the P. conspicillatus
group. However, this result should be interpreted with caution since we found very low support for this relationship.
Canedo and Haddad (2012), using a more comprehensive dataset of Terrarana and a different set of molecular mark-

532 · Zootaxa 5100 (4) © 2022 Magnolia Press Roberto et al.

ers, including a mitochondrial fragment of 12S -tVAl and 16S genes and two nuclear genes, recovered this lineage as
the sister species to P. gaigei (also with low support). A multilocus analysis of a larger sampling of the P. conspicil-
latus group is needed to clarify the relationships of P. relictus within the group. Despite the lack of resolution of
phylogenetic relationships within the P. conspicillatus species group, it is interesting to note that its species richness
remains underestimated and deserving of further future work and taxonomic revision. For instance, Trevisan et al.
(2020) investigated the history of the diversification of the Atlantic Forest clade using molecular markers and found
nine distinct clades, indicating the presence of deep divergent lineages as well as very structured populations within
named species. In addition, our phylogenetic inference show at least one putative new specie in the Atlantic forest
clade sister to all other species (Figure 1; Pristimantis aff. vinhai AFlab0445).
The advertisement call of P. relictus has the same pattern as other species in the P. conspicillatus group, being
composed of pulsatile and amplitude-modulated notes (Padial et al. 2016, Octaven et al. 2017; Oliveira et al. 2020;
Taucce et al. 2020). The advertisement call of P. relictus is similar to those of P. ramagii (Octaven et al. 2017) and
P. cf. paulodutrai (Heyer & Carvalho 2000), with the only difference being the lower number of pulses for P. relic-
tus. However, the complete acoustic repertoire and reproductive behavior of the species is more complex, with the
presence of distinctive calls that still need to be recorded and described in more detail (I.J. Roberto pers. obs). Such
natural history information is extremely useful to understand the evolutionary processes within different groups
of amphibians (Malagoli et al. 2021) and could help to understand how the diversity of Pristimantis in Northeast
Brazil was shaped, especially in the Atlantic Forest where different cryptic lineages occur in sympatry (Roberto et
al. 2017; Trevisan et al. 2020).
Since the species is abundant in all of the mountain ranges that we studied (see Loebmann & Haddad 2010;
Castro et al. 2019), and there are conservation units, such as Ubajara National Park, and other private protected
areas, we consider this species as Least Concern following the guidelines of the International Union for Conserva-
tion of Nature (IUCN).

Acknowledgments

We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing a research fellow-
ship to DL (#310859/2020-4), CFBH (#304943/2018-5) and RWA (# 303622/2015-6; 305988/2018-2; 307722/2021-
0), and a postdoc fellowship (#159999/2019-7) to IJR. We also thank FUNCAP (Chamada 18/2017—CNPq/ICMBio,
Process ICM-0132-00006.01.00/18), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–CAPES,
Fundação Cearense de Apoio Científico e Tecnológico FUNCAP, and CNPq (chamada CNPQ / ICMBIO / FAPs n
18/2017—process n 421350 / 2017-2) for partial financial support. We thank K. de Queiroz (USNM), D. O. Mes-
quita, F. R. Delfim (UFPB), P. M. S. Nunes (UFPE), P. I. Simões (UFPE), E. M. Santos (UFRPE) and F. F. Curcio
(UFMT) for access to collections and institutional specimen loans. We thank J. Streicher (BMNH) for the holotype
photos of Pristimantis ramagii. CFBH and MLL thank Fundação de Amparo Pesquisa do Estado de São Paulo for
financial support (#2013/50741-7; 2017/26162-8 and # 2018/03428-5). IJR and RWA thank K.C. Araújo for shar-
ing personal data about the species. The field expeditions were performed under permits # 12545-1, 12545-2 and
29613-5, issued by SISBio / Instituto Chico Mendes de Conservação da Biodiversidade. We thank Erik Wild for
revising the English of the manuscript.

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Appendix 1. List of sequences used in phylogenetic inferences, including GenBank accession numbers and
sample ID as in GenBank.
Species GenBank Acc. Num. GenBank Sample ID Group
Pristimantis relictus sp nov MW263916 CFBHT11321 P. conspicillatus
Pristimantis relictus sp nov MW263917 CFBHT11329 P. conspicillatus
Pristimantis relictus sp nov MW263918 CFBH25888 P. conspicillatus
Pristimantis relictus sp nov MW263919 CFBH25893 P. conspicillatus
Pristimantis relictus sp nov JX267386 MRT4514 P. conspicillatus
Pristimantis cf. paulodutrai JX267296 MNRJ35338 P. conspicillatus
Pristimantis cf. paulodutrai JX267297 MNRJ46880 P. conspicillatus
Pristimantis cf. paulodutrai JX267299 MNRJ46788 P. conspicillatus
Pristimantis cf. paulodutrai JX267360 MNRJ40299 P. conspicillatus
Pristimantis cf. paulodutrai KU495302 CFBHT04112 P. conspicillatus
Pristimantis cf. paulodutrai JX267484 MNRJ49745 P. conspicillatus
Pristimantis ramagii JX267318 MNRJ36751 P. conspicillatus
Pristimantis ramagii KU495303 CFBHT11142 P. conspicillatus
Pristimantis ramagii JX267319 MNRJ50249 P. conspicillatus
Pristimantis ramagii JX267380 AFlab426 P. conspicillatus
Pristimantis cf. vinhai JX267362 MTR13417 P. conspicillatus
Pristimantis cf. vinhai JX267363 MTR13504 P. conspicillatus
Pristimantis cf. vinhai JX267365 MTR13631 P. conspicillatus
Pristimantis cf. vinhai JX267364 MTR13607 P. conspicillatus
Pristimantis cf. vinhai JX267343 AFlab0923 P. conspicillatus
Pristimantis vinhai MN954210 UFMG19855 P. conspicillatus
Pristimantis vinhai MN954211 UFMG19856 P. conspicillatus
Pristimantis vinhai MN954212 UFMG19858 P. conspicillatus
Pristimantis aff. vinhai JX267492 AFlab0445 P. conspicillatus
Pristimantis moa MK992552 209 P. conspicillatus
Pristimantis moa MK992553 210 P. conspicillatus
Pristimantis moa MK992554 LA77 P. conspicillatus
Pristimantis rupicola MN954203 MZFSDAR4460 P. conspicillatus
Pristimantis rupicola MN954205 UFMG20558 P. conspicillatus
Pristimantis rupicola MN954206 UFMG20568 P. conspicillatus
Pristimantis rupicola MN954209 UFMG20575 P. conspicillatus
Pristimantis achatinus EF493660 KU217809 P. conspicillatus
Pristimantis achatinus JN371033 UVC15953 P. conspicillatus
Pristimantis ardilae MN215424 MCNUPH117 P. conspicillatus
Pristimantis ardilae MN215426 MCNUPH253 P. conspicillatus
Pristimantis ardilae MN215428 MCNUPH163 P. conspicillatus
Pristimantis bipunctatus EF493702 KU291638 P. conspicillatus
Pristimantis bipunctatus KY006089 MUSM31120 P. conspicillatus
Pristimantis bipunctatus KY594758 MUSM31179 P. conspicillatus
Pristimantis buccinator EU712630 MNCNDNA9504 P. conspicillatus
Pristimantis buccinator KY652650 MUSM33269 P. conspicillatus
Pristimantis buccinator MG820140 EB223 P. conspicillatus
Pristimantis chiastonotus JN691280 351PG P. conspicillatus
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 APPENDIX 1. (Continued)
Species GenBank Acc. Num. GenBank Sample ID Group
Pristimantis chiastonotus JN691278 AF0937 P. conspicillatus
Pristimantis chiastonotus JN691293 366MC P. conspicillatus
Pristimantis citriogaster EF493700 KU212278 P. conspicillatus
Pristimantis condor EF493701 KU217857 P. conspicillatus
Pristimantis conspicillatus EF493529 QCAZ28448 P. conspicillatus
Pristimantis fenestratus EU192273 MNKA6629 P. conspicillatus
Pristimantis fenestratus EU192276 MNCN43031 P. conspicillatus
Pristimantis fenestratus EU192277 MHNC3130 P. conspicillatus
Pristimantis gaigeae FJ784385 KRL0825 P. conspicillatus
Pristimantis gaigeae FJ784490 KRL1202 P. conspicillatus
Pristimantis gaigei JN991448 CH6471 P. conspicillatus
Pristimantis gaigei JN991449 nrps0009 P. conspicillatus
Pristimantis giorgii KU495457 CFBHT12087 P. conspicillatus
Pristimantis giorgii JX267540 MZUSP139440 P. conspicillatus
Pristimantis giorgii MK992557 CAX19 P. conspicillatus
Pristimantis giorgii MK992567 LZA1365 P. conspicillatus
Pristimantis gutturalis JN691313 577PG P. conspicillatus
Pristimantis incertus EU186650 P. conspicillatus
Pristimantis koehleri EU192278 MNCN42990 P. conspicillatus
Pristimantis koehleri EU192279 MNKA6627 P. conspicillatus
Pristimantis koehleri EU192282 MNCN43013 P. conspicillatus
Pristimantis latro KX242519 LZATM467 P. conspicillatus
Pristimantis latro KX242522 LZATM277 P. conspicillatus
Pristimantis latro KX926011 MPEG177 P. conspicillatus
Pristimantis lymani EF493392 KU218019 P. conspicillatus
Pristimantis malkini EU186663 QCAZ28296 P. conspicillatus
Pristimantis pictus MK992508 ABAM2129 P. conspicillatus
Pristimantis pictus MK992511 ABAM2151 P. conspicillatus
Pristimantis pictus MK992512 LA53 P. conspicillatus
Pristimantis pluvian MK992575 ABAM2173 P. conspicillatus
Pristimantis pluvian MK992576 ABAM742 P. conspicillatus
Pristimantis pluvian MK992577 ABAM1348 P. conspicillatus
Pristimantis samaipatae EU192290 MNCN42988 P. conspicillatus
Pristimantis samaipatae EU192291 MNCN42989 P. conspicillatus
Pristimantis samaipatae EU192292 MNCN42987 P. conspicillatus
Pristimantis skydmainos EF493393 P. conspicillatus
Pristimantis vilarsi KP149278 AJC3944 P. conspicillatus
Pristimantis vilarsi KP149333 AJC3957 P. conspicillatus
Pristimantis vilarsi KP149384 AJC3946 P. conspicillatus
Pristimantis zeuctotylus JN691245 1681BPN P. conspicillatus
Pristimantis zeuctotylus JN691257 80AFC P. conspicillatus
Pristimantis zeuctotylus JQ742171 VUB3624 P. conspicillatus
Pristimantis appendiculatus EF493524 KU177637 Outgroup
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New species of Pristimantis Zootaxa 5100 (4) © 2022 Magnolia Press · 539
 APPENDIX 1. (Continued)
Species GenBank Acc. Num. GenBank Sample ID Group
Pristimantis ocreatus EF493682 KU208508 Outgroup
Pristimantis actites EF493696 KU217830 Outgroup
Pristimantis duellmani AY326003 WED53050 Outgroup
Pristimantis chloronotus AY326007 WED52959 Outgroup
Pristimantis ridens EF493355 AMNHA124551 Outgroup
Pristimantis unistrigatus EF493387 KU218057 Outgroup
Pristimantis versicolor EF493389 KU218096 Outgroup
Holoaden luederwaldti EU186710 MZUSP131872 Outgroup
Craugastor daryi EF493531 UTAA57940 Outgroup
Lynchius flavomaculatus EU186667 KU218210 Outgroup
Yunganastes mercedesae FJ539066 ZFMK72572 Outgroup

Appendix 2. List of additional specimens examined for morphological characters. CHUFC: UFMT-A, NHMW,
URCA-H, CFBH, USNM, CHUFPB, CHUFPE, BMNH.

Pristimantis chiastonotus: BRAZIL: Amapá, Santana, CHUFC 2505. Pristimantis dundeei: BRAZIL: Mato Grosso, Cha-
pada dos Guimarães, UFMT-A 8215, 8217. Pristimantis fenestratus: BRAZIL: Amazonas, Borba, NHMW 19940:2 (Paralec-
totype); Rondônia, Mamoré River NHMW 19940:1 (Lectotype). Pristimantis giorgii: BRAZIL: Pará, Curionópolis, URCA-H
2113, 2127, 2130. Pristimantis latro: BRAZIL: Pará, Portel, CHUFC 2591. Pristimantis moa: BRAZIL: Maranhão, Bal-
sas, UFMT-A 11234-35, 11248, Tocantins, Araguaína, URCA-H 13534-35. Pristimantis cf. paulodutrai: BRAZIL: Alagoas,
Campo Alegre, CFBH 18614, 18617, 18619, 18630, Bahia, Aurelino Leal, CFBH 18745, 18774, Ilhéus (type locality), CFBH
13318, Mata de São João, CFBH 27785-89, 27798. Pristimantis pictus: BRAZIL: Pará, Jacareacanga, UFMT-A 9833, 9872.
Pristimantis pluvian: BRAZIL: Mato Grosso, Paranaíta, UFMT-A 9811, 9819. Pristimantis ramagii: BRAZIL: Paraíba,
Areia, CHUFPB 20047, João Pessoa, CHUFPB 4504–4505, 4938, 5014, 8358, 9417, Mamanguape, CHUFPB 12667, 12885–
12886, 46840, Santa Rita, CHUFPB 16851, 19503, Pernambuco, Igarassu (type locality), CFBH 2480-81, 2549; BMNH
1947.2.15.97 (holotype–photo), Abreu e Lima, CHUFPE A609–612, A617, Paudalho, CHUFPE A616, Tamandaré, CHUFPE
A851–852, Timbaúba, CHUFPE A1116–1119, Rio Grande do Norte, Espírito Santo, CHUFPB 20572, Tibau do Sul, CHUFPB
20185. Pristimantis reichlei: PERU: Huanuco Department, Manu National Park, USNM 298900–01 (Paratypes). Pristimantis
cf. vinhai: BRAZIL: Bahia, Igrapiúna, CHUFPB 20046.

Appendix 3. Estimates of uncorrected p-distances for the 16S rRNA gene fragment between species of Pristi-
mantis conspicillatus group present in Northeast Brazil.
1 2 3 4 5
1 P. relictus
2 P. cf. paulodutrai 15.9%
3 P. ramagii 16.3% 7.1%
4 P. cf. vinhai 15.9% 8.2% 8.8%
5 P. aff. vinhai 15.0% 7.5% 8.5% 7.0%
6 P. rupicola 14.8% 11.8% 13.7% 11.5% 9.0%

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