a new large canopy-dwelling species of phyllodytes wagler ... · in the forest canopy, hampering...

Submitted 18 September 2019Accepted 27 January 2020Published 23 June 2020

Corresponding authorIuri R Dias iurirdiashotmailcom

Academic editorMax Lambert

Additional Information andDeclarations can be found onpage 20

DOI 107717peerj8642

Copyright2020 Dias et al

Distributed underCreative Commons CC-BY 40

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A new large canopy-dwelling speciesof Phyllodytes Wagler 1930 (AnuraHylidae) from the Atlantic Forest of thestate of Bahia Northeastern BrazilIuri R Dias1 Gabriel Novaes-e-Fagundes1 Antonio Mollo Neto2 JulianaZina3 Caroline Garcia3 Renato Sousa Recoder2 Francisco Dal Vechio2 MiguelTrefaut Rodrigues2 and Mirco Soleacute14

1Programa de Poacutes-Graduacatildeo em Zoologia Universidade Estadual de Santa Cruz Ilheacuteus Bahia Brazil2 Instituto de Biociecircncias Universidade de Satildeo Paulo Satildeo Paulo Satildeo Paulo Brazil3Departamento de Ciecircncias Bioloacutegicas Universidade Estadual do Sudoeste da Bahia Jequieacute Bahia Brazil4Herpetology Section Zoologisches Forschungsmuseum Alexander Koenig Bonn Germany

ABSTRACTThe known diversity of treefrogs of the genus Phyllodytes has rapidly increased in recentyears currently comprising 14 species Recent field work in the Atlantic Rainforest ofthe state of Bahia lead to the discovery of a new large species of Phyllodytes whichis herein described based on multiple evidence including morphological acousticaland genetic data Phyllodytes sp nov is one of the largest species within the genusand presents immaculate yellowish dorsum and limbs The advertisement call ofthe species is composed of 7ndash31 notes (half pulsedpulsatile-half harmonic) withfrequency-modulated harmonics Phyllodytes sp nov has a karyotype of 2n = 22chromosomes as also found in other species of the genus Genetic distance values of the16S mitochondrial rRNA among Phyllodytes sp nov and its congeners range between64 to 102 The description of another new species for this state reinforces the needfor further taxonomic work with Phyllodytes in this region that has been revealed as apriority area for research and conservation of this genus

Subjects Biodiversity Taxonomy ZoologyKeywords Amphibia Biodiversity Bromeliad Integrative taxonomy Lophyohylini

INTRODUCTIONThe genus Phyllodytes Wagler 1830 assembles 14 species distributed mainly throughoutthe morphoclimatic domain of the Atlantic Forest most of them with occurrence inthe northeast of Brazil (Orrico Dias amp Marciano-Jr 2018 Frost 2019) These small tomedium-sized tree frogs (182ndash485 mm SVL) are characterized mainly by the presence ofodontoids on their mandibles and association with bromeliads where they complete theirentire life cycle (Bokermann 1966 Peixoto 1995 Cruz Feio amp Cardoso 2006)

Although some species of Phyllodytes can use terrestrial bromeliads (Ferreira Schineideramp Teixeira 2012 Cunha amp Napoli 2016) most calling males are heard from bromeliadsin the forest canopy hampering the sampling of representatives of this genus Thus thispeculiar life history trait may explain the scarcity of studies dealing with its taxonomy

How to cite this article Dias IR Novaes-e-Fagundes G Neto AM Zina J Garcia C Recoder RS Vechio FD Rodrigues MT Soleacute M2020 A new large canopy-dwelling species of PhyllodytesWagler 1930 (Anura Hylidae) from the Atlantic Forest of the state of BahiaNortheastern Brazil PeerJ 8e8642 httpdoiorg107717peerj8642

ecology and phylogeny (Marciano-Jr Lantyer-Silva amp Soleacute 2017) Fortunately this scenariois changing due to the greater attention that the genus has received recently with half ofthe species within the genus (ie seven species) having been described in the last 15 years(Frost 2019)

The state of Bahia is a diversity hotspot for Phyllodytes with nine of the 14 known species(Orrico Dias amp Marciano-Jr 2018) Notwithstanding these numbers may underestimatethe actual number of species as inventories carried out in coastal areas of the south ofthe state indicate difficulties in assigning species names to collected individuals (Dias etal 2014 Dias Mira-Mendes amp Soleacute 2014 Mira-Mendes et al 2018) suggesting that thediversity within the genus may be even larger This assumption has been corroboratedby the description of three new endemic species from southern Bahia in the last threeyears (Marciano-Jr Lantyer-Silva amp Soleacute 2017 Voumlroumls Dias amp Soleacute 2017 Orrico Dias ampMarciano-Jr 2018)

During recent field trips to the Atlantic Forest of southern Bahia we collected specimensof Phyllodytes (adults and tadpoles) from both terrestrial and epiphytic bromeliads Acloser examination revealed that these specimens correspond to an undescribed speciesHerein we describe this new species based on morphology of adults and tadpolesbioacoustics cytogenetic and molecular evidence In addition we compile and discussavailable information on bioacoustic features and tadpole morphology of the genus

MATERIALS amp METHODSMorphological dataWe analyzed specimens housed at herpetological collections of Museu de Zoologia daUniversidade Estadual de Santa Cruz Ilheacuteus state of Bahia Brazil (MZUESC) Museu deHistoacuteriaNatural de Jequieacute ColecatildeoHerpetoloacutegica Jequieacute state of Bahia Brazil (MHNJCH)andMuseu de Zoologia da Universidade de Satildeo Paulo state of Satildeo Paulo Brazil (MZUSP)Examined specimens are listed in Appendix 1 For comparisons with Phyllodytes brevirostrisP edelmoi and P gyrinaethes we used data available in the literature (Peixoto amp Cruz 1988Peixoto Caramaschi amp Freire 2003) Specimens collected for this workwere obtained underIBAMA 12920-1 and ICMBIO 13708-1 and 35068 permits This research was approvedby the ethics committee on the use of animals (CEUA-UESC 00212)

We took the following measurements of adult specimens SVL (snout-vent length) HL(head length) HW (head width) IND (internarial distance) END (eye-nostril distance)ED (eye diameter) IOD (interorbital distance) TD (tympanum diameter) THL (thighlength) TBL (tibia length) TAL (tarsus length) FL (foot length) HAL (hand length) DF3(width of disc on finger III) and 4TD (toe IV disc diameter) We measured SVL HL HWTHL TL and FL with calipers to the nearest 002 mm The remaining measurements weremadewith calipers under a stereomicroscopeMeasurements followedKok amp Kalamandeen(2008) with the exception of IOD that was measured between the anterior corners of theeyes in order to enhance repeatability Snout profile terminology followed Heyer et al(1990) texture of dorsal skin is described according to Kok amp Kalamandeen (2008) andwebbing formula notation followed Savage amp Heyer (1967) and Savage amp Heyer (1997)

Dias et al (2020) PeerJ DOI 107717peerj8642 227

measured on left hand and foot Colour in life was described based on photographs of livespecimens taken during the day

Cranial osteologyWe scanned two individuals of the new species one adult (MZUSP 157524) and onejuvenile (MZUSP 157526) with a SkyScan digital microtomograph with a resolution of18micromand of 9microm respectively The images were processed in CTAnalyzer v111 softwareThe resulting 3D models were visualized on CTvox v33 software and the osteologicaldescriptions were made based on three-dimensional images Terminology of osteologicalelements follows Trueb (1973) and Duellmann amp Trueb (1986)

TadpoleTwo tadpoles (MZUSP 157525) of the new species were obtained together with an adultmale (MZUSP 157524) in a large bromeliad (ca 100 cm diameter) on a trunk of a large treeat 8mheight at Estacatildeo Ecoloacutegica Estadual deWenceslauGuimaratildeesWenceslauGuimaratildeesmunicipality State of Bahia Brazil (1335prime418primeprimeS 3943prime105primeprimeW)Mitochondrial sequencesobtained from a piece of the tail fin of one tadpole at stage 27 of Gosner (1960) confirmedtheir conspecificity (tadpoles obtained with MTR 22178 referred to as Phyllodytes sp 2in Blotto et al 2020) Tadpoles were euthanized in 5 lidocaine fixed and preserved in10 formalin Terminology for external morphology follows Altig amp McDiarmid (1999)Tadpole description and illustrations are based on the specimen at developmental stage 35of Gosner (1960) and compared with the specimen at stage 27

We measured 13 morphometric variables with a Digimess Rcopy digital caliper(precision plusmn 001 mm) and a micrometer ocular in a Nikon SMZ645 stereomicroscopefollowing Altig amp McDiarmid (1999) total length (TL) body length (BL) body height(BH) body width (BW) interorbital distance (IOD) internarial distance (IND) eyediameter (ED) eye-nare distance (END) nare-snout distance (NSD) tail length (TAL)maximum tail height (MTH) tail muscle height (TMH) and tail muscle width (TMW)The drawings were prepared with the aid of digital photograph obtained with an OlympusDP72 digital camera attached to an Olympus SZX12 stereomicroscope

Bioacoustic dataWe recorded the advertisement call of two individuals at their natural habitat beforecollecting The holotype was recorded with a digital recorder (Tascam DR1) with adirectional microphone (Sennheiser ME45) on March 2nd 2015 between 0040 amndash0200am at a distance of sim05 m and air temperature of 219 C The animal was calling from aterrestrial bromeliad and no conspecifics were heard nearby For the analysis of notes weused only the notes from 10 randomly selected calls

One paratype (MZUESC 18265) was recordedwith a digital recorder (Marantz PMD660)with a shotgun microphone (Sennheiser-ME66) on June 18th 2014 around 0010 am ata distance of sim1 m and air temperature of around 19 C The animal was calling from anisolated epiphytic bromeliad (Hohenbergia sp) around 31 m above the ground Duringthe recording we imitated the frog call (lsquolsquovocal playbackrsquorsquo) to stimulate its response Forthis reason we did not measure the interval between calls as they would be uninformative


In eight occasions the male emitted a different type of call which we named lsquolsquocall type IIrsquorsquoWe deposited all recordings at the Fonoteca Neotropical Jacques Vielliard (FNJV 4099741382ndash41384)

All callings were recorded in uncompressed PCM format with a sample rate of 441 kHzand 16 bits Sound analysis wasmade with Raven Pro 14 Time components weremeasuredfrom the waveform while spectral components were measured from the spectrogram Weset the spectrographic parameters as following window type Hann window size 1024samples 3dB filter bandwidth 619 Hz time grid overlap 90 time grid size 102 samplesfrequency grid DFT size 1024 samples frequency grid spacing 431 Hz When necessarywe filtered the frequencies below 150 Hz to reduce wind sound interference and above5000 Hz to reduce insect sound interference using the lsquolsquoFilter out active selectionrsquorsquo toolCategorization of sound types follows Beeman (1998) modified by Koumlhler et al (2017)We used a note-centered terminology (sensu Koumlhler et al 2017) for the call elementsnomenclature

The following parameters were measured from the calls call duration (from peakamplitude of the first note to the peak of the last note of each call) interval between calls(from peak amplitude of the last note of a call to the peak amplitude of the first note ofnext call) and number of notes of the call For the analysis of the notes we delimited theselection borders of each note using the threshold of 5 of the maximum amplitude of thenote (see Littlejohn 2001) Based on this selection the following automated measurements(see Charif Waack amp Strickman 2010) were taken duration 90 dominant frequencybandwidth 90 frequency 5 and frequency 95 Then we made additional smallerselections to measure the initial and final frequency of the first harmonic (the peakfrequency of the first 0015 s and of the last 0015 s of the first harmonic) Finally wemeasured the inter-note interval note emission rate and note shape (note rise time dividedby note duration)

For the comparisons with the calls of the congeneric species we used the data availablein the published call descriptions Based on the literature and our personal observationswe assume functional homology between the herein defined notes and those described forother species of the genus Phyllodytes

Cytogenetic dataWe karyotyped one paratype (MZUESC 18265) which was injected with colchicine 05six hours before euthanasia with a lethal dose of lidocaine Mitotic chromosomes wereobtained from intestinal epithelium after 30 min under distilled water and 24 h in coldCarnoy fixer (31) following a modified protocol from King amp Rofe (1976)

Chromosomic classification followed Levan Fredga amp Sandberg (1964) by adopting thefollowing limits for the Arm Ratio (AR) AR = 100 to 170 metacentric AR = 171ndash300submetacentric AR= 301 ndash700 subtelocentric AR= greater than 700 acrocentric Theywere arranged in decreasing order of size Metacentric submetacentric and subtelocentricchromosomes have two arms the acrocentric ones a single one

The C-banding was obtained as described by Sumner (1972) with the followingmodifications the blades were immersed in HCl 02 N for 13 min at room temperature and


then immersed in Ba(OH)2 solution at 60 C for 35 s and in 2X SSC at 60 C for 30 minNucleolar organizing regions (NORs) were identified by silver nitrate impregnationfollowing Howell amp Black (1980) and FISH with 18S rDNA probe according to PinkelStraume amp Gray (1986) and Hatanaka amp Galetti (2004)

At least 30 metaphase spreads were analyzed to confirm the 2n and karyotype structureresults Images were captured using an Olympus BX50 microscope (Olympus CorporationIshikawa Japan) with CoolSNAP camera and the images processed using Image Pro Plus41 software (Media Cybernetics Silver Spring MD United States)

Molecular dataWe sequenced the final part of fragment of the mitochondrial 16S ribosomal RNA gene(16S rRNA) from the holotype (MZUESC 18264) and from a paratype (MZUESC 18265)We also sequenced the same fragment for one topotype of Phyllodytes kautskyi (Museude Biologia Mello Leitatildeo - MBML 8818) from Domingos Martins State of Espiacuterito Santo(GenBank MN648397ndashMN648399 Document S1) following protocols of Faivovich et al(2005) and using the primers 16SC (F) 5prime-GTRGGCCTAAAAGCAGCCAC-3prime(Darst ampCannatella 2004) and 16SBr-H (R) 5prime-CCGGTCTGAACTCAGATCACGT-3prime(Palumbi etal 2001)

Phylogenetic analysesSequence alignment was carried out using the MAFFT algorithm with L-INS-i strategy(Katoh amp Toh 2008) Phylogenetic reconstruction was performed using the Bayesianinference with MrBayes 323 (Huelsenbeck amp Ronquist 2001) after the best model ofevolution was determined using jModelTest 213 (Darriba et al 2012) based on Akaikeinformation criterion to be GTR+I+G Bayesian analyses included two independent runseach with four chains and sampling every 1000 generations for 70 million generationsWe examined trace plots and effective sample size in Tracer 17 (Rambaut et al 2018)to check MCMC mixing and convergence We removed trees from the first 20 of thesamples as burn-in A consensus of the post burn-in trees was visualized in FigTree142 (httptreebioedacuksoftwarefigtree) These analyses were performed at CIPRESScience Gateway (Miller Pfeiffer amp Schwartz 2010) We included homologous sequencesavailable in Genbank of all species of Phyllodytes and of at least one species from all generaof Lophyohylini tribe (sensu Faivovich et al 2005) We used Cophomantini Nesorohylakanaima to root the tree following the results of a previous phylogenetic analysis (Faivovichet al 2005 Duellman Marion amp Hedges 2016) To estimate the genetic distance wecalculated uncorrected p-distances among the species of Phyllodytes in MEGA 606(Tamura et al 2013) considering dtransitions + transversions uniform rates amongsites and gapsmissing data as complete deletion

Nomenclatural actsThe electronic version of this article in Portable Document Format (PDF) will represent apublished work according to the International Commission on Zoological Nomenclature(ICZN) and hence the new names contained in the electronic version are effectivelypublished under that Code from the electronic edition alone This published work


and the nomenclatural acts it contains have been registered in ZooBank the onlineregistration system for the ICZN The ZooBank LSIDs (Life Science Identifiers) can beresolved and the associated information viewed through any standard web browser byappending the LSID to the prefix httpzoobankorg The LSID for this publication isas follows urnlsidzoobankorgpubFD2CACD2-F59F-4B75-947D-B576701424D9 Theonline version of this work is archived and available from the following digital repositoriesPeerJ PubMed Central and CLOCKSS

RESULTSGeneric placementmdash The new species can be allocated to the genus Phyllodytes by thepresence of odontoids in the mandible and phylogenetic reconstructions indicate closerelationship to other species in this genus also supporting this placement (see below)

Phyllodytes magnus sp nov

Phyllodytes kautskyimdash(Freitas 2015)Phyllodytes cf kautskyimdash(Freitas et al 2018)urnlsidzoobankorgactD8DF9024-57C1-413B-9D05-B0CF05B7929C

Holotypemdash MZUESC 18264 adult male (Figs 1 2 3A and 3C) found in a terrestrialbromeliad on March 2nd 2015 by IRD and Carlos Augusto Souza-Costa at Serra da Jiboacuteia(1252prime1908primeprimeS 3928prime5390primeprimeW 490 m asl WGS84) between the municipalities of SantaTerezinha and Eliacutesio Medrado State of Bahia BrazilParatypesmdash MZUESC 18265 (ex-MHNJCH 914) adult male (Fig 3B and 3D) collectedin an epiphytic giant bromeliad (Hohenbergia sp) at sim310 m above the ground at ParqueEstadual da Serra do Conduru (1429prime3680primeprimeS 3908prime1070primeprimeW 201 m asl WGS84)municipality of Urucuca State of Bahia Brazil on June 19th 2014 byGNF and Joedison dosSantos Rocha MZUSP 157524 (adult male) and MZUSP 157526 (juvenile) both collectedin Estacatildeo Ecoloacutegica Estadual de Wenceslau Guimaratildees (1335prime418primeprimeS 3943prime105primeprimeW530 m asl WGS84) municipality of Wenceslau Guimaratildees State of Bahia Brazil onDecember 14th 2011 by MTR RSR Mauro Teixeira Junior and FDVEtymologymdash The specific epithet is an adjective from Latin meaning lsquolsquogreatrsquorsquo or lsquolsquolargersquorsquo(Lewis amp Short 1891) and refers to the large size of the adult males collected from thisspecies which are among the largest known in the genusDiagnosismdash A large species (SVL 364 to 411 mm in males n= 3) characterized by(1) snout mucronate in dorsal view acute in profile (2) mandible with two anteriorlarge odontoids on each side in adults (3) adults males with dorsum of body and limbsimmaculate uniformly pale yellow (4) dorsal skin granular (5) ventral skin cream andevenly granular lacking distinct rows of tubercles (6) a row of tubercles along lateral surfaceof forearm and tarsus (7) tubercle subarticular from second segment of finger IV singleand rounded shape (8) canthus rostralis immaculate (9) tympanum size correspondingto 66ndash71 of SVL (10) advertisement call consisting of a series of 7ndash31 composite notes(half pulsedpulsatilendashhalf harmonic)


Figure 1 Holotype of Phyllodytes magnus sp nov (MZUESC 18264 SVL 364 mm) (A) Dorsal and (B)ventral view of the body Scale bar= 10 mm

Full-size DOI 107717peerj8642fig-1

Description of holotypemdash An adult male in good state of preservation with a pieceof muscle removed from the right thigh for molecular analyses Raw measurements aredetailed in Table S1 Body robust head wider than long (HL 894 of HW HW 390 ofSVL HL 349 of SVL) snout mucronate in dorsal view and acute in profile (Figs 1 2Aand 2B) nostrils small elliptical directed anterolaterally nearer to the tip of snout thanto eye canthus rostralis slightly concave loreal region oblique slightly concave internarialdistance smaller than eyendashnostril distance (IND 675 of END) eyes large (ED 276 ofHL 246 of HW 875 of END) prominent situated laterally directed anterolaterallytympanum evident relatively medium (TD 71 of SVL and TD 205 of HL) nearlycircular separated from posterior border of eye by approximately the half of diameter oftympanum tympanum diameter smaller than eye to nostril distance (TD 65 of END)eye diameter (TD 743 of ED) interorbital distance (TD 313 of IOD) slightly smallerthan internarial distance (TD 963 of IND) its diameter larger than width of discs onthird finger (TDDF3 = 124) and of fourth toe (TD4TD = 136) tympanic annulusevident supratympanic fold developed covering dorsal edge of tympanum nearly straightposteriorly slightly curved downwards and extending until near the insertion of armvocal sac single subgular poorly developed vomerine teeth in two patches oblique andbarely separated positioned below and between choanae each side of mandible with twolarge anterior and smaller discrete and subequal odontoids pupil horizontal with distinctmeniscus

Arms slightly thicker than forearms (forearmarm= 945) lateral margin of forearmscrenate with four outer tubercles those closest to hand smaller and less evident handlarge (Fig 2C) 316 of SVL slightly crenate laterally fingers in the following order oflength IltIIsim=IVltIII subarticular tubercles rounded the second of the fourth finger single


Figure 2 Holotype of Phyllodytes magnus sp nov (MZUESC 18264) (A) Dorsal view of the head (B)Lateral view of the head (left side) (C) Plantar view of the left hand and (D) Plantar view of the left footScale bar= two mm


(Fig S1A) a few small supranumerary tubercles palmar tubercle well developed roundedthenar tubercle large elliptical adhesive discs developed first fingerrsquos disc smaller thanthe others diameter of disc of third finger equivalent to 600 of eye diameter fingersfringed webbing formula I ndashII 2 ndash3+ III 3 ndash212 IV

Hind limbs long tibia slightly longer than thigh (THL 956 of TBL) sum of tibiaand thigh lengths 967 of SVL a row of evident tubercles along lateral surface oftarsus tarsus length smaller than foot length (TAL 651 of FL) foot length smallerthan thigh and tibia lengths (FL 849 of THL FL 811 of TBL) plantar surface withsupernumerary tubercles subarticular tubercles rounded inner metatarsal tubercle welldeveloped elongated projecting laterally outer metatarsal tubercle small and rounded


Figure 3 Phyllodytes magnus sp nov (A B C D) P maculosus (E) and P kautskyi (F) in lifePhyllodytes magnus sp nov holotype (MZUESC 18264) in (A) dorsal and (C) ventral view and paratype(MZUESC 18265) in (B) dorsal and (D) ventral view In (E) P maculosus and (F) P kautskyi


discs on toe I slightly smaller than those of other toes toes fringed webbing formula I 2ndash312 II 2minus ndash3 + III 2minus ndash3 + IV 3minus ndash112 V

Dorsal surface of body forearms and tibia very granular belly granular without distinctrows of tubercles throat anterior and posterior surfaces of thighs and ventral surface ofshanks smooth ventral surface of thighs granular with distinct round tubercles of which apair near thigh insertion is more prominent

Dorsum immaculate uniformly pale yellow ventral surface of body limbs and thighscream white iris yellow with black horizontal band projecting from pupilVariationmdash The measurements of the specimens from the type series are summarized inTable S1 Overall the type series is morphologically congruent with the holotype However


one paratype (MZUESC 18265) shows the following differences the snout outline is morerounded in dorsal and profile view because the apical tubercle is less developed and theupper jaw more protruding in life the dorsum the throat and the ventral surfaces of limbsare orange-yellow and the belly is golden-yellow (Figs 3B and 3D) webbing formula forthe left hand is I ndashII 2minus ndash3+ III 3 ndash2+ IV and for the left foot is I 2minus ndash3 II 112 ndash3minus III 112ndash3minus IV 212 ndash1 + V The juvenile (MZUSP 157526) has black spots on dorsum which areabsent on adults This suggests that this species passes through ontogenetic pattern changeComparisons with congenersmdash Phyllodytes magnus is promptly distinguished from mostof other congeners by the larger size of its males (364ndash411 mm vs 156ndash287 mm) exceptfor P maculosus (397ndash435 mm) and P kautskyi (380ndash420 mm) The single and roundedshape of the subarticular tubercle in the second segment of finger IV distinguishes Pmagnus from P kautskyi and P maculosus (tubercles elongated and bifid in these speciesndashFig S1) In addition the dorsal skin is granular in P magnus and smooth or shagreenedin P maculosus and P kautskyi (Fig S2)

The absence of dorsolateral dark stripes distinguishes Phyllodytes magnus from Pamadoi P kautskyi (Fig 3F) P luteolus P maculosus (Fig 3E) P melanomystax Ppraeceptor P punctatus P tuberculosus and Pwuchereri (all have a pair of dorsolateral darkstripes extending from the posterior corner of the eyes towards the inguinal region thoughthe length and density of the stripes varies according to the species) The immaculatedorsum of body and limbs distinguishes Phyllodytes magnus from P amadoi and Ppraeceptor (small irregular brown patches) P gyrinaethes (dorsal surfaces of body andlimbs with marbled pattern) P maculosus (cream colored dorsum with anastomosedbrown blotches) P melanomystax (black round spots in the dorsum not present inall individualspopulations) P punctatus and P tuberculosus (distinctive brown dots ondorsum) and P wuchereri (a pair of dorsolateral longitudinal white stripes and in someindividualspopulations dorsum of hind limbs and central area of dorsum of body mottledbrown)

Phyllodytes magnus like P gyrinaethes P kautskyi P maculosu s and P melanomystaxhas an evenly granular venter without the distinctive rows of ventral tubercles presentin P acuminatus P amadoi P brevirostris P edelmoi P luteolus P megatympanum Ppunctatus P praeceptor P tuberculosus and P wuchereri The immaculate canthus rostralisand snout also distinguishes P magnus from P melanomystax (thick dark brown stripe onthe snout and canthus rostralis) Furthermore the absence of highlighted color in groinalso distinguishes P magnus from P megatympanum (yellow groin) and P gryrinaethes(red groin) In addition the relative tympanum size corresponding to 67ndash71 of SVLdistinguishes P magnus from P megatympanum and P acuminatus (TD 773ndash816 ofSVL)Cranial osteologymdash Dermal roofing bones (Fig 4A) Skull is broad and relatively flatwider than long In dorsal view nasals paired trapezoidal overlying the olfactory region ofthe skull Sphenethmoid diamond-shaped fused anteriorly with posteromedial margins ofnasals and posteriorly with anterior margins of frontoparietals Frontoparietals presentpaired longer than wide wider anteriorly constricted medially The anterior terminus ofthe pterygoid appears at the first third of the orbit


Figure 4 Skull of Phyllodytes magnus sp nov (A - dorsal B - ventral and C - front view)=Adult (MZUSP 157524) and (D - front view)= juvenile (MZUSP 157526) Scale bar= two mmAbbreviations al p pm alary process of premaxilla angspl angulosplenial dent dentary exo exoccipitalfp frontoparietal max maxilla mmk mentomeckelian bone nas nasal opt f optic foramen pmpremaxilla pro prootic pro f prootic foramen prsph parasphenoid pt pterygoid qj quadratojugalsph sphenethmoid sq squamosal vom vomer vt vomerine teeth


Maxillary arch (Fig 4B) Premaxillae paired anteromedial longer than high with a seriesof 10 teeth at the anteriormost segments of each maxillary arch contacting laterally lowerpart of maxilla Alary process of premaxilla paired oriented dorsally well developed twicelonger than correspondent dentary process Maxilla paired longer than premaxillarieslonger than high higher anteriorly with a series of approximately 36 teeth decreasing insize posteriorly Quadratojugals are positioned posterior to the maxillae and articulateswith the angulosplenial

In ventral view (Fig 4C) paired vomers dentigerous and associated with the anteriormargins of the sphenethmoid in the palate The parasphenoid extends posteriorly as aT-shaped ornamented bone

Lower jaw (Fig 4B) with paired mentomeckelian bones dentaries and angulosplenialsMentomeckelian bone with a very developed fanglike odontoid oriented dorsally slightly


smaller than the allary process of premaxilla A secondwell developed odontoid occurs in thedentary posteriorly oriented no dentition between these two projections Angulosplenialwith a thin row of five odontoids similar to the correspondent part of maxilla Odontoidsare absent in the juvenile (Fig 4D)Tadpole descriptionmdash (Fig 5) Body depressed (wider than high) ovoid in all views withmaximum height and width at the middle third of the body body length corresponding toapproximately 361 of total length Snout uniformly rounded in all views Eyes dorsallypositioned and dorsolaterally directed eye diameter representing 134 of body lengthNarial apertures rounded anterolaterally directed closer to snout than the eyes internarialdistance representing 267 of body width Spiracle single sinistral at the middle thirdof the body length and below the midline of the body height directed posterodorsallywith a circular opening inner wall present as slight ridge fused to body Vent tube centralattached to ventral fin with opening facing the right side

Oral apparatus (Fig 5A) in anteroventral position non-emarginated surrounded bya single row of small marginal papillae with an anterior gap on the upper labia Labialtooth row formula (LTRF) is 2(2)6 A1 shorter than A2 P1 and P2 of equal size slightlylonger than P3ndashP6 Jaw sheaths pigmented with finely serrated edges Upper jaw sheatharc-shaped and lower jaw sheath U-shaped

Tail musculature height represents 50 of body height and tapers towards the tip ofthe tail myomeres slightly visible in lateral view and clearly visible in dorsal view along theentire length of tail Dorsal and ventral fins approximately equal in size dorsal fin beginson body and continues to the tip of the tail ventral fin begins at the end of the body andcontinues towards the tip of the tail which is slightly pointed The lateral line system is notvisible Measurements are provided in Table S2

In formalin the body is translucent with brown spots distributed on dorsum ventraland lateral regions The intestine is visible in all surfaces The tail is also translucent withbrown spots distributed over the surface

Variationmdash In stage 27 body length corresponding to 364 of total length eyediameter representing 12 of body length internarial distance representing 272 of bodywidth The LTRF is still not developed with only five posterior tooth rows the marginalpapillae appear in same quantity Tail musculature height represents 47 of body heightand tapers towards the tip of the tail The maximum tail height is reached in stage 27(Table S2)Tadpole comparisons with congenersmdash Nine of the 14 species of Phyllodytes havedescribed tadpoles (Table S3) Like all their congenerics tadpoles of P magnus have adepressed body (wider than high) Furthermore they have no constriction a conditionsimilar to P acuminatus P brevirostris P edelmoi P melanomystax and P wuchereriwhich contrast to P gyrinaethes which shows anterior and lateral body constrictions and Pluteolus P praeceptor and P tuberculosus which show lateral body constrictions The LTRFof P magnus is similar to P brevirostris and P edelmoi and differs from the remainingspecies that show a LTRF 2(2)3 or 2(2)4 The most differentiated LTRF appears tooccur in P praeceptor (LTRF 12) and in P gyrinaethes (LTRF 1(1)5) The single row ofmarginal papillae on the oral apparatus of P magnus is similar to P gyrinaethes P luteolus


Figure 5 Tadpole of Phyllodytes magnus sp nov Tadpole at Gosner stage 35 (MZUSP 157525) fromWenceslau Guimaratildees municipality State of Bahia Brazil (A) oral apparatus (B) lateral (C) dorsal and(D) ventral views Scale bar= one mm (A) and five mm (B C and D)


P melanomystax P tuberculosus and P wuchereri and differs from the remaining tadpolesthat show at least two posterior rows of marginal papillae

The dorsal fin of P magnus originating on body is similar to that of P brevirostris andP edelmoi and differs from P acuminatus P gyrinaethes P praeceptor and P wuchereri(origin at body-tail junction) and from P luteolus P melanomystax and P tuberculosus(origin at tail musculature) The ventral fin originating on body is similar to all species ofPhyllodytes except P melanomystax (at the tail musculature)


Table 1 Quantitative measurements of the advertisement call and of the putative agonistic call (lsquolsquocall type IIrsquorsquo) of Phyllodytes magnus sp novData is presented as meanplusmn standard deviation (minndashmax) See text for explanation of measured parameters ndash not measured see text for explana-tion n of advertisement calls (for the first three parameters) is 48 and 6 for MZUESC 18264 and MZUESC 18265 respectively n of notes of adver-tisement calls (for the other parameters) is 91 and 63 for MZUESC 18264 and MZUESC 18265 respectively For call type II n of calls is eight and nof notes is 24

MZUESC 18264 (holotype) MZUESC 18265 (paratype)

Acoustic parameters Advertisement call Call type II

Call duration (s) 595plusmn 074 (385ndash762) 960plusmn 127(754ndash1098) 092plusmn 080 (014ndash209)Call interval (s) 8561plusmn 5559 (5151ndash43025) ndash ndashNotes per call 933plusmn 100 (7ndash11) 2730plusmn 408 (20ndash31) 3plusmn 214 (1ndash6)Note duration (s) 0096plusmn 0012 (0055ndash0125) 0086plusmn 0012 (0056ndash0101) 0114plusmn 005 (0052ndash0241)Duration 90 (s) 0044plusmn 0005 (0027ndash0054) 0056plusmn 0012 (0021ndash0068) 0079plusmn 0041 (0028ndash0199)Interval between notes (s) 0615plusmn 0146 (0181ndash0960) 0278plusmn 0018 (0233ndash0344) 0281plusmn 0052 (0209ndash0379)Note emission rate (notes) 142plusmn 040 (094ndash361) 275plusmn 015 (229ndash316) 254plusmn 039 (185ndash338)Note shape 077plusmn 005 (067ndash092) 052plusmn 013 (026ndash081) 036plusmn 021 (001ndash073)Dominant frequency (kHz) 103plusmn 004 (086ndash129) 082plusmn 006 (078ndash086)

215plusmn 010 (185ndash237)112plusmn 053 (043ndash215)

Frequency 5 (kHz) 088plusmn 03 (078ndash095) 165plusmn 040 (073ndash189) 072plusmn 027 (047ndash189)Frequency 95 (kHz) 173plusmn 019 (151ndash276) 247plusmn 010 (220ndash263) 202plusmn 033 (151ndash250)Bandwidth 90 (kHz) 085plusmn 019 (069ndash181) 082plusmn 037 (052ndash168) 130plusmn 032 (047ndash189)

The spiracle position of P magnus at midbody in lower half is similar to those of Pacuminatus and P wuchereri but differs from P brevirostris P luteolus P melanomystaxand P tuberculosus (at midbody at midline) and from P edelmoi P praeceptor and Pgyrinaethes (at bodyrsquos last third in lower half) The eye dorsally positioned is similar toP acuminatus P brevirostris P luteolus P tuberculosus and P wuchereri in contrast to Pedelmoi P praeceptor and P melanomystax (dorso-laterally) and P gyrinaethes (laterally)Bioacoustics resultsmdashQuantitative measurements of call parameters and comparisonsare summarized in Tables 1 and 2 Like most Phyllodytes species the advertisement callof P magnus is composed by a series of notes of the same type (Fig 6) Calls are emittedinfrequently separated by up to seven minutes of silence (Table 1) The note begins witha series of well-spaced low-amplitude pulses which gradually raise in amplitude andemission rate until fusing into the main body of the note (Fig 6) In the second half of thenote it changes into a sparse-harmonic sound with at least four harmonics and ascendingfrequency modulation (Fig 6) The fundamental frequency is also the dominant (Table 1)The amplitude is ordinarily ascendant along the note length reaching its maximum in thesecond part near the note ending (note shape always higher than 05 Table 1)

The advertisement call of the paratype MZUESC 18265 has some differences incomparison to the holotypersquos call (Table 1) It has more notes per call and higher noteemission rate It also has two discernible parts but in this case the first part is much moreconspicuous and it has a faster rise of amplitude which is marked by intense modulationsthat give a lsquolsquonoisyrsquorsquo (pulsatile) aspect to the waveform and produces many sidebands in thespectrogram (multiples of sim04 kHz) (Fig 6) The transition from the first to the secondpart of the note is well marked by a constriction in the waveform ie an abrupt decay


Table 2 Acoustic parameters from the advertisement calls of Phyllodytes species When available data is presented as meanplusmn standard deviation (minndashmax) Whenthe dominant frequency varies between two or more bands the values of the different bands are separated by lsquolsquorsquorsquo A sole em dash (mdash) means data is not available or notapplicable Two adjacent bars () indicates an upward modulation from the frequency at the left to the frequency at the right of the bars

Species Call duration (s) Inter-calls interval (s) Notes per call Note duration (s) Inter-notes interval (s) Dominant frequency(kHz)

1st harmonic (kHz) PresentsHarmonics

Reference

Phyllodytes magnussp nov

636plusmn 141 (385ndash1098) 8561plusmn 5559(5151ndash43025) 1133plusmn 592 (7ndash31) 0092plusmn 0013 (0055ndash0125) 0470plusmn 0200(0181ndash0960) 147plusmn 056(078ndash237) 051plusmn 005 (039ndash060) 108plusmn 004 (095ndash116)

Yes This paper

P amadoi 341plusmn 028(299ndash411) 534plusmn 85(41ndash68) 145plusmn 1(13ndash17) 0043plusmn 0021(0008ndash0119) 0204plusmn 002(0137ndash0285) 376plusmn 040(241ndash431) mdash No Voumlroumls Dias amp Soleacute (2017)

P acuminatus mdash 1899plusmn 1118 (603ndash534) 1ndash4 010plusmn 003(003ndash017) 035plusmn 005 (027ndash047) 214plusmn 009 (207ndash233) 428plusmn 015 (405ndash457)

214plusmn 009 (207ndash233) Yes Campos et al (2014)

P edelmoi 52plusmn 044 (428ndash573) 4827plusmn 833 (3811ndash6483) 2646plusmn 23 (22ndash29) 01plusmn 0003 (004ndash016) mdash 284plusmn 016(149ndash332) mdash No Lima Lingnau amp Skuk (2008)

P gyrinaethes 17plusmn 03(13ndash23) 524plusmn 257 (212ndash887) 49plusmn 06(4ndash6) 004plusmn 001 (002ndash007) 04plusmn 003(03ndash05) 275plusmn 016(253ndash309) mdash No Roberto amp Aacutevila (2013)

P kautskyi 355plusmn 019 4666plusmn 1145 21 0085plusmn 0012 0066ndash0120 137(087 181) 137(087 181) Yes Simon amp Gasparini (2003)

P luteolus 5 mdash 8ndash15 0125 mdash 2ndash6 mdash No Weygoldt (1981)

P megatympanum 591plusmn 456 (32ndash2363) 309plusmn 1031 (696ndash4758) 1337plusmn 256 (10ndash19) 0092plusmn 008 (0009ndash0245) 0305plusmn 01(01ndash062) 398plusmn 0136 (356ndash412) 198 Yes Marciano-Jr Lantyer-Silva amp Soleacute (2017)

P melanomystax 007plusmn 004 2881plusmn 1248 (1118ndash5429) 1 007plusmn 004 2881plusmn 1248 (1118ndash5429) 139plusmn 005 311plusmn 025 139plusmn 005 Yes Nunes Santiago amp Juncaacute (2007)

P praeceptor 534plusmn 153(302ndash941) 485plusmn 212(295ndash992) 839plusmn 155(6ndash12) 0295plusmn 0075(0141ndash0538) 0389plusmn 0102(0236ndash1057) 305plusmn 012(293ndash327) mdash No Orrico Dias amp Marciano-Jr (2018)

P tuberculosus 672plusmn 173 (465ndash935) mdash 1860plusmn 336 (14ndash23) 0167plusmn 0047 (0068ndash0246) 0214plusmn 0048 (0067ndash0357) 246plusmn 045 (168ndash327) mdash No Juncaacute et al (2012)

P wuchereri 28ndash68 134plusmn 10 (120ndash143) 10ndash21 005ndash032 009ndash021 112ndash146 267ndash353 mdash No Cruz Marciano amp Napoli (2014) andMagalhatildees Juncaacute amp Garda (2015)

NotesThese values separated by en dash are not minimum and maximum in the statistical sense instead they represent approximately the minimum and maximum of a frequency bandwidth

Dias

etal(2020)PeerJDO

I107717peerj86421527

Figure 6 Advertisement call of Phyllodytes magnus sp nov Graphic representations of the advertise-ment call of the paratype MZUESC 18265 (AndashD) and of the holotype MZUESC 18264 (EndashH) of Phyl-lodytes magnus sp nov A and E waveform of an entire call B and F waveform and spectrogram of onenote vertical arrow in waveform in B indicates the constriction of the waveform (see text for explanation)horizontal arrows in the spectrogram indicate the harmonics vertical dashed lines in the spectrograms in-dicate the position of the spectrogram slices C and G spectrogram slice of the first part of the note (in-dicated by the left vertical line) in B and F respectively arrows in C indicate the sidebands multiples ofsim04 kHz (see text for explanation) D and H spectrogram slice of the second part of the note (indicatedby the right vertical line) in B and F respectively arrows indicate the harmonics multiples ofsim107 kHz(see text for explanation) Spectrographic views settings window type Hann window size 512 samples 3dB filter bandwidth 124 Hz time grid overlap 90 time grid size 51 samples frequency grid DFT size512 samples frequency grid spacing 861 Hz Call voucher ndashFNJV 40997 (holotype) and FNJV 41382-41384 (MZUESC 18265)


followed by a subsequent rise of amplitude (Fig 6) The note shape is way more variablethan that of the holotype (Table 1) because the maximum amplitude is reached sometimesnear the beginning of note and sometimes near its ending

We believe the differences between the calls of the two recorded individuals are due totheir different levels of motivation or due to geographic variation Further studies shouldaddress the variation in advertisement calls of this species

A second type of call was emitted exclusively in response to our imitations suggestingan agonistic function This call is composed by a single note or a series of up to sixnotes with decreasing duration The first notes (lsquolsquoA notesrsquorsquo) have conspicuous secondaryamplitude modulation giving a lsquolsquomultipulsedrsquorsquo fashion to the waveform with up to fiveamplitude peaks (lsquolsquopulsesrsquorsquo) (Fig S3) The spectrogram of this type of note has harmonicsand fast A-shaped frequency modulations (Fig S3) The last notes of the series (lsquolsquoB notesrsquorsquo)resemble the notes of the advertisement call This type of call was emitted with much lower


amplitude only about one tenth of the maximum amplitude of the advertisement call Theduration of note of call type II is slightly longer than the duration of advertisement callnote but regarding the dominant frequency there is no great difference between the twotypes of call (Table 1)Bioacoustic comparisons with congenersmdash The advertisement call of Phyllodytes magnusis unique in the genus by having composite notes with two components of approximatelysame duration the first pulsedpulsatile and the second harmonic (Table 2) Though Pmelanomystax also presents composite notes its harmonic component is much longer thanthe noisy (pulsatile) component which is present both in the beginning and in the end of thenote (Fig S4) The other species calls have either exclusively pulsedpulsatile (P amadoiP edelmoi P gyrinaethes P luteolus P tuberculosus P praeceptor and P wuchereri) orharmonic (P acuminatus P kautskyi P megatympanum) notes

Furthermore the dominant frequency in Phyllodytes magnus (078ndash237 kHz) is lowerthan inP amadoiP gyrinaethesP megatympanum andP praeceptor (combined dominantfrequencies 241ndash431 kHz) as well as the frequency of the first harmonic in P magnus(039ndash116 kHz) is lower than in P acuminatus P megatympanum and P melanomystax(combined first harmonic frequencies 139ndash233 kHz) The number of notes per call in Pmagnus (7ndash31 notes) is higher than in P acuminatus (1ndash4 notes) P gyrinaethes (4ndash6 notes)and P melanomystax (single-note call) The advertisement call of Phyllodytes magnus canbe distinguished from P kautskyi by lacking a downward frequency modulation at the endof the note (marked in P kautskyi - see Simon amp Gasparini 2003) and by a longer intervalbetween the notes (P magnus = 0181ndash0960 s versus P kaustskyi = 0066ndash0120 s)Molecular resultsmdash Our results recovered Phyllodytes as a well-supported monophyleticgroup (Fig 7) in a polytomy together with Tepuihyla Osteopilus and Osteocephalus +Dryaderces The relationships of the main clades recovered within Lophyohylini receivedlow support as well as within Phyllodytes The new species was recovered as sister to otheranalyzed species of Phyllodytes (Fig 7) Values of genetic distance between Phyllodytesmagnus and the other analyzed species of the genus range between 64 to 102 beinglowest compared to P luteolus (Table S4)Cytogenetic resultsmdash We analyzed 35 somatic metaphases and defined the diploidnumber of 2n= 22 chromosomes and NF = 44 (Fig 8A) Chromosome pairs 1 7 8 910 and 11 are metacentric pairs 2 3 5 and 6 submetacentric and pair 4 subtelocentric Cbanding identified a small amount of constitutive heterochromatin as conspicuous blockspositioned pericentrically in all chromosomes The NORs are euchromatic (Fig 8B) Theimpregnation with silver nitrate showed single NORs placed terminally in the long arms ofpair 2 The number and position of NORs sites were confirmed after FISH with 18S rDNAprobe (Fig 8C Fig 8D)Geographic Distribution and Natural historymdash Phyllodytes magnus is known from threelocalities from the Atlantic Rainforest of Bahia (Fig S5) Calls of this species have also beenheard in the municipalities of Almadina Ilheacuteus Igrapiuacutena and Camacan state of Bahiabut as no vouchers or recordings were obtained these records need to be confirmed

Although one specimen of Phyllodytes magnus was in a ground bromeliad most callingmales were in canopy bromeliads At Parque Estadual da Serra do Conduru Phyllodytes


Figure 7 Phylogenetic relationship of genus Phyllodytes through 16Smitochondrial rRNA fragmentgene (791 bp) Bayesian posterior probabilities values are indicated close to the branches


Figure 8 Karyotype of Phyllodytes magnus sp nov Giemsa staining (A) and C-banding (B)Highlighted are the NOR-bearing chromosomes after silver nitrate staining (C) and FISH with 18SrDNA probe (D)


magnus is syntopic with four congeners (P maculosus P megatympanum P melanomystaxand P praeceptor) although apparently less abundant than its relatives In an area of 52ha we heard only five males in a one-night search All were calling from epiphytic giantbromeliads of the genus Hohenbergia with diameter superior to 150 m and at heightsbetween 310 m and 1130 m (x = 772 m plusmn 356 m) We found a male of Phyllodytesmelanomystax in the same bromeliad where we collected the paratype MZUESC 18265though in a different axil

DISCUSSIONPhyllodytes magnus can be readily distinguished from their congeners by morphological(large size and immaculate dorsum) acoustical (advertisement call with 7 to 31 notesamplitude modulated notes with the first part pulsedpulsatile and the second partsparse-harmonic) and molecular characteristics (gt6 genetic distance from all analyzedcongeners)


We remark for the first time the presence of composite notes (ie a note composed by aharmonic part plus a pulsedpulsatile part) in the advertisement call of a Phyllodytes speciesWhen comparing the calls of congeners we found that this feature occurs in Phyllodytesmelanomystax as well This can be observed in the waveform as an intense amplitudemodulated (noisy) section and in the spectrogram as a dense and broad range of sidebandsboth in the beginning and in the end of the note while the midnote is comprised by a lessnoisy waveform and harmonics in the spectrogram (Fig S4) We believe that what NunesSantiago amp Juncaacute (2007) interpreted as being artifacts in the call of P melanomystax mayactually represent these natural components We recommend that further call descriptionsshould pay attention to this feature as it seems to be an important character and can bemore widespread in the genus as well as in other frogs (eg Hyla gratiosa see Gerhardt1981)

Although there are proposals to allocate Phyllodytes species into groups based on colorpattern (Peixoto Caramaschi amp Freire 2003 Caramaschi Peixoto amp Rodrigues 2004) orbioacoustic characteristics (Roberto amp Aacutevila 2013) it has only been possible to test theseproposals very recently within a more comprehensive phylogenetic context and none ofthem have been recovered as monophyletic (Blotto et al 2020) Another question thathangs on Phyllodytes is itrsquos relationship with Phytotriades Most of the studies did notrecover this genus as sister taxon (eg Jowers Downie amp Cohen 2008 Wiens et al 2010Pyron amp Wiens 2011 Pyron 2014 Ron et al 2016 Jetz amp Pyron 2018) although otherauthors obtained this result (Moen ampWiens 2009 Duellman Marion amp Hedges 2016)Even when we increase the number of analyzed species of Phyllodytes (previously therewere only two species of this genus with sequences available for analysis) we did notrecover these genera as close relatives The same result was obtained in a phylogeneticstudy contemplating a larger sampling of mitochondrial and nuclear genes from morethan 96 of the species of the Lophyohylini tribe and all recognized Phyllodytes speciesincluding P magnus as P sp 2 (Blotto et al 2020)

Phyllodytes magnus presents 2n= 22 chromosomes and single NORs placed in theterminal region of the long arms of pair 2 the same condition described to P luteolusand P edelmoi (Gruber Haddad amp Kasahara 2012) the only two species cytogeneticallystudied However P magnus has a different macrochromosome arrangement The mostfrequent chromosome number in Hylidae is 2n= 24 however variations of this diploidnumber are not rare for this family (Baldissera Jr Oliveira amp Kasahara 1993 Catroli ampKasahara 2009) Gruber Haddad amp Kasahara (2012) proposed that the diploid numberreduction observed in Phyllodytes should be considered as a synapomorphy for thegenus a result of a chromosomal fusion involving the nucleolar chromosomes Ourdata confirmed this proposal and the few differences in the macro - (pairs 4 and 6) andmicrochromosome constitution (C-banding pattern) between these species are probablyresults of non-robertsonian rearrangements Too few is known about cytogenetic of speciesof Phyllodytes This lack of knowledge does not allow a more robust discussion about thepatterns of chromosomal evolution in the genus


CONCLUSIONSAlmost 60 (nine species) of the known diversity of Phyllodytes can be found in the AtlanticForest of southern Bahia the region with the highest concentration of representatives ofthis genus Over half of these species are endemic to the state P wuchereri P praeceptor Pmegatympanum P amadoi and P magnus the latter three are known from a few localitiesor only from the type locality (Cruz Marciano amp Napoli 2014 Marciano-Jr Lantyer-Silvaamp Soleacute 2017 Voumlroumls Dias amp Soleacute 2017 this study) The high diversity of Phyllodytes in thispart of the central corridor of the Atlantic Forest (Carnaval et al 2009) is not unique Othergenera like Adelophryne (Fouquet et al 2012 Lourenco-de Moraes et al 2018) Adenomera(Fouquet et al 2014) Gastrotheca (Teixeira-Jr et al 2012) or the gymnophthalmid lizardgenus Leposoma (Rodrigues et al 2013) to cite a few have their highest species diversity inthis part of the biome The high diversity of Phyllodytes and endemism brings support tothis pattern and highlights the evolutionary importance of this area Furthermore togetherwith the scarce available biological information and habitat specificity (bromeliads) itmakes the Atlantic Forest of the state of Bahia a priority area for research and conservationof Phyllodytes

ACKNOWLEDGEMENTSWe thank Carlos Augusto Souza-Costa and Joedison dos Santos Rocha for their supportin the field and Ana Paula Barbosa for her support in lab work We are also grateful to thelsquolsquoGrupo Ambientalista da Bahiarsquorsquo - GAMBA for lodging and logistic support in Serra daJiboacuteia We also thank Rogeacuterio Miranda Ribeiro and the staff from SEMA for the permitsto work at Estacatildeo Ecoloacutegica Wenceslau Guimaratildees (2011-020194) We thank RodrigoBarbosa Ferreira Luciana Fusinatto and one anonymous referee for their careful readingof the manuscript and suggestions

ADDITIONAL INFORMATION AND DECLARATIONS

FundingScholarships were provided by the Coordenacatildeo de Aperfeicoamento de Pessoal de NiacutevelSuperior and Conselho Nacional de Desenvolvimento Cientiacutefico e Tecnoloacutegico (CNPqndashProject 4068992017-7 process 1673872017-0 and 1551982018-1) to Iuri Ribeiro DiasScholarships were provided by Fundacatildeo de Amparo aacute Pesquisa do Estado da Bahia(FAPESB No BOL17672013) and by CAPES (masters degree scholarship between 2016ndash2017) to Gabriel Novaes e Fagundes Mirco Soleacute was funded by a lsquolsquoBolsa de Produtividadeem Pesquisarsquorsquo (CNPqndash3049992015ndash6) a lsquolsquoUniversalrsquorsquo project (CNPqndash4499302014ndash9)and a lsquolsquoPROTAXrsquorsquo project (CNPqndash4406152015ndash1) Fundacatildeo O Boticaacuterio de Protecatildeoagrave Natureza (0991_20132) funded this study Miguel Trefaut Rodrigues received fundingfrom the Fundacatildeo de Amparo agrave Pesquisa do Estado de Satildeo Paulo (project 201150146-6)and CNPq Antonio Mollo Neto received funding from Fundacatildeo de Amparo agrave Pesquisado Estado de Satildeo Paulo (project 201715025-0) There was no additional external funding


received for this study The funders had no role in study design data collection and analysisdecision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsCoordenacatildeo de Aperfeicoamento de Pessoal de Niacutevel Superior and Conselho Nacionalde Desenvolvimento Cientiacutefico e Tecnoloacutegico (CNPqndashProject 4068992017-7 Process1673872017-0 and 1551982018-1)Fundacatildeo de Amparo aacute Pesquisa do Estado da Bahia No BOL17672013CAPES (masters degree scholarship between 2016ndash2017)Bolsa de Produtividade em Pesquisarsquorsquo (CNPqndash3049992015ndash6)A lsquolsquoUniversalrsquorsquo project (CNPqndash4499302014ndash9)A lsquolsquoPROTAXrsquorsquo project (CNPqndash4406152015ndash1)Fundacatildeo O Boticaacuterio de Protecatildeo agrave Natureza (0991_20132)Fundacatildeo de Amparo agrave Pesquisa do Estado de Satildeo Paulo project 201150146-6 project201715025-0

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Iuri R Dias Gabriel Novaes-e-Fagundes Antonio Mollo Neto and Caroline Garciaconceived and designed the experiments performed the experiments analyzed the dataprepared figures andor tables authored or reviewed drafts of the paper and approvedthe final draftbull Juliana Zina Renato Sousa Recoder Francisco Dal Vechio Miguel Trefaut Rodriguesand Mirco Soleacute conceived and designed the experiments authored or reviewed drafts ofthe paper and approved the final draft

Animal EthicsThe following information was supplied relating to ethical approvals (ie approving bodyand any reference numbers)

This research was approved by the ethics committee on the use of animals (CEUA-UESC00212) Specimens collected for this manuscript were obtained under IBAMA 12920-1and ICMBIO 13708-1 and 35068 permits

DNA DepositionThe following information was supplied regarding the deposition of DNA sequences

New sequences of Phyllodytes magnus sp nov (MZUESC 18264 and 18265) and Pkautskyi (MBML 8818) are available at GenBank MN648397 to MN648399

Raw sequence data is available in the Supplemental File

Data AvailabilityThe following information was supplied regarding data availability


All recordings are available at the Fonoteca Neotropical Jacques Vielliard (httpswww2ibunicampbrfnjv) FNJV 40997 41382-41384 and in the Supplemental Files

Additional data is available at MorphosourcehttpswwwmorphosourceorgDetailMediaDetailShowmedia_id47746

New Species RegistrationThe following information was supplied regarding the registration of a newly describedspecies

Publication LSID urnlsidzoobankorgpubFD2CACD2-F59F-4B75-947D-B576701424D9

Phyllodytes magnus LSID urnlsidzoobankorgactD8DF9024-57C1-413B-9D05-B0CF05B7929C

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj8642supplemental-information

REFERENCESAltig R McDiarmid RW 1999 Body plan development and morphology In Mc-

Diarmid RW Altig R eds Tadpoles the biology of anuran larvae Chicago TheUniversity of Chicago Press 24ndash51

Baldissera Jr FA Oliveira PSL Kasahara S 1993 Cytogenetics of four BrazilianHyla species (Amphibia ndashAnura) and description of a case with a supernumerarychromosome Brazilian Journal of Genetics 16335ndash345

Beeman K 1998 Digital signal analysis editing and synthesis In Hopp SL Owren MJEvans CS eds Animal acoustic communication sound analysis and research methodsBerlin Springer Verlag 59ndash103

Blotto BL Lyra ML CardosoMCS Rodrigues MT Dias IR Marciano-Jr E VechioFD Orrico VGD Brandatildeo RA Assis CL Lantyer-Silva ASF RutherfordMGGagliardi-Urrutia G Soleacute M Baldo D Nunes I Cajade R Torres A Grant TJungfer KH Silva HR Haddad CFB Faivovich J 2020 he phylogeny of the Casque-headed Treefrogs (Hylidae Hylinae Lophyohylini) Cladistics Epub ahead of print2020 27 March DOI 101111cla12409

BokermannWCA 1966 O gecircnero PhyllodytesWagler 1830 (Anura Hylidae) Anais daAcademia Brasileira de Ciecircncias 38335ndash344

Campos TF LimaMG Nascimento AC Santos EM 2014 Larval morphology andadvertisement call of Phyllodytes acuminatus Bokermann 1966 (Anura Hylidae)from northeastern Brazil Zootaxa 377993ndash100 DOI 1011646zootaxa3779110

Caramaschi U Peixoto OL Rodrigues MT 2004 Revalidation and redescription ofPhyllodytes wuchereri (Peters 1873) (Amphibia Anura Hylidae) Arquivos do MuseuNacional Nova Seacuterie Zoologia 62185ndash191


Carnaval AC HickersonMJ Haddad CFB Rodrigues MT Moritz C 2009 Stability pre-dicts genetic diversity in the Brazilian Atlantic Forest hotspot Science 323785ndash789DOI 101126science1166955

Catroli GF Kasahara S 2009 Cytogenetic data on species of the family Hylidae (Am-phibia Anura) results and perspectives Publicatio UEPG Ciecircncias Bioloacutegicas e daSauacutede 1567ndash86

Charif RAWaack AM Strickman LM 2010 Raven Pro 14 Userrsquos Manual IthacaCornell Lab of Ornithology

Cruz CAG Feio RN CardosoMCS 2006 Description of a new species of PhyllodytesWagler 1830 (Amphibia Anura Hylidae) from the Atlantic Rain Forest of theState of Minas Gerais and Bahia Arquivos do Museu Nacional do Rio de Janeiro64321ndash324

Cruz D Marciano JRE Napoli MF 2014 Advertisement and courtship calls of Phyl-lodytes wuchereri (Peters 1873) (Anura Hylidae) Zootaxa 37747ndash100

CunhaMS Napoli MF 2016 Calling site selection by the bromeliad-dwelling treefrogPhyllodytes melanomystax (Amphibia Anura Hylidae) in a coastal sand dunehabitat Studies on Neotropical Fauna and Environment 51144ndash151

Darriba D Taboada GL Doallo R Posada D 2012 jModelTest 2 more models newheuristics and parallel computing Nature Methods 98772

Darst CR Cannatella DC 2004 Novel relationships among hyloid frogs inferred from12S and 16S mitochondrial DNA sequencesMolecular Phylogenetics and Evolution31462ndash475 DOI 101016jympev200309003

Dias IR Medeiros T NovaMV Soleacute M 2014 Amphibians of Serra Bonita southernBahia a new hotpoint within Brazilrsquos Atlantic Forest hotspot ZooKeys 449105ndash130DOI 103897zookeys4497494

Dias IR Mira-Mendes CV Soleacute M 2014 Rapid inventory of herpetofauna at the APA(environmental protection area) of the Lagoa Encantada and Rio Almada SouthernBahia Brazil Herpetology Notes 7627ndash637

DuellmanWE Marion AB Hedges SB 2016 Phylogenetics classification and bio-geography of the treefrogs (Amphibia Anura Arboranae) Zootaxa 41041ndash109DOI 1011646zootaxa410411

DuellmannWE Trueb L 1986 Biology of amphibians New York McGraw-HillFaivovich J Haddad CFB Garcia PCA Frost DR Campbell JA WheelerWC 2005 Sys-

tematic review of the frog family Hylidae with special reference to Hylinae phylo-genetic analysis and taxonomic revision Bulletin of the American Museum of NaturalHistory 2941ndash240 DOI 1012060003-0090(2005)294[0001SROTFF]20CO2

Ferreira RB Schineider JA Teixeira RL 2012 Diet fecundity and use of bromeliads byPhyllodytes luteolus (Anura Hylidae) in Southeastern Brazil Journal of Herpetology4619ndash24 DOI 10167009-040

Fouquet A Cassini CS Haddad CFB Pech N Rodrigues MT 2014 Species delimita-tion patterns of diversification and historical biogeography of the Neotropical froggenus Adenomera (Anura Leptodactylidae) Journal of Biogeography 41855ndash870DOI 101111jbi12250


Fouquet A Loebmann D Castroviejo-Fisher S Padial JM Orrico VGD Lyra MLRoberto IJ Kok PJR Haddad CFB Rodrigues MT 2012 From Amazonia to theAtlantic forest Molecular phylogeny of Phyzelaphryninae frogs reveals unexpecteddiversity and a striking biogeographic pattern emphasizing conservation challengesMolecular Phylogenetics and Evolution 65547ndash561 DOI 101016jympev201207012

Freitas MA 2015Herpetofauna no Nordeste Brasileiro Rio de Janeiro Technical BooksEditora

Freitas MA Abegg AD Dias IR Moraes EPF 2018Herpetofauna from Serra da Jiboacuteiaan Atlantic Rainforest remnant in the state of Bahia northeastern Brazil HerpetologyNotes 1159ndash72

Frost DR 2019 Amphibian species of the world an online reference ElectronicDatabase Version 60 New York USA American Museum of Natural HistoryAvailable at http researchamnhorgherpetologyamphibia indexhtml (accessed on20 August 2019)

Gerhardt HC 1981Mating call recognition in the Barking Treefrog (Hyla gratiosa)responses to synthetic calls and comparisons with the Green Treefrog (Hyla cinerea)Journal of Comparative Physiology 14417ndash25 DOI 101007BF00612793

Gosner KL 1960 A simplified table for staging anuran embryos and larvae with notes onidentification Herpetologica 16183ndash190

Gruber SL Haddad CF Kasahara S 2012 Karyotype analysis of seven species of thetribe Lophiohylini (Hylinae Hylidae Anura) with conventional and molecularcytogenetic techniques Comparative Cytogenetics 6409ndash423DOI 103897compcytogenv6i43945

Hatanaka T Galetti PM 2004Mapping of the 18S and 5S ribosomal RNA genes in thefish Prochilodus argenteus Agassiz 1829 (Characiformes Prochilodontidae) Genetica122239ndash244 DOI 101007s10709-004-2039-y

HeyerWR Rand AS Cruz CAG Peixoto OL Nelson CE 1990 Frogs of BoraceacuteiaArquivos de Zoologia 31231ndash410

Howell WM Black DA 1980 Controlled silver staining of nucleolar organizer regionswith a protective colloidal developer a 1-step method Experientia 361014ndash1015DOI 101007BF01953855

Huelsenbeck JP Ronquist F 2001MrBayes bayesian inference of phylogenetic treesBioinformatics 17754ndash755 DOI 101093bioinformatics178754

JetzW Pyron RA 2018 The interplay of past diversification and evolutionary isolationwith present imperilment across the amphibian tree of life Nature Ecology andEvolution 2850ndash858 DOI 101038s41559-018-0515-5

Jowers MJ Downie JR Cohen BL 2008 The Golden Tree Frog of Trinidad Phyllodytesauratus (Anura Hylidae) systematic and conservation status Studies on NeotropicalFauna and Environment 43181ndash188 DOI 10108001650520801965490

Juncaacute FA Napoli MF Cedraz J Nunes I 2012 Acoustic characteristics of the advertise-ment and territorial calls of Phyllodytes tuberculosus Bokermann 1966 (AmphibiaAnura Hylidae) Zootaxa 350687ndash88


Katoh K Toh H 2008 Recent developments in the MAFFT multiple sequence alignmentprogram Briefings in Bioinformatics 9276ndash285 DOI 101093bibbbn015

KingM Rofe R 1976 Karyotype variation in the Australian gekko Phyllodactylusmarmoratus (Gray) (Gekkonidae Reptilia) Chromosoma 5475ndash87DOI 101007BF00331835

Koumlhler J JansenM Rodriacuteguez A Kok PJR Toledo LF EmmrichM Glaw F HaddadCFB Roumldel MO Vences M 2017 The use of bioacoustics in anuran taxonomytheory terminology methods and recommendations for best practice Zootaxa42511ndash124 DOI 1011646zootaxa425111

Kok PJR KalamandeenM 2008 Introduction to the taxonomy of the amphibians ofKaieteur national Park Guyana Abc Taxa 51ndash278

Levan A Fredga K Sandberg AA 1964 Nomenclature for centromeric position onchromosomes Hereditas 52201ndash220

Lewis CT Short C 1891 A New Latin Dictionary Founded on Andrewsrsquo edition ofFreundrsquos Latin dictionary Revised enlarged and in great part rewritten New YorkOxford Clarendon Press

LimaMG Lingnau R Skuk GO 2008 The Advertisement call of Phyllodytes edelmoi(Anura Hylidae) South American Journal of Herpetology 3118ndash121DOI 1029941808-9798(2008)3[118TACOPE]20CO2

LittlejohnMJ 2001 Patterns of differentiation in temporal properties of acoustic signalsof anurans In Ryan M ed Anuran communication Washington and LondonSmithsonian Institution Press 102ndash120

Lourenco-de Moraes R Dias IR Mira-Mendes CV Oliveira RM Barth A Ruas DSVences M Soleacute M Bastos RP 2018 Diversity of miniaturized frogs of the genusAdelophryne (Anura Eleutherodactylidae) A new species from the Atlantic Forestof northeast Brazil PLOS ONE 139e0201781

Magalhatildees FDM Juncaacute FA Garda AA 2015 Tadpole and vocalisations of Phyllodyteswuchereri (Anura Hylidae) from Bahia Brazil Salamandra 5183ndash90

Marciano-Jr E Lantyer-Silva AS Soleacute M 2017 A new species of PhyllodytesWagler1830 (Anura Hylidae) from the Atlantic Forest of southern Bahia Brazil Zootaxa4238135ndash142 DOI 1011646zootaxa4238111

Miller MA PfeifferW Schwartz T 2010 Creating the CIPRES Science Gateway forinference of large phylogenetic trees In Proceedings of the Gateway ComputingEnvironments Workshop (GCE) New Orleans 1ndash8

Mira-Mendes CB Ruas DS Oliveira RM Castro IM Dias IR Baumgarten JE JuncaacuteFA Soleacute M 2018 Amphibians of the Reserva Ecoloacutegica Michelin a high diversitysite in the lowland Atlantic Forest of southern Bahia Brazil ZooKeys 7531ndash21DOI 103897zookeys75321438

Moen DSWiens JJ 2009 Phylogenetic evidence for competitively driven diver-gence body-size evolution in caribbean treefrogs (Hylidae Osteopilus) Evolution63195ndash214 DOI 101111j1558-5646200800538x


Nunes I Santiago RS Juncaacute FA 2007 Advertisement calls of four hylid frogs from thestate of Bahia northeastern Brazil (Amphibia Anura Hylidae) South American Jour-nal of Herpetology 289ndash96 DOI 1029941808-9798(2007)2[89ACOFHF]20CO2

Orrico VGD Dias IR Marciano-Jr E 2018 Another new species of Phyllodytes (AnuraHylidae) from the Atlantic Forest of northeastern Brazil Zootaxa 4407101ndash110DOI 1011646zootaxa440716

Palumbi S Martin A Romano S McMillanWO Stice L Grabowski G 2001 Thesimple foolrsquos guide to PCR Version 2 Honolulu University of Hawaii 47 Availableat http palumbistanfordeduSimpleFoolsMasterpdf

Peixoto OL 1995 Associacatildeo de anuros a bromeliaacuteceas na Mata Atlacircntica RevistaUniversidade Rural Seacuterie Ciecircncias da Vida 1775ndash83

Peixoto OL Caramaschi U Freire EMX 2003 Two new species of Phyllodytes (AnuraHylidae) from the state of Alagoas northeastern Brazil Herpetologica 59235ndash246DOI 1016550018-0831(2003)059[0235TNSOPA]20CO2

Peixoto OL Cruz CAG 1988 Descricatildeo de duas espeacutecies novas do gecircnero PhyllodytesWagler (Amphibia Anura Hylidae) Revista Brasileira de Biologia 48265ndash272

Pinkel D Straume T Gray JW 1986 Cytogenetic analysis using quantitative highsensitivity fluorescence hybridization Proceedings of the National Academy ofSciences of the United States of America 832934ndash2938 DOI 101073pnas8392934

Pyron RA 2014 Biogeographic analysis reveals ancient continental vicarianceand recent oceanic dispersal in amphibians Systematic Biology 63779ndash797DOI 101093sysbiosyu042

Pyron RAWiens JJ 2011 A large-scale phylogeny of Amphibia including over 2800species and a revised classification of extant frogs salamanders and caeciliansMolecular Phylogenetics and Evolution 61543ndash583 DOI 101016jympev201106012

Rambaut A Drummond AJ Xie D Baele G SuchardMA 2018 Posterior summarisa-tion in Bayesian phylogenetics using Tracer 17 Systematic Biology 67(5)901ndash904

Roberto IJ Aacutevila RW 2013 The advertisement call of Phyllodytes gyrinaethesPeixoto Caramaschi amp Freire 2003 (Anura Hylidae) Zootaxa 3669193ndash196DOI 1011646Zootaxa3669213

Rodrigues MT Teixeira M Recoder RS Vechio F Damasceno R Pellegrino KCM2013 A new species of Leposoma (Squamata Gymnophthalmidae) with four fingersfrom the Atlantic Forest central corridor in Bahia Brazil Zootaxa 3635459ndash475DOI 1011646zootaxa363547

Ron SR Venegas PJ Ortega-Andrade HM Gagliardi-Urrutia G Salerno PE 2016Systematics of Ecnomiohyla tuberculosa with the description of a new species andcomments on the taxonomy of Trachycephalus typhonius (Anura Hylidae) Zookeys630115ndash154 DOI 103897zookeys6309298

Savage JM HeyerWR 1967 Variation and distribution in the tree frog genus Phyllome-dusa in Costa Rica central America Beitrage zur Neotropischen Fauna 5111ndash131DOI 10108001650526709360400

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Simon JE Gasparini JL 2003 Descricatildeo da vocalizacatildeo de Phyllodytes kautskyi Peixoto eCruz 1988 (Amphibia Anura Hylidae) Boletim do Museu de Biologia Mello Leitatildeo1647ndash54

Sumner AT 1972 A simple technique for demonstrating centromeric heterocromatioExperimental Cell Research 75304ndash306 DOI 1010160014-4827(72)90558-7

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 molecu-lar evolutionary genetics analysis version 60Molecular Biology and Evolution30122725ndash2729

Teixeira-Jr M Vechio FDal Recoder RS Carnaval ACOQ Strangas M DamascenoRP SenaMA Rodrigues MT 2012 Two new species of marsupial tree-frogs genusGastrotheca Fitzinger 1843 (Anura Hemiphractidae) from the Brazilian AtlanticForest Zootaxa 34371ndash23 DOI 1011646zootaxa343711

Trueb L 1973 Bones frogs and evolution In Vial JL ed Evolutionary biology ofthe anurans Contemporary research on major problems Columbia University ofMissouri Press 65ndash132

Voumlroumls J Dias IR Soleacute M 2017 A new species of Phyllodytes (Anura Hylidae)from the Atlantic Rainforest of Southern Bahia Brazil Zootaxa 4337584ndash594DOI 1011646zootaxa433749

Weygoldt P 1981 Beobachtungen zur Fortpflazungsbiologie von Phyllodytes luteolus(Wied 1824) im Terrarium (AmphibiaSalientia Hylidae) Salamandra 171ndash11

Wiens JJ Kuczynski CA Hua X Moen DS 2010 An expanded phylogeny of treefrogs(Hylidae) based on nuclear and mitochondrial sequence dataMolecular Phylogenet-ics and Evolution 55871ndash882 DOI 101016jympev201003013


ecology and phylogeny (Marciano-Jr Lantyer-Silva amp Soleacute 2017) Fortunately this scenariois changing due to the greater attention that the genus has received recently with half ofthe species within the genus (ie seven species) having been described in the last 15 years(Frost 2019)

The state of Bahia is a diversity hotspot for Phyllodytes with nine of the 14 known species(Orrico Dias amp Marciano-Jr 2018) Notwithstanding these numbers may underestimatethe actual number of species as inventories carried out in coastal areas of the south ofthe state indicate difficulties in assigning species names to collected individuals (Dias etal 2014 Dias Mira-Mendes amp Soleacute 2014 Mira-Mendes et al 2018) suggesting that thediversity within the genus may be even larger This assumption has been corroboratedby the description of three new endemic species from southern Bahia in the last threeyears (Marciano-Jr Lantyer-Silva amp Soleacute 2017 Voumlroumls Dias amp Soleacute 2017 Orrico Dias ampMarciano-Jr 2018)

During recent field trips to the Atlantic Forest of southern Bahia we collected specimensof Phyllodytes (adults and tadpoles) from both terrestrial and epiphytic bromeliads Acloser examination revealed that these specimens correspond to an undescribed speciesHerein we describe this new species based on morphology of adults and tadpolesbioacoustics cytogenetic and molecular evidence In addition we compile and discussavailable information on bioacoustic features and tadpole morphology of the genus

MATERIALS amp METHODSMorphological dataWe analyzed specimens housed at herpetological collections of Museu de Zoologia daUniversidade Estadual de Santa Cruz Ilheacuteus state of Bahia Brazil (MZUESC) Museu deHistoacuteriaNatural de Jequieacute ColecatildeoHerpetoloacutegica Jequieacute state of Bahia Brazil (MHNJCH)andMuseu de Zoologia da Universidade de Satildeo Paulo state of Satildeo Paulo Brazil (MZUSP)Examined specimens are listed in Appendix 1 For comparisons with Phyllodytes brevirostrisP edelmoi and P gyrinaethes we used data available in the literature (Peixoto amp Cruz 1988Peixoto Caramaschi amp Freire 2003) Specimens collected for this workwere obtained underIBAMA 12920-1 and ICMBIO 13708-1 and 35068 permits This research was approvedby the ethics committee on the use of animals (CEUA-UESC 00212)

We took the following measurements of adult specimens SVL (snout-vent length) HL(head length) HW (head width) IND (internarial distance) END (eye-nostril distance)ED (eye diameter) IOD (interorbital distance) TD (tympanum diameter) THL (thighlength) TBL (tibia length) TAL (tarsus length) FL (foot length) HAL (hand length) DF3(width of disc on finger III) and 4TD (toe IV disc diameter) We measured SVL HL HWTHL TL and FL with calipers to the nearest 002 mm The remaining measurements weremadewith calipers under a stereomicroscopeMeasurements followedKok amp Kalamandeen(2008) with the exception of IOD that was measured between the anterior corners of theeyes in order to enhance repeatability Snout profile terminology followed Heyer et al(1990) texture of dorsal skin is described according to Kok amp Kalamandeen (2008) andwebbing formula notation followed Savage amp Heyer (1967) and Savage amp Heyer (1997)














































































Reference



Yes This paper














Dias

etal(2020)PeerJDO

I107717peerj86421527








































































































































































































Reference



Yes This paper














Dias

etal(2020)PeerJDO

I107717peerj86421527




























































































































a new large canopy-dwelling species of phyllodytes wagler ... · in the forest canopy, hampering...

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