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Recent genetic studies identified three deep genetic lin-eageswith up to . divergence (Kimura -parameter
distance) in the mitochondrial COI gene between sam-ples o P.caryophyllaceusrom Costa Rica, central Panama,and eastern Panama,respectively (C et al. ,P-S et al. ). C et al. () con-templated apossible co-occurrence o at least three candi-date species thatwere concealed under the name P. caryo-phyllaceusat El Cop (C etal. ). Pristimantiscaryophyllaceus is indeed an old lineage, whichoriginat-ed in South America about million years ago andsub-sequently evolved and expanded its range into CentralAmericaprior to the closure o the Isthmus o Panama(P-S et al. ). As aconsequence, the diver-gence o different lineages was triggered bythe dynamicgeological history in the region and the rise o theIsthmuso Panama, which ultimately resulted in variousisolationprocesses over time.
Ecological inormation on Pristimantis caryophylla-ceus (sensulato) (D , M , H
, S , L et al. ) is mostly available onpopulations rom westernPanama and eastern Costa Rica,whereas there is only littleinormation on the natural histo-ry o eastern Panamanian orColombian populations (M- ). Herein, we contribute new inormationby de-scribing or the first time the advertisem*nt call o P.caryo-phyllaceusrom eastern Panama, and providing additionaldata onits ecology, biogeography and morphology in anintegrative taxonomicapproach to potential species deline-ation within the P.caryophyllaceuscomplex in Panama.
Materials and methods
Fieldwork was carried out in the mountain ranges oDarin,Jingurud, Maj, Pirre, San Blas, and Sapo in east-
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ern Panama, and the abasar and alamanca mountainranges inwestern Panama (Fig. ). We evaluated the abun-
dance o members o the Pristimantis caryophyllaceus com-plex byopportunistic search or rogs in the lea litter andundergrowth alongtrails. Search transect lengths were cal-culated with the trackingunction o a Garmin GPSmapCSx. All georeerences were recorded in theWGS datum ormat. Maps and transects were created and calcu-latedusing ArcGIS (ESRI ). Collected specimenswere euthanised with ,fixed with ml o ormalin() in l o ethanol (), and subsequentlystored inethanol (). All figures incorporated herein have been
digitally improved and combined using Adobe PhotoshopCS. Forcandidate species and their delimitation, we ol-low the integrativeconcept o V et al. ().
Molecular laboratory and phylogenetic inerences
MtDNA was extracted rom resh muscle or liver tissue.Temitochondrial S mtDNA was amplified using a Mas-
tercycler pro S (Eppendor, Hamburg, Germany) by per-orming aninitial denaturation or min at C ollowedby steps with denaturationor s at C, annealingor s at C, and elongation or . min at C.Finalelongation proceeded or min at C. For the nuclearRAG(Recombination Activating Gene ) we used cycle: min at C; cycles: sat C, s at C, minat C; cycle: min at C. Te reaction mix consistedol mtDNA template, . l Reaction Buffer x (Peq-
Gold), l . mM dNPs, . l (containing . units)aq Polymerase(PeqLab), . l HO, l mM MgCl
,
and or S l per primer (containing pmol, orward:L CGCCGACAAAAACA; reverse:
were ound to be variable and sites parsimony-inorm-ative. Onlyten samples o combined mitochondrial genes
and the nuclear RAG gene were obtained (excluding out-groups),consisting o bp, o which sites were vari-able and wereparsimony-inormative. Using MEGA( et al.), we computed uncorrectedpairwisegenetic distances or COI and S both separately andcom-bined. For each gene and or the combined-gene data set,weconducted Maximum Likelihood (ML) analyses, usingtheamura--Parameter, with , bootstrap replicates.Prior to model-basedphylogenetic inerences, JModeltest.. (P ) was used under thecorrected Akaike
Inormation Criterion (AICc) to select the substitutionmodel orthe Bayesian analysis. We ran a Bayesian phylo-genetic analysis inMrBayes .. (H R- ) or ,, generations with our deaultchains,sampling every thgeneration and subsequentlydiscarding as burn-in.o test species delimitation inthe case o P. educatoris P.caryophyllaceus, we appliedtwo different methods. First, weconducted a statisticalparsimony network analysis with gapsconsidered as a fifh
character state (only or COI) in CS v. (C et al.). In order toconnect all haplotypes we set the connec-tion limit to steps.Second, we used the Automatic Bar-code Gap Discovery (ABGD)algorithm (P etal. ) under the ollowing settings: steps = ,distance =Kimura -parameter model with atransversion/transitionratio o ., and the setting or the minimumrelative gapwidth (X) varied at values between and .. TeMOUsphylogenetic relationships and divergence times were esti-
mated or the mtDNAs S and COI, and the nDNA RAG( individuals and, bp), using the program BEAS.. (D R ), with a relaxedclock,allowing substitution rates to vary according to an uncor
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en as mean standard deviation and range in parenthe-sis(Supplementary ab. S). Specimens were deposited
in the Museo Herpetolgico de Chiriqu (MHCH) at theUniversidadAutnoma de Chiriqu, Panama, and the Sen-ckenberg Research Instituteand Nature Museum (SMF),Frankurt, Germany. Morphological data osimilar Pristi-mantisspecies used or comparisons were taken romtherespective original descriptions.
Te ollowing morphometric measurements were taken(withabbreviations): snoutvent length (SVL); head length(HL), diagonallyrom angle o jaw to tip o snout; headwidth (HW), between angles ojaws; interorbital distance
(IOD); eye length (EL), rom anterior to posterior edgeoexternally accessible eye; eye to nostril distance (END),romanterior edge o eye to posterior corner o nostril; in-ternarialdistance (IND), between centres o nostrils; ore-arm length (FAL),rom proximal edge o palmar tubercleto outer edge o flexed elbow;hand length (HAL), romproximal edge o palmar tubercle to tip othird finger; tibialength (L), as the distance rom the knee to thedistal endo the tibia; oot length (FL), rom proximal edge oout-
er metatarsal tubercle to tip o ourth toe; width o thirdfinger(FW), at penultimate phalanx just anterior to disk;width o disk othird finger (FD), at greatest width; widtho ourth toe (W), atpenultimate phalanx just anteriorto the disk; width o disk o ourthtoe (D), at greatestwidth; and tympanum diameter (YMP), measuredhori-zontally, supposing an approximately circular tympanum.Wedetermined the sexes o adults by differences in SVL(males smallerthan emales), the presence o vocal slits in
males, and the presence o eggs in emales. Specimens witha SVL< mm were classified as juveniles and excludedrom themorphological analyses.
We sorted genetic lineages into Molecular Operational
analysed and the power spectra calculated in Raven Pro .(Window:Blackman, DF: samples, dB filter band-
width: Hz; Grid spacing . Hz; overlap .; Cet al. ). Lowest andhighest requencies were measured db below peak requency. erminologyused in the ad-vertisem*nt call description ollows D (). ocalculate call rates, we divided the average callduration by (sec.)plus the average o call intervals.
Colour variation
Generalized colouration summaries were derived directlyrom livespecimens or indirectly rom photos o live speci-mens. Within thestandardized colour descriptions o se-lected individuals, thecapitalized colours and colour codes(the latter in parentheses) arethose o S ().
Results
Based on the lineages resulting rom our phylogeneticanalysis(see section on molecular phylogenetics below),we assigned eachspecimen to one o three MOUs. TeMOUs were largely consistent with ageographical pat-tern and defined as ollows (Fig. ): MOU containsspeci-mens rom western Panama, MOU rom eastern Atlan-tic Panama(Daren and San Blas mountain ranges), andMOU rom eastern PacificPanama (Jingurud, Maj,Pirre and Sapo mountain ranges). aking intoaccount
the congruence between the phylogenetic results andthebiogeographical pattern, we assigned measured speci-mens totheir respective MOUs, with to MOU, to MOU and seven to MOU In theollowing we
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sample rom Donoso (MOU) with eight mutationalsteps between themin the S network and our in the COI.
Te samples rom Altos del Mara, Panama and Ro Gacho,Costa Rica,were connected to nine unsampled haplotypesin the S network, butwere not connected in the COInetwork; one sample rom apant, CostaRica, was con-nected to the samples rom Ro Changena and RoClari-to, Bocas del oro, with unsampled haplotypes betweenthem(only one o these samples was included or COI).In MOU, only thesamples rom El Cop were groupedin the same haplotype, in both S andCOI. In MOU,the samples rom the Maj and Jingurud mountainranges
(S and COI), as well as those rom the Cana field station(COI)were connected. Our ABGD analysis generated fivegroups or S with adivergence threshold o . with arelative width o the barcoding gap o. in the X-value.For COI, it produced eight groups, assuming an apriori in-traspecific divergence threshold o . with a relativegapwidth o . (X-value), whereas ten groups resulted whenboth geneswere combined (threshold o .). Our threeanalyses (S, COI, and bothgenes combined) lumped all
samples in one unit, with a priori intraspecific divergenceso .,., and ., respectively. With ABGD, allsamples rom MOU and almostall rom MOU or theCOI and S sequences (Supplementary Figs SS)weregrouped in their corresponding geographic MOU. TeS ABGDassigned all samples o MOU to one cluster,
but also included samples rom Maj that were placed inMOUaccording to our phylogenetic analyses. One sam-
ple rom El Cop (USNM ) did not nest within anycluster in the Sanalysis, but took a place within MOUwhen using COI and both genescombined.
According to our divergence time estimates, the MO-Us started todiverge rom other species o the subge-nus Hypodictyon C, (sensuHedges et al. ),which were present in Central America in theOligocene. Mya (with a credibility interval, CI, o .. Ma). Te crownage o the MOUs dates to . Mya(CI: .. Mya) during the Miocene, whenMOU
and MOU + MOU split; a second break between theancestors o MOUand MOU occurred . Mya, ol-lowed by several splitting processeswithin the respectiveMOUs between . and . Mya. When all geneswereused in the phylogenetic analysis, the ML analysis yield-ed aconsensus tree that was topologically congruent withthedivergence-time tree. However, in the divergence-timeanalyses, wheneach MOU was treated as a monophylet-ic group, the Maj samples werenested within MOU,
showing a divergence-time o . Mya. Tis is not sup-ported (p = .,see Supplementary Fig. S), however, al-though when the MOUs werenot supposed to be mono-phyletic, the posterior probability or thedivergence-timeanalysis was not supported either (p = ., Fig. andSup-plementary Fig. S).
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Morphometrics
In Supplementary able S, we present the morphometricvariablesused to evaluate the differences between lineag-es within thePristimantis caryophyllaceus complex. Ourmorphological analysisrevealed differences between thethree MOUs (Fig. ). A DiscriminantFunction Analysis(DFA) classified . o the specimens according tooura priori groupings (. MOU; . MOU; .
MOU). Te principal morphological variables contrib-
uting to the grouping were ) YMP/SVL, ) D/W,) IOD, ) FD; thefirst unction is: DS = . * YMP/SVL + . D/W + . IOD + -. FD; andthesecond unction is DS = -. YMP/SVL + . D/W + . IOD + . FD. Tespecimensincluded in MOU are usually larger than those romMOU andMOU, respectively. Likewise, MOU andMOU seem to be more similar toeach other (MOU MOU: .) than either o them is to MOU (.
and ., respectively; see Figs + ).
able 1. amura 3-parameter distances among the Pristimantiscaryophyllaceus (includingP. educatoris)specimensused in thisstudy.MOU: MOU1 contains specimens rom western Panama, MOU2 romeastern Atlantic Panama (Daren and San Blas mountainranges), andMOU3 rom eastern Pacific Panama (Jingurud, Maj, Pirre and Sapomountain ranges).
GenesAverage
genetic distance
Distance within MOUs Distance between MOUs
MOU1 MOU2 MOU3 MOU1-MOU2 MOU1-MOU3 MOU2-MOU3
16S 6.5 4.02 1.49 6.1 7.15 7.53 8.9
COI 14.98 6.86 7.23 9.91 19.96 18.13 15.75
16S + COI 12.43 5.4 5.53 9.12 16.05 14.8 13.76
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Our analysis o differences between specimens romwest o Fortuna,alias Pristimantis caryophyllaceus, andeast o Fortuna, alias P.educatorissensu R et al.(),showed differences between each other,both according togeography (MANOVA, Pillais trace = ., F
. = .,
P < .) and based on the condition o subarticular tu-bercles(MANOVA, Pillais trace = ., F
. = ., P =
.). Te DFA classified specimens on the basis o geo-graphy romwestern and eastern Panama correctly, with. probability, into theirrespective groups (DFA = . FD/FW + . IOD/HW + . EL + -. FD +. D/W),and when using the condition o subar-
ticular tubercles, the DFA classified the two MOUs with.probability into their original groups (DFA = . EL + . D/W + -.SVL).
Colour variation
Pristimantis caryophyllaceus is one o the most poly-chromaticspecies in its genus. Te general dorsal colour-ation varied romyellow to reddish with various brown-ish tonalities, with orwithout black chevron marks onthe dorsum, and sometimes withsulphur-yellow spots on
Figure 4. Discriminant unction analysis o MOUs; see text ord til
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the dorsum and/or limbs (Figs ). Groin and posteriorthigh variedrom not contrasting in colour to yellow orred Pristimantiscaryophyllaceus also had a highly varia
Simultaneously, a third male was observed, calling rom adistanceo approximately m, but was not recorded. Tenext day (// see ab ) werecorded a ourth male
able 2. Spectral and temporal parameters o the advertisem*ntcall o Pristimantis caryophyllaceus. Te specimen MHCH 3022 wastheonly one collected, recordings 13 correspond to males that were notcaptured (see Vocalisation section or explanation). Averagesor theparameters are given in the Resume column.
Variables Males Resume
MHCH 3022 Recording 1 Recording 2 Recording 3
Length recording (min) 00:20:10 00:20:10 00:11:09 00:08:2600:59:55
Date 05-Dec-12 05-Dec-12 06-Dec-12 26-Aug-13
ime 19:12 19:12 17:40 19:18
SVL (mm) 22.50
emperature (C) 19.4 21.6 21.6 22 21.15
Humidity (%) 83 63 63 52.25Number o calls 1 2 3 1 7
Number o call intervals n/a 1 2 n/a 3
Number o pulses 6 14 23 8 51
Number o pulse intervals 5 12 20 7 44
Call duration (s) 0.04 0.04 0.04 0.05 0.0400.004(0.0350.046)
Call interval (s) 181.87 135.55 15126.74 (135.45181.87)
Call rate (call/min) 0.44 0.33 0.390.08 (0.330.44)
Pulses/call (s) 6.00 7.00 7.67 8.00 7.280.76 (68)
Pulses/duration (s) 0.003 0.003 0.004 0.003 3.34x10-30.7x10-3(2.0x10-34.0x10-3)
Pulses/interval (s) 0.003 0.003 0.002 0.003 2.41x10-30.75x10-3(1.0x10-34.0x10-3)
Lowest Freq (kHz) 2.48 2.30 2.24 2.41 2.320.11 (2.182.48)
Highest Freq (kHz) 3.25 3.69 3.16 3.31 3.340.25 (3.133.77)
Delta Freq (kHz) 0.76 1.38 0.92 0.90 1.030.26 (0.761.4)
Energy (dB) 85.30 82.90 88.13 107.60 89.019.52 (80.3107.60)
Max. Freq (kHz) 2.76 3.01 2.56 2.80 2.750.23 (2.433.17)
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Natural history notes
Frogs o the Pristimantis caryophyllaceuscomplex are pri-marilyinhabitants o pristine orest, but are also oundat the edges oorests. During seven nighttime transectsearches in eastern Panama(Jingurud, Pirre and Sapomountain ranges), we observed individuals(see Sup-plementary ab. S). Te relative abundance was .indv./ mtrail transect; most specimens were ound atheights between m abovethe ground perched on
items (one each): two crickets and one arachnid, with anaveragevolume o . + . mm.
Discussion
Our results demonstrate a high level o genetic diversityin thePristimantis caryophyllaceus complex, comprisingthree main lineageswithin its currently known distribu-tion (this study C et al PS
Figure 9. Oscillogram (top) and spectrogram (bottom) o theadvertisem*nt call o a Pristimantiscaryophyllaceus-complex-male,
recorded at Cerro Sapo (MHCH 3022).
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in their zones o contact and/or evaluating whether theseMOUsbehave/evolve as independent entities.
Te ABGD has grouped successully almost all sampleso MOU and MOUor both mitochondrial genes. Temost incongruent result obtained inthe species delimita-tion test (ABGD) was the placement o thesamples romthe Maj mountain range (MOU) with the samples romwesternPanama (MOU) in the same group (Supple-mentary Fig. S). Teover-splitting detected by the ABGDanalysis in MOU (SupplementaryFigs SS) is likelydue to the great genetic distances ound withinthat group,as reflecting the inclusion o specimens rom threediffer-
ent isolated regions (Maj mountain range, Jingurud-Sa-pomountains, and Pirre lowlands). In our analyses, theABGD lumped allMOUs with a low prior intraspeci-ic divergence (. or S, . or COI,and . bothgenes combined), compared to the maximum values o theapriori threshold or conspecific divergence that yieldeda primaryspecies hypothesis in the closely related Pristi-mantis museosus(.or S, . or COI and whenboth genes combined; C et al. ).
Earlier studies (R et al. ) and our genetic ana-lysis couldsupport splitting the Pristimantis caryophylla-ceus complex into P.caryophyllaceus andP. educatoris, oreven into three speciesaccording to the MOUs recoveredin this study. We also detectedmorphological, yet non-sig-nificant, variation among MOUs thatshould not be dis-regarded until larger samples can be tested toevaluate apotential clinal variation that orms a continuum withthevariability detected within the MOUs (M ),corresponding tointerbreeding between the MOUs (e.g.,at El Cope), or corresponds toa geographical pattern. Forexample, the specimens rom Ro Changenaand Ro Clari-to (MOU) share the same haplotype but the Ro Clari
ure ,but a P. ridens-like rog and not a member o theP.caryophyllaceuscomplex in Figure . Our comprehen-sive photographicdata and colour descriptions confirm awide colour variation withinand between MOUs. Speci-mens with different eye colours rom thesame populationsdid not exhibit other morphological differences.Tereore,eye colouration has no diagnostic value, and we areconsid-ering specimens with different eye colourationsoccurringwithin a MOU as conspecific and/or members o thesamelineage, even though we have no genetic data to corrobo-ratethis (no samples with red eyes were sequenced). Ret al. ()mentioned the presence o yellowish wart-like
spots on the dorsum as being characteristic oPristimantiseducatorisrom eastern Panama. However, we alsooundspecimens with such yellowish marks in extreme westernPanama,which otherwise agree with characters o P. caryo-phyllaceus inhaving rounded pads and disk covers, andlow and roundedsubarticular tubercles.
Te most common polymorphic traits (F ,M ) ound in populations odifferent erraranaspecies (G , H B , S
E ) relate to characteristics o the dorsal skintexture, colourpattern, and iris colour (S ). InP. caryophyllaceus, polymorphismis most prominent in thedorsal colour pattern, iris colour anddigital disk. It seemsto be a balanced polymorphism (F ), inasmuchasit appears to be maintained in the different MOUs. Col-ourpattern in errarana rogs could be inherited by a sim-ple Mendeliangenetic mechanism (G , , S- et al. , ON B ), perhaps main-tained bythe heterogeneity o colour compositions andshapes predominant inthe habitat, selective orces exert-ed by visually guided predators,and fitness-related traits(H B S E
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to acilitate improved climbing in an arboreal environmentandwould prove beneficial also or semiarboreal activitiesin erraranaspecies (S , H et al. ).Although the exact unction o the digitaldisks in P. caryo-phyllaceus has not been investigated yet, onepossibilitycould be that the differences in their widths reflectdiffer-ent degrees o arboreality among MOUs. However, thedifferentdegrees o arboreality appear to be not the mainreason or thisvariation, since all specimens were ound inthe understorey between. and . m above the groundon leaves and branches.
Te polymorphism ound in P. caryophyllaceus (sensu
lato) appears to be complex and it is not properly reflect-ed bya two-species taxonomy sensu R et al. () orthree species as ound inthis study; i.e., by splitting the spe-cies into P.caryophyllaceusor western Panama (MOU)and two P. educatoristaxa oreastern Panama (Caribbean-MOU and Pacific-MOU). Based on themolecular in-ormation available as o now, all three MOUs co-occuratEl Cop. However, there is no additional evidence or im-plementinga three-species taxonomy, and sensu V et
al. (), we would need at least one diagnostic morpho-logicaldifference, a character trait that is o low intraspeci-icvariability and o high value, to discriminate among taxa(here:MOUs). Te morphological differences betweenthese MOUs areinsufficient to support them as distinctspecies, however.Furthermore, there are currently no bio-acoustic data rom El Cop totest or putative interspecificdifferences.
R et al. () used the SVL to differentiate betweenP.caryophyllaceusand P. educatoris. However, rather thana cleardifference in SVL, our data suggest a smooth clinaltransition o theSVL rom east to west (Fig. ). Te condi-tion o a projectingsubarticular tubercle as suggested as a
morphological variation within the particular MOUs andthemorphological similarities between the MOUs, weound no additionalevidence to prove the three MOUsas respective Confirmed CandidateSpecies (V et al.). Moreover, at this point, we have no evidence oras-signing P. educatoristo either MOU or MOU, and ourdata do notsupport the biogeographical concept suggestedby R et al. (). Fornow, we suppose to see the threeMOUs within the P.caryophyllaceus-educatoris-complexas geographically and geneticallydistinct lineages in ac-cordance with the definition o a DeepConspecific Lineageby V et al. () with a small contact zonearound
El Cop. Consequently, we reject P. educatoris as a validspeciesand place it in the synonymy o P. caryophyllaceus.
Te phylogenetic history o the MOUs studied here islinked to thecomplex biogeography o the Isthmus o Pan-ama and northwestern SouthAmerica, more precisely othe Choco Block (D-C ab, C et al., ),ormed by the Maj-Baud Arc (Maj, Sapo,Jingurud and Pirre massis) andthe Dabeiba Arc (Dar-ien and San Blas massis). Pristimantiscaryophyllaceushas
originated in South America (D , Het al. , P-S et al. ), and itsdispersal toCentral America is consistent with the recenthypothesis oan earlier ormation o the Isthmus o Panama (F etal. , Met al. ), dating well beore . Mya aswas previously thought (C O , WR , S et al. , W et al. ). TeMOUs show a geographical pattern withalmost all MO-Us (except or the El Cop populations) beingdistributedin different geographic areas (Figs ). According toourresults, the split between MOU and MOUMOU( Mya) supports theproposed connection o the Isth-mus o Panama with South Americawhich would have a
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MOUs in the time divergence analysis. Tis would meanthat MOU romeastern Panama had available a consid-erable period o time toexpand into western Panama andmix with MOU there.
Even though we here present a lot o new inormationon thedistribution pattern o Pristimantis caryophyllaceusand itsvariation in morphology, genetics, colour pattern,as well asadvertisem*nt calls, it is apparently still notenough to clariy thetaxonomic status o the species. De-tailed molecular analyses atpopulation level, using nucle-ar markers such as microsatellites todetect gene flow andpast demographic bottlenecks, includingpopulations rom
central Panama (especially rom the Piedras-Pacora moun-tainrange) into the analysis o morphology, and statisti-cally supportedbioacoustics data rom various populationsover wide areas are stillneeded. Further studies should in-clude correlation analysesbetween geographic and geneticdistances to test whether theuncovered differences expressjust a clinal variation among thepopulations, and to evalu-ate the existence and role o previous andcurrent intro-gressions among the MOUs, particularly at El Cop.
Acknowledgements
Scientific permits or (SC/A--, SC/A--), (SC/A--), (SC/A--), andexport permits (SC/A--) and (SEX/A--) were provided by ANAM,Panama,and . Q (Cacique General del rea de Samb) rom theDespachodel Cacique Regional Comarca Ember-Wounaan,Panama. Special thanksgo to the indigenous people o the Em-bera rom Puerto Indio andPavarand, especially to D
B (Secretary o the Ember-Wounaan congress, Sam-b), and L C (Nokoo the Pavarando village) andhis amily who allowed us to enter theirautonomous territory
and FY), and the Chinese Academy o Science (No.KSCX-EW-Z-).
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Supplementary material
Additional inormation is available in the online version othis
article at http://www.salamandra-journal.com
4 Supplementary figures and 6 Supplementary tables:
Figure S1. Maximum likelihood consensus tree o the COImtDNA othe Pristimantis caryophyllaceuscomplex.
Figure S2. Maximum likelihood consensus tree o the 16S mtDNAothe Pristimantis caryophyllaceuscomplex.
Figure S3. Maximum likelihood consensus tree o mitochondrial(16S& COI mDNA) and nuclear (RAG1 DNA) genes combined
o the Pristimantis caryophyllaceuscomplex.Figure S4. Chronogramo the Pristimantis caryophyllaceuscom-plex based on Rag1, 16S andCOI genes, using P. cerasinus andP. cruentusas outgroups.
able S1. Morphological variables taken rom 78 specimens usedinthe analyses.
able S2. Mean genetic distances in the 16S mtDNA gene be-tweenthe Pristimantis caryophyllaceus samples used in the phylo-geneticanalysis.
able S3. Mean genetic distances in the COI mtDNA gene be-
tween the Pristimantis caryophyllaceus samplesused in thephylo-genetic analysis.
able S4. Mean genetic distances in the COI and 16S mtDNAgenescombined between the Pristimantis caryophyllaceus sam-ples used inthe phylogenetic analysis.
able S5. Details o sample and museum voucher numbers.
able S6. ransect details or Pristimantiscaryophyllaceus(MOU3).
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Supplementary material
B, A., A. H, G. K, K. M M. V: Phylogeny, shapes, colours, andphylogeography of frogs
related to Pristimantis caryophyllaceus
(Anura: errarana: Craugastoridae) in Panama. Salamandra, 50(3):155171.
Supplementary Figures and Supplementary ables.
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Supplementary Material to B et al. (2014) Salamandra 50(3):155171
Supplementary Table S1.Morphological variables taken from 78specimens used in the analyses. Number of specimens measured inparenthesis (see main text for explanation).
TraitMOTU1 MOTU2 MOTU3
Females (20) Males (33) Females (6) Males (1) Females (5) Males(13)
SVL 25.533.09 (21.9032.60) 20.131.87 (16.1022.70) 29.022.56(25.0031.90) 22.1 29.321.59 (27.2031.30) 22.541.83 (18.5025.20)
HW 9.891.23 (8.4013.00) 7.800.80 (6.309.40) 11.381.02(10.1012.60) 8.1 11.580.31 (11.1011.90) 8.870.70 (7.2010.00)HL 9.381.16 (8.1012.30) 7.640.78 (6.009.10) 11.271.02 (9.6012.40) 8.211.340.51 (10.7012.00) 8.820.91 (7.1010.10)
IND 2.030.28 (1.602.70) 1.650.15 (1.401.90) 2.380.16 (2.202.60)1.6 2.320.16 (2.102.50) 1.940.12 (1.802.10)
IOD 3.060.41 (2.503.90) 2.490.29 (2.003.00) 3.950.27 (3.804.50)2.9 3.940.40 (3.604.50) 3.060.34 (2.603.50)
TYMP 0.81 0.29 (0.501.70) 0.690.14 (0.501.00) 1.050.08(1.001.20) 0.8 1.160.23 (0.901.50) 1.010.19 (0.701.40)
EL 3.330.32 (2.904.10) 2.840.32 (2.203.40) 3.720.22 (3.404.00)3.5 3.940.36 (3.404.30) 3.280.38 (2.503.80)
END 3.270.50 (2.604.60) 2.610.35 (2.003.20) 4.280.44 (3.704.90)3.1 4.120.41 (3.504.50) 3.070.36 (2.503.60)
TL 15.031.72 (13.1019.00) 12.101.15 (10.0014.10) 17.471.01(15.6018.20) 12.8 17.020.97 (15.7018.00) 13.281.07 (10.7014.90)
FL 11.161.37 (9.4014.50) 8.851.08 (7.0010.90) 12.030.86(10.7013.00) 9.9 12.140.46 (11.4012.50) 9.921.05 (7.2011.10)
4TW 0.560.09 (0.500.90) 0.490.06 (0.400.60) 0.580.04 (0.500.60)0.4 0.580.04 (0.500.60) 0.480.07 (0.400.60)
4TD 1.010.19 (0.701.40) 0.890.19 (0.601.30) 1.250.14 (1.101.40)0.9 1.200.14 (1.001.40) 0.960.17 (0.601.20)
FAL 5.380.60 (4.506.80) 4.380.47 (3.205.10) 6.080.58 (5.607.20)4.6 6.680.50 (5.807.00) 4.950.52 (4.105.70)
HAL 6.901.12 (5.609.80) 5.510.70 (4.406.90) 7.530.57 (6.908.30)5.5 7.981.12 (7.209.90) 6.230.74 (4.407.10)
3FW 0.570.07 (0.500.70) 0.490.07 (0.400.60) 0.620.08 (0.500.70)0.5 0.600.07 (0.500.70) 0.480.07 (0.400.60)
3FD 1.130.25 (0.801.60) 0.930.20 (0.601.40) 1.400.25 (1.101.70)1.1 1.260.18 (1.101.50) 1.080.23 (0.601.40)
TYMP/SVL 0.030.01 (0.020.05) 0.030.01 (0.020.05) 0.040.01(0.030.04) 0.04 0.040.01 (0.030.05) 0.040.01 (0.040.06)
TL/SVL 0.590.03 (0.540.63) 0.600.03 (0.510.66) 0.600.03(0.560.64) 0.58 0.580.03 (0.540.62) 0.590.03 (0.54)0.66
FL/SVL 0.440.03 (0.390.48) 0.440.03 (0.380.49) 0.420.02(0.380.44) 0.45 0.350.17 (0.050.44) 0.440.03 (0.390.49)
HW/SVL 0.390.02 (0.360.42) 0.390.02 (0.360.44) 0.390.01(0.370.41) 0.37 0.400.02 (0.380.41) 0.390.02 (0.350.42)
HL/SVL 0.130.01 (0.110.14) 0.130.01 (0.110.16) 0.150.00(0.140.15) 0.14 0.140.02 (0.120.16) 0.140.01 (0.120.15)
END/SVL 0.130.01 (0.110.14) 0.130.01 (0.110.16) 0.150.00(0.140.15) 0.14 0.140.02 (0.120.16) 0.140.01 (0.120.15)
4TD/IVTW 1.810.23 (1.402.20) 1.810.29 (1.402.60) 2.140.20(1.832.33) 2.25 2.070.15 (2.002.33) 2.020.25 (1.502.50)
3FD/IIIFW 1.970.30 (1.332.67) 1.910.35 (1.33) 2.310.60(1.833.40) 2.2 2.110.26 (1.832.50) 2.300.53 (1.403.50)
IOD/HW 0.310.03 (0.270.38) 0.320.03 (0.280.38) 0.350.02(0.330.38) 0.36 0.340.03 (0.310.38) 0.350.04 (0.280.42)HL/HW0.950.04 (0.851.01) 0.980.05 (0.841.16) 0.990.04 (0.941.06) 1.010.980.02 (0.951.01) 0.990.06 (0.911.11)
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Supplementary Material to B et al. (2014) Salamandra 50(3):155171
Supplementary Table S2.Mean genetic distances in the 16S mtDNAgene between the Pristimantis caryophyllaceus samples used in thephylogenetic analysis (Tamura-3-parameter-distances).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1 USNM572335 {MOTU1}
2 USNM572331 {MOTU1} 0.0
3 AJC1138 {MOTU1} 2.8 2.84 UCR16434 {MOTU1} 4.3 4.3 3.3
5 USNM572330 {MOTU1} 0.0 0.0 2.8 4.3
6 SMF97035 {MOTU1} 6.3 6.3 5.2 3.8 6.3
7 SMF 97037 {MOTU1} 6.3 6.3 5.2 3.8 6.3 0.0
8 USNM572329 {MOTU1} 0.0 0.0 2.8 4.3 0.0 6.3 6.3
9 MVZ203810 {MOTU1} 4.8 4.8 3.8 0.5 4.8 4.3 4.3 4.8
10 SMF50938 {MOTU1} 1.4 1.4 2.3 3.8 1.4 5.8 5.8 1.4 4.3
11 MHCH3039 {MOTU2} 7.3 7.3 7.3 5.8 7.3 8.3 8.3 7.3 6.3 6.8
12 SMF50939 {MOTU2} 7.3 7.3 6.2 6.8 7.3 7.8 7.8 7.3 7.3 6.82.3
13 USNM572343 {MOTU2} 7.3 7.3 7.3 6.2 7.3 7.8 7.8 7.3 6.7 6.82.3 1.9
14 MVUP1925 {MOTU2} 7.3 7.3 7.3 6.2 7.3 7.8 7.8 7.3 6.7 6.8 2.31.9 0.0
15 SMF50943 {MOTU2} 7.8 7.8 7.8 6.7 7.8 8.3 8.3 7.8 7.3 7.3 1.91.4 0.5 0.5
16 SMF50933 {MOTU3} 4.7 4.7 5.7 4.3 4.7 7.3 7.3 4.7 4.8 5.2 6.37.8 7.8 7.8 8.3
17 MHCH3022 {MOTU3} 9.3 9.3 9.3 9.3 9.3 10.4 10.4 9.3 9.9 8.89.8 9.8 9.8 9.8 10.3 8.8
18 MHCH3017 {MOTU3} 5.2 5.2 6.2 4.8 5.2 7.8 7.8 5.2 5.3 5.7 6.88.3 8.3 8.3 8.8 0.5 9.3
19 SMF50936 {MOTU3} 9.8 9.8 9.9 8.8 9.8 9.9 9.9 9.8 9.4 10.411.5 11.4 11.4 11.4 12.0 8.8 2.3 9.3
20 MHCH3042 {MOTU3} 8.8 8.8 8.8 8.8 8.8 9.9 9.9 8.8 9.4 8.3 9.39.3 9.3 9.3 9.8 8.3 0.5 8.8 1.9
21 SMF50944 {MOTU3} 4.7 4.7 5.7 4.3 4.7 7.3 7.3 4.7 4.8 5.2 6.37.8 7.8 7.8 8.3 0.0 8.8 0.5 8.8 8.3
22 CH6367 {MOTU3} 7.8 7.8 6.7 7.8 7.8 7.8 7.8 7.8 8.3 7.2 8.37.2 8.2 8.2 8.8 7.7 6.2 8.3 7.8 5.8 7.7
23 SMF97033 {MOTU1} 3.8 3.8 4.8 4.8 3.8 7.8 7.8 3.8 5.3 4.3 6.36.8 6.8 6.8 7.3 3.8 9.9 4.3 10.4 9.3 3.8 8.8
24 USNM572338 {MOTU1} 4.8 4.8 4.7 4.7 4.8 6.7 6.7 4.8 5.2 5.35.7 6.3 6.2 6.2 6.8 4.7 10.9 5.2 11.4 10.4 4.7 9.3 3.8
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Supplementary Material to B et al. (2014) Salamandra 50(3):155171
Supplementary Table S3.Mean genetic distances in the COI mtDNAgene between the Pristimantis caryophyllaceus samples used in thephylogenetic analysis (Tamura-3-parameter-distances).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 USNM572331 {MOTU1} 0.0
2 USNM572330 {MOTU1} 0.0
3 USNM572329 {MOTU1} 0.0 0.0
4 SMF50938 {MOTU1} 0.9 0.9 0.9
5 USNM572335 {MOTU1} 0.0 0.0 0.0 0.9
6 UCR16434 {MOTU1} 16.4 16.4 16.4 15.9 16.4
7 AJC1138 {MOTU1} 8.4 8.4 8.4 8.2 8.4 16.9
8 MHCH3039 {MOTU2} 20.1 20.1 20.1 20.4 20.1 16.9 21.5
9 SMF50939 {MOTU2} 22.0 22.0 22.0 21.8 22.0 17.7 21.5 11.4
10 SMF50943 {MOTU2} 20.1 20.1 20.1 20.1 20.1 14.6 20.9 8.86.9
11 MVUP1925 {MOTU2} 19.8 19.8 19.8 19.8 19.8 16.2 21.2 9.8 8.04.4
12 USNM572343 {MOTU2} 20.1 20.1 20.1 20.1 20.1 16.4 20.9 10.18.2 4.6 0.2
13 SMF50933 {MOTU3} 19.7 19.7 19.7 20.5 19.7 15.5 20.5 16.6 17.316.8 17.0 17.3
14 MHCH3042 {MOTU3} 19.3 19.3 19.3 19.3 19.3 15.9 20.3 14.8 17.714.8 15.8 16.0 12.315 CH6367 {MOTU3} 17.0 17.0 17.0 17.5 17.0 14.119.5 13.9 16.0 13.7 14.6 14.8 11.6 11.5
16 USNM572338 {MOTU3} 17.2 17.2 17.2 17.2 17.2 13.5 17.2 15.817.7 14.4 15.0 15.3 10.5 10.3 11.1
17 MHCH3017 {MOTU3} 19.7 19.7 19.7 20.5 19.7 15.5 20.5 16.6 17.316.8 17.0 17.3 0.0 12.3 11.6 10.5
18 SMF50936 {MOTU3} 18.8 18.8 18.8 18.8 18.8 15.5 19.8 15.3 17.714.8 15.8 16.0 12.3 0.4 12.0 10.3 12.3
19 MHCH3019 {MOTU3} 17.2 17.2 17.2 17.7 17.2 14.3 19.7 13.7 15.713.4 14.3 14.6 11.8 11.8 0.2 11.3 11.8 12.2
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Supplementary Material to B et al. (2014) Salamandra 50(3):155171
Supplementary Table S4.Mean genetic distances in the COI and 16SmtDNA genes combined between the Pristimantiscaryophyllaceussamples used in the phylogenetic analysis.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 USNM572329 {MOTU1}
2 AJC1138 {MOTU1} 6.8
3 UCR16434 {MOTU1} 12.7 12.74 USNM572335 {MOTU1} 0.0 6.812.7
5 USNM572331 {MOTU1} 0.0 6.8 12.7 0.0
6 USNM572330 {MOTU1} 0.0 6.8 12.7 0.0 0.0
7 SMF50938 {MOTU1} 1.1 6.5 12.2 1.1 1.1 1.1
8 CH6367 {MOTU3} 14.2 15.6 12.3 14.2 14.2 14.2 14.4
9 MHCH3017 {MOTU3} 15.2 16.1 12.3 15.2 15.2 15.2 15.9 10.6
10 SMF50936 {MOTU3} 16.1 16.8 13.5 16.1 16.1 16.1 16.3 10.811.4
11 MHCH3042 {MOTU3} 16.1 16.9 13.8 16.1 16.1 16.1 16.0 9.8 11.20.8
12 SMF50933 {MOTU3} 15.1 15.9 12.1 15.1 15.1 15.1 15.7 10.5 0.111.2 11.1
13 USNM572338 {MOTU3} 13.4 13.4 10.9 13.4 13.4 13.4 13.6 10.68.9 10.6 10.3 8.8
14 MVUP1925 {MOTU2} 16.0 16.9 13.2 16.0 16.0 16.0 15.8 12.7 14.414.5 13.8 14.2 12.4
15 MHCH3039 {MOTU2} 16.2 17.1 13.6 16.2 16.2 16.2 16.2 12.2 13.614.2 13.2 13.5 12.8 7.6
16 SMF50943 {MOTU2} 16.4 16.9 12.2 16.4 16.4 16.4 16.2 12.2 14.414.0 13.4 14.3 12.1 3.2 6.7
17 USNM572343 {MOTU2} 16.2 16.7 13.4 16.2 16.2 16.2 16.0 12.914.6 14.6 14.0 14.4 12.6 0.1 7.8 3.4
18 SMF50939 {MOTU2} 17.5 16.8 14.4 17.5 17.5 17.5 17.1 13.3 14.615.8 15.2 14.4 14.2 6.2 8.7 5.3 6.3
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Supplementary Material to B et al. (2014) Salamandra 50(3):155171
Supplementary Table S5.Details of sample and museum vouchernumbers (where available), collecting localities, and GenBankaccession numbers for all samples used in this study.
Voucher Species Locality Province Country Genbank accessionnumber Coordinates elev. (m)
16S COI RAG1 N W
AJC1138 P. caryophyllaceus* Panama, corregimiento de Chame,Altos del Maria, ~7.5 km NEof El Valle de Anton, corregimiento,Chame
Panama Panama JN991435.1 JN991364 JQ025176 8.6330 -80.0770
CH6367 P. caryophyllaceus* Panama, Darin, Distrito de Pinogana,Cana, Laguna Darin Panama JN991436.1 JN991365 JQ025175 7.7220-77.6560MHCH3183 P. caryophyllaceus Fortuna/Westhang Pata de MachoChiriqu Panama 8.6710 -82.1967 1420
MHCH3184 P. caryophyllaceus Fortuna/Westhang (=western slope)Pata de Macho Chiriqu Panama 8.6710 -82.1967 1420
MHCH3185 P. caryophyllaceus Fortuna/Westhang (=western slope)Pata de Macho Chiriqu Panama 8.6775 -82.1980 1760
MHCH3186 P. caryophyllaceus La Nevera/Cerro Santiago Westhang(=western slope) Comarca Ngbe Bugl Panama 8.5011 -81.7694 1580
MHCH3187 P. caryophyllaceus La Nevera/Cerro Santiago Westhang(=western slope) Comarca Ngbe Bugl Panama 8.5010 -81.7691 1600
MHCH3188 P. caryophyllaceus La Nevera/Cerro Santiago Westhang(=western slope) Comarca Ngbe Bugl Panama 8.5010 -81.7691 1600
MHCH3189 P. caryophyllaceus Cerro Mariposa Veraguas Panama8.5145 -81.1207 880
MHCH3190 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5122 -81.1214 935
MHCH3191 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5001 -81.1170 1255
MHCH3192 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5001 -81.1173 1261
MHCH3193 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5111 -81.1214 916
MHCH3194 P. caryophyllaceus Fortuna Chiriqu Panama 8.6739-82.2188 1292MHCH3017 P. caryophyllaceus Serrania de Maj, Ambroya,Chepo Panam Panama KJ201960 KJ201949 KJ201970 8.8921 -78.5604911
MHCH3018 P. caryophyllaceus Serrania de Maj, Ambroya, ChepoPanam Panama 8.8916 -78.5617 886
MHCH3019 P. caryophyllaceus Panama, Darin, Distrito de Pinogana,Cana, Laguna Darin Panama KJ201961 KJ201950
MHCH3020 P. caryophyllaceus Ro Sambu, Serrana de Jingurudo,Samb. Darin Panama 7.6842 -78.0387 971
MHCH3021 P. caryophyllaceus Cerro Sapo, Garachin Darin Panama7.9762 -78.3626 1169
MHCH3022 P. caryophyllaceus Cerro Sapo, Garachin Darin PanamaKJ201952 KJ201942 7.9762 -78.3628 1168
MHCH3037 P. caryophyllaceus Ro Taintidu, Chucunaque WargandiPanama 9.0355 -78.0264 289
MHCH3038 P. caryophyllaceus Ro Taintidu, Chucunaque WargandiPanama 9.0593 -77.9842 553
MHCH3039 P. caryophyllaceus Ro Tuquesa, Embera-WaounaanEmbera-Wounaan Panama KJ201967 8.4800 -77.5194 859
MHCH3040 P. caryophyllaceus Ro Tuquesa, Embera-WaounaanEmbera-Wounaan Panama 8.4791 -77.5280 718
MHCH3041 P. caryophyllaceus Ro Tuquesa, Embera-WaounaanEmbera-Wounaan Panama 8.4791 -77.5280 718
MHCH3042 P. caryophyllaceus Ro Sambu, Serrana de Jingurudo,Samb. Embera-Wounaan Panama KJ201969 7.7640 -78.1006 655MHCH3043 P.caryophyllaceus Chucant ridge, ro Congo, Chepigana Darin Panama8.7977 -78.4623 1295
MHCH3044 P. caryophyllaceus Chucant ridge, ro Congo, ChepiganaDarin Panama 8.7965 -78.4630 1342
MHCH3045 P. caryophyllaceus Rancho Fro Field station, PinoganaDarin Panama 7.9595 -77.7037 1230
MHCH3046 P. caryophyllaceus Rancho Fro Field station, PinoganaDarin Panama 7.9595 -77.7037 1230
MHCH457 P. caryophyllaceus El pianista, Bocas del Toro, PanamBocas del Toro Panama 8.8714 -82.4159
MHCH523 P. caryophyllaceus Qda Arena, Fortuna, Chiriqu, PanamChiriqu Panama 8.7180 -82.2284 1074
MVUP1925 P. caryophyllaceus* Parque Nacional G. D. Omar TorrijosH., El Cope, Corregimientois Ola, Distrito La Pintada, Cocl
Cocl Panama FJ784473.1 FJ766776 8.6670 -80.5920 800
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Voucher Species Locality Province Country Genbank accessionnumber Coordinates elev. (m)
16S COI RAG1 N W
MVZ203810 P. caryophyllaceus* Costa Rica: Cartago, 2.5 km STapanti Bridge across Rio Grandede Orosi
Cartago CostaRica
EU186686.1 na
SMF 89976 P. caryophyllaceus Cerro Negro/PN Santa Fe VeraguasPanama 8.5706 -81.1043 800
SMF 89977 P. caryophyllaceus Cerro Negro/PN Santa Fe VeraguasPanama 8.5663 -81.0988 690
SMF 89978 P. caryophyllaceus Cerro Negro/PN Santa Fe VeraguasPanama 8.5706 -81.1043 800SMF 89979 P. caryophyllaceus CerroNegro/PN Santa Fe Veraguas Panama 8.5769 -81.0973 900
SMF 89980 P. caryophyllaceus Cerro Mariposa Veraguas Panama8.6757 -81.1228 1385
SMF 89981 P. caryophyllaceus I Brazo Mulaba Veraguas Panama8.5186 -81.1332 700
SMF 97027 P. caryophyllaceus La Nevera Panama 8.4996 -81.77101650
SMF 97028 P. caryophyllaceus Cerro Mariposa Veraguas Panama8.5145 -81.1207 880
SMF 97029 P. caryophyllaceus La Nevera/Cerro Santiago WesthangComarca Ngbe Bugl Panama 8.4953 -81.7673 1800
SMF 97030 P. caryophyllaceus Llano Tugri Comarca Ngbe BuglPanama 8.5082 -81.7162 1600
SMF 97031 P. caryophyllaceus Willi Mazu Comarca Ngbe Bugl Panama8.7885 -82.2016 799
SMF 97032 P. caryophyllaceus Willi Mazu Comarca Ngbe Bugl Panama8.7885 -82.2016 799
SMF 97033 P. caryophyllaceus Willi Mazu Comarca Ngbe Bugl PanamaKJ476734 8.7885 -82.2016 799
SMF 97034 P. caryophyllaceus Changena Trail/Oberes Camp Bocasdel Toro Panama 8.9505 -82.7094 1968
SMF 97035 P. caryophyllaceus Changena Trail/Oberes Camp Bocasdel Toro Panama KJ476733 8.9505 -82.7094 1968SMF 97036 P.caryophyllaceus Rio Clarito Bocas del Toro Panama 9.0090 -82.66441258
SMF 97037 P. caryophyllaceus Rio Clarito Bocas del Toro PanamaKJ476732 9.0090 -82.6644 1258
SMF 97039 P. caryophyllaceus Cerro Guayaba Chiriqu Panama 8.7657-82.2528 1565
SMF 97040 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5122 -81.1214 935
SMF 97041 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5061 -81.1196 1108
SMF 97042 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.4966 -81.1164 1356
SMF 97043 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.4997 -81.1168 1264
SMF 97044 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5000 -81.1170 1257
SMF 97045 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5000 -81.1170 1257
SMF 97046 P. caryophyllaceus Cerro Mariposa, Santa Fe VeraguasPanama 8.5024 -81.1191 1163
SMF 97047 P. caryophyllaceus Lost and Found/Fortuna ChiriquPanama 8.6757 -82.2128 1364
SMF 97048 P. caryophyllaceus Lost and Found/Fortuna ChiriquPanama 8.6773 -82.2103 1288SMF50931 P. caryophyllaceus Serrania deMaje, Ambroya, Chepo Panam Panama 8.8920 -78.5609 901
SMF50932 P. caryophyllaceus Serrania de Maje, Ambroya, ChepoPanam Panama 8.8920 -78.5609 901
SMF50933 P. caryophyllaceus Serrania de Maje, Ambroya, ChepoPanam Panama KJ201953 KJ201943 KJ201963 8.8919 -78.5608 911
SMF50934 P. caryophyllaceus Rancho Fro Field station, PinoganaDarin Panama 7.9891 -77.7073 1136
SMF50935 P. caryophyllaceus Ro Sambu, Serrana de Jingurudo,Samb. Darin Panama 7.6841 -78.0387 962
SMF50936 P. caryophyllaceus Ro Sambu, Serrana de Jingurudo,Samb. Darin Panama KJ201954 KJ201944 KJ201964 7.6837 -78.0384961
SMF50937 P. caryophyllaceus Ro Sambu, Serrana de Jingurudo,Samb. Darin Panama 7.6691 -78.0380 1133
SMF50938 P. caryophyllaceus Qda Valle Grande, Donoso Coln PanamaKJ201958 KJ201947 KJ201968 8.8216 -80.6632 211
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Voucher Species Locality Province Country Genbank accessionnumber Coordinates elev. (m)
16S COI RAG1 N W
SMF50939 P. caryophyllaceus Ro Taintidu, Chucunaque WargandiPanama KJ201962 KJ201951 KJ201971 9.0355 -78.0264 289
SMF50940 P. caryophyllaceus Ro Taintidu, Chucunaque WargandiPanama 9.0355 -78.0264 289
SMF50941 P. caryophyllaceus Serrana de San Blas Guna Yala Panama9.0611 -77.9797 340
SMF50942 P. caryophyllaceus Ro Sambu, Serrana de Jingurudo, SambEmbera-Wounaan Panama 7.7590 -78.0923 643
SMF50943 P. caryophyllaceus Ro Terable, El Llano, Chepo PanamaPanama KJ201965 9.2840 -78.9838 322
SMF50944 P. caryophyllaceus Chucant ridge, ro Congo, ChepiganaDarin Panama KJ201956 KJ201966 8.7977 -78.4623 1295
SMF50945 P. caryophyllaceus Chucant ridge, ro Congo, ChepiganaDarin Panama 8.7977 -78.4623 1295
SMF50946 P. caryophyllaceus Rancho Fro Field station, PinoganaDarin Panama 8.0168 -77.7297 133
SMF50947 P. caryophyllaceus Rancho Fro Field station, PinoganaDarin Panama 7.9595 -77.7037 1230
SMF85008 P. caryophyllaceus RF Fortuna Chiriqu Panama 8.7264-82.2615 1100
SMF85010 P. caryophyllaceus RF Fortuna Chiriqu Panama 8.7264-82.2615 1100
SMF85011 P. caryophyllaceus RF Fortuna Chiriqu Panama 8.7264-82.2615 1100
SMF85012 P. caryophyllaceus RF Fortuna Chiriqu Panama 8.7264-82.2615 1100
SMF85014 P. caryophyllaceus BP Palo seco, Los tucanes trailBocas del Toro Panama 8.7817 -82.2122 1120
SMF85015 P. caryophyllaceus Fortuna Town Chiriqu Panama 8.7313-82.2534 1300
SMF85016 P. caryophyllaceus La Nevera Comarca Ngbe Bugl Panama8.4996 -81.7710 1600
SMF85018 P. caryophyllaceus La Nevera Comarca Ngbe Bugl Panama8.4996 -81.7710 1600
UCR 16429 P. cerasinus* Vuelta de Queque, Rio Siquirres trail,Guayacan Limn CostaRica
JN991437 JN991366 JQ025177 10. 0400 -83.5500
UCR16434 P. caryophyllaceus* Costa Rica: San Jose, Rio Gacho,Los Juncos, Cascajal, CantonVazquez de Coronado
San Jos CostaRica
JN991434.1 JN991363 9.9800 -83.8400
UCR16447 P. cruentus* Tapant, Cantn, Paraiso CartagoCostaRica
JN991441 JN991370 JQ025179 9.6500 -83.8500 1200
USNM572329 P. caryophyllaceus* Parque Nacional G. D. OmarTorrijos H., El Cope, Corregimientois Ola, Distrito La Pintada,Cocle
Cocl Panama FJ784397.1 FJ766771 8.6670 -80.5920 800
USNM572330 P. caryophyllaceus* Parque Nacional G. D. OmarTorrijos H., El Cope, Corregimientois Ola, Distrito La Pintada,Cocle
Cocl Panama FJ784421.1 FJ766774 8.6670 -80.5920 800
USNM572331 P. caryophyllaceus* Parque Nacional G. D. OmarTorrijos H., El Cope, Corregimientois Ola, Distrito La Pintada,Cocle
Cocl Panama FJ784422.1 FJ766773 8.6670 -80.5920 800
USNM572335 P. caryophyllaceus* Parque Nacional G. D. OmarTorrijos H., El Cope, Corregimientois Ola, Distrito La Pintada,Cocle Cocl Panama FJ784589.1 FJ766770 8.6670 -80.5920 800
USNM572338 P. caryophyllaceus* Parque Nacional G. D. OmarTorrijos H., El Cope, Corregimientois Ola, Distrito La Pintada,Cocle
Cocl Panama FJ784491.1 FJ766775 8.6670 -80.5920 800
USNM572343 P. caryophyllaceus* Parque Nacional G. D. OmarTorrijos H., El Cope, Corregimientois Ola, Distrito La Pintada,Cocle
Cocl Panama FJ784375.1 FJ766772 8.6670 -80.5920 800
GK1452 P. caryophyllaceus RF Fortuna Chiriqu Panama 8.7264-82.2615 1100
GK1469 P. caryophyllaceus BP Palo seco Los tucanes Bocas delToro Panama 8.7817 -82.2122 1120
GK1595 P. caryophyllaceus La nevera Comarca Ngbe Bugl Panama8.4996 -81.7710 1600
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Supplementary Table S6.Transect details for Pristimantiscaryophyllaceus (MOTU3).
Locality Indiv. Date Length (m) Duration Climatic condition T(C) Humidity (%)Altitude Coordinates
(m) N W
Serrania de Pirre 8 10/08/2011 240 03:30:00 cloudy 22.3 76 11377.98845 77.7076
Serrania de Pirre 10 11/08/2011 300 02:53:45 rainy 20.5 79 11107.9791 77.7086Serrania de Jingurud 7 26/09/2011 200 03:20:00 clear20.9 84 943 7.68338 78.0384
Serrania de Jingurud 8 27/09/2011 418 03:46:00 clear 23.5 79 9537.68035 78.0387
Serrania de Jingurud 3 29/09/2011 280 03:00:00 cloudy 22.6 81865 7.69312 78.0423
Serrania de Sapo 4 05/12/2011 172 03:10:00 cloudy 21.4 72 11607.97589 78.3625
Serrania de Sapo 6 06/12/2011 160 03:50:00 cloudy 19.4 83 9177.97749 78.3592
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Supplementary Text S6.Pristimantis caryophyllaceuscomplex colourdescriptions.
MOTU1:Alto de Piedra, Veraguas (MHCH3189, no photo): Dorsal basecolour Buff (124)with dark Brownish Olive (129) v-shapedtransversal stripes and dark Brownish Olive (129)mottling inbetween. Posterior surface of thigh Gem Ruby (110). Ventral surfaceSalmonColour (106) with small dark spots in the gular region andsmall white spots between axillaand groin.
Reserva Forestal La Fortuna, western slope of Cerro Pata deMacho, Chiriqu (MHCH3184):Dorsal base colour Cinnamon (123A) with asuggestion of Yellow Ochre (123C), bordered bySepia (119); dorsaltubercles Buff Yellow (53); lateral surfaces transparent lgcsparsely mottledwith Buff Yellow (53) and fading dorsally into BuffYellow (53) on transparent ground; dlcdorsal surfaces of limbs BuffYellow (53) on transparent ground speckled withOlive-Green(Auxiliary) (47) auxiliary lines; vgc ventral groundcolour dirty white fading into Smalt Blue(70) towards lateraledges; vlc ventral surfaces of hands and feet transparent withspots ofBuff Yellow (53), and tubercles in Sepia (119); Ic Iriscolouration Pale Pinkish Buff (121D),bordered with Sepia (119) andRobins Egg Blue (93).
MOTU2:Ro Tuquesa, Darin Mountain Range (MHCH3039, Fig. 8E):Dorsal colourChamois (84) with Warm Sepia (40) irregular blotchesand spots; Warm Sepia (40) interor-bital band; upper surface ofthigh with Warm Sepia (40) bars; groin and posterior surfaceofthigh suffused with Geranium (66); upper iris region MediumChrome Orange (75); lower
iris region Cream White (52), iris centre Dark Salmon (59); irisperiphery Jet Black (300);eye periphery Smoky White (261).
Nurra, San Blas Mountain range (SMF50939, Fig 8G): Dorsal colourRusset (44) withsmall Warm Sepia (40) spots; upper surface of thighwith Pale Pinkish Buff (3) bars; groin andposterior surface ofthigh suffused with Geranium (66); upper iris region SpectrumOrange(9); lower iris region Light Lavender (201), iris centre DarkSalmon (59); iris periphery JetBlack (300); eye periphery PearlGray (262). SMF50940 (Fig. 8H) same as SMF50939, butwith the upperand lower iris regions Spectrum Red (67), and the eye peripheryLight SkyBlue (191).
Nurra, San Blas mountain range (MHCH3037, Juvenile, Fig. 8F):Dorsal colour Sepia(279) with a mid-dorsal line in Medium Fawn(257); a series of delicate Medium Fawn (257)transverse lines ondorsum; dorsolateral line from the tip of snout to the groin Beige(254);upper and lower iris regions Spectrum Red (67); irisperiphery Jet Black (300); eye periphery
Pearl Gray (262).MOTU3:Pirre Mountain range (SMF50934, 1149):Dorsal colour Buff (5), frontal region
and some blotches on the rest of the dorsum Pale Buff (1); nocontrasting pattern on groinor posterior surface of thigh; upperand lower iris regions Light Buff (2), iris centre WalnutBrown(27); iris periphery Jet Black (300); eye periphery Smoky White(261).
Pirre mountain range (SMF50946, Fig. 9F) Dorsal colour Buff (5),with small, scatteredblack spots; Pale Buff (1) spots on dorsum; nocontrasting pattern on groin or posteriorsurface of thigh; upperand lower iris regions Light Buff (2), iris centre Walnut Brown(27);iris periphery Jet Black (300); eye periphery Smoky White(261).
Pirre Mountain range (MHCH3045, Fig. 9G): Dorsal colour RawUmber (22) with aLight Yellow Ochre (13) dorsolateral line from thetip of the snout to the groin, separatingthe dorsal from thelateral colouration; face Tawny Olive (17), lateral region behindthe eyesLight Yellow Ochre (13); groin and p osterior surface ofthigh Buff Yellow (6); upper surfaceof thigh with Ground Cinnamon(270) bars. Upper and lower iris region Light Buff (2); iriscentreWalnut Brown (27); iris periphery Jet Black (300); eye peripherySmoky White (261).
Cerro Garra Garra, Jingurud (MHCH3042, Fig. 8C): Dorsal colourFlesh (249) with
Warm Sepia (40) irregular blotches and spots; Warm Sepia (40)interorbital band; uppersurface of thigh with Warm Sepia (40) bars;groin and posterior surface of thigh suffusedwith Geranium (66);upper and lower iris region Olive Horn (16), iris centre WalnutBrown(27); iris periphery Jet Black (300); eye periphery SmokyWhite (261).
Ambroya, Maje (specimen not collected, Fig. 9A): Dorsal colourChamois (84) with smallWarm Sepia (40) spots; iris Spectrum Red(67); iris periphery Jet Black (300); eye peripherySmoky White(261). Chucanti, SMF50945 (Fig. 9B), dorsal colour Chesnut (30)with someDusky Brown (285) blotches; dorsolateral line from the tipof snout to the groin Flesh (249);lateral colour Salmon (58); uppersurface of thigh with Dusky Brown (285) bars; groin andposteriorsurface of thigh Flame Scarlet (73); upper and lower iris regionSpectrum Red (67);iris periphery Jet Black (300); eye peripheryLight Lavender (201). Chucanti, MHCH3043(Fig. 9C), dorsal colourDeep Vinaceous (248) with the frontal region in Pale Pinkish Buff(3);groin and posterior surface of thigh Flame Scarlet (73); irisSpectrum Red (67); iris periphery
Jet Black (300); eye periphery Light Lavender (201).Chucanti(specimen not collected, Fig. 9D), dorsal colour Clay (18) with adorsolateral
line from the tip of snout to the groin in Cream (12); groin andposterior surface of thighFlame Scarlet (73); iris Pale NeutralGray (296); iris periphery Jet Black (300); eye peripheryLightLavender (201).
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