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ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN 1175-5334 (online edition)
Accepted by M. Rivera-Correa: 16 Oct. 2019; published: 11 Dec. 2019 531
Zootaxa 4706 (4): 531–545
https://www.mapress.com/j/zt/
Copyright © 2019 Magnolia Press Article
https://doi.org/10.11646/zootaxa.4706.4.3
http://zoobank.org/urn:lsid:zoobank.org:pub:0DD32E1A-566D-4D25-95FE-E0DEA3759614
A new species of Ischnocnema Reinhardt and Lütken, 1862
(Anura: Brachycephalidae) of the I. lactea species series
from southeastern Brazil
PEDRO P. G. TAUCCE1,4, BÁRBARA F. ZAIDAN2, HUSSAM ZAHER3 & PAULO C. A. GARCIA2,4
1Instituto de Biociências, UNESP – Universidade Estadual Paulista, Câmpus Rio Claro, Departamento de Zoologia e Centro de Aqui-
cultura (CAUNESP), Cx. Postal 199, 13506-900, Rio Claro, SP, Brazil
2Instituto de Ciências Biológicas, UFMG – Universidade Federal de Minas Gerais, Departamento de Zoologia, Laboratório de Her-
petologia, Avenida Antônio Carlos, 6627, Pampulha, 31270-910, Belo Horizonte, MG, Brazil
3Museu de Zoologia da Universidade de São Paulo, Avenida Nazaré 481, São Paulo, SP, CEP 04263-000, Brazil
4Corresponding authors. E-mail: pedrotaucce@gmail.com, pcagarcia@gmail.com
Abstract
We describe a new species of Ischnocnema from the Serra da Bocaina mountain range, state of São Paulo, southeastern
Brazil, based on morphological, bioacoustic, and mtDNA data. The new species is retrieved with high support values
within the I. lactea species series as the sister species of I. spanios. Ischnocnema bocaina sp. nov. is characterized by its
medium size (18.6–19.0 mm), a smooth venter, a rounded snout in dorsal view and acuminate in lateral view, a slightly
expanded subgular, single vocal sac, a round and whitish, poorly-developed glandular-appearing nuptial pad on the dorsal
surface of the thumb, and a nonpulsed advertisement call with 9 to 18 notes. We raise to 38 the number of Ischnocnema
species, the 12th described in the past 10 years.
Key words: Atlantic Rainforest, Bioacoustics, Brachycephaloidea, Integrative taxonomy, Molecular phylogeny
Introduction
The Neotropical genus Ischnocnema Reinhardt & Lütken, 1862 is a taxonomically challenging group of ground-
dwelling direct-developing frogs that encompasses some morphologically cryptic species complexes (Gehara et al.
2013, 2017; Taucce et al. 2018a). Much of its diversity (about 30%) has been described during the past ten years,
and presently it comprises 37 species (Frost 2018) , from which 34 are allocated in five species series (Taucce et
al. 2018b). Among these, the I. lactea series currently contains ten described species (Canedo & Haddad 2012):
I. concolor Targino, Costa & Carvalho-e-Silva 2009, I. gehrti (Miranda-Ribeiro 1926), I. holti (Cochran 1948), I.
lactea (Miranda-Ribeiro 1923), I. melanopygia Targino, Costa & Carvalho-e-Silva 2009, I. nigriventris (Lutz 1925),
I. paranaensis (Langone & Segalla 1996), I. randorum (Heyer 1985), I. spanios (Heyer 1985), and I. vizottoi Mar-
tins & Haddad, 2010. The series is endemic to the Brazilian Atlantic rainforest, occurring in the Brazilian states of
Paraná, São Paulo, Minas Gerais, and Rio de Janeiro.
Hedges et al. (2008) were the first to define the I. lactea species series, based on the former Eleutherodactylus
lacteus group of Lynch & Duellman (1997) and considering several morphological characters. However, some of
the species they included in the series have been posteriorly reallocated in other groups, like “Eleutherodactylus”
bilineatus Bokermann “1975” 1974, currently considered incertae sedis within Holoadeninae (Canedo & Haddad
2012); I. bolbodactyla (Lutz 1925); currently allocated in the I. verrucosa series (Canedo & Haddad 2012); I. ve-
nancioi (Lutz 1958), currently allocated in the I. venancioi series (Taucce et al. 2018b); and I. manezinho (Garcia
1996) and I. sambaqui (Castanho & Haddad, 2000), both currently unassigned to any series (Canedo & Haddad
2012). Despite consisting a phylogenetically solid group currently (Canedo & Haddad 2012; Taucce et al. 2018a; b),
these recent rearrangements, along with new information on the morphology of some members of the I. lactea series
(Targino & Carvalho-e-Silva 2008; Berneck et al. 2013; Silva-Soares et al. 2018), suggest that the morphological
TAUCCE ET AL.
532 · Zootaxa 4706 (4) © 2019 Magnolia Press
characters proposed by Hedges et al. (2008) are now outdated and not applicable anymore. All these aspects make
classic morphology-based taxonomy approaches difficult to be applied to the group as the only line of evidence.
Because of the taxonomic difficulties faced with the genus and its species series, we based our description herein on
an integrative approach that combines three lines of evidence: mtDNA sequences, morphological, and bioacoustic
characters.
Material and methods
Molecular analyses. In order to allocate the new species in one of the five currently recognized species series,
we used a matrix that includes 28 of the 37 described species of Ischnocnema and two species of Brachycephalus
Günther, 1858 as outgroups (Appendix I). The sister-group relationship between these two genera has been exten-
sively tested (Canedo & Haddad 2012; Padial et al. 2014; Taucce et al. 2018a; b). We chose a fragment of the 16S
ribosomal RNA gene (16S), which is commonly used in anuran taxonomy (Fouquet et al. 2007; Lyra et al. 2017;
Vences et al. 2005), to perform our analyses. We used the primers 16S-AR (CGCCTGTTTATCAAAAACAT) and
16S-BR (GACCTGGATTACTCCGGTCTGA) to amplify our fragment (Palumbi et al. 1991).
We extracted whole cellular DNA from 99.5% ethanol liver tissue using a standard ammonium precipitation
method (Lyra et al. 2017), and performed PCR amplification using Taq DNA Polymerase Master Mix (Ampliqon
S/A, Denmark) and Axygene Maxygene thermocyclers. The PCR program consisted of a 3-min initial denaturing
step at 95°C, followed by 35 cycles of 20 s at 95°C, 20 s at 50°C, and 45 s at 72°C, followed by a final extension
step of 5 min at 72°C. We purified PCR product following Lyra et al. (2017) and sequenced it in both directions
with a BigDye Terminator Cycle Sequencing Kit (version 3.0, Applied Biosystems) in an ABI 3730 automated DNA
sequencer (Applied Biosystems) at Macrogen Inc. (Seoul, South Korea).
We performed the alignment with MAFFT v7.273 (Katoh & Standley 2013) using the E-INS-i algorithm (--
genafpair command), which is adapted for sequences with conserved domains flanked by regions rich in gaps. We
conducted a search for the best-fitting nuclear model with PartitionFinder v2.1.1 (Lanfear et al. 2017) using the
corrected Akaike information criterion (AICc; Hurvich & Tsai 1989) and considering the whole 16S fragment as a
single partition. PartitionFinder starts its analysis constructing a maximum likelihood tree, and we used PhyML 3.0
(Guindon et al. 2010) implemented on it for this purpose. The resulting best-fitting nucleotide substitution model
was the general time-reversible (GTR) with proportion of invariant sites estimates and a gamma distribution of rates
across sites.
We reconstructed phylogenetic trees using both Bayesian inference and maximum likelihood optimality crite-
ria. For the first we used two independent runs of 1.0 x 107 generations, starting with random trees and four Markov
chains (one cold), sampled every 1,000 generations in MrBayes 3.2.6 (Ronquist et al. 2012). We discarded 25% of
generations and trees as burnin. We used the standard deviation of split frequencies (should be less than 0.01) and
Estimated Sample Size (should be larger than 200) to assess run convergence. We performed maximum likelihood
analysis using RAxML 8.2.10 (Stamatakis 2014), searching the most likely tree 100 times. To assess support, we
conducted 1,000 nonparametric bootstrap replicates. We computed uncorrected pairwise distances using R v3.3.3
(R Core Team 2018) with the packages APE v3.4 (Paradis et al. 2004) and SPIDER v1.3-0 (Brown et al. 2012). In
order to reduce the effect of the alignment on the genetic distances, we deleted the sites with gaps in a pairwise way
(using pairwise.deletion=T in the dist.dna command).
Morphological analyses. The specimens used in the morphological analyses are housed in the following in-
stitutions: CFBH (Coleção de Anfíbios Célio F. B. Haddad, Universidade Estadual Paulista, Rio Claro, São Paulo,
Brazil), MZUSP (Museu de Zoologia da Universidade de São Paulo, São Paulo, São Paulo, Brazil), and UFMG
(Coleção de Anfíbios da Universidade Federal de Minas Gerais, Universidade Federal de Minas Gerais, Belo Hori-
zonte, Minas Gerais, Brazil). A complete list of specimens examined is given in Appendix II.
We took following measurements to the nearest 0.05 mm with a Mitutoyo® digital caliper under a stereomicro-
scope: snout-vent length (SVL), head length (HL), head width (HW), forearm length (FAL), hand length (HAL),
thigh length (THL), tibia length (TL), tarsal length (TAL), foot length (FL), eye diameter (ED), tympanum diameter
(TD), eye to nostril distance (END), internarial distance (IND), distance between the anterior margins of the eyes
(AMD), maximum width of disk on third Finger (3FD), and maximum width of disk on fourth Toe (4TD). SVL,
HL, HW, FAL, TL, FL, ED, TD, END, and IND follow Duellman (1970); 3FD and 4TD follow Heyer (1984); HAL,
ISCHNOCNEMA BOCAINA SP. NOV. FROM BRAZIL Zootaxa 4706 (4) © 2019 Magnolia Press · 533
THL, and TAL follow Heyer et al. (1990); and AMD follows Garcia et al. (2003). Sex was determined by the ob-
servation of secondary sexual characters of male specimens (presence of nuptial pads and vocal slits) and by direct
observation of eggs through the belly wall in female specimens. Morphological nomenclature follows previous
literature on Brachycephaloidea (Duellman & Lehr 2009; Hedges et al. 2008; Heyer 1984; Heyer et al. 1990).
Call analyses. We recorded advertisement calls from the new species using a Marantz PMD 222 tape recorded
coupled to an AudioTechnica AT835b unidirectional microphone. We carried out recordings at 44.1 kHz sampling
frequency and we analyzed them with the software Raven pro 1.5 (Bioacoustics Research Program 2011). We pro-
duced spectrograms using window size of 512 samples, 99% overlap, DFT of 512 samples, and window type Hann.
We obtained spectrogram and oscillogram figures using tuneR version 1.0 (Ligges et al. 2013) and seewave ver-
sion 2.0.2 (Sueur et al. 2008) packages of R platform version 3.3.3 (R Core Team 2018). We produced spectrogram
figures with window length of 512 samples, 99% overlap, and window name Hanning. Voucher specimen (MZUSP
138663) is housed at MZUSP and call recordings (CBUFMG 1015–1018) are housed Coleção Bioacústica da Uni-
versidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais (CBUFMG).
We took the following acoustic parameters: call duration (Köhler et al. 2017), call rise time (Hepp & Canedo
2013), dominant frequency (Köhler et al. 2017), notes per call, note (repetition) rate (Taucce et al. 2018b), and note
repetition rate acceleration (Gehara et al. 2013). Call and note concepts follow Köhler et al. (2017).
Results
Ischnocnema bocaina sp. nov.
urn:lsid:zoobank.org:act:64C9B836-EA8B-496F-B3B6-2BCFFE0CD1BA
Holotype. MZUSP 138663, adult male, collected at Estação Ecológica do Bananal, municipality of Bananal, state
of São Paulo, southeastern Brazil, by H. Zaher and P.C.A. Garcia on 18 January 2008.
Paratopotype. MZUSP 138664, adult male, collected with the holotype.
Diagnosis. In the I. lactea species series by phylogenetic placement (Fig. 1). Ischnocnema bocaina sp. nov. is
distinguished from all other species of the I. lactea series by the following combination of characters: (1) medium
size (SVL in males 18.6–19.0 mm); (2) snout rounded in dorsal view (Fig. 2); (3) tip of the snout acuminate in lat-
eral view (Fig. 3); (4) dorsum smooth with granular flanks; (5) venter smooth; (6) head longer than wide; (7) eyelid
tubercles absent; (8) dentigerous process of the vomer present; (9) vocal sac single, subgular, slightly expanded;
(10) tips of Fingers II–IV expanded, truncated; (11) glandular-appearing nuptial pad on dorsal surface of the thumb,
rounded, poorly-developed, whitish, extending to the posterior half of the thenar tubercle; (12) toe lengths formula
I < II < V < III < IV; (13) calcar tubercle present, well-developed; (14) 9–18 notes per call; (15) advertisement call
duration 1000–2010 ms; (16) note rate 8.1–9.0 notes/s; (17) dominant frequency 2325–2746 Hz; (18) nonpulsed
advertisement call.
Comparison with other species. We based the comparisons both on collection specimens and literature data
(references follow in parenthesis at the end of each species comparison). The new species differs from I. concolor
by its larger size (I. bocaina sp. nov. males 18.6–19.0 mm SVL; I. concolor males 15.0–18.4 mm SVL); smooth
venter (slightly areolate in I. concolor); rounded snout in dorsal view (subacuminate in I. concolor); acuminate
snout in lateral view (rounded in I. concolor); slightly expanded vocal sac (moderately expanded in I. concolor);
tips of Fingers II–IV well-developed and truncate (moderately developed and rounded in I. concolor); toe lengths
formula I < II < V < III < IV (I < II < III < V < IV in I. concolor); and well-developed calcar tubercle (calcar tubercle
absent in I. concolor; Targino et al. 2009). Ischnocnema bocaina sp. nov. differs from I. gehrti by its rounded snout
in dorsal view (truncate in I. gehrti) and its acuminate snout in lateral view (subacuminate in I. gehrti; Pombal &
Cruz 1999). From I. holti, the new species differs by its smooth venter (slightly areolate in I. holti); head longer
than wide (wider than long in I. holti); acuminate snout in lateral view (rounded in I. holti); slightly expanded vocal
sac (not expanded in I. holti); whitish nuptial pad (translucent in I. holti); and toe lengths formula I < II < V < III <
IV (I < II < III < V < IV in I. holti; Targino & Carvalho-e-Silva 2008). Ischnocnema bocaina sp. nov. differs from
I. lactea by its smaller size (I. bocaina sp. nov. males 18.6–19.0 mm SVL; I. lactea males 19.6–26.7 mm SVL); its
smooth dorsum and venter (rugose and moderately granular in I. lactea); acuminate snout in lateral view (obtuse in
I. lactea); the absence of upper eyelid tubercles (present in I. lactea); its single nuptial pad (double in I. lactea); its
toe lengths formula I < II < V < III < IV (I < II < III < V < IV in I. lactea); and the advertisement call with 9–18 non-
TAUCCE ET AL.
534 · Zootaxa 4706 (4) © 2019 Magnolia Press
pulsed notes (one pulsed note in I. lactea; Silva-Soares et al. 2018). The new species differs from I. melanopygia by
its smooth venter (venter slightly areolate in I. melanopygia); snout rounded in dorsal and acuminate in lateral views
(respectively subacuminate and rounded in I. melanopygia); and its slightly expanded vocal sac (not expanded in
I. melanopygia; Targino et al. 2009). Ischnocnema bocaina sp. nov. differs from I. nigriventris by its smooth dor-
sum and venter (respectively shagreen with tubercles or warts and weakly areolate in I. nigriventris); head longer
than wide (wider than long in I. nigriventris); snout rounded in dorsal and acuminate in lateral views (respectively
nearly-rounded and rounded in I. nigriventris); by the absence of upper eyelid tubercles (present in I. nigriventris);
by the slightly expanded vocal sac (not expanded in I. nigriventris); its toe lengths formula I < II < V < III < IV (I
< II < III = V < IV in I. nigriventris); and the advertisement call with 9–18 notes and call duration of 1000–2010
ms (2–4 notes and 194–565 ms in I. nigriventris; Berneck et al. 2013). The head longer than wide (as long as wide
in I. paranaensis); snout acuminate in lateral view (rounded in I. paranaensis); vomerine teeth present (absent in I.
paranaensis); toe lengths formula I < II < V < III < IV (I < II < III < V < IV in I. paranaensis); and well-developed
calcar tubercle differ I. bocaina sp. nov. from I. paranaensis (calcar absent in I. paranaensis; Langone & Segalla
1996). Ischnocnema bocaina sp. nov. differs from I. randorum by its larger size (I. bocaina sp. nov. males 18.6–19.0
mm SVL; I. randorum males 11.8–15.0 mm SVL); its smooth venter (weakly granular in I. randorum); rounded
snout in dorsal and acuminate in lateral views (respectively subovoid and rounded in I. randorum); the presence of
dentigerous processes of the vomer (absent in I. randorum); its slightly expanded vocal sac (greatly expanded in
I. randorum); its toe lengths formula I < II < V < III < IV (I < II < III < V < IV in I. randorum); and the nonpulsed
advertisement call with 9–18 notes, note rate 8.1–9.0 notes/s, and dominant frequency 2325–2756 Hz (pulsed single
note advertisement call with note rate 1.3–2.6 and dominant frequency 3800–5200 Hz in I. randorum; Heyer 1985;
Heyer et al. 1990). Ischnocnema bocaina sp. nov differs from I. spanios, its sister species, by its larger size (I.
bocaina sp. nov. males 18.6–19.0 mm SVL; I. spanios males 14.7–15.3 mm SVL); its rounded snout in dorsal and
acuminate in lateral views (respectively subovoid and rounded in I. spanios); its slightly expanded vocal sac (not
expanded in I. spanios); the presence of a nuptial pad (absent in I. spanios); and its toe lengths formula I < II < V <
III < IV (I < II < III = V < IV in I. spanios; Heyer 1985; Heyer et al. 1990). Finally, I. bocaina sp. nov. differs from
I. vizottoi by its larger size (I. bocaina sp. nov. males 18.6–19.0 mm SVL; I. vizottoi males 13.3–16.6 mm SVL); its
smooth dorsum and venter (respectively slightly rugose and granular in I. vizottoi); its rounded snout in dorsal and
acuminate in lateral views (respectively sub-elliptical and acuminate-rounded in I. vizottoi); its slightly expanded
vocal sac (moderately expanded in I. vizottoi); its toe lengths formula I < II < V < III < IV (I < II < III = V < IV in
I. vizottoi); its well-developed calcar tubercle (small or absent in I. vizottoi); and its advertisement call with 9–18
notes, call duration 1000–2010 ms and dominant frequency 2325–2756 Hz (advertisement call with a single note,
call duration 38–72 ms, and dominant frequency 3417–3763 Hz in I. vizottoi; Martins & Haddad 2010).
Genetic distances of mitochondrial 16S rRNA fragment within and between members of the Ischnocnema lac-
tea species series are given in Table 1.
Description of holotype. Adult male with vocal slits, subgular vocal sac, and nuptial pads; medium-size (SVL
= 18.6 mm). Head slightly longer than wide; head length 36% of the SVL, head width 34% of the SVL; snout round-
ed in dorsal view, acuminate in lateral view; nostril elliptical, slightly protuberant, oriented laterally, located near
the tip of snout; canthus rostralis slightly distinct, curved; loreal region slightly concave; two poorly-developed,
rounded, postrictal tubercles on each side about the same size; eye protuberant, oriented laterally; eye diameter 42%
of head length; palpebral tubercles absent; tympanum barely distinct, rounded; tympanic membrane undifferenti-
ated; annulus present, barely visible externally, its dorsal portion hidden; tympanum diameter 45% of eye diameter;
supratympanic fold absent; vocal slits present; vocal sac subgular, distinct, one visible fold on each side of the jaw;
tongue large, elliptical, without posterior notch; choanae elliptical, larger than the dentigerous processes of the vo-
mer; dentigerous processes of the vomer round, located posteromedially to choanae, medially separated by a gap
approximately the same size as one dentigerous process; vomerine teeth present.
Forelimb slender; palmar tubercle barely distinct, cordiform, its diameter approximately equal that of the thenar
tubercle; thenar tubercle barely distinct, elliptical; glandular-appearing nuptial pad on dorsal surface of the thumb,
round, poorly-developed, whitish, extending to the posterior half of the thenar tubercle; palm smooth; supernumer-
ary tubercles absent; single subarticular tubercles flat, rounded, large; fingers slender, without fringes; tip of Finger
I slightly expanded, rounded; tip of Finger II moderately expanded, truncated; tips of Fingers III and IV fairly ex-
panded, truncated, with ungual flaps indented; Finger I half the size of Finger II; finger lengths formula I < II < IV
< III.
ISCHNOCNEMA BOCAINA SP. NOV. FROM BRAZIL Zootaxa 4706 (4) © 2019 Magnolia Press · 535
TABLE 1. Uncorrected pairwise genetic distances (given in percentage) of mitochondrial 16S rRNA fragment (ca. 600bp)
within (in bold) and between members of the Ischnocnema lactea species series. Within species distances are in bold.
Data are shown as min–max where appropriate.
I. bocaina
sp. nov.
I. spanios I. nigriven-
tris
I. randorum I. concolor I. vizottoi I. melano-
pygia
I. holti I.
lactea
I. bocaina
sp. nov.
NA
I. spanios 6.3–6.9 0.0–1.9
(n = 6)
I. nigriventris 13.6–13.7 12.0–12.5 0.0–0.2
(n = 3)
I. randorum 12.1 12.0–12.2 10.0–10.1 0.0
(n = 2)
I. concolor 13.6 12.2–12.7 7.0 8.6 0.0
(n = 2)
I. vizottoi 14.1 12.5–13.1 6.7–7.0 8.9 1.4 0.0
(n = 2)
I. melanopy-
gia
10.7 10.9–11.1 10.5–10.7 9.8 10.0 10.2 0.0
(n = 2)
I. holti 13.6 15.1–15.5 13.9–14.5 12.6 13.7 12.9 12.0 0.0
(n = 2)
I. lactea 13.9–14.8 14.9–16.2 15.5–16.4 12.6–13.2 13.1–14.2 12.9–14.0 12.7–13.2 9.2–10.2 0.0–2.7
(n = 7)
Hindlimb slightly robust; shank slightly longer than thigh; tibia length 49% of SVL; thigh length 48% of SVL;
moderately-developed, conical calcar tubercle present; tarsal folds absent; foot length 47 % of SVL; inner meta-
tarsal tubercle elliptical, twice as large as outer metatarsal tubercle; outer metatarsal tubercle rounded; sole of foot
smooth; supernumerary tubercles absent; single subarticular tubercles present, flat, rounded; toes long, slender,
without fringes; tips of Toes I and V slightly expanded, rounded; tips of Toes II–IV moderately expanded, truncated,
ungual flaps indented on Fingers III and IV; toe lengths formula I < II < V < III < IV.
Dorsal skin smooth; venter smooth; flanks granulated; posterior portion of ventral surface of thighs coarsely
shagreen; discoidal and thoracic folds present.
Coloration of holotype. Dorsum diffused marbled rust, brown, and dark-brown, with two medial white spots at
the shoulder girdle (Fig. 4); head with clear brown stripe between eyes; lips with sparse white spots; flanks diffused
marbled rust, brown, and dark-brown; posterior half with sparse white spots; dorsal surfaces of the limbs banded
brown and dark-brown with sparse rust blotches. Venter, venter surfaces of the limbs, and gular region marbled
light-brown, brown, and rust; gular region darker than rest of the ventral region. Posterior surfaces of thighs brown.
In preservative, rust becomes nut-brown and all other colors become lighter.
Measurements of holotype (in millimeters). SVL 18.6; HL 6.6; HW 6.3; ED 2.8; TD 1.3; END 1.5; IND 1.8;
AMD 3.8; FAL 3.7; HAL 5.3; 3FD 1.1; THL 8.9; TL 9.1; TAL 4.7; FL 8.8; 4TD 0.8.
Variation. The paratopotype is somewhat faded, but it is overall similar to the holotype, except that its dentiger-
ous processes of the vomer are barely distinct (distinct in the holotype). It also lacks the two medial white spots at
the shoulder girdle. Measurements of the paratopotype (in millimeters): SVL 19.0; HL 7.3; HW 7.0; ED 2.7; TD 1.1;
END 2.1; IND 2.0; AMD 3.4; FAL 3.6; HAL 5.6; 3FD 1.1; THL 9.4; TL 9.4; TAL 5.1; FL 8.8; 4TD 1.0.
Phylogenetic relationships and genetic distances. The Bayesian inference and the maximum likelihood anal-
yses yielded similar topologies (Fig. 1). We recovered the I. lactea species series with high support (1.0 of posterior
probability and 98% of bootstrap), with I. bocaina sp. nov. as the sister species of I. spanios, also with high support
(1.0 of posterior probability and 98% of bootstrap). The uncorrected pairwise distance of partial 16S rRNA between
these two species was among the lowest of all species pairs (6.3–6.9%, Table 1).
Etymology. The specific epithet refers to the Bocaina Mountain Range (Serra da Bocaina, in Portuguese),
where the type locality of the species is located, in recognition of the great biodiversity importance of this mountain
range. The name is used here as a noun in apposition.
Vocalization. We recorded two types of calls, call 1 (n = 11; Fig. 5A; Table2) and call 2 (n = 10; Fig. 5B; Table
2). The first one was composed of 9 to 18 non-pulsed notes ( = 15.6 ± 2.3), with the energy gradually increasing in
each note through the call, until reaching a peak typically on the penultimate or last note (Fig. 5A). Call 1 duration
TAUCCE ET AL.
536 · Zootaxa 4706 (4) © 2019 Magnolia Press
ranged from 1000 to 2010 ms ( = 1760 ± 260) and call rise time ranged from 91.6–99.4% ( = 97.8 ± 2.2) of the
call. Note repetition rate was 8.1–9.0 notes/s ( = 8.3± 0.3) and note repetition rate acceleration ranged from –13.2
to 0.2 ( = –6.1 ± 4.4). Dominant Frequency was 2325–2756 Hz ( = 2576 ± 146). Call 2 was composed of one
non-pulsed note, with duration ranging from 13 to 14 ms ( = 13.7 ± 0.0) and Dominant Frequency ranging from
2756–2842 Hz ( = 2834 ± 27). We considered call 1 the advertisement call and call 2 the territorial call because of
the similarities of each call with the advertisement and territorial calls of I. nigriventris (Berneck et al. 2013).
FIGURE 1. The 50% majority rule consensus tree from Bayesian inference of mitochondrial 16S rRNA gene (16S) showing
the relationships within the Ischnocnema lactea species series. Numbers above branches indicate posterior probabilities and
numbers below branches indicate maximum likelihood nonparametric bootstrap values. We only show bootstrap values above
50%.
ISCHNOCNEMA BOCAINA SP. NOV. FROM BRAZIL Zootaxa 4706 (4) © 2019 Magnolia Press · 537
FIGURE 2. Dorsal (left) and ventral (right) views of Ischnocnema bocaina sp. nov. holotype (MZUSP 138663). Scale bar = 10
mm. Photos by B. F. Zaidan.
TABLE 2. Call parameters comparing the members of the Ischnocnema lactea species series. Data are given as ranges
(mean ± SD) where appropriate.
I. bocaina sp. nov. I. nigriventris I. lactea I. randorum I. vizottoi
Call 1* Call 2 Call 1* Call 2 Call 1* Call 1* Call 1*
Notes per call 9–18
(15.6 ± 2.3)
1 2–4 1 1 3–8 1
Call duration (ms) 1000–2010
(1760 ± 260)
13–14
(13.7 ± 0.0)
194–565 30–41 635–1060 2000–5000 38–72
(52.7 ± 10.2)
Note rate (notes/s) 8.1–9.0
(8.4±0.3)
– – – – 1.3–2.6 –
Note rate acceleration (%) -13.2 to 0.2
(-6.1 ± 4.4)
– – – – – –
Call rise time (%) 91.6–99.4
(97.8–2.2)
– – – – – –
Dominant Frequency (Hz) 2325–2756
(2576 ± 146)
2756–2842
(2834 ± 27)
1955–3932 2004–3685 2240–2756 3800–5200 3417–3763
(3611 ± 103)
Pulsed/non-pulsed Non-pulsed Non-pulsed Non-pulsed Non-pulsed Pulsed Pulsed Non-pulsed
*Advertisement call
Natural history notes. The collectors found the two type specimens calling perched on trees about 50 cm
above the ground, right after a heavy rain. The call activity started about one hour before dusk, together with two
other Ischnocnema species from the I. guentheri and I. parva species series, but the new species ceased to call ear-
lier, about one hour after dusk.
Geographic distribution. Ischnocnema bocaina sp. nov. is known only from the type locality: Estação Ecológi-
ca do Bananal, municipality of Bananal, state of São Paulo, southeastern Brazil (Fig. 6).
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FIGURE 3. Lateral view of the head (above), ventral view of the foot (below, left), and ventral view of the hand (below, right)
of Ischnocnema bocaina sp. nov. holotype (MZUSP 138663). Scale bar = 2 mm. Photos by B. F. Zaidan.
ISCHNOCNEMA BOCAINA SP. NOV. FROM BRAZIL Zootaxa 4706 (4) © 2019 Magnolia Press · 539
FIGURE 4. Live specimen of Ischnocnema bocaina sp. nov., holotype (MZUSP 138663). Photo by P. C. A. Garcia.
Discussion
Since the proposition of the Ischnocnema lactea species series (Hedges et al. 2008), its content has changed signifi-
cantly (see introduction) and the morphological definition of the series is outdated. However, we recovered I. bocai-
na within the I. lactea series as it is currently recognized with high support in both analyses, as the sister species of
I. spanios. This result shows that the new species undoubtedly should be allocated to the I. lactea series. Nine of the
eleven species of the I. lactea species series have their phylogenetic position known, but I. gehrti and I. paranaensis
are still tentatively assigned to it. Both species are known only from their holotypes (I. gehrti holotype poorly
preserved; Pombal Jr. & Cruz 1999), and a taxonomic revision and reevaluation of the morphological characters
supporting the I. lactea species series is needed in order to clarify the phylogenetic affinities of these two species.
Moreover, efforts towards the rediscovery of I. gehrti and I. paranaensis in their type localities and surroundings are
paramount for getting molecular, acoustic, and new morphological data from these two poorly known species.
In addition to recovering the I. lactea series monophyletic, we also recovered the I. venancioi and I. guentheri
series monophyletic, both with moderate to high support (0.97 of posterior probability and 66% of bootstrap to the
former and 1.0 of posterior probability and 99% of bootstrap to the latter). Unlike previous studies (Canedo & Had-
dad 2012; Taucce et al. 2018a; b), we did not recover the I. verrucosa series monophyletic, and the relationships
within the species series, despite overall similar, are not the same as previous studies. These differences are probably
due to our small matrix (one mitochondrial fragment, final alignment of 554 bp) and, since we only wanted to posi-
tion I. bocaina within the I. lactea series, discussing these relationships is beyond the scope of this paper. The only
member of the I. parva series present in our phylogenetic analyses was I. parva.
The nuptial pad has been an important character when it comes to the taxonomy of Ischnocnema. It is a highly
variable character in the genus, and the presence of a single large, conspicuous, glandular-appearing nuptial pad was
proposed to be a putative synapomorphy of the clade composed by the I. parva, the I. venancioi, and the I. guentheri
species series (Taucce et al. 2018b). In the I. lactea species series it can be absent (I. spanios), whitish and poorly-
developed (I. bocaina sp. nov., I. randorum), double (I. lactea; Silva-Soares et al. 2018), and translucent (I. nigri-
ventris, I. holti). The character state is unknown in I. concolor, but it is present in some males (Targino et al. 2009).
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540 · Zootaxa 4706 (4) © 2019 Magnolia Press
FIGURE 5. Spectrogram (above) and oscillogram (below) of the two types of calls of Ischnocnema bocaina sp. nov.: (A) ad-
vertisement call and (B) territorial call. Respectively recordings CBUFMG 1017 and 1016, 18 January 2008.
ISCHNOCNEMA BOCAINA SP. NOV. FROM BRAZIL Zootaxa 4706 (4) © 2019 Magnolia Press · 541
FIGURE 6. Map showing the type locality of Ischnocnema bocaina sp. nov. (indicated by a black square): Estação Ecológica
do Bananal, municipality of Bananal, state of São Paulo, southeastern Brazil. Elevations above 500 and 1000 m are shaded
gray.
Martins & Haddad (2010) state that it is absent in I. vizottoi, but we examined the type series and it is present and
translucent in several males. When the nuptial pad is not large and conspicuous it may be difficult to see, especially
when it is translucent, and we recommend care on checking this character on the specimens. It may be easier to see
this character after drying the thumb of the specimens a little bit. Understanding the character states of the nuptial
pads throughout the genus Ischnocnema, both macroscopically and microscopically, will be paramount for the un-
derstanding of the taxonomy and the phenotypic evolution of the genus.
Acknowledgements
We thank C. F. B. Haddad (CFBH) and T. Grant (MZUSP) for making available specimens under their care. For
support at CFBH we thank N. C. Pupin and at MZUSP we thank A. S. Benetti. M. L. Lyra helped with laboratory
procedures. We are grateful to Centro de Estudos de Insetos Sociais (CEIS; São Paulo State University [UNESP])
for allowing us the use of the molecular laboratory. This research was supported by resources supplied by the Cen-
ter for Scientific Computing (NCC/GridUNESP) of UNESP and Cyberinfrastructure for Phylogenetic Research
(CIPRES). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
- Brasil (CAPES) - Finance Code 001. PPGT thanks FAPESP and CAPES grant #2014/05772-4 for the PhD fel-
lowship (2014-2018) and FAPESP grant #2019/04076-8 for the Postdoctoral fellowship (currently) and financial
support. PCAG thanks FAPESP grants #2005/56228-3 and #2006/57853-1 for financial support and the Young
Researcher fellowship. BFZ thanks CAPES for the PhD fellowship. HZ and PCAG thank Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) for financial support.
TAUCCE ET AL.
542 · Zootaxa 4706 (4) © 2019 Magnolia Press
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APPENDIX I. List of terminals and accession numbers of sequences used in the phylogenetic and genetic distance
analyses.
Species GenBank ID In the phylogenetic matrix? In the genetic distance matrix?
Brachycephalus pombali HQ435700 yes no
Brachycephalus ephippium HQ435693 yes no
Ischnocnema abdita JX267472 yes no
Ischnocnema bocaina MK967680 yes yes
Ischnocnema bolbodactyla JX267476 yes no
Ischnocnema colibri MH538418 yes no
Ischnocnema concolor JX267366 yes yes
Ischnocnema concolor JX267493 no yes
Ischnocnema erythromera JX267340 yes no
Ischnocnema feioi MF957166 yes no
Ischnocnema garciai MF957163 yes no
Ischnocnema guentheri JX267503 yes no
Ischnocnema henselii JX267478 yes no
Ischnocnema hoehnei JX267507 yes no
Ischnocnema holti JX267306 yes yes
Ischnocnema holti JX267509 no yes
Ischnocnema juipoca JX267349 yes no
Ischnocnema lactea JX267342 yes yes
Ischnocnema lactea JX267308 no yes
Ischnocnema lactea JX267514 no yes
Ischnocnema lactea JX267515 no yes
Ischnocnema lactea JX267516 no yes
Ischnocnema lactea JX267517 no yes
Ischnocnema lactea JX267518 no yes
Ischnocnema cf. manezinho JX267481 yes no
Ischnocnema melanopygia JX267292 yes yes
Ischnocnema melanopygia JX267519 no yes
Ischnocnema nanahallux KC569986 yes no
......continued on the next page
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APPENDIX I. (Continued)
Species GenBank ID In the phylogenetic matrix? In the genetic distance matrix?
Ischnocnema nasuta JX267311 yes no
Ischnocnema nigriventris JX267483 yes yes
Ischnocnema nigriventris JX267482 no yes
Ischnocnema nigriventris JX267359 no yes
Ischnocnema octavioi JX267521 yes no
Ischnocnema oea JX267313 yes no
Ischnocnema parnaso MH538421 yes no
Ischnocnema parva KY399230 yes no
Ischnocnema randorum JX267361 yes yes
Ischnocnema randorum JX267381 no yes
Ischnocnema sambaqui JX267531 yes no
Ischnocnema spanios JX267490 yes yes
Ischnocnema spanios JX267489 yes yes
Ischnocnema spanios JX267488 yes yes
Ischnocnema spanios JX267487 yes yes
Ischnocnema spanios JX267486 yes yes
Ischnocnema spanios JX267485 yes yes
Ischnocnema venancioi JX267321 yes no
Ischnocnema verrucosa JX267538 yes no
Ischnocnema vizottoi JX267350 yes yes
Ischnocnema vizottoi JX267352 no yes
APPENDIX II. Specimens examined (all from Brazil).
Ischnocnema holti: MINAS GERAIS: Itatiaia: MZUSP 97343–97371
Ischnocnema nigriventris: SÃO PAULO: Paranapiacaba, municipality of Santo André: CFBH 23478–23479; Parque das Neb-
linas, municipality of Bertioga: CFBH 28951–28952.
Ischnocnema randorum: SÃO PAULO: Boracéia, municipality of Salesópolis: MZUSP 23665–23670, 36865, 37555, 49644
(paratypes).
Ischnocnema spanios: SÃO PAULO: Carlos Botelho: MZUSP 135942; Jaceguava: MZUSP 135942; Mogi das Cruzes: MZUSP
133910–133913; Paranapiacaba, municipality of Santo André: MZUSP 142792–142795, 145271–145272, 145255,
145275–145277, 145263, 153754–153764; Furnas, muncipality of Piedade: MZUSP 136443–136449.
Ischnocnema vizottoi: SÃO PAULO: Campos do Jordão: CFBH 8205–8221 (paratypes).