CONGRUENCE BETWEEN ACOUSTIC TRAITS AND ...Guina 7 North 6805051.900 72852045.800 –– 2 2 Pa´ramo...

Herpetologica, 68(4), 2012, 523–540� 2012 by The Herpetologists’ League, Inc.

CONGRUENCE BETWEEN ACOUSTIC TRAITS AND GENEALOGICALHISTORY REVEALS A NEW SPECIES OF DENDROPSOPHUS (ANURA:

HYLIDAE) IN THE HIGH ANDES OF COLOMBIA

CARLOS E. GUARNIZO1,3,4, CAMILO ESCALLON

2, DAVID CANNATELLA1, AND ADOLFO AMEZQUITA

3

1Section of Integrative Biology and Texas Natural Science Center, University of Texas at Austin,1 University Station, CO930, Austin, TX 78712, USA

2Department of Biological Sciences, Virginia Tech, 2119 Derring Hall, Blacksburg, VA 240613Departamento de Ciencias Biologicas, Universidad de los Andes, Carrera 1E No. 18a–10,

PO Box 4976, Santafe de Bogota DC, Colombia

ABSTRACT: The High-Andean Frog (Dendropsophus labialis) is distributed along the Eastern Andes ofColombia between 1900 and 3600 m. We analyzed multiple traits to determine if acoustics and morphologycovary with genealogical history or if they evolved following independent trajectories. We generated aphylogeny of D. labialis populations with the use of mitochondrial ribosomal gene (12S, val-RNA, 16S) andnuclear gene (proopiomelanocortin A [POMC]) sequences. Two well-supported clades that correspond togeographic distribution were recovered. Acoustic variation diverges according to genealogical history, butexternal morphology does not follow this pattern. The strong and congruent divergence observed in acousticand genetic characters indicates that these two clades correspond to morphologically cryptic, parapatricspecies. We describe the Northern Clade as a new species (Dendropsophus luddeckei; the Southern Claderetains the name D. labialis). The low level of molecular divergence between two outgroup species,Dendropsophus pelidna and Dendropsophus meridensis, suggests D. pelidna is a junior subjective synonymof D. meridensis.

Key words: Acoustic divergence; Dendropsophus; labialis group; Morphological variation; New species;Tropical Andes

THE NEOTROPICS are characterized by highendemism (Myers et al., 2000; Orme et al.,2005). Historically, most tropical species havebeen described as morphospecies. Inferringspecies boundaries by morphology alone (orany phenotypic trait) is problematic whengenealogical history and that trait do notevolve in a coupled fashion, as may happenwhen selection breaks correlation patternsbetween phenotypic traits (Lougheed et al.,2006). Even if phenotypes evolve underneutral processes, problems with speciesdelimitation still may occur if the phenotypictrait has not fully diverged after a speciationevent (de Queiroz, 2007). As a result, thenumber of morphospecies will tend to under-estimate species diversity based on moleculardata.

We used the High-Andean Frog Dendrop-sophus labialis (Peters, 1863) to contrastpatterns of evolution of multiple characters(mitochondrial DNA, nuclear DNA, acoustics,and morphology) following recent divergence,

and to test for the possible existence of crypticspecies.

The nominal species D. labialis is distribut-ed along the Eastern Andes of Colombiabetween 3.5 and 6.38N and 1900 and 3600 min elevation (Ruiz-Carranza et al., 1996).Mitochondrial and nuclear DNA sequencesindicate two strongly differentiated cladeswith parapatric distribution within D. labialis(Guarnizo et al., 2009). However, taxonomicanalyses have treated these populations as asingle species (Cochran and Goin, 1970).Even though some studies have analyzed theeffect of elevation on the phenotype, physiol-ogy, and life history of populations within theSouthern Clade (Amezquita, 1999; Luddecke,2002; Luddecke and Sanchez, 2002), nostudies have compared the phenotypic varia-tion between the Northern and SouthernClades.

We conducted a multitrait analysis todetermine whether acoustics and morphologycodiverged according to genealogical history.We also tested if the climatic characteristicsassociated with each clade were statisticallydifferent. These analyses allowed us to deter-4CORRESPONDENCE: e-mail, [email protected]

523

mine the degree to which acoustic andmorphological traits differentiate because ofdivergence events that occurred in the veryyoung northern Andes (Fjeldsaa and Lovett,1997; Young et al., 2002).

MATERIALS AND METHODS

DNA Sequencing

Tissue samples from the nominal species D.labialis were obtained from 10 populations

(Fig. 1). Three of these populations geograph-ically correspond to the Southern Clade(Guarnizo et al., 2009), and seven to theNorthern Clade (Table 1). Tissues wereobtained by toe-clipping adult frogs (IACUCprotocol 07101901). Total genomic DNA wasextracted with the use of a Viogene Blood andTissue Genomic DNA Extraction MiniprepSystem (Viogene, Inc., Taipei, Taiwan). Fourprimer pairs (Symula et al., 2008) were usedto amplify the almost-complete 12S–16S

FIG 1.—Map of the localities included in this study. The vertical line delineates the contact zone between theNorthern and Southern Clades. The Tachira Depression is a low-elevation region (, 900 m) that separates the Andes ofColombia and Venezuela. Names and geographic coordinates of each locality are given in Table 1. Stars indicate thelocalities used to build the 12S–16S and proopiomelanocortin A phylogenies. Only elevations above 1000 m are shown.

524 [Vol. 68, No. 4HERPETOLOGICA

rRNA region. One primer pair (Guarnizo etal., 2009) was used to amplify the nuclear geneproopiomelanocortin A (POMC); we se-quenced additional populations that werenot included in Guarnizo et al. (2009).

The polymerase chain reaction (PCR) cyclefor the 12S–16S region included an initialdenaturing step of 2 min at 948C, followed by35 amplification cycles (30 s at 948C, 30 s at 488C,60 s at 728C), and a final extension of 7 min at

728C. For POMC we used the PCR cycle

protocol in Guarnizo et al. (2009). PCR products

were purified with the use of a Viogene Gel

Purification Kit and were sequenced at the

University of Texas ICMB Core Research

Facility with ABI 3730 DNA sequencers. Each

fragment was sequenced in both directions to

confirm base calls. Nucleotide sequences were

aligned with the use of MUSCLE (Edgar, 2004).

TABLE 1.—Sampling sites, map codes, assigned clade, geographic coordinates, and the number of individuals analyzedfor acoustics, morphology, and the phylogenetic analysis (12S-16S and proopiomelanocortin A [POMC]). Localities areordered from north to south. Asterisks indicate additional localities sequenced earlier for cytb and CO1 in Guarnizoet al. (2009). The North–South Clade corresponds to a locality that contained haplotypes from both clades (sympatric

locality).

Locality Map code Clade Latitude N Longitude WAcoustics

(n individuals)Morphology

(n individuals)

n individualssequenced for

12S–16S

n individualssequenced for

POMC

Los Suarez,Merida

1 Dendropsophusmeridensis

8838034.7 00 71822055.9 00 – – 1 –

Betania,Tachira

2 Dendropsophuspelidna

7824038.2 00 72825032.2 00 – – 1 –

Laureles 3 North 6824054.8 00 72826021.7 00 – – 2 2Cocuy 4 North 6824019.3 00 72826047.6 00 – – 2 2Cueva 5 North 6824015.2 00 72822055.9 00 – – 2 2Sativa Norte 6 North 6808000.2 00 72842031.3 00 – 2 – –Guina 7 North 6805051.9 00 72852045.8 00 – – 2 2Paramo

de la Rusia8 North 5859020.3 00 73805010.7 00 – 3 – –

Vado Hondo 8b North 5829052.6 00 72844052.2 00 24 – –Duitama 9 North 5849035.9 00 73802017.0 00 – 5 – –Paipa 10 North 5846054.5 00 73808001.6 00 – 3 2 2Arcabuco 11 North 5845014.2 00 73826040.3 00 – – 2 2Manzano 12 North 5845000.5 00 73810040.0 00 22 – 2* –Santa Sofıa 13 North 5842025.5 00 73836022.4 00 – 5 – –Villa de Leyva 14 North 5838055.2 00 73831056.1 00 – – 1 1Combita 15 North 5838056.8 00 72826057.9 00 – 5 – –Sutamarchan 16 North 5837044.2 00 73836041.6 00 9 – – –Chiquinquira 17 South 5836027.7 00 73846010.9 00 7 – 1 1San Carlos 18 North–South 5835053.9 00 73843000.1 00 7 – 6* –Cucaita 19 North 5832044.9 00 73827000.4 00 25 – 2* –Tunja 20 North 5832003.8 00 73822013.9 00 – 5 – –Aquitania 21 North 5831003.8 00 72852050.1 00 – 5 – –Paramo

de Guerrero22 South 581301.48 00 73859034.7 00 – 5 – –

Choconta 23 North 5810016.2 00 73840005.5 00 9 – 1* –Tenerıa 24 South 580509.80 00 73846027.1 00 8 – – –Cucunuba 25 South 5800036.7 00 73847044.8 00 11 – 3* –La Caro 26 South 4851048.9 00 74801043.6 00 – 3 – –Cota 27 South 4848034.9 00 74800036.6 00 12 – 2* –Encenillo 28 South 4847043.2 00 73854038.9 00 21 – – –Chingaza 29 South 4841025.0 00 73848023.0 00 11 – 2* –Bogota 29b South 4847009.8 00 74802030.3 00 – 16 – –La Granja 30 South 4836052.2 00 74810038.1 00 – 5 – –Guadalupe 31 South 4835044.2 00 74801049.7 00 – – 3 3Paramo Cruz

Verde32 South 4833026.0 00 74803032.7 00 – 2 – –

Las Brisas 33 South 4825058.6 00 73855013.1 00 34 – 1 1

December 2012] 525HERPETOLOGICA

Phylogenetic Analyses

Nucleotide diversity (a measure of geneticdifferences within a group of sequences; Neiand Li, 1979), was calculated with the use ofDNAsp (Rozas and Rozas, 1999). Geneticdistances between clades were estimated withthe program MEGA 5.0 (Tamura et al., 2011).To reconstruct the haplotypes for the nuclearPOMC locus, we used PHASE 2.1 (Stephensand Donnelly, 2003) as implemented inDNASP v. 5 (Librado and Rozas, 2009).Phylogenetic trees were constructed with theuse of both Bayesian and maximum likelihood(ML) methods. The Akaike Information Cri-terion (AIC) implemented in jModelTest 0.1.1(Posada, 2008) was used to select the optimalnucleotide substitution model for the Bayes-ian analysis. For each independent gene weused MrBayes 3.1.2 (Ronquist and Huelsen-beck, 2003) to generate two runs of 5,000,000generations implementing Metropolis-cou-pled Markov chain Monte Carlo (MCMC).Each run contained four incrementally heatedMarkov chains (temperature¼ 0.05), sampledevery 1000 generations, and preliminaryanalyses indicated a burn-in of 25% to beenough for discarding the first proportion ofthe Markov chain. Convergence of the tworuns was indicated when the average standarddeviation of the split frequencies was less than0.01. ML trees were generated with RAxML-VI-HPC (Stamatakis, 2006) with the use of aGTRGAMMA model of evolution (GTR þ C,following the recommendation of Stamatakis,2006) and implementing a standard bootstrapsearch with 1000 replicates.

We included in the phylogenetic analysistwo other members of the labialis group(Duellman, 1989): Dendropsophus meridensisfrom Merida (2200 m) Venezuela (12S–16Sand POMC sequenced by CEG), and Den-dropsophus pelidna from Tachira (2800 m)Venezuela (GenBank accession AY819434);no GenBank POMC sequences were availablefor D. pelidna. The most distant outgroup wasD. carnifex (12S and 16S only; GenBankAY843616; following Faivovich et al., 2005,and Wiens et al., 2010). GenBank numbers fornew sequences are JF422585–JF422622.

Acoustic data were obtained from fieldworkand morphometric data were collected frommuseum specimens. Unfortunately, no mor-

phometric data were collected from individu-als used for acoustic analysis because thepurpose of the companion study (Guarnizo etal., 2009) was to assess the relation ofecological variation to phylogeny. Therefore,each individual was analyzed either foracoustic or morphologic variation, but notboth. Table 1 includes sample sizes, localities,and analyses performed.

Acoustic Analyses

We analyzed the calls of 176 individualsfrom 12 localities across most of the geo-graphic range of the species (Fig. 1; Table 1).We included four localities in the NorthernClade, seven in the Southern Clade, and onefrom a locality with haplotypes from bothclades (San Carlos, Locality 18; see alsoGuarnizo et al., 2009). The call data fromseven localit ies were obtained fromAmezquita (2002) as averages for each popu-lation. Between 9 and 14 consecutive adver-tisement calls from each of 8–12 males perlocality were recorded by placing a SennheiserME67 directional microphone in the frontalplane of a calling male. The distance betweenthe microphone and the male was . 50 cm.The calls were recorded on TDK MA90 metalbias tapes with a Sony WM D6C ProfessionalWalkman. Each male was recorded callingfrom shallow water at the edges of ponds. Airtemperature (used to correct certain callvariables) was measured with a digital ther-mometer placed within 1 m of the male.

Terminology for call parameters followsCocroft and Ryan (1995). Male calls weredigitized at 44.5 KHz and 16 bits, and wereanalyzed with Cool Edit (Syntrillium SoftwareCorporation, USA). Oscillograms were used tomeasure call duration and number of pulsesper call; these call traits display the strongestlevels of geographic variation in D. labialis(Amezquita, 2002). Pulse rate was calculatedas the number of pulses/call duration. Toremove the effect of ambient temperature inamong-locality comparisons of call traits(range of 8.2–20.08C), we used the residualsof pulse rate on temperature regressions astemperature-independent call descriptors.

For each of the three call variables (callduration, number of pulses/call, and pulserate), we used a two-tailed t-test to test for


differences in means between the Northernand Southern Clades. The Central Clade wasincluded within the Southern Clade.

To reduce the number of variables neededto describe overall variation in acousticparameters, we performed a principal-com-ponent analysis (PCA) with the use of thecorrelation matrix. Analysis of variance (AN-OVA) was used to test for differences betweenthe PC factor scores using clade (Northern orSouthern) as the grouping variable.

It is important to control for the confound-ing effect of geographic distance on acousticdifferentiation, because phenotypic analysesthat include a spatial component may sufferfrom spatial autocorrelation (Legendre andFortin, 1989). We performed a partial Manteltest to determine if acoustic distance (esti-mated as pairwise Euclidean distances usingscores of the first principal component) wassignificantly correlated with clade correspon-dence (a binary matrix where 0 indicatesintraclade and 1 interclade comparisons),controlling for the effect of linear geographicdistance. The test was performed with theprogram zt (Bonnet and Van de Peer, 2002)using 10,000 permutations. Although someuses of partial mantel tests have beencriticized, the use of these tests for analysisof pairwise distance data is appropriate(Legendere and Fortin, 2010).

Morphometric Analyses

We performed morphometric analyses of102 individuals from 16 localities (Fig. 1,Table 1), 7 in the Southern Clade and 9 in theNorthern Clade. Measurements were takenwith a digital caliper (precision of 0.01 mm)from preserved frogs in the collections of theInstituto de Ciencias Naturales at the Uni-versidad Nacional de Colombia (ICN) and theUniversity of Kansas Biodiversity Institute(KU). We collected data from eight externalvariables following Ron et al. (2005): snout–vent length, tibia length, foot length, headlength, head width, eye diameter, tympanumdiameter, and eye–tympanum distance. Allmeasurements were taken from the right sideof the specimen and log10 transformed. Giventhe small sample size of females, only maleswere analyzed. We used individual scores onPC1 (assuming the presence of uniform

loadings of the same sign) to reflect overallsize (Bookstein, 1989). Although residualsfrom variables regressed on SVL are oftenused as size-free variables, we did not use thisapproach because these residuals often do notcompletely remove the effect of size (Book-stein et al., 1985).

To determine if morphometric variationdiffers significantly between clades, excludingthe North–South locality, we performed aPCA with the use of the correlation matrix. Atwo-tailed t-test was used to test for differ-ences between scores from the first two PCfactors grouped by clade membership. Mor-phological distance between individuals wasestimated as the pairwise Euclidean distancebetween factor scores. Partial Mantel testswere performed to detect correlation betweenmorphological distance and clade member-ship, controlling for the effect of geographicdistance between populations, as describedabove.

Climatic Analysis

An association between genetic and climaticvariation might indicate ecological mecha-nisms in the process of diversification (Ogdenand Thorpe, 2002). To determine whetherlocalities grouped by clade can be differenti-ated by climatic properties of the sampledlocalities, we used the program DIVA–GIS(Hijmans et al., 2001) to extract 30-arcsecresolution layers of annual temperature andprecipitation averages (Hijmans et al., 2005)from the localities. ANOVA was used todetermine if there were significant differencesin mean precipitation and mean temperaturevariables between the two clades. Eachlocality was used as a data point; n ¼ 19 forthe Northern Clade and n ¼ 12 for theSouthern Clade.

RESULTS

Phylogenetic Analysis

We sequenced 2369 base pairs (bp) of the12S–16S region of mitochondrial DNA and490 bp of the nuclear gene POMC. All sites inboth genes were included because no siteswere ambiguously aligned. The Bayesian(best-fit model GTRþCþ I) and ML analysisof the 12S–16S region recovered the sameclades (Southern and Northern) found by


Guarnizo et al. (2009), who used the faster-evolving mitochondrial genes cytochrome band cytochrome oxidase 1 (Fig. 2). Thespecies D. meridensis, D. pelidna, and indi-viduals from the Serranıa del Cocuy, whichlies north of the Northern Clade (Localities 3,4, and 5; Fig. 1). in Colombia formed a thirdclade (here termed the meridensis Clade).These three clades are individually wellsupported (. 80% bootstrap), however, thebranch that groups the Northern and South-ern Clade has a bootstrap value of 55% Themaximum p distance (uncorrected) betweenthe Northern and Southern Clades (12S–16S)was 2.7%, and that between the meridensis-Northern and meridensis-Southern Cladeswere 2.5% and 2.2%, respectively. TheSouthern Clade contains haplotypes fromlocalities southwest of Chiquinquira (Locality17, Fig. 1), and the Northern Clade includeslocalities northeast of the city. The meridensisClade, as mentioned earlier, included individ-uals from the Venezuelan Andes and theSerranıa del Cocuy in Colombia. POMC alsorecovered the Southern Clade (Fig. 2), butwas unable to resolve the Northern andmeridensis Clades.

Guarnizo et al. (2009) recovered a centralclade. In this analysis, which includes addi-tional data and localities, the distinctiveness of

this clade is less apparent, and we havesubsumed it into the Southern Clade.

Acoustic Analyses

Call duration was highly correlated withnumber of pulses per call (Pearson’s r¼ 0.78,P , 0.0001, n¼176 individuals). Therefore, toavoid redundancies in later analyses we usedonly pulse rate and pulses per call. Frogscalling at higher temperatures produced callswith slightly fewer pulses per call (linearregression, r2 ¼ 0.06, F ¼ 11.3, P ¼ 0.009, n¼ 186 individuals) produced at a higher pulserate (r2 ¼ 0.21, F ¼ 50.0, P , 0.0001). Toremove the effect of ambient temperature inamong-locality comparisons of call features(range of 8.2–20.08C), we used the residuals ofthese regressions as temperature-independentcall descriptors for subsequent analyses.

Basic statistics for the three call variablesare summarized in Table 3. The analysis ofcovariance (ANCOVA) between the Northernand Southern Clades (using temperature as acovariate) showed that call duration wassignificantly different (F ¼ 41.12, P , 0.001,n ¼ 21 for Northern, n ¼ 21 for Southern);number of pulses/call was significantly differ-ent (F¼ 17.30, P , 0.001), and pulse rate wassignificantly different (F ¼ 33.66, P , 0.001).

Call data for the two clades were pooled forthe PCA. Two principal components summa-rized more than 99% of the acoustic variance(Table 2). The highest loading for PC1 waspulses per call, and the highest loading forPC2 was pulse rate. After the effects of thetemperature were statistically controlled, wefound significant differences between the twoclades when using the PC1 as a descriptor ofthe acoustic variation (ANOVA, F ¼ 26.67, P, 0.001). Also, we found a significantcorrelation between acoustic (Euclidean) dis-tance and clade assignment when controllingfor geographic distance (partial Mantel test, r¼ 0.19, P , 0.001), which indicates that thevariation in calls is dependent on clade,independent of geographic distance.

Morphometric Analyses

Two components with eigenvalues higherthan 0.9 were extracted from the PCA andaccount for 71% of the variance. The highestloadings for PC1 were SVL and foot length,

TABLE 2.—Summary of the principal-component analysison the correlation matrix of acoustic and morphologicalcharacters of Dendropsophus labialis. All acoustic vari-

ables are corrected for temperature when appropriate.

Factor loadings

PC1 PC2

Acoustics

Eigenvalue 2.325 0.658Percent of variation 77.500 21.950Pulses per call 0.651 –Pulse rate – �0.793

Morphology

Eigenvalue 4.758 0.919Percen of variation 59.471 11.488Log snout–vent length 0.916 0.146Log foot length 0.904 0.020Log tibia length 0.887 �0.079Log head width 0.814 �0.031Log head length 0.800 �0.118Log eye–tympanum 0.596 �0.506Log eye diameter 0.479 0.785Log tympanum diameter 0.654 �0.056


whereas the highest loadings for PC2 were eyediameter, eye–tympanum distance, and tym-panum diameter (Table 2). We found broadoverlap in morphological space (Fig. 3) and nostatistical differences among males betweenthe clades (PC1: t¼�0.93, P¼ 0.36; PC2: t¼0.31, P ¼ 0.76). We did not find a significantcorrelation between morphological (Euclide-an) distance and clade assignment whencontrolling for geographic distance (partialMantel test, r ¼ 0.07, P ¼ 0.20), whichindicates that morphological variation is notdependent on clade origin even after control-ling for geographic distance.

Climatic Analyses

We did not find statistical differences inannual averages of temperature and precipita-tion across the sampled localities between thetwo clades (ANOVA; temperature: F¼ 1.07, P¼ 0.32; precipitation: F¼ 2.22, P¼ 0.17).

DISCUSSION

Genealogical History of D. labialis

The tree topology obtained with 12S–16Sindicates D. labialis is composed of two highlydivergent clades distributed across the East-ern Cordillera in Colombia, confirming theresults of Guarnizo et al. (2009), who usedfaster-evolving mitochondrial genes. We alsofound a third clade including D. meridensis,

D. pelidna (Venezuela), and individuals fromSerranıa del Cocuy in Colombia. POMC isunable to resolve the polytomy that includesthe Northern and meridensis clades. This issimilar to patterns expected in recentlydiverged sister species (Guayasamın et al.,2008; Gvozdık et al., 2010; Rodrıguez et al.,2010) where there is not enough time orsufficiently small effective population size ofthe nuclear genes to counteract incompletelineage sorting (Moore, 1995).

Given the geographic distribution of thesampled individuals, it might first appearthat the Southern and Northern Cladesresult from the sampling gap between them(Fig. 1). However, the relationship betweengeographic and genetic distances (Fig. 4)indicates that this gap does not explain thedivergence between the two clades, becauselarge interclade genetic distances (. 2%)completely overlap low intraclade geneticdistances (, 1%) along the geographicdistance axis. A meaningful effect of aspatial sampling gap on genetic divergencewould be indicated if large intercladegenetic distances did not overlap with lowintraclade genetic distances along this axis.

Phenotypic Divergence Between the Northernand Southern Clades

We found that acoustic (temporal callcharacteristics) but not morphological traits

TABLE 3.—Acoustic and morphological measurements of Dendropsophus labialis and Dendropsophus luddeckei. Samplesizes (n, below in table) are given in number of individuals distributed in seven localities. All morphometric data are in

millimeters. SD¼ standard deviation.

Dendropsophus labialis (Southern Clade) Dendropsophus luddeckei (Northern Clade)

Mean (n ¼ 105) SD Range Mean (n ¼ 66) SD Range

Call duration (s) 197.4 23.94 152–252.1 140.5 14.65 108.2�212.7Pulse rate/s 75.2 6.38 61.8–88.2 92.9 2.96 71.2–106.3Pulses per call 14.5 0.74 13.4–17.2 12.8 0.98 11.1–15.6

Mean (n ¼ 31) SD Range Mean (n ¼ 57) SD Range

Snout–vent length 46.2 0.96 32.6–61.0 38.6 0.69 26.4–50.0Tibia length 24.6 0.92 16.4–55.4 19.6 0.33 13.7–25.4Foot length 21.8 0.51 14.7–30.4 18.0 0.38 11.2–23.5Head length 13.8 0.25 9.9–17.9 11.9 0.21 8.5–18.0Head width 13.7 0.24 10.2�17.9 11.5 0.19 8.4–14.8Interorbital distance 11.8 0.22 8.8–15.8 10.3 0.16 7.5–13.5Eyes–nostril distance 3.6 0.06 2.8–4.7 3.0 0.05 2.1–4.1Eye diameter 4.6 0.05 3.7–5.8 3.8 0.04 3.0–4.9Tympanum diameter 2.8 0.06 2.1–4.1 2.3 0.05 1.6–4.3Eye–tympanum distance 2.8 0.06 1.9–4.0 2.0 0.05 1.1–3.0


differ statistically when individuals weregrouped according to their clade membership.We found a significant difference in acoustictraits between individuals grouped by clade.However, we did not find a differencebetween clades in morphometric traits. As-suming that the mtDNA tree reflects popula-tion divergence, two alternative hypothesesare: Either acoustic traits diverge according tomtDNA history and morphological traits donot exhibit divergence (e.g., they are con-strained by selection), or both acoustic andmorphological traits track mtDNA history, butthe rate of acoustic divergence is faster. Thus,the two clades are distinguishable based onacoustic traits but not by morphology. Both

character complexes suggest that the likelyscenario for D. labialis is that morphology andacoustic variation covary with mtDNA popu-lation history, but morphology is diverging at aslower rate than acoustic traits.

For frogs, it is well established in somespecies that morphological traits underesti-mate the amount of divergence and/or repro-ductive isolation between closely relatedpopulations or cryptic species, whether thereis character displacement (Blair, 1955; Lit-tlejohn and Martin, 1964) or not (Padial et al.,2009). Thus, morphological traits seem toprovide a conservative delineation of speciesboundaries. However, it can also be arguedthat morphological characters appear to be

FIG. 2.—Maximum-likelihood gene trees with the mitochondrial 12S–16S (left) and the nuclear proopiomelanocortinA genes (right). Numbers above the branches are Bayesian posterior probabilities and below are bootstrap supportvalues. Numbers after locality names indicate the specimen identifier number. Numbers in parentheses correspond tothe map codes in Table 1. Grey boxes delineate the Dendropsophus meridensis Clade (top), Dendropsophus labialisNorthern Clade (middle), and D. labialis Southern Clade (bottom).


conservative simply because they are notthoroughly studied. For example, with theuse of mtDNA, Elmer and Cannatella (2008)delineated three new species formerly includ-ed under Pristimantis ‘‘ockendeni.’’ Given thephylogeny, they were able a posteriori toidentify phenotypic traits that distinguishedthe three species.

Multitrait divergence as evidence of specia-tion.—Dendropsophus labialis exhibits anabrupt (~30 km) change in haplotype fre-quencies and acoustic traits (Fig. 5), whichsuggests a secondary contact zone (Barton andHewitt, 1985). The species also displayssignificant differences in temporal call char-acters (particularly pulse rate) between theSouthern Clade and the Northern Clade. This

FIG. 3.—Principal-component analysis plot that summarizes variation in call parameters (top) and variation in externalmorphology (bottom) among frogs grouped in two clades. White dots: Northern Clade (Dendropsophus luddeckei),black dots: Southern Clade (Dendropsophus labialis), white triangles: sympatric occurrence.


agreement between two independent charac-ter systems, temporal acoustic signals—crucialcues in species recognition—and mitochon-drial genes, indicates that the two clades aremorphologically cryptic species with para-patric distributions. For this reason wedesignate the Northern Clade as a new species(see species description below). Given that theholotype of D. labialis (ZMB 4913) geograph-ically corresponds to the Southern Clade(‘‘vicinity of Bogota’’ in Cundinamarca), thename D. labialis remains associated with theSouthern Clade.

Are the Northern and Southern CladesExchanging Alleles?

Our evidence indicates that there is nomovement of mitochondrial alleles either westor east of the locality of Chiquinquira(Locality 17; Fig. 1). In other words, thereare no mitochondrial haplotypes from theSouthern Clade within the geographic rangeof the Northern Clade and vice versa.

Fifty-nine percent of the individuals wereheterozygous for POMC. After phasing thislocus, we found nine alleles restricted to theNorthern Clade and five alleles restricted tothe Southern Clade. However, two heterozy-gous individuals contained alleles from both

clades, suggesting limited hybridization.These two individuals are from Villa de Leyva(Locality 14) and Arcabuco (Locality 11; Fig.1), which are within 30 km of the regionwhere the two clades meet (the contact zone;Fig. 1). Interestingly, we found that the twohybrids had nuclear alleles from both clades,but only mitochondrial haplotypes from theNorthern Clade. In other words, these twohybrids seem to have Northern Clade mothersand Southern Clade fathers.

Based on these very limited data, we offersome speculative remarks; these are meant topoint out areas of needed research rather thanto suggest definitive answers to the questionsthat are posed.

Are hybrids under a selective disadvantage?We found only 2 hybrid individuals out of 18sequenced for both 16S and POMC, suggest-ing that they are uncommon. Barton andHewitt (1985) indicate that it is hard todistinguish whether parapatric forms remaindistinct because they are adapted to differentenvironments or because hybrids are selectedagainst. We did not find annual temperatureor precipitation differences between the twospecies at a macrogeographical scale (althoughour data are admittedly coarse). If this trulyindicates a lack of local ecological differences

FIG. 4.—Scatter plot depicting geographic vs. genetic distances in Dendropsophus labialis using 12S–16S. Black dotsindicate intraclade (Southern vs. Southern and Northern vs. Northern) and white dots indicate interclade (Southern vs.Northern) pairwise comparisons of individuals in Fig. 2. None of the individuals of the meridensis clade was included.


FIG. 5.—Variation of the Southern and Northern Clades across the contact zone for (A) pulse rate, (B) pulses per call,and (C) haplotype frequencies, based on the populations sampled by Guarnizo et al. (2009). The dotted line correspondsto the contact zone that passes through the sympatric locality San Carlos (map code 18). The negative values in the x-axiscorrespond to the region southwest of the contact zone (Southern Clade) and the positive values to the region northeastof the contact zone (Northern Clade). From left to right, the map codes in A and B are 27, 33, 28 29, 25, 24, 17 18, 23,16, 19, and 12.


between the two regions, then perhapshybrids are uncommon as a result of aselective disadvantage.

Hybrids may be subject to postzygoticbarriers that reduce fitness. Ulloa (2003)found that Southern Clade individuals matewith Northern Clade individuals under labo-ratory conditions. To test for postzygoticbarriers, she measured larval survival (% ofliving larvae per day) of crosses between andwithin the two clades. Although she did notfind a significant difference (Mann–Whitney:Z ¼ �1.46, P ¼ 0.14, n ¼ 11 clutches;Appendix), she found that the median hybridlarval survival between the two clades waslower than the median larval survival in theintraclade crosses.

If hybrid individuals are being selectedagainst, we would expect to find acousticcharacter displacement preventing maladap-tive hybridization. Our very limited dataindicate that one locality (San Carlos, Locality18; Guarnizo et al., 2009) contains mtDNAhaplotypes of both clades in sympatry. How-ever, individuals from this locality show noevidence of character displacement in callparameters; they are located clearly within theprincipal-component space delimited by theNorthern and Southern Clades (white trian-gles in Fig. 3, top graph). Hybrid inviabilitybetween sister species calling sympatricallydoes not mean, however, that acoustic char-acter displacement is occurring. Niche parti-tioning (spatial or temporal) might preventfemales of one species from syntopic contactwith the males of the other species. Our datado not contain fine-grained spatial or temporalacoustic information; therefore we cannotmake firm conclusions.

As an alternative, behavioral prezygoticmechanisms such as assortative mating mightexplain rarity of hybrids, but we do not havedata needed to make firm conclusions. A morecomplete description of the genetic variationacross the contact zone, together with exper-iments that may reveal an effect of prezygoticbarriers (such as phonotaxis experiments), orthe effect of postzygotic barriers (proportionof fertilized eggs in interspecific crosses andfitness assessment of the F1 and F2 genera-tions), are needed to characterize the mech-anisms that prevent exchange of alleles.

Comments on phylogenetic relationshipsand biogeography of the meridensis clade.—We found surprisingly low 12S–16S se-quence divergence (maximum uncorrectedpairwise difference of 0.9%) between D.meridensis and D. pelidna, compared to2.7% between D. luddeckei and D. labialis(Fig. 6). Given that the tissue samples ofboth species are from localities relativelyclose to their type localities, it seems likelythat the sequences are correctly associatedwith their nominal species. This low level ofdivergence suggests that D. pelidna and D.meridensis are conspecific. If so, D. pelidnais a junior subjective synonym of D.meridensis. However, we do not formallypropose this taxonomic reassignment be-cause our sample size is low and other datasuch as advertisement calls and morphomet-ric analysis are highly desirable in makingthese assessments.

If D. meridensis and D. pelidna areconspecific, then D. meridensis sensu latooccurs on both sides of the point where theEastern Andes reaches its lowest elevation(Tachira Depression: 900 m), which is belowthe current altitudinal range of the species(1200–2400 m; La Marca, 2004). A possibleexplanation for such a distribution is thatoriginally D. meridensis was found only westof the Tachira depression. Because ofPleistocene glaciations, populations of D.meridensis might have been pushed down-wards to warmer elevations, allowing themto cross the altitudinal barrier. Followingthis, interglacial warmer periods might have,again, displaced populations upward andisolated a small relict of D. meridensis inthe Merida Andes in Venezuela. This wouldexplain why D. meridensis has a restricteddistribution and D. pelidna is very abundant(La Marca, 2004). However, this scenario isspeculative.

Equally interesting is the fact that somespecimens (Laureles 08 at Locality 3, Cueva14 at Locality 5, and Cocuy 239 and 255 atLocality 4; Figs. 1 and 2) were syntopic withthe Northern Clade (Laureles 09 and Cueva07, both from Locality 3; Figs. 1 and 2), butclustered within the D. meridensis clade.However, we have not referred thesespecimens to D. meridensis. Comparing


the morphology between the individuals ofthe D. meridensis and D. luddeckei cladesfrom the syntopic locality would be inter-esting and informative; unfortunately, we donot have access to the specimens of thislocality. If further studies determine that allsamples in the meridensis clade prove to bea single species, this would indicate that thedistribution of D. meridensis extends fromthe Merida Andes in Venezuela to theSerranıa del Cocuy in Colombia, crossingthe Tachira Depression.

This study provides an additional exampleof a widespread frog species, abundant inurban areas, that was delimited by the lack ofmorphometric divergence among populations(presumably because morphological traitsevolve slowly), but is found to have otheraspects of the phenotype (acoustic traits) andgenetic diversity that reveal cryptic species.

SPECIES ACCOUNT

Dendropsophus luddeckei sp. nov.

Holotype.—Deposited at Museo de HistoriaNatural Andes, Universidad de los Andes,Bogota, Colombia. Catalogue number AN-DES-A 0603 (Fig. 7). An adult male fromSotaquira, (05845 002.8 00N; 73816 039.7 00W),2765 m elevation, Departamento de Boyaca,Colombia, collected 16 June 2010 by C.Escallon.

Paratypes.—57 (Male adult: ANDES-A0604, collected at the type locality(05845002.8 00N; 73816039.7 00W) with the holo-type by C. Escallon, 33 adults from thecollection of Instituto de Ciencias Naturales,Universidad Nacional de Colombia: ICN5588, 5658, 33263, 5661, 5686: Aquitania,Departamento de Boyaca. ICN 33615, 33626,33633, 33618, 33629: Combita, Boyaca. ICN12585, 12563, 12584, 12568, 12585: Duitama,Boyaca. ICN 52895, 52897, 52896: Paipa,Boyaca. ICN 4423, 959, 4424: Paramo de laRusia, Boyaca. ICN 52941, 52944, 52945,52942, 52943: Santa Sofia, Boyaca. ICN 5856,5810: Sativa Norte, Boyaca. ICN 10296,10294, 33638, 10299, 10297: Tunja, Boyaca;and 23 adults from the collection of theUniversity of Kansas Biodiversity Institute:KU 169521–169546: Vado Hondo, Boyaca.

Diagnosis.—Dendropsophus luddeckei canbe acoustically differentiated from D. labialis.It has calls of significantly shorter durationthan D. labialis, higher pulse rate, and fewerpulses per call (see statistics in text and Table3). At the molecular level, D. luddeckei differsfrom D. labialis by 13 fixed nucleotidedifferences in 12S–16S, 30 in cytochromeoxidase I (CO1), 27 in cytochrome b (cytb),and 5 in POMC (Guarnizo et al., 2009).

Description of the holotype.—An adultmale, SVL 34.7 mm; body of medium width;head about as wide as body; head as wide aslong, HW/HL 1.06, widest at level of

FIG. 6.—Elevation profiles and the level of sequence divergence (for the 12S–16S genes) within the species pairsDendropsophus labialis (Southern Clade)–Dendropsophus luddeckei (Northern Clade) and Dendropsophus pelidna–Dendropsophus meridensis. Gray areas depict the presumed elevation range of each species based on their geographicdistribution. The arrow points to the Tachira Depression, which reaches elevations (900 m) below the minimumelevation registered for any of the four species.


tympanum; head nearly rounded in dorsalview, snout rounded in lateral profile; eye–nostril distance shorter than diameter of eye,E–N/ED 0.69; canthus rostralis concave indorsal view, rounded in section; loreal regionflat; lips thin, not flared; internarial region

slightly concave; nostrils slightly protuberant,directed anterolaterally. Interorbital area flat,IOD/ED 2.11, IOD/HW 0.87; eyes of mediumsize and moderately protuberant, ED/HL 0.43,ED/HW 0.41. Palpebral membrane transpar-ent. Supratympanic fold conspicuous, slightly

FIG. 7.—Right foot (A), and right hand (B) of paratype (KU 169536; ventral view), oscillogram (C), sonogram (D),holotype (ANDES-A 0603) (E), and typical habitat (F), of Dendropsophus luddeckei. Pa ¼ pascal, KHz ¼ kilohertz.Photos (A) and (B) by A. Berrıo, (E) by C. Escallon, and (F) by C. Guarnizo.


obscuring the posteroventral edge of tympa-num; tympanic annulus contacting supratym-panic fold; tympanum moderately large, distinct,directed dorsolaterally and separated from eyeby a distance equal to half of the width of thetympanum; diameter of tympanum about one-half diameter of eye, diameter TD/ED 0.55.Arm short and robust, lacking an axillarymembrane; ulnar fold very weak, consisting offew tubercles, extending from base of handalmost to elbow; fingers short with rounded discsslightly wider than finger; relative length offingers I, II, IV, III; fingers bearing lateralfringes; subarticular tubercles slightly distinct,low in profile, not bifid except for distalsubarticular tubercle of digit IV; subarticulartubercles rounded in ventral view, distal sub-articular tubercles more prominent than proxi-mal tubercles, most prominent on digit IV;supernumerary tubercles small; palmar tuber-cles indistinct; inner metacarpal tubercle low,small, flat; outer metacarpal tubercle smaller;nuptial pad large and prominent, stronglydelineated by surrounding skin, extending frombase of thumb, narrowing toward distal sub-articular tubercle; webbing absent betweenfingers I and II; webbing basal between fingersII and III, and III and IV. Hind limbs ofmoderate length; shank robust; TL/SVL 0.52;heels not in contact when hind limbs are heldperpendicular; inner tarsal fold distinct, extend-ing from base of inner metatarsal tuberclealmost to heel; outer tarsal fold absent; calcarsand heel tubercles absent; toes short, bearingsmall round discs, slightly smaller than discs onfingers, approximately same width as toes,relative lengths of toes I, II, V, III, IV; toesbearing lateral fringes; subarticular tuberclesmoderately large, round in ventral view, round-ed in profile; distal subarticular tubercles of toesIV and V bifid, supernumerary tubercles absent;outer metatarsal tubercle small, indistinct; innermetatarsal tubercle distinct, elliptical in ventralview, flat in profile, in contact with inner tarsalfold and medial fringe of first toe; toes almostfully webbed, webbing moderately thick; web-bing extensive between toes III, IV, and V. Skinof all dorsal surfaces smooth; skin of flanks withscattered low warts; skin in axilla, groin, and onanterior surface of thighs smooth; skin of throat,chest, belly, and ventral surfaces of thighscoarsely areolate, skin of tibia and concealed

surface of tarsus smooth. Cloacal opening withslight dorsal flap, directed posteriorly at upperlevel of thighs; cloacal sheath distinct; cloacaltubercles slightly distinct, scattered. Tonguecordiform, filling buccal cavity, barely free onposterior end, posterior margin slightly notched;dentigerous processes of vomers prominent, intwo slightly angled series, each bearing three–four teeth, narrowly separated medially; choa-nae of moderate size, lateral to and narrowlyseparated from vomers; vocal slits moderatelylong, extending from midlength of tongue,almost reaching to angle of jaws; vocal sac single,median, subgular covering almost all of the chin.

Measurements of the holotype (mm).—Snout–vent length 34.7, tibia length 18.2,foot length 14.3, head length 9.8, head width10.4, interorbital distance 9.1, eye–nostrildistance 3.0, eye diameter 4.3, tympanumdiameter 2.4, eye–tympanum distance 1.8.Measurements of the paratypes are given inTable 3.

Coloration of holotype in life.—Dorsumbright green; throat pale green, chest andbelly grey with multiple pale blue spots;extremities with pale grey and fewer paleblue spots; iris golden.

Coloration of holotype in preservative.—Gray dorsum with faint black spots. Ventralpale gray with cream spots. Venter with palecream spots. Legs display similar color asarms, having fewer cream spots than chest andthroat.

Variation.—In the paratype series, indi-viduals from higher elevations (Aquitaniaand Paramo de la Rusia) are larger, onaverage, than individuals from lower eleva-tions, replicating the phenomenon observedin the sister species D. labialis, where thereis a strong correlation between elevation andbody size (Amezquita, 1999). The smallsample size of females prevents analysis ofsize dimorphism; however, the availableadult female specimens are larger thanmales. One from Sativanorte is 50.0 mm,and from Paramo de la Rusia 49.6 mm).There is geographic variation within andbetween populations in the dorsal colorationof individuals in life, which may be variousshades of pale green, grading into darkbrown. Dorsal coloration is lost in preservedspecimens, but not the pattern of dorsal


markings, which display extensive variationamong individuals.

Distribution and ecology.—Dendropso-phus luddeckei is a highland Andean speciesfound northeast of the cities Chiquinquira,Boyaca (5810 016.21 00N, 73840 005.52 00W; da-tum ¼WGS84) and Choconta, Cundinamar-ca (5810 016.21 00N, 73840 005.52 00W), reachingDepartamento de Norte de Santander inColombia. One of the few hylid speciesknown from high elevations, its elevationalrange goes from 2000 m (Villa de Leyva,Boyaca) to 4100 m (Serranıa del Cocuy,Boyaca). It is abundant and easy to find neartemporary or permanent ponds, as well asvery humid places in urban areas. Maleshave been seen calling near large roads orhighly populated areas. Even though D.luddeckei has nocturnal reproductive activ-ity, it is a heliothermic species and thermo-regulates in open areas during the day.

Etymology.—This species epithet is apatronym in recognition of Professor HorstLuddecke Dr. Rer. Nat. For more than 30yr, he produced important contributions tothe areas of behavior, physiology, andbiogeography of high Andean anurans ofColombia, especially D. labialis. He encour-aged and served as a model to manygenerations of Colombian biologists.

All nominal species that are now juniorsubjective synonyms of D. labialis (Peters)—Hyla creolica Werner 1899, Hyla servalinaWerner 1899, Hyla vilsoniana Cope 1899,Hyla gularis Werner 1916, Hyla vilsonianakrausi Hellmich 1940, Hyla labialis labialis(see Cochran and Goin, 1970), and Hylalabialis krausi (see Cochran and Goin,1970)—were described from holotypes thatgeographically correspond to the SouthernClade (Cochran and Goin, 1970). Nonominal species are based on holotypesfrom within the range of the NorthernClade. There exists the possibility that someof the holotypes that are reported tooriginate from Bogota in fact were collectedfrom localities further north, but there is nodirect evidence for this.

Acknowledgments.—Funding was provided to C. Guar-nizo by the L. I. Stengl and Hamilton GraduateFellowships from University of Texas at Austin. Fundingto D. Cannatella was provided by National ScienceFoundation grant EF 0334952. We thank the people

from the Grupo de Ecofisiologıa del Comportamiento yHerpetologıa (GECOH) at the Universidad de los Andesfor assistance with fieldwork, especial O. Ramos, O.Ballestas, A. Delgadillo, R. Guerrero, S. Reyes, and M. P.Quintero. We also thank M.C. Caro for helping withlogistics during the fieldwork. We thank J. D. Lynch,Instituto de Ciencias Naturales, Universidad Nacional deColombia, and R. Brown, University of Kansas Biodiver-sity Institute, for access to the specimens. The Canna-tella–Hillis–Bull lab members, especially P. Salerno,provided helpful comments on the early versions of themanuscript. Two anonymous reviewers helped to improvethe final version. Samples of Dendropsophus labialis wereobtained under permit number 01187 (15 November2007) issued to C. Guarnizo by the Ministerio de MedioAmbiente, Vivienda, y Desarrollo Territorial, Colombia.We especially thank C. Ulloa for kindly providing thefigure in the Appendix, J. Wiens for providing the tissue ofDendropsophus pelidna, and C. Barrio for providingtissue of Dendropsophus meridensis, collected withpermit number 2231 to Fundacion AndigenA by theMinisterio del Ambiente de Venezuela.

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Accepted: 15 July 2012Associate Editor: Christopher Raxworthy

APPENDIX

Box plot showing the effect of type of cross on larval survival in Dendropsophus labialis. Figure obtained from Ulloa(2003) with her permission. The localities from the Northern Clade were 14 and 19 (Table 1). The localities from theSouthern Clade were 25, 27 (Table 1) and another two that do not appear in Table 1: Suesca (05800030.60 00N,73846037.92 00W) and Mondonedo (0484100.38 00N, 74815049.00 00W). The horizontal line within the gray boxes indicatesthe median. Above and below the median are the upper and lower quartiles, respectively. The error bars indicate thesmallest and largest observations, and the circles indicate outliers.


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