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Recent genetic studies identified three deep genetic lin-eages with 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 a possible co-occurrence o at least three candi-date species that were concealed under the name P. caryo-phyllaceusat El Cop (C et al. ). Pristimantiscaryophyllaceus is indeed an old lineage, which originat-ed in South America about million years ago and sub-sequently evolved and expanded its range into CentralAmerica prior to the closure o the Isthmus o Panama(P-S et al. ). As a consequence, the diver-gence o different lineages was triggered by the dynamicgeological history in the region and the rise o the Isthmuso Panama, which ultimately resulted in various isolationprocesses over time.

Ecological inormation on Pristimantis caryophylla-ceus (sensu lato) (D , M , H

, S , L et al. ) is mostly available onpopulations rom western Panama and eastern Costa Rica,whereas there is only little inormation on the natural histo-ry o eastern Panamanian or Colombian populations (M- ). Herein, we contribute new inormation by de-scribing or the first time the advertisement call o P. caryo-phyllaceusrom eastern Panama, and providing additionaldata on its ecology, biogeography and morphology in anintegrative taxonomic approach 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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Morphological variation and phylogeography o Pristimantis caryophyllaceus

ern Panama, and the abasar and alamanca mountainranges in western Panama (Fig. ). We evaluated the abun-

dance o members o the Pristimantis caryophyllaceus com-plex by opportunistic search or rogs in the lea litter andundergrowth along trails. Search transect lengths were cal-culated with the tracking unction o a Garmin GPSmapCSx. All georeerences were recorded in the WGS datum ormat. Maps and transects were created and calcu-lated using ArcGIS (ESRI ). Collected specimenswere euthanised with , fixed with ml o ormalin() in l o ethanol (), and subsequently stored inethanol (). All figures incorporated herein have been

digitally improved and combined using Adobe PhotoshopCS. For candidate species and their delimitation, we ol-low the integrative concept o V et al. ().

Molecular laboratory and phylogenetic inerences

MtDNA was extracted rom resh muscle or liver tissue.Te mitochondrial S mtDNA was amplified using a Mas-

tercycler pro S (Eppendor, Hamburg, Germany) by per-orming an initial denaturation or min at C ollowedby steps with denaturation or 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: s at C, s at C, minat C; cycle: min at C. Te reaction mix consistedo l 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. Only ten 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 were parsimony-inormative. Using MEGA( et al.), we computed uncorrected pairwisegenetic distances or COI and S both separately and com-bined. For each gene and or the combined-gene data set,we conducted Maximum Likelihood (ML) analyses, usingthe amura--Parameter, with , bootstrap replicates.Prior to model-based phylogenetic inerences, JModeltest.. (P ) was used under the corrected Akaike

Inormation Criterion (AICc) to select the substitutionmodel or the Bayesian analysis. We ran a Bayesian phylo-genetic analysis in MrBayes .. (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, we conducted a statisticalparsimony network analysis with gaps considered as a fifh

character state (only or COI) in CS v. (C et al.). In order to connect 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 a transversion/transitionratio o ., and the setting or the minimum relative gapwidth (X) varied at values between and .. Te MOUsphylogenetic 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 relaxed clock,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 theUniversidad Autnoma de Chiriqu, Panama, and the Sen-ckenberg Research Institute and Nature Museum (SMF),Frankurt, Germany. Morphological data o similar Pristi-mantisspecies used or comparisons were taken rom therespective original descriptions.

Te ollowing morphometric measurements were taken(with abbreviations): snoutvent length (SVL); head length(HL), diagonally rom angle o jaw to tip o snout; headwidth (HW), between angles o jaws; interorbital distance

(IOD); eye length (EL), rom anterior to posterior edge oexternally accessible eye; eye to nostril distance (END),rom anterior edge o eye to posterior corner o nostril; in-ternarial distance (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 o third finger; tibialength (L), as the distance rom the knee to the distal endo the tibia; oot length (FL), rom proximal edge o out-

er metatarsal tubercle to tip o ourth toe; width o thirdfinger (FW), at penultimate phalanx just anterior to disk;width o disk o third finger (FD), at greatest width; widtho ourth toe (W), at penultimate phalanx just anteriorto the disk; width o disk o ourth toe (D), at greatestwidth; and tympanum diameter (YMP), measured hori-zontally, supposing an approximately circular tympanum.We determined the sexes o adults by differences in SVL(males smaller than 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 the morphological 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 and highest requencies were measured db below peak requency. erminology used in the ad-vertisement call description ollows D (). o calculate call rates, we divided the average callduration by (sec.) plus the average o call intervals.

Colour variation

Generalized colouration summaries were derived directlyrom live specimens or indirectly rom photos o live speci-mens. Within the standardized colour descriptions o se-lected individuals, the capitalized colours and colour codes(the latter in parentheses) are those o S ().

Results

Based on the lineages resulting rom our phylogeneticanalysis (see section on molecular phylogenetics below),we assigned each specimen to one o three MOUs. TeMOUs were largely consistent with a geographical pat-tern and defined as ollows (Fig. ): MOU contains speci-mens rom western Panama, MOU rom eastern Atlan-tic Panama (Daren and San Blas mountain ranges), andMOU rom eastern Pacific Panama (Jingurud, Maj,Pirre and Sapo mountain ranges). aking into account

the congruence between the phylogenetic results and thebiogeographical pattern, we assigned measured speci-mens to their respective MOUs, with to MOU, to MOU and seven to MOU In the ollowing we


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sample rom Donoso (MOU) with eight mutationalsteps between them in 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, but were not connected in the COInetwork; one sample rom apant, Costa Rica, was con-nected to the samples rom Ro Changena and Ro Clari-to, Bocas del oro, with unsampled haplotypes betweenthem (only one o these samples was included or COI).In MOU, only the samples rom El Cop were groupedin the same haplotype, in both S and COI. In MOU,the samples rom the Maj and Jingurud mountain ranges

(S and COI), as well as those rom the Cana field station(COI) were connected. Our ABGD analysis generated fivegroups or S with a divergence threshold o . with arelative width o the barcoding gap o . in the X-value.For COI, it produced eight groups, assuming an a priori in-traspecific divergence threshold o . with a relative gapwidth o . (X-value), whereas ten groups resulted whenboth genes were combined (threshold o .). Our threeanalyses (S, COI, and both genes combined) lumped all

samples in one unit, with a priori intraspecific divergenceso ., ., and ., respectively. With ABGD, allsamples rom MOU and almost all rom MOU or theCOI and S sequences (Supplementary Figs SS) weregrouped in their corresponding geographic MOU. TeS ABGD assigned all samples o MOU to one cluster,

but also included samples rom Maj that were placed inMOU according to our phylogenetic analyses. One sam-

ple rom El Cop (USNM ) did not nest within anycluster in the S analysis, but took a place within MOUwhen using COI and both genes combined.

According to our divergence time estimates, the MO-Us started to diverge rom other species o the subge-nus Hypodictyon C, (sensu Hedges et al. ),which were present in Central America in the Oligocene. Mya (with a credibility interval, CI, o .. Ma). Te crown age o the MOUs dates to . Mya(CI: .. Mya) during the Miocene, when MOU

and MOU + MOU split; a second break between theancestors o MOU and MOU occurred . Mya, ol-lowed by several splitting processes within the respectiveMOUs between . and . Mya. When all genes wereused in the phylogenetic analysis, the ML analysis yield-ed a consensus tree that was topologically congruent withthe divergence-time tree. However, in the divergence-timeanalyses, when each MOU was treated as a monophylet-ic group, the Maj samples were nested within MOU,

showing a divergence-time o . Mya. Tis is not sup-ported (p = ., see Supplementary Fig. S), however, al-though when the MOUs were not supposed to be mono-phyletic, the posterior probability or the divergence-timeanalysis was not supported either (p = ., Fig. and Sup-plementary Fig. S).


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Morphometrics

In Supplementary able S, we present the morphometricvariables used to evaluate the differences between lineag-es within the Pristimantis caryophyllaceus complex. Ourmorphological analysis revealed differences between thethree MOUs (Fig. ). A Discriminant Function Analysis(DFA) classified . o the specimens according to oura priori groupings (. MOU; . MOU; .

MOU). Te principal morphological variables contrib-

uting to the grouping were ) YMP/SVL, ) D/W,) IOD, ) FD; the first unction is: DS = . * YMP/SVL + . D/W + . IOD + -. FD; andthe second unction is DS = -. YMP/SVL + . D/W + . IOD + . FD. Te specimensincluded in MOU are usually larger than those romMOU and MOU, respectively. Likewise, MOU andMOU seem to be more similar to each other (MOU MOU: .) than either o them is to MOU (.

and ., respectively; see Figs + ).

able 1. amura 3-parameter distances among the Pristimantis caryophyllaceus (includingP. educatoris)specimensused in this study.MOU: MOU1 contains specimens rom western Panama, MOU2 rom eastern Atlantic Panama (Daren and San Blas mountainranges), and MOU3 rom eastern Pacific Panama (Jingurud, Maj, Pirre and Sapo mountain 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 rom western and eastern Panama correctly, with. probability, into their respective 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-chromatic species in its genus. Te general dorsal colour-ation varied rom yellow to reddish with various brown-ish tonalities, with or without black chevron marks onthe dorsum, and sometimes with sulphur-yellow spots on

Figure 4. Discriminant unction analysis o MOUs; see text ord t il


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the dorsum and/or limbs (Figs ). Groin and posteriorthigh varied rom not contrasting in colour to yellow orred Pristimantis caryophyllaceus also had a highly varia

Simultaneously, a third male was observed, calling rom adistance o approximately m, but was not recorded. Tenext day (// see ab ) we recorded a ourth male

able 2. Spectral and temporal parameters o the advertisement call o Pristimantis caryophyllaceus. Te specimen MHCH 3022 wasthe only one collected, recordings 13 correspond to males that were not captured (see Vocalisation section or explanation). Averagesor the parameters 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 00:08:26 00:59:55

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-marily inhabitants o pristine orest, but are also oundat the edges o orests. 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./ m trail transect; most specimens were ound atheights between m above the ground perched on

items (one each): two crickets and one arachnid, with anaverage volume o . + . mm.

Discussion

Our results demonstrate a high level o genetic diversityin the Pristimantis caryophyllaceus complex, comprisingthree main lineages within its currently known distribu-tion (this study C et al P S

Figure 9. Oscillogram (top) and spectrogram (bottom) o the advertisement call o a Pristimantis caryophyllaceus-complex-male,

recorded at Cerro Sapo (MHCH 3022).


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in their zones o contact and/or evaluating whether theseMOUs behave/evolve as independent entities.

Te ABGD has grouped successully almost all sampleso MOU and MOU or both mitochondrial genes. Temost incongruent result obtained in the species delimita-tion test (ABGD) was the placement o the samples romthe Maj mountain range (MOU) with the samples romwestern Panama (MOU) in the same group (Supple-mentary Fig. S). Te over-splitting detected by the ABGDanalysis in MOU (Supplementary Figs SS) is likelydue to the great genetic distances ound within that group,as reflecting the inclusion o specimens rom three differ-

ent isolated regions (Maj mountain range, Jingurud-Sa-po mountains, and Pirre lowlands). In our analyses, theABGD lumped all MOUs with a low prior intraspeci-ic divergence (. or S, . or COI, and . bothgenes combined), compared to the maximum values o thea priori threshold or conspecific divergence that yieldeda primary species 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 could support splitting the Pristimantis caryophylla-ceus complex into P. caryophyllaceus andP. educatoris, oreven into three species according to the MOUs recoveredin this study. We also detected morphological, yet non-sig-nificant, variation among MOUs that should not be dis-regarded until larger samples can be tested to evaluate apotential clinal variation that orms a continuum with thevariability detected within the MOUs (M ),corresponding to interbreeding between the MOUs (e.g.,at El Cope), or corresponds to a geographical pattern. Forexample, the specimens rom Ro Changena and 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 photographic data and colour descriptions confirm awide colour variation within and between MOUs. Speci-mens with different eye colours rom the same populationsdid not exhibit other morphological differences. Tereore,eye colouration has no diagnostic value, and we are consid-ering specimens with different eye colourations occurringwithin a MOU as conspecific and/or members o the samelineage, even though we have no genetic data to corrobo-rate this (no samples with red eyes were sequenced). Ret al. () mentioned the presence o yellowish wart-like

spots on the dorsum as being characteristic o Pristimantiseducatorisrom eastern Panama. However, we also oundspecimens with such yellowish marks in extreme westernPanama, which otherwise agree with characters o P. caryo-phyllaceus in having rounded pads and disk covers, andlow and rounded subarticular tubercles.

Te most common polymorphic traits (F ,M ) ound in populations o different erraranaspecies (G , H B , S

E ) relate to characteristics o the dorsal skintexture, colour pattern, and iris colour (S ). InP. caryophyllaceus, polymorphism is most prominent in thedorsal colour pattern, iris colour and digital disk. It seemsto be a balanced polymorphism (F ), inasmuch asit appears to be maintained in the different MOUs. Col-our pattern in errarana rogs could be inherited by a sim-ple Mendelian genetic mechanism (G , , S- et al. , ON B ), perhaps main-tained by the heterogeneity o colour compositions andshapes predominant in the 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 environmentand would prove beneficial also or semiarboreal activitiesin errarana species (S , H et al. ).Although the exact unction o the digital disks in P. caryo-phyllaceus has not been investigated yet, one possibilitycould be that the differences in their widths reflect differ-ent degrees o arboreality among MOUs. However, thedifferent degrees o arboreality appear to be not the mainreason or this variation, 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 by a two-species taxonomy sensu R et al. () orthree species as ound in this study; i.e., by splitting the spe-cies into P. caryophyllaceusor western Panama (MOU)and two P. educatoristaxa or eastern Panama (Caribbean-MOU and Pacific-MOU). Based on the molecular in-ormation available as o now, all three MOUs co-occur atEl Cop. However, there is no additional evidence or im-plementing a three-species taxonomy, and sensu V et

al. (), we would need at least one diagnostic morpho-logical difference, a character trait that is o low intraspeci-ic variability and o high value, to discriminate among taxa(here: MOUs). Te morphological differences betweenthese MOUs are insufficient to support them as distinctspecies, however. Furthermore, there are currently no bio-acoustic data rom El Cop to test or putative interspecificdifferences.

R et al. () used the SVL to differentiate betweenP. caryophyllaceusand P. educatoris. However, rather thana clear difference in SVL, our data suggest a smooth clinaltransition o the SVL rom east to west (Fig. ). Te condi-tion o a projecting subarticular tubercle as suggested as a

morphological variation within the particular MOUs andthe morphological similarities between the MOUs, weound no additional evidence to prove the three MOUsas respective Confirmed Candidate Species (V et al.). Moreover, at this point, we have no evidence or as-signing P. educatoristo either MOU or MOU, and ourdata do not support the biogeographical concept suggestedby R et al. (). For now, we suppose to see the threeMOUs within the P. caryophyllaceus-educatoris-complexas geographically and genetically distinct lineages in ac-cordance with the definition o a Deep Conspecific Lineageby V et al. () with a small contact zone around

El Cop. Consequently, we reject P. educatoris as a validspecies and place it in the synonymy o P. caryophyllaceus.

Te phylogenetic history o the MOUs studied here islinked to the complex biogeography o the Isthmus o Pan-ama and northwestern South America, more precisely othe Choco Block (D-C ab, C et al., ), ormed by the Maj-Baud Arc (Maj, Sapo,Jingurud and Pirre massis) and the Dabeiba Arc (Dar-ien and San Blas massis). Pristimantis caryophyllaceushas

originated in South America (D , Het al. , P-S et al. ), and its dispersal toCentral America is consistent with the recent hypothesis oan earlier ormation o the Isthmus o Panama (F etal. , M et al. ), dating well beore . Mya aswas previously thought (C O , W R , S et al. , W et al. ). TeMOUs show a geographical pattern with almost all MO-Us (except or the El Cop populations) being distributedin different geographic areas (Figs ). According to ourresults, the split between MOU and MOUMOU( Mya) supports the proposed connection o the Isth-mus o Panama with South America which would have a


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MOUs in the time divergence analysis. Tis would meanthat MOU rom eastern Panama had available a consid-erable period o time to expand into western Panama andmix with MOU there.

Even though we here present a lot o new inormationon the distribution pattern o Pristimantis caryophyllaceusand its variation in morphology, genetics, colour pattern,as well as advertisement calls, it is apparently still notenough to clariy the taxonomic status o the species. De-tailed molecular analyses at population level, using nucle-ar markers such as microsatellites to detect gene flow andpast demographic bottlenecks, including populations rom

central Panama (especially rom the Piedras-Pacora moun-tain range) into the analysis o morphology, and statisti-cally supported bioacoustics data rom various populationsover wide areas are still needed. Further studies should in-clude correlation analyses between geographic and geneticdistances to test whether the uncovered differences expressjust a clinal variation among the populations, and to evalu-ate the existence and role o previous and current intro-gressions among the MOUs, particularly at El Cop.

Acknowledgements

Scientific permits or (SC/A--, SC/A--), (SC/A--), (SC/A--), and export permits (SC/A--) and (SEX/A--) were provided by ANAM, Panama,and . Q (Cacique General del rea de Samb) rom theDespacho del Cacique Regional Comarca Ember-Wounaan,Panama. Special thanks go to the indigenous people o the Em-bera rom Puerto Indio and Pavarand, especially to D

B (Secretary o the Ember-Wounaan congress, Sam-b), and L C (Noko o the Pavarando village) andhis amily who allowed us to enter their autonomous territory

and FY), and the Chinese Academy o Science (No.KSCX-EW-Z-).

References

A, F., K. C. W M. V (): Ecological cor-relates o eye colour and pattern in mantellid rogs. Salaman-dra, : .

B, . (): Proceedings o the New England ZoologicalClub, : .

B, A. (): Comer o Cantar? Implicaciones del tamaocorporal sobre la importancia de la dieta y el canto en un en-

samble de especies de Anuros (Amphibia: Anura: errarana). Unpubl. MSc thesis.

C, R. A., C. W. C K. M. F (): Raven .Users Manual. Cornell Laboratory o Ornithology, Ithaca,NY.

C, M., D. P K. C (): CS: a compu-ter program to estimate gene genealogies. Molecular Ecolo-gy Notes, : .

C, A. G. J. A. O (): Te geologic evolution o

the Central American Isthmus. pp. in: J, J. B.C., A. F. B A. G. C (eds): Evolution and Environ-ment in ropical America. University o Chicago Press, Chi-cago.

C, A. G., M. P. A, W. A. B, L. S. C M.K (): Early Neogene history o the Central Americanarc rom Bocas del oro, western Panama. Geological Soci-ety o American Bulletin, : .

C, A. G., L. S. C, M. P. A W. A. B(): Te geology o the Darien, Panama, and the late Mi-

ocene-Pliocene collision o the Panama arc with northwesternSouth America. Geological Society o American Bulletin,: .


16/30

A B et al.

D, A. J. A. R (): BEAS: Bayesian evo-lutionary analysis by sampling trees. BioMed Central, Evo-lutionary Biology, : .

D, W. E. (): Te hylid rogs o Middle America, Vol.. Contributions to Herpetology, : .

D, W. E. E. L (): errestrial breeding rogs(Strabomantidae) in Peru. Natur und ier-Verlag, Naturwis-senschaf, Mnster, pp.

D, W. E. L. (): Biology o Amphibians. Te Johns Hopkins University Press, Baltimore and London, pp.

D, E. R. (): Te amphibian and reptilian auna o brome-

liads in Costa Rica and Panama. Copeia, :.ESRI (Environmental Systems Resource Institute) (): Arc-

Map . ESRI, Redlands, Caliornia.

F, D. W., C. J, G. B, S. A. R-M, C. M, A. C, A. M, R. J. S-, M. D. G V. V (): Fracturing othe Panamanian Isthmus during initial collision with SouthAmerica. Geology, :.

F, E. B. (): Polymorphism and taxonomy. Heredity, :.

G, F. M. V (): Anuran eye colouration: defini-tions, variation, taxonomic implications and possible unc-tions. pp. in: B, W., W. B . Z- (eds): Herpetologia Bonnensis, Proceedings o the th Or-dinary General Meeting o the Societas Europaea Herpetolo-gica, August . Societas Europaea Herpetologica,Deutsche Gesellschaf r Herpetologie und errarienkundeand Zoologisches Forschungsinstitut und Museum AlexanderKoenig, Bonn.

G, C. J. (): Colour pattern inheritance in some rogs o thegenus Eleutherodactylus. Bulletin o the Chicago Academyo Sciences, : .

G C J ( ) P tt i ti i th El th d t l

L, K. R., J. D. R L. R. W (): Ecological traitspredicting amphibian population declines in Central Ameri-ca. Conservation Biology, : .

L, J. D. (): Systematic status and distribution o somepoorly known rogs o the genus Eleutherodactylusrom theChocoan lowlands o South America. Herpetologica :.

L, J. D. W. E. D (): Frogs o the genusEleutherodactylusin western Ecuador. University o KansasSpecial Publication, : .

M, J. (): Hybridization, ecological races and the na-ture o species: empirical evidence or the ease o speciation. Philosophical ransactions o the Royal Society B: BiologicalSciences, : .

M, E. (): Animal species and evolution. Cambridge,MA: Harvard University Press.

M, M. M. (): Central American Frogs Allied toEleutherodactylus cruentus: Allozyme and MorphologicalData. Journal o Herpetology, : .

M, C. G., A. B, A. C, D. M. B, C. A. S-, S. E. M, N. H, D. A. R, C. A. J V. V (): Arc-continent collision and orocline

ormation: closing o the Central American Seaway. Jour-nal o Geophysical Research, : DOI: ./JB.

M, C. W. (): Te ecological geography o cloud orest inPanama. American Museum Novitates, :

OD R. P. K. E. M (): wo rog species or one?A multi-marker approach to assessing the distinctiveness ogenetic lineages in the Northern Leopard Frog, Rana pipiens. Conservation Genetics, :

ON, E. M. K. H. B (): Genetic basis o a colourpattern polymorphism in the coqui rog Eleutherodactyluscoqui. Journal o Heredity, : .

P, J. M. I. D R (): Integrative taxonomy re-l ti A i i P i ti ti (A


17/30


R, M. J., K. R. L, J. . G (): New spe-cies o Pristimantis (Anura: errarana: Stabomantinae) romlower Central America. Journal o Herpetology, : .

S, J. C., L. A. C, K. S, J. P. C, R.R, D. C. C (): Amazonian amphibian di-

versity is primarily derived rom Late Miocene Andean line-ages. Public Library o Science, Biology, : .

S, J. M. (): Te Amphibians and Reptiles o Costa Rica:A Herpetoauna between two Continents, between two Seas. University o Chicago Press, Chicago, pp.

S, J. M. S. B. E (): Central American rogsallied to Eleutherodactylus bransfordii (Cope): a problem opolymorphism. Copeia, : .

S, F. B. (): Naturalists Colour guide. Part I. Col-our guide. Colour swatches. American Museum o Natu-ral History, New York, New York, U.S.A.

S, K., . W. C, . K (): Cross-breed-ing o distinct color morphs o the strawberry poison rog(Dendrobates pumilio) rom the Bocas del oro Archipelago,Panama. Journal o Herpetology, : .

, K. (): Estimation o the number o nucleotide sub-stitutions when there are strong transition-transversion and

G + C-content biases. Molecular Biology and Evolution, :

, K., D. P, N. P, G. S, M. N S. K (): MEGA: Molecular Evolutionary GeneticsAnalysis using Maximum Likelihood, Evolutionary Distance,and Maximum Parsimony. Molecular Biology and Evolu-tion, :

V, M., J. K, D. R. V F. G (): Molecu-lar and bioacoustic differentiation o deep conspecific lineageso the Malagasy treerogs Boophis tampoka and B. luteus.

Herpetology Notes, : .V, D. R., K. C. W, F. A, J. K, F.

G M V (): Vast underestimation o Mada-

Supplementary material

Additional inormation is available in the online version o this

article at http://www.salamandra-journal.com

4 Supplementary figures and 6 Supplementary tables:

Figure S1. Maximum likelihood consensus tree o the COImtDNA o the Pristimantis caryophyllaceuscomplex.

Figure S2. Maximum likelihood consensus tree o the 16S mtDNAo the 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. Chronogram o the Pristimantis caryophyllaceuscom-plex based on Rag1, 16S and COI genes, using P. cerasinus andP. cruentusas outgroups.

able S1. Morphological variables taken rom 78 specimens usedin the analyses.

able S2. Mean genetic distances in the 16S mtDNA gene be-tween the Pristimantis caryophyllaceus samples used in the phylo-genetic analysis.

able S3. Mean genetic distances in the COI mtDNA gene be-

tween the Pristimantis caryophyllaceus samplesused in the phylo-genetic analysis.

able S4. Mean genetic distances in the COI and 16S mtDNAgenes combined between the Pristimantis caryophyllaceus sam-ples used in the phylogenetic analysis.

able S5. Details o sample and museum voucher numbers.

able S6. ransect details or Pristimantis caryophyllaceus(MOU3).


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Supplementary material

B, A., A. H, G. K, K. M M. V: Phylogeny, shapes, colours, and phylogeography 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 78 specimens used in the analyses. Number of specimens measured in parenthesis (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 9.38 1.16 (8.1012.30) 7.640.78 (6.009.10) 11.271.02 (9.6012.40) 8.2 11.340.51 (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/HW 0.950.04 (0.851.01) 0.980.05 (0.841.16) 0.990.04 (0.941.06) 1.01 0.980.02 (0.951.01) 0.990.06 (0.911.11)


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Supplementary Table S2.Mean genetic distances in the 16S mtDNA gene between the Pristimantis caryophyllaceus samples used in the phylogenetic 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 6.8 2.3

13 USNM572343 {MOTU2} 7.3 7.3 7.3 6.2 7.3 7.8 7.8 7.3 6.7 6.8 2.3 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 2.3 1.9 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 1.9 1.4 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 6.3 7.8 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 8.8 9.8 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 6.8 8.3 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 10.4 11.5 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 9.3 9.3 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 6.3 7.8 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 8.3 7.2 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 6.3 6.8 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 5.3 5.7 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 Table S3.Mean genetic distances in the COI mtDNA gene between the Pristimantis caryophyllaceus samples used in the phylogenetic 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 8.8 6.9

11 MVUP1925 {MOTU2} 19.8 19.8 19.8 19.8 19.8 16.2 21.2 9.8 8.0 4.4

12 USNM572343 {MOTU2} 20.1 20.1 20.1 20.1 20.1 16.4 20.9 10.1 8.2 4.6 0.2

13 SMF50933 {MOTU3} 19.7 19.7 19.7 20.5 19.7 15.5 20.5 16.6 17.3 16.8 17.0 17.3

14 MHCH3042 {MOTU3} 19.3 19.3 19.3 19.3 19.3 15.9 20.3 14.8 17.7 14.8 15.8 16.0 12.315 CH6367 {MOTU3} 17.0 17.0 17.0 17.5 17.0 14.1 19.5 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 15.8 17.7 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 17.3 16.8 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 17.7 14.8 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 15.7 13.4 14.3 14.6 11.8 11.8 0.2 11.3 11.8 12.2


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Supplementary Table S4.Mean genetic distances in the COI and 16S mtDNA genes combined between the Pristimantis caryophyllaceussamples 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 6.8 12.7

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 10.8 11.4

11 MHCH3042 {MOTU3} 16.1 16.9 13.8 16.1 16.1 16.1 16.0 9.8 11.2 0.8

12 SMF50933 {MOTU3} 15.1 15.9 12.1 15.1 15.1 15.1 15.7 10.5 0.1 11.2 11.1

13 USNM572338 {MOTU3} 13.4 13.4 10.9 13.4 13.4 13.4 13.6 10.6 8.9 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 14.4 14.5 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 13.6 14.2 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 14.4 14.0 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 12.9 14.6 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 14.6 15.8 15.2 14.4 14.2 6.2 8.7 5.3 6.3


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Supplementary Table S5.Details of sample and museum voucher numbers (where available), collecting localities, and GenBank accession numbers for all samples used in this study.

Voucher Species Locality Province Country Genbank accession number 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 Macho Chiriqu 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



MHCH3189 P. caryophyllaceus Cerro Mariposa Veraguas Panama 8.5145 -81.1207 880

MHCH3190 P. caryophyllaceus Cerro Mariposa, Santa Fe Veraguas Panama 8.5122 -81.1214 935




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 -78.5604 911

MHCH3018 P. caryophyllaceus Serrania de Maj, Ambroya, Chepo Panam 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 Panama 7.9762 -78.3626 1169

MHCH3022 P. caryophyllaceus Cerro Sapo, Garachin Darin Panama KJ201952 KJ201942 7.9762 -78.3628 1168

MHCH3037 P. caryophyllaceus Ro Taintidu, Chucunaque Wargandi Panama 9.0355 -78.0264 289

MHCH3038 P. caryophyllaceus Ro Taintidu, Chucunaque Wargandi Panama 9.0593 -77.9842 553

MHCH3039 P. caryophyllaceus Ro Tuquesa, Embera-Waounaan Embera-Wounaan Panama KJ201967 8.4800 -77.5194 859

MHCH3040 P. caryophyllaceus Ro Tuquesa, Embera-Waounaan Embera-Wounaan Panama 8.4791 -77.5280 718

MHCH3041 P. caryophyllaceus Ro Tuquesa, Embera-Waounaan Embera-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 Panama 8.7977 -78.4623 1295

MHCH3044 P. caryophyllaceus Chucant ridge, ro Congo, Chepigana Darin Panama 8.7965 -78.4630 1342

MHCH3045 P. caryophyllaceus Rancho Fro Field station, Pinogana Darin Panama 7.9595 -77.7037 1230

MHCH3046 P. caryophyllaceus Rancho Fro Field station, Pinogana Darin Panama 7.9595 -77.7037 1230

MHCH457 P. caryophyllaceus El pianista, Bocas del Toro, Panam Bocas del Toro Panama 8.8714 -82.4159

MHCH523 P. caryophyllaceus Qda Arena, Fortuna, Chiriqu, Panam Chiriqu Panama 8.7180 -82.2284 1074

MVUP1925 P. caryophyllaceus* Parque Nacional G. D. Omar Torrijos H., El Cope, Corregimientois Ola, Distrito La Pintada, Cocl

Cocl Panama FJ784473.1 FJ766776 8.6670 -80.5920 800


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16S COI RAG1 N W

MVZ203810 P. caryophyllaceus* Costa Rica: Cartago, 2.5 km S Tapanti Bridge across Rio Grandede Orosi

Cartago CostaRica

EU186686.1 na

SMF 89976 P. caryophyllaceus Cerro Negro/PN Santa Fe Veraguas Panama 8.5706 -81.1043 800

SMF 89977 P. caryophyllaceus Cerro Negro/PN Santa Fe Veraguas Panama 8.5663 -81.0988 690

SMF 89978 P. caryophyllaceus Cerro Negro/PN Santa Fe Veraguas Panama 8.5706 -81.1043 800SMF 89979 P. caryophyllaceus Cerro Negro/PN Santa Fe Veraguas Panama 8.5769 -81.0973 900

SMF 89980 P. caryophyllaceus Cerro Mariposa Veraguas Panama 8.6757 -81.1228 1385

SMF 89981 P. caryophyllaceus I Brazo Mulaba Veraguas Panama 8.5186 -81.1332 700

SMF 97027 P. caryophyllaceus La Nevera Panama 8.4996 -81.7710 1650

SMF 97028 P. caryophyllaceus Cerro Mariposa Veraguas Panama 8.5145 -81.1207 880

SMF 97029 P. caryophyllaceus La Nevera/Cerro Santiago Westhang Comarca Ngbe Bugl Panama 8.4953 -81.7673 1800

SMF 97030 P. caryophyllaceus Llano Tugri Comarca Ngbe Bugl Panama 8.5082 -81.7162 1600

SMF 97031 P. caryophyllaceus Willi Mazu Comarca Ngbe Bugl Panama 8.7885 -82.2016 799

SMF 97032 P. caryophyllaceus Willi Mazu Comarca Ngbe Bugl Panama 8.7885 -82.2016 799

SMF 97033 P. caryophyllaceus Willi Mazu Comarca Ngbe Bugl Panama KJ476734 8.7885 -82.2016 799

SMF 97034 P. caryophyllaceus Changena Trail/Oberes Camp Bocas del Toro Panama 8.9505 -82.7094 1968

SMF 97035 P. caryophyllaceus Changena Trail/Oberes Camp Bocas del Toro Panama KJ476733 8.9505 -82.7094 1968SMF 97036 P. caryophyllaceus Rio Clarito Bocas del Toro Panama 9.0090 -82.6644 1258

SMF 97037 P. caryophyllaceus Rio Clarito Bocas del Toro Panama KJ476732 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 Veraguas Panama 8.5122 -81.1214 935







SMF 97047 P. caryophyllaceus Lost and Found/Fortuna Chiriqu Panama 8.6757 -82.2128 1364

SMF 97048 P. caryophyllaceus Lost and Found/Fortuna Chiriqu Panama 8.6773 -82.2103 1288SMF50931 P. caryophyllaceus Serrania de Maje, Ambroya, Chepo Panam Panama 8.8920 -78.5609 901

SMF50932 P. caryophyllaceus Serrania de Maje, Ambroya, Chepo Panam Panama 8.8920 -78.5609 901

SMF50933 P. caryophyllaceus Serrania de Maje, Ambroya, Chepo Panam Panama KJ201953 KJ201943 KJ201963 8.8919 -78.5608 911

SMF50934 P. caryophyllaceus Rancho Fro Field station, Pinogana Darin 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 -78.0384 961

SMF50937 P. caryophyllaceus Ro Sambu, Serrana de Jingurudo, Samb. Darin Panama 7.6691 -78.0380 1133

SMF50938 P. caryophyllaceus Qda Valle Grande, Donoso Coln Panama KJ201958 KJ201947 KJ201968 8.8216 -80.6632 211


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16S COI RAG1 N W

SMF50939 P. caryophyllaceus Ro Taintidu, Chucunaque Wargandi Panama KJ201962 KJ201951 KJ201971 9.0355 -78.0264 289

SMF50940 P. caryophyllaceus Ro Taintidu, Chucunaque Wargandi Panama 9.0355 -78.0264 289

SMF50941 P. caryophyllaceus Serrana de San Blas Guna Yala Panama 9.0611 -77.9797 340

SMF50942 P. caryophyllaceus Ro Sambu, Serrana de Jingurudo, Samb Embera-Wounaan Panama 7.7590 -78.0923 643

SMF50943 P. caryophyllaceus Ro Terable, El Llano, Chepo Panama Panama KJ201965 9.2840 -78.9838 322

SMF50944 P. caryophyllaceus Chucant ridge, ro Congo, Chepigana Darin Panama KJ201956 KJ201966 8.7977 -78.4623 1295

SMF50945 P. caryophyllaceus Chucant ridge, ro Congo, Chepigana Darin Panama 8.7977 -78.4623 1295



SMF85008 P. caryophyllaceus RF Fortuna Chiriqu Panama 8.7264 -82.2615 1100




SMF85014 P. caryophyllaceus BP Palo seco, Los tucanes trail Bocas 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 Panama 8.4996 -81.7710 1600

SMF85018 P. caryophyllaceus La Nevera Comarca Ngbe Bugl Panama 8.4996 -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 Cartago CostaRica

JN991441 JN991370 JQ025179 9.6500 -83.8500 1200

USNM572329 P. caryophyllaceus* Parque Nacional G. D. Omar Torrijos H., El Cope, Corregimientois Ola, Distrito La Pintada, Cocle

Cocl Panama FJ784397.1 FJ766771 8.6670 -80.5920 800


Cocl Panama FJ784421.1 FJ766774 8.6670 -80.5920 800


Cocl Panama FJ784422.1 FJ766773 8.6670 -80.5920 800

USNM572335 P. caryophyllaceus* Parque Nacional G. D. Omar Torrijos H., El Cope, Corregimientois Ola, Distrito La Pintada, Cocle Cocl Panama FJ784589.1 FJ766770 8.6670 -80.5920 800


Cocl Panama FJ784491.1 FJ766775 8.6670 -80.5920 800


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 del Toro Panama 8.7817 -82.2122 1120

GK1595 P. caryophyllaceus La nevera Comarca Ngbe Bugl Panama 8.4996 -81.7710 1600


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Supplementary Table S6.Transect details for Pristimantis caryophyllaceus (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 1137 7.98845 77.7076

Serrania de Pirre 10 11/08/2011 300 02:53:45 rainy 20.5 79 1110 7.9791 77.7086Serrania de Jingurud 7 26/09/2011 200 03:20:00 clear 20.9 84 943 7.68338 78.0384

Serrania de Jingurud 8 27/09/2011 418 03:46:00 clear 23.5 79 953 7.68035 78.0387

Serrania de Jingurud 3 29/09/2011 280 03:00:00 cloudy 22.6 81 865 7.69312 78.0423

Serrania de Sapo 4 05/12/2011 172 03:10:00 cloudy 21.4 72 1160 7.97589 78.3625

Serrania de Sapo 6 06/12/2011 160 03:50:00 cloudy 19.4 83 917 7.97749 78.3592


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Supplementary Text S6.Pristimantis caryophyllaceuscomplex colour descriptions.

MOTU1:Alto de Piedra, Veraguas (MHCH3189, no photo): Dorsal base colour Buff (124)with dark Brownish Olive (129) v-shaped transversal stripes and dark Brownish Olive (129)mottling in between. Posterior surface of thigh Gem Ruby (110). Ventral surface SalmonColour (106) with small dark spots in the gular region and small white spots between axillaand groin.

Reserva Forestal La Fortuna, western slope of Cerro Pata de Macho, Chiriqu (MHCH3184):Dorsal base colour Cinnamon (123A) with a suggestion of Yellow Ochre (123C), bordered bySepia (119); dorsal tubercles Buff Yellow (53); lateral surfaces transparent lgc sparsely mottledwith Buff Yellow (53) and fading dorsally into Buff Yellow (53) on transparent ground; dlcdorsal surfaces of limbs Buff Yellow (53) on transparent ground speckled with Olive-Green(Auxiliary) (47) auxiliary lines; vgc ventral ground colour dirty white fading into Smalt Blue(70) towards lateral edges; vlc ventral surfaces of hands and feet transparent with spots ofBuff Yellow (53), and tubercles in Sepia (119); Ic Iris colouration Pale Pinkish Buff (121D),bordered with Sepia (119) and Robins Egg Blue (93).

MOTU2:Ro Tuquesa, Darin Mountain Range (MHCH3039, Fig. 8E): Dorsal colourChamois (84) with Warm Sepia (40) irregular blotches and spots; Warm Sepia (40) interor-bital band; upper surface of thigh with Warm Sepia (40) bars; groin and posterior surface ofthigh suffused with Geranium (66); upper iris region Medium Chrome Orange (75); lower

iris region Cream White (52), iris centre Dark Salmon (59); iris periphery Jet Black (300);eye periphery Smoky White (261).

Nurra, San Blas Mountain range (SMF50939, Fig 8G): Dorsal colour Russet (44) withsmall Warm Sepia (40) spots; upper surface of thigh with Pale Pinkish Buff (3) bars; groin andposterior surface of thigh suffused with Geranium (66); upper iris region Spectrum Orange(9); lower iris region Light Lavender (201), iris centre Dark Salmon (59); iris periphery JetBlack (300); eye periphery Pearl Gray (262). SMF50940 (Fig. 8H) same as SMF50939, butwith the upper and lower iris regions Spectrum Red (67), and the eye periphery Light 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 on dorsum; dorsolateral line from the tip of snout to the groin Beige (254);upper and lower iris regions Spectrum Red (67); iris periphery 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); no contrasting pattern on groinor posterior surface of thigh; upper and 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; no contrasting pattern on groin or posteriorsurface of thigh; upper and 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 Raw Umber (22) with aLight Yellow Ochre (13) dorsolateral line from the tip of the snout to the groin, separatingthe dorsal from the lateral colouration; face Tawny Olive (17), lateral region behind the eyesLight Yellow Ochre (13); groin and p osterior surface of thigh Buff Yellow (6); upper surfaceof thigh with Ground Cinnamon (270) bars. Upper and lower iris region Light Buff (2); iriscentre Walnut Brown (27); iris periphery Jet Black (300); eye periphery Smoky White (261).

Cerro Garra Garra, Jingurud (MHCH3042, Fig. 8C): Dorsal colour Flesh (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 Walnut Brown(27); iris periphery Jet Black (300); eye periphery Smoky White (261).

Ambroya, Maje (specimen not collected, Fig. 9A): Dorsal colour Chamois (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 tip of snout to the groin Flesh (249);lateral colour Salmon (58); upper surface of thigh with Dusky Brown (285) bars; groin andposterior surface of thigh Flame Scarlet (73); upper and lower iris region Spectrum Red (67);iris periphery Jet Black (300); eye periphery Light Lavender (201). Chucanti, MHCH3043(Fig. 9C), dorsal colour Deep Vinaceous (248) with the frontal region in Pale Pinkish Buff (3);groin and posterior surface of thigh Flame Scarlet (73); iris Spectrum Red (67); iris periphery

Jet Black (300); eye periphery Light Lavender (201).Chucanti (specimen not collected, Fig. 9D), dorsal colour Clay (18) with a dorsolateral

line from the tip of snout to the groin in Cream (12); groin and posterior surface of thighFlame Scarlet (73); iris Pale Neutral Gray (296); iris periphery Jet Black (300); eye peripheryLight Lavender (201).

batista et al-0640 incl si

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