COLOR DIFFERENCES AMONG CLOSELY RELATED SPECIES OF RED-BREASTED MEADOWLARKS (STURNELLA

COLOR DIFFERENCES AMONG CLOSELY RELATED SPECIESOF RED-BREASTED MEADOWLARKS (STURNELLA)

PILAR BENITES1, PABLO L. TUBARO

1,4, DARIO A. LIJTMAER1, STEPHEN C. LOUGHEED

2,AND MUIR D. EATON

3

1Division de Ornitologıa, Museo Argentino de Ciencias Naturales ‘‘Bernardino Rivadavia,’’ Av. Angel Gallardo470, C1405DJR, Buenos Aires, Argentina

2Department of Biology, Queen’s University, Kingston, Ontario, K7L 3N6, Canada3University of Kansas, Natural History Museum, 1345 Jayhawk Blvd., Lawrence, KS 66045-7561

Abstract. Interspecific differences in sexually selected traits may be important formaintaining reproductive isolation among closely related species living in sympatry. Wepresent the first study of plumage color differences among males of partially sympatricspecies of South American red-breasted meadowlarks—the White-browed Blackbird(Sturnella superciliaris), the Pampas Meadowlark (S. defilippii), and the Long-tailedMeadowlark (S. loyca)—using reflectance spectrophotometry and the avian visual modelof Vorobyev and Osorio (1998). Reflectance values of sexually dichromatic red plumagepatches were measured on study skins. Total reflectance, reflectance in the shortwavelength part of the spectrum, and several measures of spectral shape were extracteddirectly from the spectra. Our analyses revealed that S. loyca and S. defilippii were brighterand had higher reflectance in the short wavelength part of the spectrum than S.superciliaris. Minimum reflectance was located at higher wavelengths in breeding than innonbreeding plumage. Interspecific distances in avian visual space obtained from theVorobyev and Osorio (1998) model were considerably higher than the threshold value forcolor discrimination, indicating that the differences found are also detectable by birds.Taken together, these results show that the red plumage patches of these three speciespresent significant color differences throughout the year, not only in the visible but also inthe UV part of the spectrum.

Key words: male plumage, meadowlarks, reflectance, spectrophotometry, Sturnella,sympatry.

Diferencias de Coloracion entre Especies Cercanamente Emparentadas de Pechos

Colorados (Sturnella)

Resumen. Las diferencias interespecıficas en caracteres sexualmente seleccionadospueden ser importantes para mantener el aislamiento reproductivo entre especiessimpatricas cercanamente emparentadas. Este es el primer estudio de las diferencias decoloracion de plumaje entre machos de especies parcialmente simpatricas de pechoscolorados sudamericanos (Sturnella superciliaris, S. defilippii, y S. loyca) usandoespectrofotometrıa de reflectancia y el modelo de percepcion visual de Vorobyev y Osorio(1998). Los valores de reflectancia de los parches rojos sexualmente dimorficos fueronmedidos en pieles de estudio. La reflectancia total, la reflectancia en la porcion de ondacorta del espectro y varias medidas de forma espectral fueron obtenidas directamente delos espectros. Nuestros analisis revelaron que S. loyca y S. defilippii fueron mas brillantes ytuvieron mayor reflectancia en la region de onda corta del espectro que S. superciliaris. Laposicion de reflectancia mınima se localizo a longitudes de onda mayores en el plumajereproductivo comparado con el no reproductivo. Las distancias interespecıficas en elespacio visual aviano obtenidas del modelo de Vorobyev y Osorio (1998) fueronconsiderablemente mayores que el valor umbral de discriminacion de color, indicando queestas diferencias son tambien detectables por las aves. Tomados en su conjunto, estosresultados muestran que los parches de plumaje rojo de estas tres especies presentandiferencias significativas en la coloracion durante todo el ano tanto en la porcion visibledel espectro como en la UV.

Manuscript received 5 July 2006; accepted 16 March 2007.4 Corresponding author: E-mail: [email protected]

The Condor 109:605–616# The Cooper Ornithological Society 2007

[605]

INTRODUCTION

A central endeavor in evolutionary biology isthe investigation of mechanisms of reproductiveisolation between closely related species. Theo-retical arguments indicate that precopulatorymechanisms, such as visual signals employed inspecies recognition and sexual selection, shouldbe among the most important reproductivebarriers (Coyne and Orr 2004). In particular,differences in sexual signals among morpholog-ically similar, closely related species should begreater in sympatry, where chances of confu-sion and hybridization are at a maximumcompared to allopatry (Miller 1982).

The genus Sturnella is comprised of sevenspecies living in open grasslands or open steppesof the Americas (Short 1968). Sturnella speciesfall into two broad categories with respect tobreast coloration: the yellow-breasted meadow-larks (Sturnella magna [Eastern Meadowlark]and S. neglecta [Western Meadowlark]) and thered-breasted meadowlarks, comprised of the fiveremaining species (S. militaris [Red-breastedBlackbird], S. superciliaris [White-browed Black-bird], S. bellicosa [Peruvian Meadowlark], S.defilppii [Pampas Meadowlark], and S. loyca[Long-tailed Meadowlark]). In this genus thereare several pairs or trios of morphologicallysimilar (sibling) species: S. magna and S. neglecta;S. bellicosa, S. defilippii, and S. loyca; and S.militaris and S. superciliaris. Some of thesespecies are sympatric over at least part of theirranges. This is true for three Argentine Sturnellaspecies, S. superciliaris, S. defilippii, and S. loyca,which coexist in the southern pampas.

Red-breasted meadowlarks are sexually di-morphic in color, mainly in the extent andintensity of the red patches located on thethroat, chest, belly, and marginal wing coverts(Gochfeld 1975). This, together with the use ofa flight display by most males of the species inthe red-breasted group, suggests that theseplumage characters might be used as speciesrecognition signals (Short 1968). If this is thecase, however, these signals must be differentamong sympatric species.

Because of the striking differences in visualsystems and color perception between humansand birds (Bennett and Cuthill 1994, Bowmakeret al. 1997, Cuthill et al. 2000a, 2000b), humandescription of plumage color is inadequate forthe study of many biological questions. To

overcome this problem, several studies haveused reflectance spectrophotometry to assessdifferences among closely related species ofbirds, such as tanagers (Johnson and Brush1972, Bleiweiss 2004a, 2005). Here we presentthe first quantitative and objective study ofcolor differences among males of the threepartially sympatric species of meadowlarksliving in the Argentine pampas by means ofreflectance spectrophotometry. In particular,we test for the existence of consistent differ-ences in the red patches of plumage that mightbe used as species recognition signals. Inaddition, we examine seasonal changes in thered-colored patches and discuss this variation inrelation to the pattern of molt and wear of thefeathers. We present the results of the re-flectance analysis alone and in combinationwith the avian visual perceptual model de-veloped by Vorobyev and Osorio (1998). Toour knowledge, this is the first study to usevisual modeling methods to quantify differencesin plumage color among bird species and itsseasonal variation. Thus, plumage is assessedindependently of human vision and differencesin plumage color are evaluated in the way inwhich birds should perceive them.

METHODS

DATA COLLECTION

This study is based on study skins deposited atthe Museo Argentino de Ciencias Naturales‘‘Bernardino Rivadavia.’’ Only specimens inperfect condition (without evidence of degra-dation due to poor storage conditions or defectsin preparation) and with complete informationon sex, locality, and date of collection wereincluded, totaling 12 S. superciliaris (seven inbreeding and five in nonbreeding plumage), 16S. defilippii (10 in breeding and six in non-breeding plumage), and 15 S. loyca (eight inbreeding and seven in nonbreeding plumage)adult males. We considered birds collectedbetween September and January to be inbreeding plumage, and those collected betweenFebruary and August to be in nonbreedingplumage. The collection dates of the individualsranged from 1896 to 1979 (mean collection year6 standard deviation for S. superciliaris 5 19316 20, for S. defilippii 5 1924 6 13, and for S.loyca 5 1940 6 25). For each study skin, wemeasured four of the most conspicuous red-

606 PILAR BENITES ET AL.

colored patches: the throat, chest, belly, andmarginal wing coverts (hereafter referred to ascoverts). These regions were chosen for threereasons: 1) they are the areas that are moreapparently dimorphic in color, suggesting thatthey could be involved in species recognition orsexual selection, 2) they appear homogeneous-ly colored and thus are amenable to spectro-photometric analysis, and 3) they are ofsufficient size to allow at least one reflectancemeasure. Three measurements were taken fromthe chest and belly, one at the center and oneto each side of the midline, and the mean ofthese measurements was used in all analyses.The coverts of both wings and the throat weremeasured only once; the mean of the right andleft wing was used in all analyses. Becausesome S. defilippii individuals were preparedwith the head pointing ventrally, we wereunable to obtain a throat measurement foreight individuals (seven in breeding and one innonbreeding plumage).

REFLECTANCE SPECTROPHOTOMETRY

Plumage reflectance was measured using anOcean Optics 2000 (Ocean Optics, Inc., Dune-din, Florida) spectrophotometer with a PX-2pulsed xenon light source (effective range ofemission from 220 to 750 nm) calibratedagainst a white standard of barium sulphate,following Osorio and Ham (2002) and Bleiweiss(2004a, 2004b, 2005). Before measuring eachspecimen, a new calibration was done to correctfor possible shifts in the performance of thespectrophotometer. Plumage was illuminatedand reflected light collected at 45u to the surfaceand from the proximal end of the feather. Thisprocedure was adopted to avoid the effect ofspecular reflectance that can result when thefeather is illuminated and reflected light col-lected perpendicular to its surface (Andersson1996). The probe was mounted in a prismaticprobe holder that was held (not pressed) overthe selected region of the study skin. Thediameter of the circular region illuminated andmeasured was approximately 6 mm, and thedistance between the probe and the plumagewas 23 mm. The spectrophotometer resolutionwas 0.35 nm. Each spectrum was the average ofthree readings with an integration time of100 msec. Boxcar smoothing was not per-formed. Measurements were done blind to theseason and year of capture of the specimens.

STATISTICAL ANALYSES

The original variables included in this studywere median reflectance values for 5 nm binsfrom 340 to 700 nm for each individual.Reflectance values below 340 nm were excludedbecause of considerable noise at these wave-lengths. For each spectrum we calculated eightvariables (Fig. 1), following Pryke et al. (2001).Brightness (R340–700, total reflectance) wascalculated as the sum of the reflectance from340 to 700 nm. We defined lRmin and lRmax asthe wavelengths of minimum and maximumreflectance in the visible part of the spectrum,respectively. Hue (lR50), the spectral locationof the reflectance band at long visible wave-lengths, was calculated as the value midwaybetween that of maximum (lRmax) and mini-mum reflectance (lRmin). Because carotenoidpigmentation can exhibit a bimodal peak of

FIGURE 1. Representative reflectance spectrum ofa red plumage patch of Sturnella, showing thedifferent variables extracted from the spectral data.Variables include R340–700 as the total reflectancefrom 340 to 700 nm, lRmin as the wavelength of thereflectance minimum, lRmax as the wavelength of thereflectance maximum, lRUVpeak as the wavelength ofthe reflectance maximum in the near-UV part of thespectrum, and lR50, calculated as the value midwaybetween the wavelength of maximum and minimumreflectance (lRmax – lRmin), which accounts for thehue (spectral location of the reflectance band atvisible wavelength). Two reflectance segments aredetermined by the wavelength of minimum reflec-tance (lRmin); one accounts for the reflectancebetween 340 nm and lRmin (R340-min), and theother for the reflectance between lRmin and 700 nm(Rmin-700). These two reflectance segments wereused in the calculation of Rcontrast as [(Rmin-700) –(R340-min)]/(R340–700). Vertical line at 400 nm indi-cates the boundary between the near-UV wave-lengths, from 340 to 400 nm (visible to birds), andthe visible wavelengths, from 400 to 700 nm (visibleto both humans and birds).

COLOR DIFFERENCES IN RED-BREASTED MEADOWLARKS 607

reflectance (Bleiweiss 2004b, 2005), we alsomeasured the wavelength of maximum reflec-tance between 340 nm and lRmin. This variablewas called lRUVpeak and was the wavelength ofmaximum reflectance in the near-UV region ofthe spectrum. The reflectance at wavelengthslower than lRmin (R340-min) and the reflectanceat wavelengths higher than lRmin (Rmin-700)were also measured and used to calculatereflectance contrast (Rcontrast, a surrogate mea-sure of chroma) as the relative differencebetween these two nonoverlapping spectralsegments using the formula [(Rmin-700) –(R340-min)]/(R340–700). We used these variablesin separate two-way ANCOVAs, with speciesand season (breeding versus nonbreeding) asmain factors and year of capture as a covariateto control for possible effects of plumage fadingwith time since capture (Endler and Thery 1996,McNaught and Owens 2002). Although wemeasured eight variables, only five of them(R340–700, lRmin, lR50, lRUVpeak, and Rcontrast)were used in the ANCOVAs, because the otherthree (lRmax, R340-min, and Rmin-700) were onlyobtained to calculate other variables.

All statistical analyses were performed usingStatistica version 6.0 (StatSoft 2001) and alltests were two-tailed as we made no a prioripredictions as to which species would havehigher values for any of the variables measured.ANCOVAs were followed by Scheffe contrastsfor those variables that differed significantlyamong species. Because we made multiplecomparisons using eight different variables fordescribing each body region, we used a Bonfer-roni-corrected a 5 0.006 to keep the global typeI error smaller than 5%.

VISUAL MODELING

We evaluated color differentiation betweenSturnella species for each feather patch, andbetween the breeding and nonbreeding seasonswithin each species, using the Vorobyev andOsorio (1998) color discrimination model. Thismodel calculates distance in avian color space(DS), defined by the quantum catches of eachreceptor type in the avian retina. Thus, DS fora given feather patch represents the relativedifference in color between species (or seasons)with respect to the avian visual system.

To calculate DS for each feather patchcomparison, we first calculated the input toeach avian cone cell type i of the average color

among individuals for each feather patch, usingthe following equation (equation 1 in Vorobyevet al. 1998):

Qi ~

ðl

Ri lð ÞS lð ÞI lð Þdl, ð1Þ

where l denotes wavelength, Ri(l) is the spectralsensitivity of cone cell type i (data taken fromthe Blue Tit [Parus caeruleus]; Hart et al. 2000),S(l) is the reflectance spectrum of a givenfeather patch, I(l) is the irradiance spectrumentering the eye, and integration is over 340–700 nm. Spectral sensitivity data, Ri(l), are notavailable for Sturnella species, therefore datafrom the Blue Tit were used as an approxima-tion. These data likely provide a good estimatefor other species, as passerine visual pigmentcharacteristics (and thus cone cell sensitivities)are highly conserved over much of the visualrange (Hart 2001). Because we were notstudying the effects of viewing homologousfeather patches in different light environments,we set I(l) 5 1 for all calculations.

The signal of each receptor type, fi, isproportional to the natural logarithm of therespective quantum catch (Qi from equation 1),which is normalized against an adapting back-ground (see equation 2 and 3 in Vorobyev et al.[1998]). The distance (DS) between each ho-mologous feather patch was then calculatedusing equation 8 in Vorobyev et al. (1998):

DSð Þ2~ v1v2ð Þ2 Df4 { Df3ð Þ2h

z v1v3ð Þ2 Df4 { Df2ð Þ2


z v2v3ð Þ2 Df4{Df1ð Þ2


z v3v4ð Þ2 Df2 { Df1ð Þ2i

7 v1v2v3ð Þ2h

z v1v2 v4ð Þ2

z v1v3 v4ð Þ2

z v2v3 v4ð Þ2i; ð2Þ

where vi is the noise-to-signal ratio (Weberfraction) for receptor type i. We used Vorobyev


et al.’s (1998) estimate of the Weber fraction forthe long-wave sensitive cone (LWS), v4 5 0.05,which is based on an empirical estimate ofbehavioral data from the Pekin Robin (Leio-thrix lutea). Relative values of vi are defined bythe relative proportions of receptor types in theeye, thus the remaining vi were derived (seeequation 10 in Vorobyev et al. [1998]) fromestimates of the relative numbers of receptorcell types, gi, based on anatomical data fromthe Blue Tit (Hart et al. 2000). Photoreceptortype proportions vary among closely relatedbird species, possibly in relation to differencesin visual ecology (Hart 2001). Thus, werecalculated all DS with Weber fractions de-rived from cone cell proportions of EuropeanStarlings (Sturnus vulgaris) and EuropeanBlackbirds (Turdus merula), species with argu-ably different visual ecologies compared to theBlue Tit (Hart 2001). Although DS values didvary when calculated from the three differentratios of photoreceptor types of these threespecies, the magnitude of these differenceschanged none of our conclusions regardinginterspecific and intraspecific color differentia-tion. Thus, all values reported and discussed aretaken from the calculations based on thephotoreceptor type ratios of the Blue Tit (seealso Siddiqi et al. 2004, Eaton 2005).

The units of DS from equation 2 are jnd (justnoticeable differences), where 1.0 jnd is thethreshold value for discrimination of colors.Thus, DS values ,1.0 jnd indicate that colordifferences are most likely not visually discern-able to birds, while values .1.0 jnd indicatea magnitude of color difference above the visualdiscriminatory abilities of birds (Vorobyev et al.1998, Vorobyev 2003, Siddiqi et al. 2004, Eaton2005). Visual performance can vary amongspecies and viewing conditions (Vorobyev et al.1998), but, in general, at jnd 5 1.0, two colorsare barely distinguishable under ideal condi-tions, and as jnd increases two colors becomemore easily discernable under worsening view-ing conditions (Siddiqi et al. 2004).

RESULTS

The shape of the reflectance curves for all threespecies showed a band of low reflectance in therange of 420–550 nm, with a minimum between455 and 485 nm depending on the body regionand species considered. This determined twosegments of higher reflectance: a primary seg-

ment situated in the long wavelength part of thevisible spectrum that accounted for most of thetotal reflectance, and a secondary and smallerpeak in the near-UV and short wavelengthportion of the visible spectrum (Fig. 2). Max-imum reflectance in the primary and secondaryregions of higher reflectance varied between28% and 58% and 3% and 11% of the lightsource, respectively. Primary and secondaryregions of higher reflectance were higher inthe coverts than in the other body patches.Maximum reflectances in the visible and UVparts of the spectra, as well as reflectances inthe short (R340-min) and long (Rmin-700) wave-lengths, covaried positively (r . 0.53, P ,

0.05). The general shape and location of thereflectance minimum, together with the highcorrelation between primary and secondaryreflectance regions, suggests that these spectraare produced by carotenoid pigments, consis-tent with studies undertaken in other passerinespecies, for example tanagers (Bleiweiss 2004b,2005).

Two-way ANCOVAs performed on the in-dividual variables of the spectra and using yearof capture as a covariate (test of parallelism, Fs# 3.4, P $ 0.04, nonsignificant after Bonferronicorrection), followed by Scheffe contrasts forthose variables that differed significativelyamong species, revealed the following patterns(Table 1, Fig. 2): 1) S. loyca and S. defilippiihad brighter (higher R340–700) coverts (P 5

0.001) and belly (P # 0.01) than S. superciliaris,2) S. loyca had a higher lRmin than S. super-ciliaris in the coverts (P 5 0.004), whiledifferences in the throat were significant onlyduring the nonbreeding season (P 5 0.03), 3) S.defilippii and S. loyca typically had higherreflectance in the short wavelength segment ofthe spectrum. This resulted in a lower Rcontrast

for S. defilippii and S. loyca than for S.superciliaris (Rcontrast also tended to be lowerfor S. defilippii than for S. loyca). In particular,S. defilippii had lower Rcontrast than the othertwo species in the belly and chest (P # 0.005 inall cases). S. defilippii also showed a lowerRcontrast in the throat than S. superciliaris (P 5

0.006), but only in the nonbreeding season.Finally, in the coverts S. loyca had a lowerRcontrast than S. defilippii (P , 0.001), and bothspecies had lower Rcontrast than S. superciliaris(P , 0.001), and 4) The secondary segment ofreflectance was centered on a lower lRUVpeak in


FIGURE 2. Reflectance spectra for the plumage of males of Sturnella superciliaris (n 5 12), S. defilippii (n 516, except for throat where n 5 8), and S. loyca (n 5 15), in the breeding and nonbreeding seasons (B and NB,respectively). Each spectrum represents the mean reflectance 6 SE (error bars). Note the different y-axis scalefor the coverts graphs. These spectra show significant color differences among species and between seasons.


S. defilippii and S. loyca than in S. superciliaris(and it tended to be lower in S. defilippii than inS. loyca). In particular, S. defilippii exhibiteda lower lRUVpeak than S. superciliaris year-roundin the coverts (P , 0.001) and belly (P , 0.001),but only during the nonbreeding season in thethroat (P 5 0.04) and chest (P , 0.001). In turn,S. loyca showed a lower lRUVpeak than S.superciliaris in the coverts (P , 0.001) all yearlong and in the throat during the nonbreedingseason (P 5 0.03), but a higher lRUVpeak than S.defilippii in the chest (P 5 0.005).

Comparisons between seasons (Table 2,Fig. 2) showed that: 1) Total reflectance andRcontrast did not change for any of the species, 2)lRmin was higher in breeding than in non-breeding plumage for the belly in all threespecies, 3) lR50 was higher in breeding plumagein the throat for S. superciliaris (P 5 0.003) andin the chest for S. defilippii (P , 0.001), and 4)the lRUVpeak was lower in the breedingcompared to the nonbreeding season in thethroat (P 5 0.001), chest (P , 0.001), and belly(P 5 0.004) for S. superciliaris and in the bellyfor S. defilippii (P 5 0.004).

Quantification of the color differences be-tween Sturnella species and between plumagesin the breeding and nonbreeding seasons using

the perceptual model of Vorobyev and Osorio(1998) corroborated all plumage differencesdescribed above, with the exception of colordifferences between seasons in the coverts of S.defilippii (Table 3, 4). Most DS values ofinterspecific color differentiation and intraspe-cific seasonal color differentiation were consid-erably higher than the threshold for avianvisual discrimination. Notably, coloration ofthe coverts showed large differences amongspecies (Table 3) and, in general, S. superciliarisexhibited more change in coloration frombreeding to nonbreeding plumages (Table 4).This resulted in S. superciliaris differing morefrom S. defilippii in the nonbreeding than in thebreeding season in all plumage areas (repeatedmeasures Student t-test 5 3.7, P , 0.05), and inS. superciliaris showing larger differences in thenonbreeding than in the breeding season inrelation to S. loyca in all plumage areas but thechest.

DISCUSSION

PATTERNS OF COLOR VARIATION ANDTHEIR POSSIBLE CAUSES

There are striking differences in the plumage ofsympatric species of red-breasted meadowlarks

TABLE 1. Species differences in plumage spectral variables for Sturnella superciliaris, S. defilippii, and S.loyca in each body region (coverts, throat, chest, and belly). See Figure 1 for definitions and illustration of thespectral variables. Values represent means 6 SD. Statistics represent the main effect of species in two-wayANCOVAs, with species and season as main factors and year of capture as a covariate. The effect of season isshown in Table 2. Significant P-values after Bonferroni correction are denoted with an asterisk.

Variable Body region S. superciliaris S. defilippii S. loyca df F P

R340–700 Coverts 955 6 224 1317 6 243 1325 6 251 2, 37 11.6 , 0.001*Throat 797 6 217 1120 6 302 1062 6 310 2, 29 3.6 0.04Chest 730 6 160 937 6 183 900 6 144 2, 37 5.3 0.009Belly 724 6 174 963 6 209 947 6 165 2, 37 6.9 0.003*

lR50 Coverts 584.62 6 4.30 587.11 6 1.96 588.34 6 4.97 2, 37 4.8 0.01Throat 584.00 6 10.39 583.11 6 8.14 587.92 6 3.80 2, 29 3.7 0.04Chest 587.56 6 5.83 586.98 6 8.70 586.77 6 4.36 2, 37 1.3 0.28Belly 588.75 6 7.50 591.76 6 4.60 587.42 6 3.21 2, 37 2.3 0.12

lRmin Coverts 468.75 6 8.59 474.10 6 3.60 477.57 6 7.58 2, 37 11.6 0.001*Throat 470.01 6 19.92 469.36 6 15.77 475.88 6 7.04 2, 29 2.4 0.11Chest 477.04 6 9.19 474.66 6 17.56 473.25 6 8.67 2, 37 2.4 0.10Belly 477.50 6 15.23 484.33 6 8.94 476.93 6 1.80 2, 37 2.7 0.08

lRUVpeak Coverts 422.17 6 1.45 398.69 6 7.01 404.83 6 8.04 2, 37 42.2 , 0.001*Throat 407.08 6 11.85 400.59 6 6.00 407.51 6 8.92 2, 29 3.8 0.04Chest 418.35 6 21.43 402.17 6 12.90 416.57 6 6.07 2, 37 10.0 , 0.001*Belly 411.27 6 13.40 397.16 6 6.44 404.49 6 9.67 2, 37 9.6 , 0.001*

Rcontrast Coverts 0.90 6 0.04 0.82 6 0.05 0.71 6 0.06 2, 37 43.2 , 0.001*Throat 0.77 6 0.08 0.68 6 0.06 0.74 6 0.06 2, 29 5.5 0.01Chest 0.83 6 0.04 0.76 6 0.05 0.82 6 0.06 2, 37 5.7 0.007Belly 0.83 6 0.04 0.72 6 0.05 0.79 6 0.05 2, 37 15.9 , 0.001*


in both the UV and human-visible parts of thespectrum. These differences persist year-roundand include not only brightness but alsospectral shape. In particular, S. loyca and S.defilippii show brighter red patches and a higherreflectance in the UV region of the spectrumthan S. superciliaris. Short (1968), based onhuman visual perception, also reported differ-ences in the red ventral coloration of thesespecies, noting that S. defilippii is redder thanthe pinker S. loyca. Our finding of higher valuesfor hue in the former species supports hisconclusion, although the differences were non-significant.

In addition to interspecific differences, wefound significant differences between seasonalplumages within species, especially in S. super-ciliaris and S. defilippii. Short (1968) reasonedthat the seasonal change in S. loyca and S.defilippii plumage color was due to fading of thebrown tips of their red feathers. The sameprocess may also account for the change in huein the red plumage patches that we measured(as estimated by longer lR50 and lRmin).Abrasion of the brown tips would be predictedto expose the red-pigmented bases of thefeathers, shifting the spectrum toward longer

visible and shorter, near-UV, wavelengths. Atest of abrasion as a cause of seasonal change incolor in Sturnella plumages will require a mi-croanatomical study of their feathers (Wil-loughby et al. 2002).

The plumage color differences among Stur-nella species that we have documented hereshould be visually detectable by these birds,because the color distances in avian perceptualspace largely exceeded the threshold for colordiscrimination, especially in the coverts, sug-gesting that this feather patch, which isconspicuously exhibited during flight displaysby S. superciliaris and S. defilippii, may be ofgreat importance as a species-specific marker.We also found that changes in color betweenbreeding and nonbreeding plumages wereabove the discrimination threshold (Vorobyevet al. 1998, Vorobyev 2003, Siddiqi et al. 2004,Eaton 2005). These patterns of seasonal changein color are a consequence of the pattern ofmolt and wear of the feathers. Sturnella speciesonly have one complete postreproductive molt(Jaramillo and Burke 1999). This molt results inlarger differences in avian visual color spacebetween S. superciliaris and the other twospecies than between S. defilippii and S. loyca.

TABLE 2. Differences in plumage spectral variables between the nonbreeding and breeding seasons forSturnella superciliaris, S. defilippii, and S. loyca for each body region (coverts, throat, chest, and belly). SeeFigure 1 for definitions and illustration of the spectral variables. Values represent means 6 SD. Statisticsrepresent the main effect of season in two-way ANCOVAs, with species and season as main factors and year ofcapture as a covariate. The effect of species is shown in Table 1. Significant P-values after Bonferronicorrection are denoted with an asterisk.

Variable Body region Nonbreeding season Breeding season df F P

R340–700 Coverts 1245 6 292 1200 6 289 1, 37 0.0 0.83Throat 1057 6 280 916 6 319 1, 29 3.6 0.13Chest 849 6 183 879 6 184 1, 37 0.1 0.79Belly 878 6 203 900 6 216 1, 37 0.0 0.96

lR50 Coverts 586.44 6 5.20 587.14 6 3.13 1, 37 4.8 0.01Throat 581.65 6 7.33 589.09 6 6.27 1, 29 16.6 , 0.001*Chest 582.68 6 6.23 590.23 6 4.67 1, 37 27.8 , 0.001*Belly 587.13 6 5.78 591.04 6 4.59 1, 37 7.8 0.008

lRmin Coverts 472.54 6 10.19 474.74 6 10.19 1, 37 8.2 0.001*Throat 465.17 6 14.16 479.19 6 11.37 1, 29 14.6 0.001*Chest 465.03 6 12.18 481.89 6 7.10 1, 37 53.4 , 0.001*Belly 474.76 6 11.04 483.51 6 7.74 1, 37 12.7 0.001*

lRUVpeak Coverts 407.50 6 11.31 407.30 6 11.94 1, 37 0.0 0.86Throat 408.69 6 9.67 403.03 6 9.12 1, 29 11.7 0.002*Chest 421.16 6 16.72 404.90 6 10.97 1, 37 28.9 , 0.001*Belly 408.06 6 11.93 400.48 6 9.72 1, 37 9.3 , 0.004*

Rcontrast Coverts 0.80 6 0.11 0.81 6 0.07 1, 37 0.1 0.74Throat 0.74 6 0.09 0.74 6 0.06 1, 29 0.3 0.58Chest 0.82 6 0.05 0.79 6 0.06 1, 37 4.3 0.05Belly 0.79 6 0.07 0.77 6 0.06 1, 37 2.4 0.13


Subsequently, feather wear of S. superciliarisproduces a greater similarity to the plumages ofthe other two species (in particular to S.defilippii), a phenomenon clearly noticeable inthe breeding season. This result is interesting,because, if color signals are important forconspecific mate choice, plumage differencesshould be larger in the breeding season.However we cannot exclude other possibleexplanations, such as color convergence facili-tating interspecific aggression and promotinginterspecific territoriality, which has been docu-mented between S. superciliaris and S. defilippii(Gochfeld 1979).

Several studies have shown that the spectralproperties of plumage in museum specimensmay change as a consequence of plumagefading with time (Endler and Thery 1996,Hausmann et al. 2003). However, the resultspresented here regarding interspecific andseasonal variation in red plumage patchesshould not be biased by this phenomenon, asyear of capture was included as a covariate inthe statistical analyses. A recent study byMcNett and Marchetti (2005) showed differ-ences in carotenoid patches between living and

museum specimens. They found that both hueand chroma decreased in the spectra of museumspecimens as a consequence of changes in colorat shorter wavelengths. This result suggests thatthe plumage of museum specimens may beunrepresentative of natural avian colors, espe-cially for variables in the UV region. Obviously,we must be aware of this possibility; however, itis possible that differences between live andmuseum specimens might also be explained bydifferences in measurement error between livebirds and study skins (e.g., because of subtlemovements of live birds while taking spectralreadings, or the arrangement of the feathers oflive birds in the hand). These possibilitiesrequire further investigation across all studiesof avian coloration.

In general, noniridescent red feathers resultfrom carotenoid pigmentation (Gray 1996,Mahler et al. 2003, Bleiweiss 2005). Carotenoidpigments have a distinctive absorption bandbetween 400 and 500 nm (Hudon and Brush1992, Ryan et al. 1994, Shawkey and Hill 2005).This results in a bimodal reflectance spectrumwith a primary band of reflectance in the longwavelength region and a secondary one in thenear-UV portion of the spectrum, stronglyreminiscent of the patterns we obtained forsome red-breasted meadowlarks. Thus, pendingdetailed biochemical study, we tentativelyassume that the red plumage patches in

TABLE 3. Color differentiation (DS) betweenSturnella superciliaris, S. defilippii, and S. loycaduring the breeding and nonbreeding seasons. DSrepresents the relative distance between species foreach body region in avian perceptual color space. Theunits of DS are jnd (just noticeable differences), with1.0 jnd being the threshold value for discriminationof colors.

Body regionSpecies

Breedingseason DS

Nonbreedingseason DS

Coverts

S. defilippii vs. S. loyca 7.1 9.4S. defilippii vs. S. superciliaris 9.7 17.6S. loyca vs. S. superciliaris 13.4 23.6

Throat


Chest


Belly


TABLE 4. Plumage color differentiation (DS)between the breeding and nonbreeding seasons,within Sturnella superciliaris, S. defilippii, and S.loyca. DS represents the relative distance between thebreeding and nonbreeding season for each bodyregion in avian perceptual color space. The units ofDS are jnd (just noticeable differences), with 1.0 jndbeing the threshold value for discrimination of colors.

SpeciesBodyregion

Breeding vs.nonbreeding season, DS

S. superciliaris Coverts 8.9Throat 8.4Chest 10.7Belly 11.1

S. defilippii Coverts 1.0Throat 6.9Chest 10.0Belly 5.1

S. loyca Coverts 2.4Throat 3.5Chest 2.9Belly 1.1


meadowlarks are indeed carotenoid based. Thepresence and magnitude of the secondaryregion of reflectance varied between species; itwas evident in S. loyca and S. defilippii, butalmost absent in S. superciliaris. Such interspe-cific variation has also been detected in tanagers(Bleiweiss 2004b, 2005). Variation in thepresence of the secondary segment of reflec-tance may be caused by several nonexclusivefactors, including: 1) the type of carotenoidpigment present and its concentration, withhigher concentration reducing the secondaryUV peak; 2) the presence of small quantities ofmelanins, pigments that absorb light differen-tially in the near-UV part of the spectrum; and3) subtle modifications in the microstructure offeathers that change the amount of shortwavelengths reflected (see Bleiweiss [2004b]and Andersson and Prager [2006] for a detaileddiscussion of these possibilities). We do not yetknow which of these explanations accounts forvariation in near-UV reflectance among thespecies we examined, but we expect a higherconcentration of carotenoids in S. superciliariscompared to S. defilippii and S. loyca, and thepresence of more melanin in the formercompared to the latter two species. This secondprediction is supported by the observation thatcolors other than red in the plumage of S.superciliaris are notably darker than in S.defilippii and S. loyca (with the exception ofthe white brow).

DO COLOR DIFFERENCES IN RED-BREASTED MEADOWLARKS PLAY A ROLEIN SPECIES RECOGNITION?

Obviously, the question of whether interspe-cific variation in color among closely relatedsympatric species of meadowlarks plays a rolein reproductive isolation cannot be answeredjust by quantifying differences in reflectancespectra. Such a conclusion will require dis-crimination and preference tests. However, thefact that almost no interspecific hybrids orhybrid pairings have been detected to date(Gochfeld 1975) is indirect evidence suggestingthat red-breasted meadowlarks can efficientlydiscriminate between species. The absence ofinterspecific territoriality and aggression be-tween S. loyca and the other two species,despite territory overlap, lends further supportto this hypothesis (Gochfeld 1975, 1979).However, S. superciliaris and S. defilippii main-

tain nonoverlapping territories by means ofinterspecific aggression. This aggression hasbeen attributed to their similarities in bothplumage and behavior, which are not a conse-quence of character convergence but rather ofa close phylogenetic relationship (Gochfeld1979).

Color differences between closely relatedspecies of meadowlarks might have evolved asa consequence of several nonexclusive pro-cesses, such as habitat differences in lightenvironment, sexual selection, or simply ran-dom drift along independent evolutionaryhistories. Given the degree of similarity in theuse of open habitats by these three species,together with their degree of sympatry andsyntopy, the former explanation is likely nottenable. In contrast, sexual selection is consid-ered a potent cause of species divergence(Andersson 1994, Panhuis et al. 2001) and islikely to have operated in Sturnella given theirhigh degree of sexual dimorphism in color, size,and behavior (Short 1968, Gochfeld 1975, 1979,Jaramillo and Burke 1999). Because of this, wetentatively interpret interspecific differences inred-colored patches among Sturnella species asthe result of sexual selection. However, wecannot exclude the possibility that selection forspecies recognition has played a role in shapinginterspecific differences in color. The presenceof reproductive character displacement, a pat-tern consisting of an increase in differencesbetween sympatric compared to allopatricpopulations of different species, would supportthis hypothesis (Panhuis et al. 2001). Sucha pattern has been shown in the plumage ofa few species of birds (Andersson 1994). We didnot plan to test for reproductive characterdisplacement, because most specimens we mea-sured came from areas of sympatry, and smallsample sizes precluded comparisons betweensympatric and allopatric populations. In addi-tion, detecting such a pattern would be chal-lenging, because: 1) the distribution of S.

defilippii is completely within that of S. super-

ciliaris, and 2) many sites are currently occupiedonly by S. superciliaris, either due to rangeexpansion or to the very recent reduction in thedistribution of S. defilippii (Tubaro and Gabelli1999). Moreover, S. loyca is a recent invader ofthe southern pampas (Gochfeld 1975), thus itssympatry with the other species of red-breasted


meadowlarks may be too recent to have detect-able evolutionary consequences.

Of course, red-breasted meadowlarks notonly differ in their red-colored patches, asshown in our study, but also in song and callrepertoires and associated behaviors (e.g., flightdisplays; Gochfeld 1975). Future work shouldassess the relative importance of interspecificvariation in acoustic and visual signals forspecies recognition and male–male or male–female interactions with the simultaneous use ofplaybacks and decoys with known reflectances.

ACKNOWLEDGMENTS

We thank Gabriela Ibarguchi for bringing thespectrophotometer from Canada to Argentina, andtwo anonymous reviewers for their valuable sugges-tions on an earlier version of the manuscript. Thiswork was supported by the Consejo Nacional deInvestigaciones Cientıficas y Tecnicas, Argentina(grant PEI 6001 to PLT), and Natural Sciences andEngineering Research Council Canada Foundationfor Innovation and Ontario Institute of Technologygrants (to SCL).

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COLOR DIFFERENCES AMONG CLOSELY RELATED SPECIES OF RED-BREASTED MEADOWLARKS (STURNELLA

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